NanoDrop Micro-UV/Vis Spectrophotometer

NanoDrop Eight
User Guide

M020 NanoDrop Eight UG Revision Date April 2023

© 2023 Thermo Fisher Scientific Inc. All rights reserved.

For U.S. Technical Support, please contact: For International Support, please contact:

Thermo Fisher Scientific http://www.thermofisher.com/

3411 Silverside Road NanoDropSupport
Tatnall Building, Suite 100 Contact your local distributor. For contact
Wilmington, DE 19810 U.S.A. information go to:
Telephone: 302 479 7707 http://www.thermofisher.com/
Toll Free: 1 877 724 7690 (U.S. & Canada only) NanoDropDistributors
E-mail: [email protected]

Thermo Fisher Scientific Inc. provides this document to its customers with a product purchase to
use in the product operation. This document is copyright protected and any reproduction of the
whole or any part of this document is strictly prohibited, except with the written authorization of
Thermo Fisher Scientific Inc.

The contents of this document are subject to change without notice. All technical information in this
document is for reference purposes only. System configurations and specifications in this document
supersede all previous information received by the purchaser.

Thermo Fisher Scientific Inc. makes no representations that this document is complete, accurate or
error-free and assumes no responsibility and will not be liable for any errors, omissions, damage or
loss that might result from any use of this document, even if the information in the document is
followed properly.

This document is not part of any sales contract between Thermo Fisher Scientific Inc. and a
purchaser. This document shall in no way govern or modify any Terms and Conditions of Sale,
which Terms and Conditions of Sale shall govern all conflicting information between the two
documents.

For Research Use Only. This instrument or accessory is not a medical device and is not intended to
be used for the prevention, diagnosis, treatment or cure of disease.

WARNING Avoid an explosion or fire hazard. This instrument or accessory is not

designed for use in an explosive atmosphere.
 C

Contents
Chapter 1 About the Spectrophotometer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7
Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8
USB-B port . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8
Accessories. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9
PR-1 Pedestal Reconditioning Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . .9
PV-8 Performance Verification Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . .9
Instrument Detection Limits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10

Chapter 2 Instrument Set up. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11

Register Your Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11
Computer Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11
Update Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12
Technical Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12
For U.S./Canada Support, please contact: . . . . . . . . . . . . . . . . . . . .12
For International Support, please contact: . . . . . . . . . . . . . . . . . . . . .12

Chapter 3 Application Measurement Ranges . . . . . . . . . . . . . . . . . . . . . . . . . .13

Detection Limits for All Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . .13
Detection limits for pre-defined dyes . . . . . . . . . . . . . . . . . . . . . . . . .15

Chapter 4 Nucleic Acid Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17

Measure dsDNA, ssDNA or RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17
Measure dsDNA, ssDNA or RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . .17
Best practices for nucleic acid measurements . . . . . . . . . . . . . . . . . .19
Nucleic Acid Reported Results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21
Settings for Nucleic Acid Measurements . . . . . . . . . . . . . . . . . . . . . .22
Calculations for Nucleic Acid Measurements . . . . . . . . . . . . . . . . . . .23
Measure Microarray. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .27
Measure Microarray Samples. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .27
Microarray Reported Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .30
Settings for Microarray Measurements . . . . . . . . . . . . . . . . . . . . . . . .31
Calculations for Microarray Measurements. . . . . . . . . . . . . . . . . . . . .35

Thermo Scientific NanoDrop Eight User Guide iii

Contents

Measure using a Custom Factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .39

Measure Nucleic Acid using a Custom Factor . . . . . . . . . . . . . . . . . .39
Custom Factor Reported Results . . . . . . . . . . . . . . . . . . . . . . . . . . . .41
Settings for Nucleic Acid Measurements using a Custom Factor . . . .42
Detection Limits for Nucleic Acid Measurements using a Custom
Factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .43
Measure Oligo DNA or Oligo RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . .45
Measure Oligo DNA or Oligo RNA . . . . . . . . . . . . . . . . . . . . . . . . . . .45
Oligo Reported Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .47
Settings for Oligo DNA and Oligo RNA Measurements . . . . . . . . . . . .49
Detection Limits for Oligo DNA and Oligo RNA Measurements. . . . . .50
Calculations for Oligo DNA and Oligo RNA Measurements. . . . . . . . .51

Chapter 5 Protein Applications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .55

Measure Protein A280. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .55
Measure Protein Concentration at A280 . . . . . . . . . . . . . . . . . . . . . .55
Best practices for protein measurements . . . . . . . . . . . . . . . . . . . . . .57
Protein A280 Reported Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . .59
Settings for Protein A280 Measurements . . . . . . . . . . . . . . . . . . . . . .60
Protein editor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .63
Detection Limits for Protein A280 Measurements. . . . . . . . . . . . . . . .65
Calculations for Protein A280 Measurements. . . . . . . . . . . . . . . . . . .65
Measure Protein A205. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .69
Measure Protein Concentration at A205 . . . . . . . . . . . . . . . . . . . . . .69
Protein A205 Reported Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . .71
Settings for Protein A205 Measurements . . . . . . . . . . . . . . . . . . . . . .72
Calculations for Protein A205 Measurements. . . . . . . . . . . . . . . . . . .73
Measure Proteins and Labels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .75
Measure Labeled Protein Samples. . . . . . . . . . . . . . . . . . . . . . . . . . .75
Proteins & Labels Reported Results . . . . . . . . . . . . . . . . . . . . . . . . . .77
Settings for Proteins and Labels Measurements. . . . . . . . . . . . . . . . .78
Detection Limits for Proteins and Labels Measurements . . . . . . . . . .80
Calculations for Proteins and Labels Measurements . . . . . . . . . . . . .81
Measure Protein BCA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .83
Measure Total Protein Concentration . . . . . . . . . . . . . . . . . . . . . . . . .83
Protein BCA Reported Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .90
Settings for Protein BCA Measurements . . . . . . . . . . . . . . . . . . . . . .92
Measure Protein Bradford . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .95
Measure Total Protein Concentration . . . . . . . . . . . . . . . . . . . . . . . . .95
Protein Bradford Reported Results . . . . . . . . . . . . . . . . . . . . . . . . . .98
Settings for Protein Bradford Measurements . . . . . . . . . . . . . . . . . .100

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 Contents

Measure Protein Lowry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .103

Measure Total Protein Concentration . . . . . . . . . . . . . . . . . . . . . . . .103
To measure Protein Lowry standards and samples . . . . . . . . . . . . .104
Protein Lowry Reported Results. . . . . . . . . . . . . . . . . . . . . . . . . . . .106
Settings for Protein Lowry Measurements . . . . . . . . . . . . . . . . . . . .108
Measure Protein Pierce 660 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .111
Measure Total Protein Concentration . . . . . . . . . . . . . . . . . . . . . . . .111
To measure Protein Pierce 660 standards and samples. . . . . . . . . .112
Protein Pierce 660 Reported Results . . . . . . . . . . . . . . . . . . . . . . . .114
Settings for Protein Pierce 660 Measurements. . . . . . . . . . . . . . . . .116

Chapter 6 Measure OD600 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .119

Measure OD600 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .119
To measure OD600 samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .121
OD600 Reported Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .123
Settings for OD600 Measurements . . . . . . . . . . . . . . . . . . . . . . . . .125
Calculations for OD600 Measurements . . . . . . . . . . . . . . . . . . . . . .127

Chapter 7 Custom Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .129

Measure UV-Vis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .130
Measure UV-Vis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .130
Best practices for UV-Vis measurements . . . . . . . . . . . . . . . . . . . . .131
UV-Vis Reported Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .132
Settings for UV-Vis Measurements. . . . . . . . . . . . . . . . . . . . . . . . . .134
Measure Custom. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .135
Measure using a Custom Method . . . . . . . . . . . . . . . . . . . . . . . . . .135
Delete Custom Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .136
Custom Method Reported Results. . . . . . . . . . . . . . . . . . . . . . . . . .137
Manage Custom Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .138

Chapter 8 Learning Center . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .149

Micro-Volume Sampling—How it Works . . . . . . . . . . . . . . . . . . . . . . .150
Set Up the Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .152
Connect Power . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .152
Connect to a Computer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .152
Operating Specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .153
Measure a Micro-Volume Sample . . . . . . . . . . . . . . . . . . . . . . . . . . . .154
Best practices for micro-volume measurements. . . . . . . . . . . . . . . .154
Recommended sample volumes . . . . . . . . . . . . . . . . . . . . . . . . . . .155
Sample Modes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .158
Module startup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .158
Sample Plate Map . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .158
Prepare Samples and Blanks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .158
Preparing Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .159
Run a Blanking Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .162

Thermo Scientific NanoDrop Eight User Guide v

Contents

Basic Instrument Operations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .164

NanoDrop Eight Home Screen. . . . . . . . . . . . . . . . . . . . . . . . . . . . .164
NanoDrop Eight Measurement Screens . . . . . . . . . . . . . . . . . . . . . .167
Measurement Screen Display Options . . . . . . . . . . . . . . . . . . . . . . .168
NanoDrop Eight General Operations . . . . . . . . . . . . . . . . . . . . . . . .181
Acclaro Sample Intelligence. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .189
Activate Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .189
View Acclaro Sample Intelligence Information. . . . . . . . . . . . . . . . . .189
Contaminant Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .191
On-Demand Technical Support . . . . . . . . . . . . . . . . . . . . . . . . . . . .194
Instrument Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .195
System Settings. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .195
Preferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .198
Dye Editor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .198
Protein Editor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .198
Measurement Screen Display Options . . . . . . . . . . . . . . . . . . . . . . . . .198

Chapter 9 Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .201

Maintenance Schedule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .202
Daily Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .202
Periodic Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .202
Every 6 Months . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .202
Maintaining the Pedestals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .203
Cleaning the Pedestals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .203
Reconditioning the Pedestals. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .205
Decontaminating the Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . .207
Instrument Diagnostics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .209
Intensity Check . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .210
Performance Verification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .212

Chapter 10 Safety and Operating Precautions . . . . . . . . . . . . . . . . . . . . . . . . .217

Operating Precautions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .218
Safety Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .219
Safety and Special Notices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .219
When the System Arrives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .221
Lifting or Moving the Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . .221
Electrical Requirements and Safety . . . . . . . . . . . . . . . . . . . . . . . . .222
Power Cords . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .222
Fire Safety and Burn Hazards . . . . . . . . . . . . . . . . . . . . . . . . . . . . .223
Optical Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .224
Hazardous Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .224

vi NanoDrop Eight User Guide Thermo Scientific

 1

About the Spectrophotometer

NanoDrop Eight Spectrophotometer

Arm

Pedestal

Well tray sample

indicator array

Note Locate the instrument away from air vents and exhaust fans to minimize
evaporation

The Thermo Scientific™ NanoDrop™ Eight is a UV-Visible spectrophotometer

developed for micro-volume analysis of a wide variety of analytes. The patented
sample retention system enables the measurement of highly concentrated samples
without the need for dilutions. The instrument accurately measures up to 8 individual
1 µl samples in one measurement cycle. The software allows the user to measure
samples using either the full 8-channel mode or a convenient single sample mode.

The NanoDrop Eight software is installed on a local PC used to control the

instrument and view data.

Thermo Scientific NanoDrop Eight User Guide 7

1 About the Spectrophotometer
Features

Note Before operating a NanoDrop Eight instrument, please read the safety and
operating precautions and then follow their recommendations when using the
instrument.

Features
TheNanoDrop Eight spectrophotometer features the patented micro-volume sample
retention system.

USB-B port
Connect the NanoDrop Eight to your PC via USB to operate the instrument using the
PC software.

8 NanoDrop Eight User Guide Thermo Scientific

 1 About the Spectrophotometer
Accessories

Accessories
This section lists the accessories included for use with the NanoDrop Eight.

PR-1 Pedestal Reconditioning Kit

Specially formulated conditioning compound
that can be applied to the pedestals to restore
them to a hydrophobic state (required to
achieve adequate surface tension for accurate
sample measurements). The kit includes
conditioning compound and applicators. For
more information, see Reconditioning the
Pedestals.

PV-8 Performance Verification Kit

Kit containing plastic consumables and liquid photometric standard used to check
instrument performance. For more information, see Performance Verification.

Thermo Scientific NanoDrop Eight User Guide 9

1 About the Spectrophotometer
Instrument Detection Limits

Instrument Detection Limits

Upper Detection Limit

Pathlength (mm) (10 mm Equivalent Absorbance)
1.0 12.5
0.2 90
0.1 200

10 NanoDrop Eight User Guide Thermo Scientific

 2

Instrument Set up

Register Your Instrument

Register your instrument to receive e-mail updates on software and accessories for
the NanoDrop Eight instrument. An Internet connection is required for registration.

To register your instrument

From any PC that is connected to the Internet, use any web browser to navigate to
www.thermofisher.com/nanodropsw.

On the website, locate the Register Your Instrument button, and follow the
instructions to register the instrument.

Computer Requirements
Required Windows Operating System
• Windows 10 Enterprise or Professional, build 1607 or greater

Minimum hardware configuration

• 2.0 GHz dual-core processor enabled
• 4 GB RAM with system managed memory enabled
• 5 GB available on drive C
• Display resolution 1366 × 768

Recommended hardware configuration

• 2.33 GHz 4-core processor enabled (or greater)
• 8 GB RAM with system managed memory enabled (or greater)
• 200 GB available on drive C (or greater)

Thermo Scientific NanoDrop Eight User Guide 11

2 Instrument Set up
Update Software

Update Software
Quickly and easily download and install the latest NanoDrop Eight software and
release notes from our website. Follow the steps to update or upgrade the
NanoDrop Eight software on a personal computer (PC). An Internet connection is
required to download software. Navigate to www.thermofisher.com/nanodropsw
and click on the NanoDrop Eight software tab. Follow the Instrument Software
Download instructions.

To install or update NanoDrop Eight software on a PC

1. Insert the USB flash drive containing the installer software into an available USB
port on your PC, or open the installation folder downloaded from the internet.
2. Launch Start.exe and click the Software button. The software installer will
run.

Technical Support
For U.S./Canada Support, please contact:
Thermo Fisher Scientific
3411 Silverside Road
Tatnall Building, Suite 100
Wilmington, DE 19810 U.S.A.

Telephone: 302 479 7707

Toll Free: 1 877 724 7690 (U.S. & Canada only)
Fax: 302 792 7155
E-mail: [email protected]
Website: www.thermofisher.com/nanodrop

For International Support, please contact:

Contact your local distributor. For contact information go to:

http://www.thermofisher.com/NanoDropDistributors

If you are experiencing an issue with your system, refer to the troubleshooting
information. If the issue persists, contact us. If you are outside the U.S.A. and
Canada, please contact your local distributor.

If your instrument requires maintenance or repair, contact us or your local distributor.

12 NanoDrop Eight User Guide Thermo Scientific

 3

Application Measurement Ranges

Detection Limits for All Applications

Note Detection limits provided in the following tables are approximate and apply
to micro-volume measurements only; they are based on the instrument’s
photometric absorbance range (10 mm equivalent) of 0.04–200 A.

Lower Detection Upper Detection

Sample Type Typical Reproducibilitya
Limit Limit
dsDNA 2.0 ng/µL 10,000 ng/µL ±2.0 ng/µL for sample concentrations
between 2.0 and 100 ng/µL samples;
±2% for samples >100 ng/µL
RNA 1.6 ng/µL 8,000 ng/µL ±2.0 ng/µL for sample concentrations
between 2.0 and 100 ng/µL samples;
±2% for samples >100 ng/µL
DNA Microarray 1.3 ng/µL 495 ng/µL ±2.0 ng/µL for sample concentrations
(ssDNA) between 2.0 and 100 ng/µL samples;
±2% for samples >100 ng/µL

Thermo Scientific NanoDrop Eight User Guide 13

3 Application Measurement Ranges
Detection Limits for All Applications

Lower Detection Upper Detection

Sample Type Typical Reproducibilitya
Limit Limit
Purified BSA by 0.06 mg/mL 300 mg/mL ±0.10 mg/mL (for 0.10–10 mg/mL
Protein A280 samples);
±2% for samples >10 mg/mL
IgG by Protein A280 0.03 mg/mL 145 mg/mL
Purified BSA by 0.06 mg/mL 19 mg/mL ±0.10 mg/mL for 0.10–10 mg/mL
Proteins & Labels samples
Protein BCA 0.2 mg/mL (20:1 8.0 mg/mL 2% over entire range
reagent/sample 0.01 mg/mL over entire range
volume);

0.01 mg/mL (1:1

reagent/sample
volume)
Protein Lowry 0.2 mg/mL 4.0 mg/mL 2% over entire range
(pedestal) (pedestal)
Protein Bradford 100 µg/mL (50:1 8000 µg/mL ±25 µg/mL for 100–500 µg/mL samples
reagent/sample ±5% for 500–8000 µg/mL samples
volume)
±4 µg/mL for 15–50 µg/mL samples
15 µg/mL (1:1 100 µg/µL ±5% for 50–125 µg/mL samples
reagent/sample
volume)
Protein Pierce 660 50 µg/mL (15:1 2000 µg/mL ±3 µg/mL for 50–125 µg/mL samples
reagent/sample ±2% for samples > 125 µg/mL
volume)
±3 µg/mL for 25–125 µg/mL samples
25 µg/mL (7.5:1 1000 µg/mL ±2% for samples >125 µg/mL
reagent/sample
volume)
a Based on five replicates (SD=ng/µL; CV=%)

14 NanoDrop Eight User Guide Thermo Scientific

 3 Application Measurement Ranges
Detection Limits for All Applications

Detection limits for pre-defined dyes

Lower Detection Upper Detection
Sample Type Typical Reproducibilityb
Limit Limita
Cy3, Cy3.5, Alexa 0.2 pmol/µL 100 pmol/µL ±0.20 pmol/µL for sample
Fluor 555, Alexa Fluor concentrations between 0.20 and
660 4.0 pmol/µL;
±2% for samples >4.0 pmol/µL
Cy5, Cy5.5, Alexa 0.12 pmol/µL 60 pmol/µL ±0.12 pmol/µL for sample
Fluor 647 concentrations between 0.12 and
2.4 pmol/µL;
±2% for samples >2.4 pmol/µL
Alexa Fluor 488, Alexa 0.4 pmol/µL 215 pmol/µL ±0.40 pmol/µL for sample
Fluor 594 concentrations between 0.40 and
8.0 pmol/µL;
±2% for samples >8.0 pmol/µL
Alexa Fluor 546 0.3 pmol/µL 145 pmol/µL ±0.30 pmol/µL for sample
concentrations between 0.30 and
6.0 pmol/µL;
±2% for samples >6.0 pmol/µL
a Values are approximate
b Based on five replicates (SD=ng/µL; CV=%)

Thermo Scientific NanoDrop Eight User Guide 15

3 Application Measurement Ranges

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16 NanoDrop Eight User Guide Thermo Scientific

 4

Nucleic Acid Applications

Measure dsDNA, ssDNA or RNA

Measures the concentration of purified

dsDNA, ssDNA or RNA samples that
absorb at 260 nm.

Measure dsDNA, ssDNA or RNA

Reported Results

Settings

Detection Limits

Calculations

Measure dsDNA, ssDNA or RNA

Use the dsDNA, ssDNA and RNA applications to quantify purified double-stranded
(ds) or RNA samples. These applications report nucleic acid concentration and two
absorbance ratios (A260/A280 and A260/A230). A single-point baseline correction
can also be used.

To measure dsDNA, ssDNA or RNA samples

NOTICE
• Do not use a squirt or spray bottle on or near the instrument as liquids will
flow into the instrument and may cause permanent damage.
• Do not use hydrofluoric acid (HF) on the pedestals. Fluoride ions will
permanently damage the quartz fiber optic cables.

Thermo Scientific NanoDrop Eight User Guide 17

4 Nucleic Acid Applications
Measure dsDNA, ssDNA or RNA

Before you begin...

Before taking pedestal measurements with the NanoDrop Eight instrument, lift the
instrument arm and clean the upper and lower pedestals. At a minimum, wipe the
pedestals with a new laboratory wipe. For more information, see Cleaning the
Pedestals.

To measure nucleic acid

1. From the Home screen, select the Nucleic Acids tab, select your sample
mode, and select dsDNA, ssDNA or RNA, depending on the samples to be
measured.
2. Specify a baseline correction if desired.
3. Pipette 1–2 µL blanking solution onto the lower pedestal and lower the arm.
4. Select Blank and wait for the measurement to complete.
Tip: If Auto-Blank is On, the blank measurement starts automatically after you
lower the arm.
5. Lift the arm and clean all pedestals with a new laboratory wipe.
6. Pipette 1-2 µL sample solution onto the pedestal and lower the arm.
7. Start the sample measurement: If Auto-Measure is On, lower arm;
if Auto-Measure is off, lower arm and select Measure .

When the sample measurement is completed, the spectrum and reported values
are displayed (see the next section).
8. When you are finished measuring samples, select End Experiment .
9. Lift the arm and clean all pedestals with a new wipe.

18 NanoDrop Eight User Guide Thermo Scientific

 4 Nucleic Acid Applications
Measure dsDNA, ssDNA or RNA

Typical nucleic acid spectrum

Comparison of nucleic acid spectra with and without two

common contaminants

Best practices for nucleic acid measurements

• Isolate and purify nucleic acid samples before measurement to remove
impurities. Depending on the sample, impurities could include DNA, RNA, free
nucleotides, proteins, some buffer components and dyes. See Preparing
Samples for more information.

Thermo Scientific NanoDrop Eight User Guide 19

4 Nucleic Acid Applications
Measure dsDNA, ssDNA or RNA

Note Extraction reagents such as guanidine, phenol, and EDTA contribute

absorbance between 230 nm and 280 nm and will affect measurement
results if present in samples (even residual amounts).

• Ensure the sample absorbance is within the instrument’s absorbance detection

limits.
• Blank with the same buffer solution used to resuspend the analyte of interest.
The blanking solution should be a similar pH and ionic strength as the analyte
solution.
• Run a blanking cycle to assess the absorbance contribution of your buffer
solution. If the buffer exhibits strong absorbance at or near the analysis
wavelength (typically 260 nm), you may need to choose a different buffer or
application. See Choosing and Measuring a Blank for more information.
• For micro-volume measurements:
– Ensure pedestal surfaces are properly cleaned and conditioned.
– If possible, heat highly concentrated or large molecule samples, such as
genomic or lambda DNA, to 63 °C (145 °F) and gently (but thoroughly) vortex
before taking a measurement. Avoid introducing bubbles when mixing and
pipetting.
– Follow best practices for micro-volume measurements.
– Use a 1-2 µL sample volume. See Recommended Sample Volumes for more
information.

Related Topics
• Measure a Micro-Volume Sample
• Best Practices for Micro-Volume Measurements
• Prepare Samples and Blanks
• Basic Instrument Operations

20 NanoDrop Eight User Guide Thermo Scientific

 4 Nucleic Acid Applications
Measure dsDNA, ssDNA or RNA

Nucleic Acid Reported Results

dsDNA measurement screen

For each measured sample, the dsDNA, ssDNA and RNA applications show the UV
absorbance spectrum and a summary of the results. Below is an example of the
measurement screen of the instrument software:
Measure sample
Application View history data
Run Blank End experiment
Load Sample ID file

Auto measure

UV spectrum

Right click graph

area to view display
options.

Baseline
correction

Menu of table
options;
click to choose
which columns to
report

Wellplate view; Sample name;

toggle to view/ hide select to edit
well plate map Well plate Nucleic acid Purity ratios Click row to select sample
options concentration and update spectrum.

Note Micro-volume absorbance measurements are normalized to a 10.0 mm

pathlength equivalent.

Thermo Scientific NanoDrop Eight User Guide 21

4 Nucleic Acid Applications
Measure dsDNA, ssDNA or RNA

dsDNA, ssDNA and RNA reported values

• sample name
• created on (date sample measurement was taken)
• nucleic acid concentration
• Species
• A260/A280
• A260/A230
• A260
• A280
• factor
• baseline correction
• monitor wavelength
• pathlength used
• contaminant
• corrected

Settings for Nucleic Acid Measurements

Baseline Correction
For the dsDNA, ssDNA or RNA, enter baseline correction wavelength in nm or use
default value (340 nm)

Optional user-defined baseline correction. Can be used to correct for any

offset caused by light scattering particulates by subtracting measured absorbance at
specified baseline correction wavelength from absorbance values at all wavelengths
in sample spectrum. As a result, absorbance of sample spectrum is zero at specified
baseline correction wavelength.

22 NanoDrop Eight User Guide Thermo Scientific

 4 Nucleic Acid Applications
Measure dsDNA, ssDNA or RNA

Calculations for Nucleic Acid Measurements

The Nucleic Acid applications use a Extinction Coefficients vs Factors
modification of the Beer-Lambert equation
(shown at right) to calculate sample Using the terms in the Beer-Lambert equation, factor (f) is
concentration where the extinction defined as:
coefficient and pathlength are combined
and referred to as a “factor.” factor (f) = 1/( * b)
where:
 = wavelength-dependent molar extinction coefficient in
ng-cm/µL
b = sample pathlength in cm

As a result, analyte concentration (c) is calculated as:

c = A * [1/( * b)]
or

c=A*f
where:
c = analyte concentration in ng/µL
A = absorbance in absorbance units (A)
f = factor in ng-cm/µL (see below)
For the dsDNA, ssDNA and RNA Factors Used
applications, the generally accepted factors
for nucleic acids are used in conjunction • dsDNA (factor = 50 ng-cm/µL)
with Beer’s Law to calculate sample
concentration. For the Custom Factor • ssDNA (factor = 33 ng-cm/µL)
application, the user-specified factor is
• RNA (factor = 40 ng-cm/µL)
used.
• Custom Factor (user entered factor between
15 ng-cm/µL and 150 ng-cm/µL

Thermo Scientific NanoDrop Eight User Guide 23

4 Nucleic Acid Applications
Measure dsDNA, ssDNA or RNA

Calculated nucleic acid concentrations are Measured Values

based on the absorbance value at 260 nm,
the factor used and the sample pathlength. Note: For micro-volume absorbance measurements the
A single-point baseline correction (or spectra are normalized to a 10 mm pathlength equivalent.
analysis correction) may also be applied.
A260 absorbance
Concentration is reported in mass units.
Calculators are available on the Internet to • Nucleic acid absorbance values are measured at 260 nm
convert concentration from mass to molar
using the normalized spectrum. This is the reported A260
units based on sample sequence.
value if Baseline Correction is not selected.
Absorbance values at 260 nm, 280 nm and
• If Baseline Correction is selected, the absorbance value
sometimes 230 nm are used to calculate
purity ratios for the measured nucleic acid at the correction wavelength is subtracted from the
samples. Purity ratios are sensitive to the absorbance at 260 nm. The corrected absorbance at
presence of contaminants in the sample, 260 nm is reported and used to calculate nucleic acid
such as residual solvents and reagents concentration.
typically used during sample purification.
A230 and A280 absorbance
• Normalized and baseline-corrected (if selected)
absorbance values at 230 nm and 280 nm are used to
calculate A260/A230 and A260/A280 ratios.
Sample Pathlength
• For micro-volume measurements, the software selects
the optimal pathlength (between 1.0 mm and 0.1 mm)
based on sample absorbance at the analysis wavelength.
• Displayed spectra and absorbance values are normalized
to a 10 mm pathlength equivalent.

24 NanoDrop Eight User Guide Thermo Scientific

 4 Nucleic Acid Applications
Measure dsDNA, ssDNA or RNA

Reported Values
• Nucleic acid concentration. Reported in selected
unit (i.e., ng/µL, µg/µl or µg/mL). Calculations are based
on modified Beer’s Law equation using corrected nucleic
acid absorbance value.

• A260/A280 purity ratio. Ratio of corrected

absorbance at 260 nm to corrected absorbance at
280 nm. An A260/A280 purity ratio of ~1.8 is generally
accepted as “pure” for DNA (~2.0 for RNA). Acidic
solutions may under represent the reported value by
0.2-0.3; the opposite is true for basic solutions.

• A260/A230 purity ratio. Ratio of corrected

absorbance at 260 nm to corrected absorbance at
230 nm. An A260/A230 purity ratio between 1.8 and 2.2
is generally accepted as “pure” for DNA and RNA.

Note: Although purity ratios are important indicators of

sample quality, the best quality indicator quality is
functionality in the downstream application of interest (e.g.,
real-time PCR).
• Factor. Used in conjunction with Beer’s Law to calculate
sample concentration
• Contaminant - If a contaminant was identified by the
Acclaro software, the contaminant will be displayed in this
column.
• Species - The species of nucleic acid contaminant
detected by the Acclaro sample intelligence technology
• A260 absorbance.
• A280 absorbance.
• Baseline correction
• Monitor wavelength - Enter an additional wavelength
whose absorbance value you want included in the report.
• Corrected - Displays the corrected analyte
concentration determined using the Acclaro software, if
one is available.

Thermo Scientific NanoDrop Eight User Guide 25

4 Nucleic Acid Applications

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26 NanoDrop Eight User Guide Thermo Scientific

 4 Nucleic Acid Applications
Measure Microarray

Measure Microarray
Measures the concentration of purified
nucleic acids that have been labeled with
up to two fluorescent dyes for use in
downstream microarray applications.

Measure Microarray Samples

Reported Results

Settings

Detection Limits

Calculations

Measure Microarray Samples

Use the Microarray application to quantify nucleic acids that have been labeled with
up to two fluorescent dyes. The application reports nucleic acid concentration, an
A260/A280 ratio and the concentrations and measured absorbance values of the
dye(s), allowing detection of dye concentrations as low as 0.2 picomole per
microliter.

To measure microarray samples

NOTICE
• Do not use a squirt or spray bottle on or near the instrument as liquids will
flow into the instrument and may cause permanent damage.
• Do not use hydrofluoric acid (HF) on the pedestals. Fluoride ions will
permanently damage the quartz fiber optic cables.

Before you begin...

Before taking pedestal measurements with the NanoDrop Eight instrument, lift the
instrument arm and clean the upper and lower pedestals. At a minimum, wipe the
pedestals with a new laboratory wipe. For more information, see Cleaning the
Pedestals.

Thermo Scientific NanoDrop Eight User Guide 27

4 Nucleic Acid Applications
Measure Microarray

To measure a microarray sample

1. From the Home screen, select the Nucleic Acids tab and select Microarray.
2. Specify the sample type and factor and the type of dye(s) used.
Tip: Select a dye from the pre-defined list or add a custom dye using the
Dye/Chromophore Editor.
3. Pipette 1–2 µL blanking solution onto the lower pedestal and lower the arm.
4. Select Blank and wait for the measurement to complete.
Tip: If Auto-Blank is On, the blank measurement starts automatically after you
lower the arm.
5. Lift the arm and clean all pedestals with a new laboratory wipe.
6. Pipette 1-2 µL sample solution onto the pedestal and lower the arm.
7. Start the sample measurement: If Auto-Measure is On, lower arm; if
Auto-Measure is off, lower arm and select Measure.
When the sample measurement is completed, the spectrum and reported values
are displayed (see the next section).
8. When you are finished measuring samples, select End Experiment.
9. Lift the arm and clean all pedestals with a new wipe.

28 NanoDrop Eight User Guide Thermo Scientific

 4 Nucleic Acid Applications
Measure Microarray

A260 absorbance peak used

to calculate nucleic acid
concentration

Dye absorbance peak used to

calculate dye concentration

Typical microarray spectrum

Related Topics
• Best Practices for Nucleic Acid Measurements
• Measure a Micro-Volume Sample
• Best Practices for Micro-Volume Measurements
• Prepare Samples and Blanks
• Basic Instrument Operations

Thermo Scientific NanoDrop Eight User Guide 29

4 Nucleic Acid Applications
Measure Microarray

Microarray Reported Results

Microarray measurement screen

For each measured sample, this application shows the absorbance spectrum and a
summary of the results. Here is an example:

View history data Samples Microarray Run Measure

displayed in Setup Blank sample Click to end
graph area experiment and
Load Sample ID file
export data

Right click
UV-Vis Spectrum graph area to
view display
options.

Click to
select unit

Click to
select data
columns

Click row to
select sample
and update
spectrum; click
Sample names; Nucleic acid Dye more rows to
Click to edit concentration concentration(s) overlay spectra

Note
• Analysis correction is performed at 340 nm (absorbance value at 340 nm is
subtracted from absorbance values at all wavelengths in sample spectrum).
• Micro-volume absorbance measurements are normalized to a 10.0 mm
pathlength equivalent.

30 NanoDrop Eight User Guide Thermo Scientific

 4 Nucleic Acid Applications
Measure Microarray

Microarray reported values

Reported values are shown in the Data table. Select which of the reported results is
shown in the data table by selecting from the data table options menu. Here are the
available reported values:

• sample name
• created on (date sample measurement was taken)
• nucleic acid concentration
• A260
• A260/A280
• dye 1/dye 2 concentration
• sample type
• analysis correction
• factor

Settings for Microarray Measurements

Microarray settings
The Microarray Setup screen appears after you select the Microarray application
from the Nucleic Acids tab on the Home screen. To show the Microarry settings from
the Microarray measurement screen, select Microarray Setup .

Setting Available Options Description

Sample type and dsDNA (with non-editable factor of Widely accepted value for double-stranded
Factor 50 ng-cm/µL) DNA
ssDNA (with non-editable factor of Widely accepted value for single-stranded DNA
33 ng-cm/µL)
RNA (with non-editable factor of Widely accepted value for RNA
40 ng-cm/µL)
Oligo DNA with non-editable Factor calculated from user-defined DNA base
calculated factor in ng-cm/µL sequence. When selected, available DNA base
units (i.e., G, A, T, C) appear as keys. Define
sequence by selecting appropriate keys. Factor
is calculated automatically based on widely
accepted value for each base unit.

Thermo Scientific NanoDrop Eight User Guide 31

4 Nucleic Acid Applications
Measure Microarray

Setting Available Options Description

Oligo RNA with non-editable Factor calculated from user-defined RNA base
calculated factor in ng-cm/µL sequence. When selected, available RNA base
units (i.e., G, A, U, C) appear as keys. Define
sequence by selecting appropriate keys. Factor
is calculated automatically based on widely
accepted value for each base unit.
Custom (with user-specified factor in Enter factor between 15 ng-cm/µL and
ng-cm/µL) 150 ng-cm/µL
Dye 1/Dye 2 Cy3, 5, 3.5, or 5.5, Select pre-defined dye(s) used to label sample
Typea Alexa Fluor 488, 546, 555, 594, 647, material, or one that has been added using Dye
or 660 Editor.
Dye 1/Dye 2 Unit picomoles/microliter (pmol/µl), Select unit for reporting dye concentrations
micromoles (uM), or millimoles (mM)
Analysis On or off Corrects sample absorbance measurement for
Correctionb any offset caused by light scattering particulates
Enter analysis correction wavelength by subtracting absorbance value at specified
in nm or use default value (340 nm) analysis correction wavelength from
absorbance value at analysis wavelength.
Corrected value is used to calculate sample
concentration.

Tip: If the sample has a modification that

absorbs light at 340 nm, select a different
correction wavelength or turn off Analysis
Correction.
a
To add a custom dye or edit the list of available dyes, use the Dye/Chromophore Editor.
b
The Analysis Correction affects the calculation for nucleic acid concentration only.

Dye/chromophore editor
Use the Dye/Chromophore Editor to add a custom dye to the list of available dyes in
Microarray Setup or Proteins & Labels Setup. You can also specify which dyes are
available in that list.

To access the Dye/Chromophore Editor, from the Home screen, select Settings >
Dye Editor.

32 NanoDrop Eight User Guide Thermo Scientific

 4 Nucleic Acid Applications
Measure Microarray

Dye Editor
Locked dye (pre-defined;
Select to add
cannot be edited or deleted)
custom dye

Select to
edit selected
custom dye

Select to delete selected

Custom dye (user-defined; custom dye
can be edited or deleted)

These operations are available from the Dye/Chromophore Editor:

Add custom dye

– tap to show Create Dye box
– enter unique Name for new dye
– select default Unit that will be used to display dye concentration
– enter dye’s Extinction Coefficient (or molar absorptivity constant) in
L/mole-cm (typically provided by dye manufacturer)
– specify Wavelength in nm (between 350 nm and 840 nm) that will be used
to measure dye’s absorbance
– specify dye’s correction values at 260 nm and 280 nm
– select Save

Thermo Scientific NanoDrop Eight User Guide 33

4 Nucleic Acid Applications
Measure Microarray

Note To determine dye correction values (if not available from dye
manufacturer):
– use instrument to measure pure dye and note absorbance at 260 nm,
280 nm and at analysis wavelength for dye (see above)
– calculate ratio of A260/Adye wavelength and enter that value for 260 nm
Correction
– calculate ratio of A280/Adye wavelength and enter that value for 280 nm
Correction

When a custom dye is selected before a measurement, the dye’s absorbance

and concentration values are reported and the corrections are applied to the
measured sample absorbance values, and to the resulting sample
concentrations and purity ratios.

Edit custom dye

Tip Dyes pre-defined in the software cannot be edited.

– select custom dye

– select edit
– edit any entries or settings
– click Save

Delete custom dye

Tip Dyes pre-defined in the software cannot be deleted.

– select custom dye

– click
NOTICE Deleting a custom dye permanently removes the dye and all
associated information from the software.

34 NanoDrop Eight User Guide Thermo Scientific

 4 Nucleic Acid Applications
Measure Microarray

Calculations for Microarray Measurements

As with the other nucleic acid applications, Available Options for Factors
the Microarray application uses a
modification of the Beer-Lambert equation • dsDNA (factor = 50 ng-cm/µL)
to calculate sample concentration where
the extinction coefficient and pathlength • ssDNA (factor = 33 ng-cm/µL)
are combined and referred to as a “factor.”
• RNA (factor = 40 ng-cm/µL)
The Microarray application offers six
options (shown at right) for selecting an • Oligo DNA (calculated from user entered DNA
appropriate factor for each measured nucleotide sequence)
sample, to be used in conjunction with
Beer’s Law to calculate sample • Oligo RNA (calculated from user entered RNA
concentration. nucleotide sequence)
If the factor is known, choose the Custom • Custom Factor (user entered factor between
Factor option and enter the factor in 15 ng-cm/µL and 150 ng-cm/µL
ng-cm/µL. Otherwise, choose the option
that best matches the sample solution. Note: See Sample Type for more information.
Tip: Ideally, the factor or extinction
coefficient should be determined
empirically using a solution of the study
nucleic acid at a known concentration
using the same buffer.

Thermo Scientific NanoDrop Eight User Guide 35

4 Nucleic Acid Applications
Measure Microarray

Calculated nucleic acid concentrations are Measured Values

based on the absorbance value at 260 nm,
the factor used and the sample pathlength. A260 absorbance
A single-point baseline correction (or
analysis correction) may also be applied. Note: The absorbance value at 850 nm is subtracted from all
wavelengths in the spectrum. As a result, the absorbance at
Concentration is reported in mass units. 850 nm is zero in the displayed spectra. Also, for
Calculators are available on the Internet to
micro-volume absorbance measurements, the spectra are
convert concentration from mass to molar
units based on sample sequence. normalized to a 10 mm pathlength equivalent.

Absorbance values at 260 nm, 280 nm and • Nucleic acid absorbance values for all Microarray sample
sometimes 230 nm are used to calculate types are measured at 260 nm using the 850-corrected
purity ratios for the measured nucleic acid and normalized spectrum.
samples. Purity ratios are sensitive to the
presence of contaminants in the sample, • If Analysis Correction is selected, the absorbance value at
such as residual solvents and reagents the correction wavelength is subtracted from the
typically used during sample purification. absorbance at 260 nm.
• If one or more dyes are selected, the dye correction
values at 260 nm are also subtracted from the
absorbance at 260 nm.
• The final corrected absorbance at 260 nm is reported
and used to calculate sample concentration.

A280 absorbance
• Analysis-corrected and normalized absorbance value at
280 nm (minus the A280 dye correction) is used to
calculate an A260/A280 ratio.

36 NanoDrop Eight User Guide Thermo Scientific

 4 Nucleic Acid Applications
Measure Microarray

Dye concentrations are calculated from the Dye absorbance

absorbance value at the dye’s analysis
wavelength, the dye’s extinction coefficient, • Dye absorbance values are measured at specific
and the sample pathlength. A sloped-line wavelengths. See Dye/Chromophore Editor for analysis
dye correction may also be used. wavelengths used.
• If Sloping Dye Correction is selected, a linear baseline is
drawn between 400 nm and 850 nm and, for each dye,
the absorbance value of the sloping baseline is
subtracted from the absorbance value at each dye’s
analysis wavelength. Baseline-corrected dye absorbance
values are reported and used to calculate dye
concentrations.

Dye correction
• Pre-defined dyes have known correction values for A260
and A280. See Dye/Chromophore Editor for correction
values used.

• A260 dye corrections are subtracted from the A260

absorbance value used to calculate nucleic acid
concentration, and from the A260 absorbance value
used to calculate the A260/A280 purity ratio.
Sample Pathlength
• For micro-volume measurements, the software selects
the optimal pathlength (between 1.0 mm and 0.1 mm)
based on sample absorbance at the analysis wavelength.
• Displayed spectra and absorbance values are normalized
to a 10 mm pathlength equivalent.

Thermo Scientific NanoDrop Eight User Guide 37

4 Nucleic Acid Applications
Measure Microarray

Reported Values
• Nucleic acid concentration. Reported in selected
unit (i.e., ng/µL, µg/µl or µg/mL). Calculations are based
on modified Beer’s Law equation using corrected nucleic
acid absorbance value.

• A260/A280 purity ratio. Ratio of corrected

absorbance at 260 nm to corrected absorbance at
280 nm. An A260/A280 purity ratio of ~1.8 is generally
accepted as “pure” for DNA (~2.0 for RNA). Acidic
solutions may under represent the reported value by
0.2-0.3; the opposite is true for basic solutions.
• Dye1/Dye2 concentration. Reported in pmol/µL.
Calculations are based on Beer’s Law equation using
(sloping) baseline-corrected dye absorbance value(s).

Note: Although purity ratios are important indicators of

sample quality, the best indicator of DNA or RNA quality is
functionality in the downstream application of interest (e.g.,
microarray).

Related Topics
• Calculations for Nucleic Acid Measurements

38 NanoDrop Eight User Guide Thermo Scientific

 4 Nucleic Acid Applications
Measure using a Custom Factor

Measure using a Custom Factor

Measures the concentration of
purified nucleic acids using a
custom factor for the
calculations.

Measure using Custom Factor

Reported Results

Settings

Detection Limits

Calculations

Measure Nucleic Acid using a Custom Factor

Use the Custom Factor application to quantify purified DNA or RNA samples that
absorb at 260 nm with a user-defined extinction coefficient or factor. The application
reports nucleic acid concentration and two absorbance ratios (A260/A280 and
A260/A230). A single-point baseline correction can also be used.

To measure nucleic acid samples using a custom factor

NOTICE
• Do not use a squirt or spray bottle on or near the instrument as liquids will
flow into the instrument and may cause permanent damage.
• Do not use hydrofluoric acid (HF) on the pedestals. Fluoride ions will
permanently damage the quartz fiber optic cables.

Before you begin...

Before taking pedestal measurements with the NanoDrop Eight instrument, lift the
instrument arm and clean the upper and lower pedestals. At a minimum, wipe the
pedestals with a new laboratory wipe. For more information, see Cleaning the
Pedestals.

To measure using a custom factor

1. From the Home screen, select the Nucleic Acids tab and select Custom
Factor.

Thermo Scientific NanoDrop Eight User Guide 39

4 Nucleic Acid Applications
Measure using a Custom Factor

2. Enter the factor to be used for the calculations and specify a baseline correction
if desired.

3. Pipette 1–2 µL blanking solution onto the lower pedestals and lower the arm.
4. Select Blank and wait for the measurement to complete.
Tip: If Auto-Blank is On, the blank measurement starts automatically after you
lower the arm.
5. Lift the arm and clean all pedestals with a new laboratory wipe.
6. Pipette 1-2 µL sample solution onto the pedestals and lower the arm.
7. Start the sample measurement: If Auto-Measure is On, lower arm; if
Auto-Measure is off, lower arm and select Measure .

When the sample measurement is completed, the spectrum and reported values
are displayed (see the next section).
8. When you are finished measuring samples, select End Experiment.
9. Lift the arm and clean all pedestals with a new wipe.

Typical nucleic acid spectrum

40 NanoDrop Eight User Guide Thermo Scientific

 4 Nucleic Acid Applications
Measure using a Custom Factor

Related Topics
• Measure a Micro-Volume Sample
• Best Practices for Micro-Volume Measurements
• Prepare Samples and Blanks
• Basic Instrument Operations

Custom Factor Reported Results

For each measured sample, this application shows the absorbance spectrum and a
summary of the results. Here is an example:

Back to View history data Run blank Measure sample

Home solution
Load Sample ID file Custom factor settings
screen End experiment
and export data

Select to toggle
With sample selected, click and drag an Auto-Measure
area to zoom. Right-click and select ON/OFF
Autoscale to fit spectra to window

Baseline
correction
wavelength;
select to edit

Sample rows

Well plate options Sample names; Custom factor

select to add or edit used to calculate
Wellplate view; nucleic acid
toggle to view/ hide concentration
well plate map
Tips:
Click sample row to select sample and update spectrum
Shift-click multiple sample rows to overlay spectra
Click a sample and hover locations on spectra to view measurement values

Thermo Scientific NanoDrop Eight User Guide 41

4 Nucleic Acid Applications
Measure using a Custom Factor

Related Topics
• Basic Instrument Operations
• Nucleic Acid Reported Results
• Nucleic Acid Calculations

Settings for Nucleic Acid Measurements using a Custom Factor

To show the Custom Factor settings, from the Custom Factor measurement screen,
select the settings icon to view the Custom Factor Setup.

Setting Available Options Description

Custom Factor Enter an integer value Constant used to calculate nucleic acid concentration in
between 15 ng-cm/µL modified Beer’s Law equation. Based on extinction
and 150 ng-cm/µL coefficient and pathlength:


f = 1/( 260 * b))

where:


f= factor
= molar extinction coefficient at 260 nm in ng-cm/µL
b = sample pathlength in cm (1 cm for nucleic acids
measured with the NanoDrop Eight instruments)
Baseline Correction On or off Optional user-defined baseline correction. Can
be used to correct for any offset caused by light scattering
Enter baseline particulates by subtracting measured absorbance at
correction wavelength specified baseline correction wavelength from absorbance
in nm or use default values at all wavelengths in sample spectrum. As a result,
value (340 nm) absorbance of sample spectrum is zero at specified
baseline correction wavelength.

NOTE: Baseline correction is selected from the

measurement screen of the instrument software and is not
shown in the Custom Factor Setup.

Related Topics
• Instrument Settings

42 NanoDrop Eight User Guide Thermo Scientific

 4 Nucleic Acid Applications
Measure using a Custom Factor

Detection Limits for Nucleic Acid Measurements using a Custom

Factor
The lower detection limits and reproducibility specifications for nucleic acids are
provided here. The upper detection limits are dependent on the upper absorbance
limit of the instrument and the user-defined extinction coefficients.

To calculate upper detection limits for nucleic acid samples

To calculate upper detection limits in ng/µL, use the following equation:

(upper absorbance limitinstrument * extinction coefficientsample)

For example, for a sample measurement using an extinction coefficient of 31, the
equation looks like this:
(200 AU * 31 ng-cm/µL) = 6,200 ng/µL

Related Topics
• Detection Limits for All Applications

Thermo Scientific NanoDrop Eight User Guide 43

4 Nucleic Acid Applications

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44 NanoDrop Eight User Guide Thermo Scientific

 4 Nucleic Acid Applications
Measure Oligo DNA or Oligo RNA

Measure Oligo DNA or Oligo RNA

Measures the concentration of purified
ssDNA or RNA oligonucleotides that
absorb at 260 nm.

Measure Oligo DNA or RNA

Reported Results

Settings

Detection Limits

Calculations

Measure Oligo DNA or Oligo RNA

Use the Oligo DNA and Oligo RNA applications to quantify oligonucleotides that
absorb at 260 nm. Molar extinction coefficients are calculated automatically based
on the user-defined base sequence of the sample. You can input unique oligo
sequences for each of the eight channels. These applications report nucleic acid
concentration and two absorbance ratios (A260/A280 and A260/A230). A
single-point baseline correction can also be used.

To measure Oligo DNA or Oligo RNA samples

NOTICE
• Do not use a squirt or spray bottle on or near the instrument as liquids will
flow into the instrument and may cause permanent damage.
• Do not use hydrofluoric acid (HF) on the pedestals. Fluoride ions will
permanently damage the quartz fiber optic cables.

Before you begin...

Before taking pedestal measurements with the NanoDrop Eight instrument, lift the
instrument arm and clean the upper and lower pedestals. At a minimum, wipe the
pedestals with a new laboratory wipe. For more information, see Cleaning the
Pedestals.

To measure an oligonucleotide sample

1. From the Home screen, select the Nucleic Acids tab and select either Oligo
DNA or Oligo RNA, as needed.

Thermo Scientific NanoDrop Eight User Guide 45

4 Nucleic Acid Applications
Measure Oligo DNA or Oligo RNA

2. Specify the Oligo base sequence for each individual channel and a baseline
correction if desired.
3. Pipette 1–2 µL blanking solution onto the lower pedestal and lower the arm.
4. Select Blank and wait for the measurement to complete.
Tip: If Auto-Blank is On, the blank measurement starts automatically after you
lower the arm.
5. Lift the arm and clean all pedestals with a new laboratory wipe.
6. Pipette 1-2 µL sample solution onto the pedestal and lower the arm.
7. Start the sample measurement: If Auto-Measure is On, lower arm; if
Auto-Measure is off, lower arm and select Measure.

When the sample measurement is completed, the spectrum and reported values
are displayed (see the next section).
8. When you are finished measuring samples, tap End Experiment .
9. Lift the arm and clean all pedestals with a new wipe.

Example Oligo DNA spectrum

Related Topics
• Best Practices for Nucleic Acid Measurements

46 NanoDrop Eight User Guide Thermo Scientific

 4 Nucleic Acid Applications
Measure Oligo DNA or Oligo RNA

• Measure a Micro-Volume Sample

• Best Practices for Micro-Volume Measurements
• Prepare Samples and Blanks
• Basic Instrument Operations

Oligo Reported Results

Oligo DNA and RNA measurement screen

For each measured sample, the Oligo DNA and Oligo RNA applications show the UV
absorbance spectrum reported results. Here is an example:
Application View history data Oligo Run Measure
RNA/RNA Blank sample Click to end
Setup experiment and
Load Sample ID file
export data

Right click
graph area to
UV spectrum view display
options.

Click to
select unit

Menu of table
options;
click to choose
which columns
to report

Click row to
select sample
and update
spectrum; click
more rows to
overlay spectra
Wellplate view; Well plate
toggle to view/ options Purity ratios
hide well plate Sample name; Nucleic acid
map Select to edit concentration

Thermo Scientific NanoDrop Eight User Guide 47

4 Nucleic Acid Applications
Measure Oligo DNA or Oligo RNA

Oligo DNA and Oligo RNA reported values

• sample name
• created on (date sample measurement was taken)
• nucleic acid concentration
• A260/A280
• A260/A230
• A260
• A280
• factor
• oligo sequence
• baseline correction
Note The five nucleotides that comprise DNA and RNA exhibit widely varying
A260/A280 ratios. See Oligo Purity Ratios for more information.

Related Topics
• Basic Instrument Operations
• Oligo Calculations

48 NanoDrop Eight User Guide Thermo Scientific

 4 Nucleic Acid Applications
Measure Oligo DNA or Oligo RNA

Settings for Oligo DNA and Oligo RNA Measurements

The Oligo setup screen appears after you select the Oligo DNA or Oligo RNA
application from the Nucleic Acids tab on the Home screen. From the Oligo DNA or
RNA measurement screen, select the settings icon to view
Oligo DNA Setup or Oligo RNA Setup.

Setting Available Options Description

Oligo Base for DNA: Use the Specify your DNA or RNA base sequences in each of the 8
Sequence G, A, T and C keys to channels.
specify the DNA base click the corresponding keys:
sequence
Add guanine Add adenine Add thymine (DNA)
or uracil (RNA)
for RNA: Use the
G, A, U and C keys to
specify the RNA base Add cytosine
sequence

Base sequences specified in each channel

You can also enter base sequence using the keyboard, or by

copy and pasting a sequence from another application.
Each time a base is added to the sequence, the software
calculates the following:
• Factor. Constant used to calculate oligonucleotide
concentration in modified Beer’s Law equation. Based on
extinction coefficient and pathlength:


f = 1/( 260 * b)

where:


f= factor
= molar extinction coefficient at 260 nm in ng-cm/µL
b = sample pathlength in cm (1 cm for nucleic acids
measured with the NanoDrop Eight instrument)

Thermo Scientific NanoDrop Eight User Guide 49

4 Nucleic Acid Applications
Measure Oligo DNA or Oligo RNA

Setting Available Options Description

• Molecular Weight of oligo calculated from user-defined
base sequence.
• Number of Bases entered.
• Molar Ext. Coefficient (260 nm). Molar extinction
coefficient of oligo (in ng-cm/µL) at 260 nm calculated from
entered base sequence.
• %GC. Percentage of guanine and cytosine residues in
total number of bases entered.
Baseline On or off Corrects for any offset caused by light scattering particulates
Correction by subtracting measured absorbance at specified baseline
Enter baseline correction wavelength from absorbance values at all
correction wavelengths in sample spectrum. As a result, absorbance of
wavelength in nm or sample spectrum is zero at specified baseline correction
use default value wavelength.
(340 nm)
Tip: If the sample has a modification that absorbs light at
340 nm, select a different correction wavelength or turn off
Baseline Correction.

Related Topics
• Instrument Settings

Detection Limits for Oligo DNA and Oligo RNA Measurements

The lower detection limits and reproducibility specifications for the oligonucleotide
sample types (ssDNA and RNA) are provided here. The upper detection limits are
dependent on the upper absorbance limit of the instrument and the extinction
coefficients for the user-defined base sequences.

To calculate upper detection limits for nucleic acid samples

To calculate upper detection limits in ng/µL, use the following equation:

(upper absorbance limitinstrument * extinction coefficientsample)

For example, for a sample measurement using an extinction coefficient of 55, the
equation looks like this:
(200 AU * 55 ng-cm/µL) = 11,000 ng/µL

50 NanoDrop Eight User Guide Thermo Scientific

 4 Nucleic Acid Applications
Measure Oligo DNA or Oligo RNA

Calculations for Oligo DNA and Oligo RNA Measurements

As with the other nucleic acid applications, the Extinction Coefficients for Oligonucleotides
Oligo applications use the Beer-Lambert
equation to correlate absorbance with The software uses the nearest neighbor method and the
concentration based on the sample’s extinction following formula to calculate molar extinction
coefficient and pathlength. Because coefficients for specific oligonucleotide base sequences:
oligonucleotides are short, single-stranded
molecules (or longer molecules of repeating
sequences), their spectrum and extinction N–1 N–1 N

coefficient ( ) are closely dependent on base  260 =  1 –  2 +  3
composition and sequence. 1 2 1

(The generally accepted extinction coefficients

and factors for single-stranded DNA and RNA where:
provide a reasonable estimate for natural,  = molar extinction coefficient in L/mole-cm
essentially randomized, sequences but not for  
1 = nearest neighbor
short, synthetic oligo sequences.) To ensure the  
2 = individual bases
most accurate results, we use the exact value of
 260 to calculate oligonucleotide concentration.
 
3 = modifications, such as fluorescent dyes

The NanoDrop software allows you to specify

the base sequence of an oligonucleotide before
it is measured. For any entered base sequence,
the software uses the equation at the right to
calculate the extinction coefficient.
Tip: The extinction coefficient is wavelength
specific for each oligonucleotide and can be
affected by buffer type, ionic strength and pH.

Thermo Scientific NanoDrop Eight User Guide 51

4 Nucleic Acid Applications
Measure Oligo DNA or Oligo RNA

Calculated nucleic acid concentrations are Measured Values

based on the absorbance value at 260 nm, the
factor used and the sample pathlength. A A260 absorbance
single-point baseline correction (or analysis
correction) may also be applied. Note: For micro-volume absorbance measurements, the
spectra are normalized to a 10 mm pathlength
Concentration is reported in mass units. equivalent.
Calculators are available on the Internet to
convert concentration from mass to molar units
• Nucleic acid absorbance values are measured at
based on sample sequence.
260 nm using the normalized spectrum. This is the
Absorbance values at 260 nm, 280 nm and reported A260 value if Baseline Correction is not
sometimes 230 nm are used to calculate purity selected.
ratios for the measured nucleic acid samples.
Purity ratios are sensitive to the presence of • If Baseline Correction is selected, the absorbance
contaminants in the sample, such as residual value at the correction wavelength is subtracted from
solvents and reagents typically used during the sample absorbance at 260 nm. The corrected
sample purification. absorbance at 260 nm is reported and used to
calculate nucleic acid concentration.

A230, A280 absorbance

• Normalized absorbance values at 230 nm, 260 nm
and 280 nm are used to calculate A260/A230 and
A260/A280 ratios.
Sample Pathlength
• For micro-volume measurements, the software
selects the optimal pathlength (between 1.0 mm and
0.1 mm) based on sample absorbance at the
analysis wavelength.
• Displayed spectra and absorbance values are
normalized to a 10 mm pathlength equivalent.

52 NanoDrop Eight User Guide Thermo Scientific

 4 Nucleic Acid Applications
Measure Oligo DNA or Oligo RNA

The five nucleotides that comprise DNA and Reported Values

RNA exhibit widely varying A260/A280 ratios.
Estimated A260/A280 ratios for each • Nucleic acid concentration. Reported in
independently measured nucleotide are selected unit (i.e., ng/µL, µg/µl or µg/mL).
provided below: Calculations are based on modified Beer’s Law
equation using corrected nucleic acid absorbance
Guanine: 1.15
value.
Adenine: 4.50
Cytosine: 1.51
Uracil: 4.00
• A260/A280 purity ratio. Ratio of corrected
Thymine: 1.47 absorbance at 260 nm to corrected absorbance at
280 nm.
The A260/A280 ratio for a specific nucleic acid
sequence is approximately equal to the • A260/A230 purity ratio. Ratio of corrected
weighted average of the A260/A280 ratios for absorbance at 260 nm to corrected absorbance at
the four nucleotides present. 230 nm.
Note: RNA will typically have a higher 260/280 Note: The traditional purity ratios (A260/A280 and
ratio due to the higher ratio of Uracil compared A260/A230), which are used as indicators of the
to that of Thymine. presence of various contaminants in nucleic acid
samples, do not apply for oligonucleotides because the
shapes of their spectra are highly dependent on their
base compositions. See side bar for more information.

Related Topics
• Calculations for Nucleic Acid Measurements

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4 Nucleic Acid Applications

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54 NanoDrop Eight User Guide Thermo Scientific

 5

Protein Applications

Measure Protein A280

Measures the concentration of purified
protein samples that absorb at 280 nm.

Measure A280 Proteins

Reported Results

Settings

Detection Limits

Calculations

Measure Protein Concentration at A280

Use the Protein A280 application to quantify purified protein samples that contain
amino acids such as tryptophan or tyrosine, or cys-cys disulfide bonds, which
exhibit absorbance at 280 nm. This application reports protein concentration
measured at 280 nm and one absorbance ratio (A260/A280). A single-point baseline
correction can also be used. This application does not require a standard curve.

To measure Protein A280 samples

NOTICE
• Do not use a squirt or spray bottle on or near the instrument as liquids will
flow into the instrument and may cause permanent damage.
• Do not use hydrofluoric acid (HF) on the pedestals. Fluoride ions will
permanently damage the quartz fiber optic cables.

Thermo Scientific NanoDrop Eight User Guide 55

5 Protein Applications
Measure Protein A280

Before you begin...

Before taking pedestal measurements with the NanoDrop Eight instrument, lift the
instrument arm and clean the upper and lower pedestals. At a minimum, wipe the
pedestals with a new laboratory wipe. For more information, see Cleaning the
Pedestals.

To measure a Protein A280 sample

1. From the Home screen, select the Proteins tab and select Protein A280.
2. Specify a sample type and baseline correction if desired.
3. Pipette 1–2 µL blanking solution onto the lower pedestal and lower the arm.

4. Click Blank and wait for the measurement to complete.

Tip: If Auto-Blank is On, the blank measurement starts automatically after you
lower the arm.
5. Lift the arm and clean all pedestals with a new laboratory wipe.
6. Pipette 2 µL sample solution onto the pedestal and lower the arm.
7. Start the sample measurement:
– If Auto-Measure is On, lower arm;
– if Auto-Measure is off, lower arm and click Measure.
When the sample measurement is completed, the spectrum and reported values
are displayed (see the next section).
8. When you are finished measuring samples, click End Experiment.
9. Lift the arm and clean all pedestals with a new wipe.

56 NanoDrop Eight User Guide Thermo Scientific

 5 Protein Applications
Measure Protein A280

High concentration BSA sample

Low concentration BSA sample

Best practices for protein measurements

• Isolate and purify protein samples before measurement to remove impurities.
Depending on the sample, impurities could include DNA, RNA and some buffer
components. See Preparing Samples for more information.
Note Extraction reagents that contribute absorbance between 200 nm and
280 nm will affect measurement results if present in samples (even residual
amounts).

• Ensure the sample absorbance is within the instrument’s absorbance detection

limits.

Thermo Scientific NanoDrop Eight User Guide 57

5 Protein Applications
Measure Protein A280

• Choosing a blank:
– For the Protein A280 application, blank with the same buffer solution used to
resuspend the analyte of interest. The blanking solution should be a similar
pH and ionic strength as the analyte solution.
• Run a blanking cycle to assess the absorbance contribution of your buffer
solution. If the buffer exhibits strong absorbance at or near the analysis
wavelength (typically 280 nm or 205 nm), you may need to choose a different
buffer or application. See Choosing and Measuring a Blank for more information.
Note Buffers such as Triton X, RIPA, and NDSB contribute significant
absorbance and are not compatible with direct A280 or A205
measurements.

• For micro-volume measurements:

– Ensure pedestal surfaces are properly cleaned and conditioned. (Proteins
tend to stick to pedestal surfaces.)
– Gently (but thoroughly) vortex samples before taking a measurement. Avoid
introducing bubbles when mixing and pipetting.
– Follow best practices for micro-volume measurements.
– Use a 2 µL sample volume. See Recommended Sample Volumes for more
information.

Related Topics
• Best practices for protein measurements
• Measure a Micro-Volume Sample
• Prepare Samples and Blanks
• Basic Instrument Operations

58 NanoDrop Eight User Guide Thermo Scientific

 5 Protein Applications
Measure Protein A280

Protein A280 Reported Results

Protein A280 measurement screen

For each measured sample, this application shows the absorbance spectrum and a
measurement results. Here is an example:

Application View history data Measure sample

Load Sample ID file Run Blank End experiment

Auto measure
UV spectrum Right click graph area
to view display options.
Absorbance at
280 nm

Baseline
correction
wavelength

Menu of table
options;
click to choose
which columns to
report

Wellplate view; Sample name; Purity ratios Click row to select sample
toggle to view/ hide select to edit and update spectrum.
well plate map
Well plate options Protein concentration

Note Micro-volume absorbance measurements are normalized to a 10.0 mm

pathlength equivalent.

Protein A280 reported values

• Protein concentration
• Absorbance at 280 nm
• Purity ratio
• Sample type
• Baseline correction wavelength

Thermo Scientific NanoDrop Eight User Guide 59

5 Protein Applications
Measure Protein A280

• Baseline correction absorbance

• created on (date sample measurement was taken)
• monitor wavelength
• pathlength used
• Extinction Coefficient
• contaminant
• corrected

Related Topics
• Basic Instrument Operations
• Protein A280 Calculations

Settings for Protein A280 Measurements

To show the Protein A280 settings, from the Protein A280 measurement screen,
select the settings icon to view Protein A280 Setup.

Protein A280 settings

The Protein A280 application provides a variety of sample type options for purified
protein analysis.

Each sample type applies a unique extinction coefficient to the protein calculations. If
the extinction coefficient of the sample is known, choose the  + MW (molar) or 1%
(mass) option and enter the value. Otherwise, calculate the extinction coefficient or
choose the option that best matches the sample solution. If you only need a rough
estimate of protein concentration and the sample extinction coefficient is unknown,
select the 1 Abs=1 mg/mL sample type option.

60 NanoDrop Eight User Guide Thermo Scientific

 5 Protein Applications
Measure Protein A280

Tip Ideally, the extinction coefficient should be determined empirically using a

solution of the study protein at a known concentration using the same buffer.

Mass Ext.
Setting Available Options Coefficient Description
(L/gm-cm)
Sample 1 Abs = 1 mg/mL General reference Recommended when extinction coefficient is
typea unknown and rough estimate of protein
concentration is acceptable for a solution with
no other interfering substances. Assumes
0.1% (1 mg/mL) protein solution produces
1.0A at 280 nm (where pathlength is 10 mm),
i.e., 1% = 10.
BSA 6.7 Calculates BSA (Bovine Serum Albumin)
protein concentration using mass extinction
coefficient () of 6.7 L/gm-cm at 280 nm for
1% (i.e., 10 mg/mL) BSA solution. Assuming
MW is 66,400 daltons (Da), molar extinction
coefficient at 280 nm for BSA is approximately
43,824 M-1cm-1.
IgG 13.7 Suitable for most mammalian antibodies (i.e.,
immunoglobulin G or IgG). Calculates protein
concentration using mass extinction
coefficient () of 13.7 L/gm-cm at 280 nm for
1% (i.e., 10 mg/mL) IgG solution. Assuming
MW is 150,000 Da, molar extinction coefficient
at 280 nm for IgG is approximately
210,000 M-1cm-1.
Lysozyme 26.4 Calculates lysozyme protein concentration
using mass extinction coefficient () of
26.4 L/gm-cm at 280 nm for
1% (i.e., 10 mg/mL) lysozyme solution.
Assumes molar extinction coefficient for egg
white lysozyme ranges between
36,000 M-1cm-1 and 39,000 M-1cm-1.

Thermo Scientific NanoDrop Eight User Guide 61

5 Protein Applications
Measure Protein A280

Mass Ext.
Setting Available Options Coefficient Description
(L/gm-cm)
Other protein User entered molar Assumes protein has known molar extinction

( + MW) extinction coefficient coefficient () and molecular weight (MW),
and molecular where:

 
weight
( molar)*10=( percent)*(MWprotein)

Enter MW in kiloDaltons (kDa) and molar

extinction coefficient () in M-1cm-1 divided by
1000 (i.e., /1000). For example, for protein
with molar extinction coefficient of 210,000
M-1cm-1, enter 210.
Other protein User entered mass Assumes protein has known mass extinction

( 1%) extinction coefficient coefficient (). Enter mass extinction
coefficient in L/gm-cm for 10 mg/mL (1%)
protein solution.
a
To add or edit a custom protein, use Protein Editor.

Mass Ext.
Setting Available Options Coefficient Description
(L/gm-cm)
Baseline On or off N/A Corrects for any offset caused by light
Correction scattering particulates by subtracting
Enter baseline measured absorbance at specified baseline
correction correction wavelength from absorbance
wavelength in nm or values at all wavelengths in sample spectrum.
use default value As a result, absorbance of sample spectrum is
(340 nm) zero at specified baseline correction
wavelength.

Tip: If the sample has a modification that

absorbs light at 340 nm, select a different
correction wavelength or turn off Baseline
Correction.

62 NanoDrop Eight User Guide Thermo Scientific

 5 Protein Applications
Measure Protein A280

Protein editor
Use the Protein Editor to add a custom protein to the list of available protein sample
types in Protein A280 Setup.

To access the Protein Editor:

• From the Home screen, select Settings > Protein Editor.
Protein Editor Click to add custom protein

Custom proteins Click to edit selected custom protein

(will appear in Sample Type
list in Protein A280 Setup Click to delete selected custom protein
and Proteins & Labels Setup)

These operations are available from the Protein Editor:

Add custom protein

1. In Protein Editor, select to show the New Protein Type
box.
2. Enter a unique Name for the new protein.
3. Enter a Description for the new protein.
4. Specify whether to enter Molar Extinction coefficient or Mass Extinction
coefficient for custom protein.
– If Mass Extinction coefficient is selected, enter mass extinction
coefficient in L/gm-cm for 10 mg/mL (1%) protein solution.

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5 Protein Applications
Measure Protein A280

– if Molar Extinction is selected,

– enter molar extinction coefficient () in M-1cm-1 divided by 1000 (that

is, /1000). For example, for protein with molar extinction coefficient
of 210,000 M-1cm-1, enter 210.
– Enter molecular weight (MW) in kiloDaltons (kDa)
5. Select Save to close the New Protein Type box.
The new custom protein appears in the Type list in Protein A280 Setup and
Proteins & Labels Setup.

Edit custom protein

1. In Protein Editor, click to select custom protein
2. Click to show the Edit Protein Type box
3. Edit any entries or settings
4. Click OK

Delete custom protein

1. In Protein Editor, click to select a custom protein to delete
2. click
Note Deleting a custom protein permanently removes the protein and all
associated information from the software.

64 NanoDrop Eight User Guide Thermo Scientific

 5 Protein Applications
Measure Protein A280

Detection Limits for Protein A280 Measurements

Detection limits and reproducibility specifications for purified BSA proteins are
provided here. The BSA lower detection limit and reproducibility values apply to any
protein sample type. The upper detection limits are dependent on the upper
absorbance limit of the instrument and the sample’s extinction coefficient.

To calculate upper detection limits for other (non-BSA) protein sample types
To calculate upper detection limits in mg/mL for proteins, use the following equation:

(upper absorbance limitinstrument /mass extinction coefficientsample) * 10

For example, if the sample’s mass extinction coefficient at 280 nm is 6.7 for a 1%
(10 mg/mL) solution, the equation looks like this:
(200 / 6.7) * 10 = 298.5 (or ~300)

Calculations for Protein A280 Measurements

The Protein A280 application uses the Beer-Lambert Equation (solved for concentration)
Beer-Lambert equation to correlate
absorbance with concentration. Solving c = A / ( * b)
Beer’s law for concentration yields the
equation at the right. where:

A = UV absorbance in absorbance units (AU)

 = wavelength-dependent molar absorptivity coefficient (or

extinction coefficient) in liter/mol-cm

b = pathlength in cm

c = analyte concentration in moles/liter or molarity (M)

Note: Dividing the measured absorbance of a sample

solution by its molar extinction coefficient yields the molar
concentration of the sample. See Published Extinction
Coefficients for more information regarding molar vs. mass
concentration values.

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5 Protein Applications
Measure Protein A280

The extinction coefficient of a peptide or Extinction Coefficients for Proteins

protein is related to its tryptophan (W),
tyrosin (Y) and cysteine (C) amino acid At 280 nm, the extinction coefficient is approximated by the
composition. weighted sum of the 280 nm molar extinction coefficients of
the three constituent amino acids, as described in this
Tip: The extinction coefficient is
wavelength specific for each protein and
equation:
can be affected by buffer type, ionic
strength and pH.  = (nW * 5500) + (nY * 1490) + (nC * 125)
where:
 = molar extinction coefficient
n = number of each amino acid residue
5500, 1490 and 125 = amino acid molar absorptivities at
280 nm
This application offers six options (shown at Available Options for Extinction Coefficient
right) for selecting an appropriate extinction
coefficient for each measured sample, to • 1 Abs = 1 mg/mL, where sample type and/or ext.
be used in conjunction with Beer’s Law to coefficient is unknown (produces rough estimate of
calculate sample concentration. protein concentration)
If the extinction coefficient of the sample is • BSA (Bovine Serum Albumin, 6.7 L/gm-cm)

known, choose the + MW (molar) or
 1% (mass) option and enter the value. • IgG (any mammalian antibody, 13.7 L/gm-cm)
Otherwise, calculate the extinction
coefficient or choose the option that best • Lysozyme (egg white lysozyme, 26.4 L/gm-cm)
matches the sample solution.
• Other protein ( + MW), user-specified molar ext.
Tip: Ideally, the extinction coefficient coefficient
• Other protein (1%), user-specified mass ext.
should be determined empirically using a
solution of the study protein at a known
concentration using the same buffer. coefficient
•

Note: See Sample Type for details.

66 NanoDrop Eight User Guide Thermo Scientific

 5 Protein Applications
Measure Protein A280

Most sources report extinction coefficients Published Extinction Coefficients

for proteins measured at or near 280 nm in
phosphate or other physiologic buffer. Published extinction coefficients for proteins may be reported
These values provide sufficient accuracy for as:
routine assessments of protein
concentration. • wavelength-dependent molar absorptivity (or extinction)
coefficient () with units of M-1cm-1
• percent solution extinction coefficient (1%) with units of
(g/100 mL)-1cm-1 (i.e., 1% or 1 g/100 mL solution
measured in a 1 cm cuvette)
• protein absorbance values for 0.1% (i.e., 1 mg/mL)
solutions

Tip: Assess published values carefully to ensure unit of

measure is applied correctly.
The equation at the right shows the Conversions Between molar and 1%
relationship between molar extinction

coefficient ( molar) and percent extinction (molar) * 10 = (1%) * (MWprotein)

coefficient ( 1%).
Example: To determine percent solution extinction coefficient
(1%) for a protein that has a molar extinction coefficient of
43,824 M-1cm-1 and a molecular weight (MW) of 66,400
daltons (Da), rearrange and solve the above equation as
follows:

1% = (molar * 10) / (MWprotein)

1% = (43,824 * 10) / 66,400 Da)
1% = 6.6 g/100 mL
To determine concentration (c) of a sample Conversions Between g/100 mL and mg/mL
in mg/mL, use the equation at the right and
a conversion factor of 10. Cprotein in mg/mL = (A / 1%) * 10
Tip: The NanoDrop Eight software
Example: If measured absorbance for a protein sample at
includes the conversion factor when
reporting protein concentrations. 280 nm relative to the reference is 5.8 A, protein
concentration can be calculated as:

Cprotein = (A / 1%) * 10
Cprotein = (5.8/6.6 g/100 mL) * 10
Cprotein = 8.79 mg/mL

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5 Protein Applications
Measure Protein A280

Calculated protein concentrations are Measured Values

based on the absorbance value at 280 nm,
the selected (or entered) extinction A280 absorbance
coefficient and the sample pathlength. A
single-point baseline correction (or analysis Note: For micro-volume absorbance measurements, the
correction) may be applied. spectra are normalized to a 10 mm pathlength equivalent.
Concentration is reported in mass units. • Protein absorbance values are measured at 280 nm
Calculators are available on the Internet to
using the normalized spectrum. If Baseline Correction is
convert concentration from mass to molar
units based on sample sequence.
not selected, this is the reported A280 value and the
value used to calculate protein concentration.
Absorbance values at 260 nm and 280 nm • If Baseline Correction is selected, the normalized and
are used to calculate purity ratios for the
baseline-corrected absorbance value at 280 nm is
measured protein samples.
reported and used to calculate protein concentration.
Purity ratios are sensitive to the presence of
contaminants in the sample, such as
residual solvents and reagents typically
used during sample purification.
Sample Pathlength
• For micro-volume measurements, the software selects
the optimal pathlength (between 1.0 mm and 0.1 mm)
based on sample absorbance at the analysis wavelength.
• Displayed spectra and absorbance values are normalized
to a 10 mm pathlength equivalent.
Reported Values
• Protein concentration. Reported in selected unit
(mg/mL or µg/mL). Calculations are based on
Beer-Lambert equation using corrected protein
absorbance value.
• A260/A280 purity ratio. Ratio of corrected
absorbance at 260 nm to corrected absorbance at
280 nm. An A260/A280 purity ratio of ~0.57 is generally
accepted as “pure” for proteins.

Note: Although purity ratios are important indicators of

sample quality, the best indicator of protein quality is
functionality in the downstream application of interest (e.g.,
real-time PCR).
• Contaminant - Displays contaminant identified by the
Acclaro software, if one is available.
• Corrected - Display the corrected analyte concentration
determined using the Acclaro software, if one is available.

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 5 Protein Applications
Measure Protein A205

Measure Protein A205

Measures the concentration
of purified protein populations
that absorb at 205 nm.

Measure A205 Proteins

Reported Results

Settings

Detection Limits

Calculations

Measure Protein Concentration at A205

Use the Protein A205 application to quantify purified peptides and other proteins that
contain peptide bonds, which exhibit absorbance at 205 nm. This application
reports protein concentration and two absorbance values (A205 and A280). A
single-point baseline correction can also be used. This application does not require a
standard curve.
Note If your samples contain mainly amino acids such as tryptophan or
tyrosine, or cys-cys disulfide bonds, use the Protein A280 application instead of
Protein A205.

To measure Protein A205 samples

NOTICE
• Do not use a squirt or spray bottle on or near the instrument as liquids will
flow into the instrument and may cause permanent damage.
• Do not use hydrofluoric acid (HF) on the pedestals. Fluoride ions will
permanently damage the quartz fiber optic cables.

Before you begin...

Before taking pedestal measurements with the NanoDrop Eight instrument, lift the
instrument arm and clean the upper and lower pedestals. At a minimum, wipe the
pedestals with a new laboratory wipe. For more information, see Cleaning the
Pedestals.

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5 Protein Applications
Measure Protein A205

To measure a Protein A205 sample

1. From the Home screen, select the Proteins tab and select Protein A205.
2. Specify a sample type and baseline correction if desired.
3. Pipette 1–2 µL blanking solution onto the lower pedestal and lower the arm.

4. Click Blank and wait for the measurement to complete.

Tip: If Auto-Blank is On, the blank measurement starts automatically after you
lower the arm.
5. Lift the arm and clean all pedestals with a new laboratory wipe.
6. Pipette 2 µL sample solution onto the pedestal and lower the arm.
7. Start the sample measurement:
– If Auto-Measure is On, lower arm;
– if Auto-Measure is off, lower arm and click Measure.
When the sample measurement is completed, the spectrum and reported values
are displayed (see the next section).
8. When you are finished measuring samples, click End Experiment.
9. Lift the arm and clean all pedestals with a new wipe.

Related Topics
• Best Practices for Protein Measurements
• Measure a Micro-Volume Sample
• Prepare Samples and Blanks
• Basic Instrument Operations

70 NanoDrop Eight User Guide Thermo Scientific

 5 Protein Applications
Measure Protein A205

Protein A205 Reported Results

Protein A205 measurement screen

For each measured sample, this application shows the absorbance spectrum and a
summary of the results. Here is an example:

Application View history data Measure sample

Load Sample ID file Run Blank End experiment

Auto measure
UV spectrum Right click graph area
to view display options.

Baseline Correction

Menu of table
options;
click to choose
which columns to
report

Click row to select

Well plate options
sample and update
Wellplate view; Sample name; spectrum.
toggle to view/ hide select to edit
Absorbance at
well plate map
280 nm
Protein concentration
Absorbance at
205 nm

Protein A205 reported values

• Protein concentration
• Absorbance at 205 nm
• Absorbance at 280 nm
• Sample type
• Baseline correction

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5 Protein Applications
Measure Protein A205

• created on (date sample measurement was taken)

• monitor wavelength
• pathlength used
• Extinction Coefficient

Related Topics
• Basic Instrument Operations
• Protein A205 Calculations

Settings for Protein A205 Measurements

To show the Protein A205 settings, from the Protein A205 measurement screen,
select Protein A205 Setup .

Mass Ext.
Setting Available Options Coefficient Description
(L/gm-cm)
Sample type 31 31 
Assumes 0.1% (1 mg/mL) at 205 nm = 31
Scopes 27 + 120 * Assumes 0.1% (1 mg/mL) at 205 nm = 27 +
(A280/A205) 120 * (A280/A205)
Other protein User entered mass Assumes protein has known mass extinction

( 1%) 
extinction coefficient coefficient ( ). Enter mass extinction

coefficient in L/gm-cm for 1 mg/mL ( 0.1%)
protein solution.
Baseline On or off N/A Corrects for any offset caused by light
Correction scattering particulates by subtracting
Enter baseline measured absorbance at specified baseline
correction correction wavelength from absorbance
wavelength in nm or values at all wavelengths in sample spectrum.
use default value As a result, absorbance of sample spectrum is
(340 nm) zero at specified baseline correction
wavelength.

Tip: If the sample has a modification that

absorbs light at 340 nm, select a different
correction wavelength or turn off Baseline
Correction.

Related Topics
• Instrument Settings

72 NanoDrop Eight User Guide Thermo Scientific

 5 Protein Applications
Measure Protein A205

Calculations for Protein A205 Measurements

As with the other protein applications, Available Options for Extinction Coefficient
Proteins A205 uses the Beer-Lambert
equation to correlate absorbance with • 31, assumes 0.1% (1 mg/mL) at 205 nm = 31
• Scopes, assumes 0.1% (1 mg/mL) at 205 nm = 27 +
concentration based on the sample’s
extinction coefficient and pathlength.
120 * (A280/A205)
This application offers three options (shown
at right) for selecting an appropriate • Other protein, enter mass extinction coefficient in
extinction coefficient for each measured L/gm-cm for 1 mg/mL (0.1%) protein solution
sample, to be used in conjunction with
Beer’s Law to calculate sample Note: See Sample Type for details.
concentration.

If the extinction coefficient of the sample is


known, choose the 1% (mass) option and
enter the value. Otherwise, calculate the
extinction coefficient or choose the option
that best matches the sample solution.

Tip: Ideally, the extinction coefficient

should be determined empirically using a
solution of the study protein at a known
concentration using the same buffer.
Calculated protein concentrations are Measured Values
based on the absorbance value at 205 nm,
the selected (or entered) extinction A205 absorbance
coefficient and the sample pathlength. A
single-point baseline correction may also Note: For micro-volume absorbance measurements, the
be applied. spectra are normalized to a 10 mm pathlength equivalent.
Concentration is reported in mass units. • Protein absorbance values are measured at 205 nm
Calculators are available on the Internet to
using the normalized spectrum. If Baseline Correction is
convert concentration from mass to molar
units based on the sample sequence.
not selected, this is the reported A205 value and the
value used to calculate protein concentration.
• If Baseline Correction is selected, the normalized and
baseline-corrected absorbance value at 205 nm is
reported and used to calculate protein concentration.

A280 absorbance
• Normalized and baseline-corrected (if selected)
absorbance value at 280 nm is also reported.

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5 Protein Applications
Measure Protein A205

Sample Pathlength
• For micro-volume measurements, the software selects
the optimal pathlength (between 1.0 mm and 0.1 mm)
based on sample absorbance at the analysis wavelength.
• Displayed spectra and absorbance values are normalized
to a 10 mm pathlength equivalent.
Reported Values
• Protein concentration. Reported in selected unit
(mg/mL or µg/mL). Calculations are based on
Beer-Lambert equation using corrected protein
absorbance value.

Related Topics
• Beer-Lambert Equation
• Protein A280 Calculations

74 NanoDrop Eight User Guide Thermo Scientific

 5 Protein Applications
Measure Proteins and Labels

Measure Proteins and Labels

Measures the concentration of purified
proteins that have been labeled with up
to two fluorescent dyes.

Measure Labeled Proteins

Reported Results

Settings

Detection Limits

Calculations

Measure Labeled Protein Samples

Use the Proteins and Labels application to quantify proteins and fluorescent dyes for
protein array conjugates, as well as metalloproteins such as hemoglobin, using
wavelength ratios. This application reports protein concentration measured at
280 nm, an A260/A280 absorbance ratio, and the concentrations and measured
absorbance values of the dyes, allowing detection of dye concentrations as low as
0.2 picomole per microliter. This information is useful for evaluating protein/dye
conjugation (degree of labeling) for use in downstream applications.

To measure labeled protein samples

NOTICE
• Do not use a squirt or spray bottle on or near the instrument as liquids will
flow into the instrument and may cause permanent damage.
• Do not use hydrofluoric acid (HF) on the pedestals. Fluoride ions will
permanently damage the quartz fiber optic cables.

Before you begin...

Before taking pedestal measurements with the NanoDrop Eight instrument, lift the
instrument arm and clean the upper and lower pedestals. At a minimum, wipe the
pedestals with a new laboratory wipe. For more information, see Cleaning the
Pedestals.

To measure a labeled protein sample

1. From the Home screen, select the Proteins tab and then select Protein &
Labels.

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5 Protein Applications
Measure Proteins and Labels

2. Specify the sample type and the type of dye(s) used.

Tip: Select a dye from the pre-defined list or add a custom dye using the
Dye/Chromophore Editor.
3. Pipette 1–2 µL of the blanking solution onto the lower pedestal and lower the
arm.
4. Tap Blank and wait for the measurement to complete.
Tip: If Auto-Blank is On, the blank measurement starts automatically after you
lower the arm.
5. Lift the arm and clean all pedestals with a new laboratory wipe.
6. Pipette 2 µL sample solution onto the pedestal and lower the arm.
7. Start the sample measurement:
– If Auto-Measure is On, lower arm;
– if Auto-Measure is off, lower arm and select Measure.
When the sample measurement is completed, the spectrum and reported values
are displayed (see the next section).
8. When you are finished measuring samples, select End Experiment.
9. Lift the arm and clean all pedestals with a new wipe.

Peptide backbone A280 absorbance peak used to

calculate protein concentration

Dye absorbance peak used to

calculate dye concentration

Typical sample spectrum measured with Proteins & Labels

76 NanoDrop Eight User Guide Thermo Scientific

 5 Protein Applications
Measure Proteins and Labels

Related Topics
• Best practices for protein measurements
• Measure a Micro-Volume Sample
• Prepare Samples and Blanks
• Basic Instrument Operations

Proteins & Labels Reported Results

Proteins & Labels measurement screen

For each measured sample, this application shows the absorbance spectrum and a
summary of the results. Below is an example of the measurement screen:

Application View history data Samples Run Blank Measure sample

displayed in
Load Sample ID file graph area Proteins & End experiment
Labels setup

Right click graph

area to view display
UV spectrum options.

Analysis Correction

Menu of table options;

click to choose which
columns to report

Click row to
select sample and
update spectrum.

Sloping Dye Sample name; Protein Dye

Correction select to edit concentration concentration(s)

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5 Protein Applications
Measure Proteins and Labels

Note
• A baseline correction is performed at 340 nm (absorbance value at 340 nm is
subtracted from absorbance values at all wavelengths in sample spectrum).
• Micro-volume absorbance measurements are normalized to a 10.0 mm
pathlength equivalent.

Proteins & Labels reported values

The initial screen that appears after each measurement (see previous image) shows
a summary of the reported values. To view all reported values, press and hold the
sample row. Here is an example:

Reported values for Proteins & Labels application

• Sample Name
• Creation date
• Protein
• A280
• Sample Type
• Dye 1/Dye 2
• Sloping Dye Correction
• Analysis Correction

Related Topics
• Basic Instrument Operations
• Proteins & Labels calculations

Settings for Proteins and Labels Measurements

To show the Proteins & Labels settings, from the Proteins & Labels measurement
screen, select Proteins & Labels Setup.

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 5 Protein Applications
Measure Proteins and Labels

Mass Ext.
Setting Available Options Coefficient Description
(L/gm-cm)
Sample 1 Abs = 1 mg/mL General reference Select Sample type for detailed description of
typea each available setting.
BSA 6.7
Each sample type applies a unique extinction
IgG 13.7 coefficient to the protein calculations. If the
extinction coefficient of the sample is known,
choose the  + MW (molar) or 1% (mass)
Lysozyme 26.4

Other protein user-entered molar option and enter the value. Otherwise,
( + MW) extinction calculate the extinction coefficient or choose
coefficient/ the option that best matches the sample
molecular weight solution. If you only need a rough estimate of
protein concentration and the sample
Other protein User entered mass extinction coefficient is unknown, select the
(1%) extinction 1 Abs=1 mg/mL sample type option.
coefficient
Tip: Ideally, the extinction coefficient should
be determined empirically using a solution of
the study protein at a known concentration
using the same buffer.
Analysis On or off N/A Corrects sample absorbance measurement
Correctionb for any offset caused by light scattering
Enter analysis particulates by subtracting absorbance value
correction wavelength at specified analysis correction wavelength
in nm or use default from absorbance value at analysis
value (340 nm) wavelength. Corrected value is used to
calculate sample concentration.

Tip: If the sample has a modification that

absorbs light at 340 nm, select a different
correction wavelength or turn off Analysis
Correction.
Dye 1/Dye 2 Cy3, 5, 3.5, or 5.5, See Select pre-defined dye used to label sample
Typec Alexa Fluor 488, 546, Dye/Chromophore material, or one that has been added using
555, 594, 647, or 660 Editor for specific Dye/Chrom. Editor.
values for each
dye
Dye 1/Dye 2 picomoles/microliter not applicable Select unit for reporting dye concentrations.
Unit (pmol/µl), micromoles
(uM), or millimoles
(mM)

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5 Protein Applications
Measure Proteins and Labels

Mass Ext.
Setting Available Options Coefficient Description
(L/gm-cm)
Sloping Dye On or off Corrects dye absorbance measurements for
Correctiond any offset caused by light scattering
particulates by subtracting absorbance value
of a sloping baseline from 400 nm to 850 nm
from absorbance value at dye’s analysis
wavelength.
a To add or edit a custom protein, use Protein Editor.
b Analysis Correction affects calculation for protein concentration only.
c To add custom dye or edit list of available dyes, use Dye/Chromophore Editor.
d Sloping Dye Correction affects calculations for dye concentration only.

Related Topics
• Instrument Settings
• Protein Editor
• Dye/Chromophore Editor

Detection Limits for Proteins and Labels Measurements

Detection limits and reproducibility specifications for purified BSA proteins and dyes
that are pre-defined in the software are provided here. The BSA lower detection limit
and reproducibility values apply to any protein sample type. The upper detection
limits are dependent on the upper absorbance limit of the instrument and the
sample’s extinction coefficient.

To calculate upper detection limits for other (non-BSA) protein sample types
To calculate upper detection limits in mg/mL for proteins, use the following equation:
(upper absorbance limitinstrument /mass extinction coefficientsample) * 10

For example, if the sample’s mass extinction coefficient at 280 nm is 6.7 for a 1%
(10 mg/mL) solution, the equation looks like this:
(200 / 6.7) * 10 = 298.5 (or ~300)

Related Topics
• Detection Limits for All Applications

80 NanoDrop Eight User Guide Thermo Scientific

 5 Protein Applications
Measure Proteins and Labels

Calculations for Proteins and Labels Measurements

As with the other protein applications, Available Options for Extinction Coefficient
Proteins & Labels uses the Beer-Lambert
equation to correlate absorbance with • 1 Abs = 1 mg/mL, where sample type and/or ext.
concentration based on the sample’s coefficient is unknown (produces rough estimate of
extinction coefficient and pathlength. protein concentration)
This application offers six options (shown at • BSA (Bovine Serum Albumin, 6.7 L/gm-cm)
right) for selecting an appropriate extinction
coefficient for each measured sample, to • IgG (any mammalian antibody, 13.7 L/gm-cm)
be used in conjunction with Beer’s Law to
calculate sample concentration. • Lysozyme (egg white lysozyme, 26.4 L/gm-cm)

If the extinction coefficient of the sample is • Other protein ( + MW), user-specified molar ext.

known, choose the + MW (molar) or coefficient
 1% (mass) option and enter the value.
• Other protein (1%), user-specified mass ext.
Otherwise, calculate the extinction
coefficient or choose the option that best coefficient
matches the sample solution.
Note: See Sample Type for details.
Tip: Ideally, the extinction coefficient
should be determined empirically using a
solution of the study protein at a known
concentration using the same buffer.
Calculated protein concentrations are Measured Values
based on the absorbance value at 280 nm,
the selected (or entered) extinction A280 absorbance
coefficient and the sample pathlength. A
single-point baseline correction (or analysis Note: The absorbance value at 850 nm is subtracted from all
correction) may be applied. wavelengths in the spectrum. As a result, the absorbance at
850 nm is zero in the displayed spectra. Also, for
Concentration is reported in mass units.
micro-volume absorbance measurements, the spectra are
Calculators are available on the Internet to
convert concentration from mass to molar normalized to a 10 mm pathlength equivalent.
units based on sample sequence.
• Protein absorbance values are measured at 280 nm
using the Analysis-corrected and normalized spectrum. If
Analysis Correction and Dye Correction are not selected,
this is the reported A280 value and the value used to
calculate protein concentration.
• If Analysis Correction is selected, the normalized and
analysis-corrected absorbance value at 280 nm is
reported and used to calculate protein concentration.
• If a Dye is used, the normalized, analysis-corrected and
dye-corrected absorbance value at 280 nm is reported
and used to calculate protein concentration.

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5 Protein Applications
Measure Proteins and Labels

Dye concentrations are calculated from the Dye absorbance

absorbance value at the dye’s analysis
wavelength, the dye’s extinction coefficient, • Dye absorbance values are measured at specific
and the sample pathlength. A sloped-line wavelengths. See Dye/Chromophore Editor for analysis
dye correction may also be used. wavelengths used.
• If Sloping Dye Correction is selected, a linear baseline is
drawn between 400 nm and 850 nm and, for each dye,
the absorbance value of the sloping baseline is
subtracted from the absorbance value at each dye’s
analysis wavelength. Baseline-corrected dye absorbance
values are reported and used to calculate dye
concentrations.

Dye correction
• Pre-defined dyes have known correction values for A260
and A280. See Dye/Chromophore Editor for correction
values used.

• A280 dye correction is subtracted from A280 absorbance

value used to calculate protein concentration.
Sample Pathlength
• For micro-volume measurements, the software selects
the optimal pathlength (between 1.0 mm and 0.1 mm)
based on sample absorbance at the analysis wavelength.
• Displayed spectra and absorbance values are normalized
to a 10 mm pathlength equivalent.
Reported Values
• Protein concentration. Reported in selected unit
(mg/mL or µg/mL). Calculations are based on
Beer-Lambert equation using corrected protein
absorbance value.

• Dye1/Dye2 concentration. Reported in pmol/µL.

Calculations are based on Beer’s Law equation using
(sloping) baseline-corrected dye absorbance value(s).

Related Topics
• Beer-Lambert Equation
• Protein A280 Calculations

82 NanoDrop Eight User Guide Thermo Scientific

 5 Protein Applications
Measure Protein BCA

Measure Protein BCA

Measures total protein concentration of
unpurified protein samples using a
bicinchoninic acid colorimetric detection
reagent.

Measure Total Protein

Reported Results

Settings

Detection Limits

Measure Total Protein Concentration

The Protein BCA assay uses bicinchoninic acid as a colorimetric detection reagent
to determine total protein concentration in unpurified protein samples. This
application is useful for measuring dilute protein solutions or proteins in the presence
of components that exhibit significant absorbance between 200 nm and 280 nm,
which rules out direct protein measurements at 280 nm or 205 nm. This application
measures absorbance at 562 nm and uses a standard curve to calculate protein
concentration. A single-point baseline correction is applied.

Theory of Protein BCA assay

The Protein BCA assay uses bicinchoninic acid (BCA) as the detection reagent for
Cu+1, which is formed when Cu+2 is reduced by certain proteins in an alkaline
environment. A purple reaction product is formed by the chelation of two molecules
of BCA with one cuprous ion (Cu+1). The resulting Cu-BCA chelate formed in the
presence of protein is measured at 562 nm and baseline-corrected using the
absorbance value at 750 nm. Pre-formulated kits of BCA reagent and CuSO4 are
available from us or a local distributor.

Protein assay kits and protocols

Please refer to the NanoDrop website for up-to-date kits and protocols for the
NanoDrop Eight instrument. Follow the assay kit manufacturer’s recommendations
for all standards and samples (unknowns). Ensure each is subjected to the same
timing and temperature throughout the assay.

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5 Protein Applications
Measure Protein BCA

Protein standards for generating a standard curve may also be provided by the kit
manufacturer. Since the NanoDrop Eight pedestals can measure higher protein
concentrations than traditional cuvette-based spectrophotometers, you may need to
supply your own protein standards at higher concentrations than provided by the
manufacturer. For example, additional standards may be required to ensure the
standard curve covers the dynamic range of the assay and the expected range of
the unknown samples.

Working with standard curves

A standard curve is required for colorimetric protein analysis.
• Each experiment requires a standard curve. You can run a new standard curve,
or import standard from a previously run experiment.
• To import a Standard curve from a previous experiment, select the Load
Standard icon, select a Standard, and click Load.
• Prepare standards and unknown samples the same way. See the kit
manufacturer’s guidelines and recommendations.
– All reference and standards solutions should be the same buffer
used to resuspend the samples plus the same volume of reagent added to
the samples.
– First standard is a reference measurement. The reference solution should
contain none of the analyte of interest. (The reference measurement is not
the same as a blank measurement. This application requires both.)
– Concentration range of the standards must cover the dynamic range
of the assay and the expected range of the unknown samples. Sample
analyte concentrations are not extrapolated beyond the concentration of the
highest standard.

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 5 Protein Applications
Measure Protein BCA

• Use the application setup to enter concentration values for the standards and to
specify how standards and samples will be measured (number of replicates, etc.).

Reference concentration

Standard concentrations

Select Done when finished

entering Standards

– Depending on the Curve Type setting, a standard curve can be generated

using two or more standards.
– The software requires one reference measurement and allows up
to 7 standards.
– Concentration values for standards can be entered in any order but
the standards must be measured in the order in which they were entered;
however, best practice dictates that standards be measured from the lowest
concentration of the standard analyte stock to the highest.
• For all colorimetric assays except Protein Pierce 660, blank the instrument
with DI H2O (deionized water). For Protein Pierce 660, blank with the reference
solution.
• Measure the reference and all standards before you start analyzing
samples. (After the first sample has been measured, no additional changes are
allowed to the standard curve.)

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5 Protein Applications
Measure Protein BCA

As you measure the standards, a measurement screen displays, similar to the

measurement screens for samples.
Select Standard Curve to see the standard curve you have built.

Here is an example:
Curve type setting R2 value (1.0 equals perfect fit)

Standard curve

Pink circles indicate data points

for standards

The R2 value indicates how well the standard curve fits the standard data points
(1.0 is a perfect fit; all points lie exactly on the curve).
Standards are listed in the lower half of the screen in the data table.

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 5 Protein Applications
Measure Protein BCA

After the minimum number of standards has been measured for the selected
curve type, a message similar to the following appears:

Remeasure standards: select to remeasure any measured standards.

Right-click on existing standard(s) and select Remeasure.

Load more standards: returns to the setup screen where you can add or edit
the concentration value for any standard and then measure the standard.
Load Sample ID File: allows for selection of Sample ID information from an
imported sample ID file.
Continue to measurements: continues to sample measurement screen,
after which standards can no longer be edited.
• You can add, edit or delete a standard any time before the first sample
measurement.
Add standard:
– from standards measurement screen, select BCA Setup
– Increment the #of standards drop-down and enter the concentration
value for the new standard
– select Done
Edit standard:
– from standards measurement screen, seelct BCA Setup

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5 Protein Applications
Measure Protein BCA

– select the Concentration field and edit the concentration value

– click Done
Delete standard:
– from standards data table, right-click the standard and select Delete.
The standard no longer appears in the table on the measurement screen and its
concentration value no longer appears on the setup screen.
Note You can use this method to delete the reference measurement;
however, a new reference must be measured immediately afterwards.

• After the minimum number of standards has been measured for the selected
curve type, the message “Invalid Curve” changes to “Valid Curve.” (This occurs
even when additional standards have been defined but not yet measured.) If the
“Invalid Curve” message remains after all entered standards have been
measured, try:
– selecting a different curve type
– remeasuring standards using the correct standard material
Valid Curve indicator: This is only an indicator that the required minimum
number of points has been established for the selected curve type. It does not
validate the integrity of the curve. For example, additional standards may be
required to cover the expected assay concentration range.

To measure Protein BCA standards and samples

NOTICE
• Do not use a squirt or spray bottle on or near the instrument as liquids will
flow into the instrument and may cause permanent damage.
• Do not use hydrofluoric acid (HF) on the pedestals. Fluoride ions will
permanently damage the quartz fiber optic cables.

Before you begin...

Before taking pedestal measurements with the NanoDrop Eight instrument, lift the
instrument arm and clean the upper and lower pedestals. At a minimum, wipe the
pedestals with a new laboratory wipe. For more information, see Cleaning the
Pedestals.

88 NanoDrop Eight User Guide Thermo Scientific

 5 Protein Applications
Measure Protein BCA

To measure Protein BCA standards and samples

1. From the Home screen, select the Proteins tab and select Protein BCA.
2. Specify a curve type and number of replicates for each standard and enter the
concentration of each standard.
Tip: For this assay, we recommend setting Curve Type to “Linear”.
3. Measure blank:
– pipette 2 µL DI H2O onto lower pedestals and lower arm.
– select Blank and wait for measurement to complete.
– lift arm and clean all pedestals with new laboratory wipe.
4. Measure reference standard:
– pipette 2 µL reference solution onto pedestal. Reference solution should
contain none of the standard protein stock, see Working With Standard
Curves for details.
– lower arm to start measurement (select Measure if Auto-Measure is off).
– lift arm and clean all pedestals with new wipe.
– if Replicates setting is greater than 1, repeat measurement with a new
sample aliquot.
5. Measure remaining standards:
– pipette 2 µL standard 1 onto pedestal.
– lower arm to start measurement (select Measure if Auto-Measure is off).
– lift arm and clean all pedestals with new wipe.
– if Replicates setting is greater than 1, repeat measurement with a new
sample aliquot.
– repeat substeps above for each additional standard (when specified
number of standards and replicates have been measured, a message asks
whether to load more standards or begin measuring samples)
– if finished measuring standards, select Load Sample ID File or
Continue to measurements (select Standard Curve from the view
drop-down to view standard curve).
6. Measure samples:
– pipette 2 µL sample 1 onto pedestal.
– lower arm to start measurement (or select Measure if Auto-Measure is off)
– lift arm and clean all pedestals with new wipe.
7. When you are finished measuring samples, select End Experiment .
8. Lift the arm and clean all pedestals with a new wipe.

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5 Protein Applications
Measure Protein BCA

Protein BCA Reported Results

Protein BCA measurement screen

For each measured sample and standard, this application shows the visible
absorbance spectrum and a summary of the results. Select “Standard Curve” to
view the Standard curve. Below is an example of the measurement screen:
Protein BCA Run Blank
Application Sample names setup
of displayed Measure sample
Graph View; Select Load
measurements
Standard Curve to Standard
End experiment
view standard curve

Right click graph

area to view display
UV spectrum options.

Menu of table
options;
click to choose
which columns to
report

Click row to select

sample and update
Sample name; Protein spectrum.
select to edit concentration
Absorbance data for
Standard replicates

Note
• A baseline correction is performed at 750 nm (absorbance value at 750 nm is
subtracted from absorbance values at all wavelengths in sample spectrum).
• Micro-volume absorbance measurements are normalized to a 10.0 mm
pathlength equivalent.

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 5 Protein Applications
Measure Protein BCA

Protein BCA standard curve screen

The standard curve screen shows graphically the relationship between the
measured standards, the calculated standard curve, and the measured absorbance
and calculated concentration for a selected sample. A horizontal line connects the
sample absorbance value on the Y-axis to the standard curve. A vertical line
connects that point to the sample concentration value on the X-axis.

The R2 value indicates how well the standard curve fits the standard data points (1.0
is a perfect fit; that is, all points lie exactly on the curve).

View standard Curve type R2 value (1.0 equals perfect fit)

curve
Line of best fit
equation

Standard curve

Selected sample
Click row to
select sample
and update
graph; select
more rows to
overlay spectra
for up to five
samples.

Pink dots indicate Orange dots Protein concentration Absorbance

standard data points indicate sample at 562 nm
data points
Standard curve view

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5 Protein Applications
Measure Protein BCA

Protein BCA reported values

Reported values are shown in the Data table. Select which of the reported results is
shown in the data table by selecting from the data table options menu. Here are the
available reported values:

Date/time measured

Protein conc.

Number of standard replicates

Absorbance at 562 nm

Average absorbance at
Baseline correction absorbance 562 nm for replicate
standard measurements
Equation of the standard curve
Additional monitored wavelength

Related Topics
• Basic Instrument Operations
• Protein A280 Calculations

Settings for Protein BCA Measurements

To show the Protein BCA settings, click the Protein BCA Setup Icon .
Note You can edit the Curve Type setting when measuring standards by
changing the list box at the top of the application measurement screen. You can
edit the concentration value for a standard from the application setup screen.
After the first sample measurement, these settings cannot be changed.

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 5 Protein Applications
Measure Protein BCA

Setting Description
Curve Type Specify type of equation used to create standard curve from standard concentration
values. Available options:
– Linear: Draws the linear least squares line through all measured standards
(requires reference measurement and at least one standard)
– Interpolation: Draws a series of straight lines to connect all measured
standards (requires reference measurement and at least one standard)
– 2nd order polynomial: Draws the 2nd order least squares polynomial
using all measured standards (requires reference measurement and at least
two standards)
– 3rd order polynomial: Draws the 3rd order least squares polynomial using
all measured standards (requires reference measurement and at least three
standards)

Replicates Enter number of measurements of the reference or the same standard that are
averaged together to produce its associated concentration value.

Note: Replicates setting cannot be changed after the first standard has been
measured.

Standards Enter actual concentration value of each standard.

Note: Concentration values can be entered in any order but the standards must be
measured in the order they were entered.

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 5 Protein Applications
Measure Protein Bradford

Measure Protein Bradford

Measures total protein concentration of
unpurified protein samples using a
Coomassie Blue dye colorimetric
detection reagent.

Measure Total Protein

Reported Results

Settings

Detection Limits

Measure Total Protein Concentration

The Protein Bradford assay uses Coomassie Blue dye as a colorimetric detection
reagent to determine total protein concentration in unpurified protein samples. This
application is useful for measuring dilute protein solutions that require lower
detection sensitivity or proteins in the presence of components that exhibit
significant absorbance between 200 nm and 280 nm, which rules out direct protein
measurements at 280 nm or 205 nm. This application measures absorbance at
595 nm and uses a standard curve to calculate protein concentration. See Working
with Standard Curves for more information. A single-point baseline correction is
applied.

Theory of Protein Bradford assay

The Protein Bradford assay uses the protein-induced absorbance shift of Coomassie
Blue dye to determine total protein concentration. The bound protein-dye complex is
measured at 595 nm and baseline-corrected using the absorbance value at 750 nm.
Pre-formulated kits of stabilized reagent mixture containing Coomassie Blue dye,
alcohol, and surfactant are available from us or a local distributor.

To maximize reliability with the Protein Bradford assay:

• Work quickly and do not allow prepared standards or samples to
sit longer than necessary. Coomassie dye-dye and Coomassie dye-protein
aggregates can form particulates with increasing development time, resulting in
significant fluctuations in absorbance readings.

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5 Protein Applications
Measure Protein Bradford

• Measure standards and samples in triplicate using a new aliquot for

each measurement. For pedestal measurements, the total analyte (protein-dye)
signal at 595 nm is limited to ~0-0.150A due to the pedestal’s 1.0 mm
pathlength, the Coomassie dye concentration, and the acidic pH.

Protein assay kits and protocols

Please refer to the NanoDrop website for up-to-date kits and protocols for the
NanoDrop Eight instrument. Follow the assay kit manufacturer’s recommendations
for all standards and samples (unknowns). Ensure each is subjected to the same
timing and temperature throughout the assay.

Protein standards for generating a standard curve may also be provided by the kit
manufacturer. Since the NanoDrop Eight pedestals can measure higher protein
concentrations than traditional cuvette-based spectrophotometers, you may need to
supply your own protein standards at higher concentrations than provided by the
manufacturer. For example, additional standards may be required to ensure the
standard curve covers the dynamic range of the assay and the expected range of
the unknown samples.

To measure Protein Bradford standards and samples

NOTICE
• Do not use a squirt or spray bottle on or near the instrument as liquids will
flow into the instrument and may cause permanent damage.
• Do not use hydrofluoric acid (HF) on the pedestals. Fluoride ions will
permanently damage the quartz fiber optic cables.

Before you begin...

Before taking pedestal measurements with the NanoDrop Eight instrument, lift the
instrument arm and clean the upper and lower pedestals. At a minimum, wipe the
pedestals with a new laboratory wipe. For more information, see Cleaning the
Pedestals.

To measure Protein Bradford standards and samples

1. From the Home screen, select the Proteins tab and select
Protein Bradford.
2. Specify a curve type and number of replicates for each standard and enter the
concentration of each standard.
Tip: For this assay, set Curve Type to “2nd Order Polynomial” and
Replicates to 3.
3. Measure blank:

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 5 Protein Applications
Measure Protein Bradford

– pipette 2 µL DI H2O onto lower pedestals and lower arm.

– select Blank and wait for measurement to complete
– lift arm and clean all pedestals with a new laboratory wipe.
4. Measure reference standard:
– pipette 2 µL reference solution onto pedestal. Reference solution should
contain none of the standard protein stock, see Working With Standard
Curves for details.
– lower arm to start measurement (or select Measure if Auto-Measure is off)
– lift arm and clean all pedestals with new wipe.
– if Replicates setting is greater than 1, repeat measurement with a new
sample aliquot.
5. Measure remaining standards:
– pipette 2 µL standard 1 onto pedestal.
– lower arm to start measurement (or select Measure if Auto-Measure is off)
– lift arm and clean all pedestals with new wipe.
– if Replicates setting is greater than 1, repeat measurement with a new
sample aliquot.
– repeat substeps above for each additional standard (when specified number
of standards and replicates have been measured, a message asks whether
to load more standards or begin measuring samples)
– if finished measuring standards, select Load Sample ID File or
Continue to measurements (select Standard Curve from the view
drop-down to view standard curve).
6. Measure samples:
– pipette 2 µL sample 1 onto pedestal.
– lower arm to start measurement (or select Measure if Auto-Measure is off)
– lift arm and clean all pedestals with new wipe,
– if Replicates setting is greater than 1, repeat measurement.
7. When you are finished measuring samples, select End Experiment .
8. Lift the arm and clean all pedestals with a new wipe.

Related Topics
• Working with standard curves
• Best practices for protein measurements

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5 Protein Applications
Measure Protein Bradford

Protein Bradford Reported Results

Protein Bradford measurement screen

For each measured sample and standard, this application shows the visible
absorbance spectrum and a summary of the results. Select “Standard Curve” to
view the Standard curve. Below is an example of the measurement screen:

Application Sample names Protein Bradford setup Run Blank

of displayed
Graph View; Select measurements Load Standard Measure sample
Standard Curve to
End experiment
view standard curve

Right click
graph area
to view
display
options.
UV spectrum Menu of
table
options;
click to
choose
which
columns
to report

Click row
to select
sample and
Sample name; Protein Absorbance data for update
select to edit concentration Standard replicates spectrum.

Note
• A baseline correction is performed at 750 nm (absorbance value at 750 nm is
subtracted from absorbance values at all wavelengths in sample spectrum).
• Micro-volume absorbance measurements are normalized to a 10.0 mm
pathlength equivalent.

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Measure Protein Bradford

Protein Bradford standard curve screen

The standard curve screen shows graphically the relationship between the
measured standards, the calculated standard curve, and the measured absorbance
and calculated concentration for a selected sample. A horizontal line connects the
sample absorbance value on the Y-axis to the standard curve. A vertical line
connects that point to the sample concentration value on the X-axis.

The R2 value indicates how well the standard curve fits the standard data points (1.0
is a perfect fit; that is, all points lie exactly on the curve).

View standard Line of best fit Curve type R2 value (1.0 equals perfect fit)
curve equation

Selected data point

Standard curve
Click row to
select sample
and update
graph; select
more rows to
overlay spectra
for up to five
samples.

Pink dots indicate Orange dots Protein Absorbance

standard data points indicate sample concentration at 595 nm
data points
Standard curve view

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5 Protein Applications
Measure Protein Bradford

Protein Bradford reported values

Reported values are shown in the Data table. Select which of the reported results is
shown in the data table by selecting from the data table options menu. Here are the
available reported values:

Date/time measured

Protein conc.
Number of standard replicates
Absorbance at 595 nm

Average absorbance at
Baseline correction absorbance 595 nm for replicate
standard measurements
Equation of the standard curve
Additional monitored wavelength

Related Topics
• Example standard curve
• Basic Instrument Operations
• Protein A280 Calculations

Settings for Protein Bradford Measurements

To show the Protein Bradford settings, click the Protein Bradford Setup
Icon.
Note You can edit the Curve Type setting when measuring standards by
changing the list box at the top of the application measurement screen. You can
edit the concentration value for a standard from the application setup screen.
After the first sample measurement, these settings cannot be changed.

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 5 Protein Applications
Measure Protein Bradford

Setting Description
Curve Type Specify type of equation used to create standard curve from standard concentration
values. Available options:
– Linear: Draws the linear least squares line through all measured standards
(requires reference measurement and at least one standard)
– Interpolation: Draws a series of straight lines to connect all measured
standards (requires reference measurement and at least one standard)
– 2nd order polynomial: Draws the 2nd order least squares polynomial
using all measured standards (requires reference measurement and at least
two standards)
– 3rd order polynomial: Draws the 3rd order least squares polynomial using
all measured standards (requires reference measurement and at least three
standards)

Replicates Enter number of measurements of the reference or the same standard or sample
that are averaged together to produce its associated concentration value.

Note: Replicates setting cannot be changed after the first standard has been
measured.

Standards Enter actual concentration value of each standard.

Note: Concentration values can be entered in any order but the standards must be
measured in the order they were entered.

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 5 Protein Applications
Measure Protein Lowry

Measure Protein Lowry

Measures total protein concentration of
unpurified protein samples using a
Folin-Ciocalteu colorimetric detection
reagent.

Measure Total Protein

Reported Results

Settings

Detection Limits

Measure Total Protein Concentration

The Protein Lowry assay uses Folin-Ciocalteu as a colorimetric detection reagent to
determine total protein concentration in unpurified protein samples. This application
is an alternative to the other colorimetric applications for measuring dilute protein
solutions or proteins in the presence of components that exhibit significant
absorbance between 200 nm and 280 nm. This application measures absorbance
at 650 nm and uses a standard curve to calculate protein concentration. See
Working with Standard Curves for more information. A single-point baseline
correction is applied.

Theory of Protein Lowry assay

The Protein Lowry assay involves the reaction of protein with cupric sulfate in alkaline
solution, resulting in the formation of tetradentate copper-protein complexes. The
Folin-Ciocalteu reagent is effectively reduced in proportion to the chelated
copper-complexes. The water-soluble blue reaction product is measured at 650 nm
and baseline-corrected using the absorbance value at 405 nm. Pre-formulated kits
of Folin-Ciocalteu reagent and CuSO4 are available from us or a local distributor.

Protein assay kits and protocols

Please refer to the NanoDrop website for up-to-date kits and protocols for the
NanoDrop Eight instrument. Follow the assay kit manufacturer’s recommendations
for all standards and samples (unknowns). Ensure each is subjected to the same
timing and temperature throughout the assay.

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5 Protein Applications
Measure Protein Lowry

To measure Protein Lowry standards and samples

NOTICE
• Do not use a squirt or spray bottle on or near the instrument as liquids will
flow into the instrument and may cause permanent damage.
• Do not use hydrofluoric acid (HF) on the pedestals. Fluoride ions will
permanently damage the quartz fiber optic cables.

Before you begin...

Before taking pedestal measurements with the NanoDrop Eight instrument, lift the
instrument arm and clean the upper and lower pedestals. At a minimum, wipe the
pedestals with a new laboratory wipe. For more information, see Cleaning the
Pedestals.

To measure Protein Lowry standards and samples

1. From the Home screen, select the Proteins tab and select Protein Lowry.
2. Specify a curve type and number of replicates for each standard and enter the
concentration of each standard.
Tip: For this assay, we recommend setting Curve Type to “2nd Order
Polynomial.”
3. Measure blank:
– pipette 2 µL DI H2O onto lower pedestal and lower arm.
– select Blank and wait for measurement to complete
– lift arm and clean all pedestals with a new laboratory wipe.
4. Measure reference standard:
– pipette 2 µL reference solution onto pedestal. Reference solution should
contain none of the standard protein stock, see Working With Standard
Curves for details.
– lower arm to start measurement (or select Measure if Auto-Measure is off)
– lift arm and clean all pedestals with a new wipe.
– if Replicates setting is greater than 1, repeat measurement with a new
sample aliquot.
5. Measure remaining standards:
– pipette 2 µL standard 1 onto pedestal.
– lower arm to start measurement (or select Measure if Auto-Measure is off)
– lift arm and clean all pedestals with new wipe.

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 5 Protein Applications
Measure Protein Lowry

– if Replicates setting is greater than 1, repeat measurement with a new

sample aliquot.
– repeat substeps above for each additional standard (when specified number
of standards and replicates have been measured, a message asks whether
to load more standards or begin measuring samples).
– if finished measuring standards, select Load Sample ID File or
Continue to measurements (select Standard Curve from the view
drop-down to view standard curve).
6. Measure samples:
– pipette 2 µL sample 1 onto pedestal.
– lower arm to start measurement (or select Measure if Auto-Measure is off)
– lift arm and clean all pedestals with new wipe.
– if Replicates setting is greater than 1, repeat measurement.
7. When you are finished measuring samples, select End Experiment .
8. Lift the arm and clean all pedestals with a new wipe.

Related Topics
• Working with standard curves
• Best practices for protein measurements
• Measure a Micro-Volume Sample
• Prepare Samples and Blanks

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5 Protein Applications
Measure Protein Lowry

Protein Lowry Reported Results

Protein Lowry measurement screen

For each measured sample and standard, this application shows the visible
absorbance spectrum and a summary of the results. Select “Standard Curve” to
view the Standard curve. Below is an example of the measurement screen:

Application Sample names Protein Lowry setup Run Blank

of displayed
Graph View; Select measurements Load Standard Measure sample
Standard Curve to
End experiment
view standard curve

Right click
graph area
to view
display
UV spectrum options.

Menu of
table
options;
click to
choose
which
columns
to report

Click row
to select
sample and
Sample name; Protein Absorbance data for update
select to edit concentration Standard replicates spectrum.

Note
• A baseline correction is performed at 405 nm (absorbance value at 405 nm is
subtracted from absorbance values at all wavelengths in sample spectrum).
• Micro-volume absorbance measurements are normalized to a 10.0 mm
pathlength equivalent.

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 5 Protein Applications
Measure Protein Lowry

Protein Lowry standard curve screen

The standard curve screen shows graphically the relationship between the
measured standards, the calculated standard curve, and the measured absorbance
and calculated concentration for a selected sample. A horizontal line connects the
sample absorbance value on the Y-axis to the standard curve. A vertical line
connects that point to the sample concentration value on the X-axis.

The R2 value indicates how well the standard curve fits the standard data points (1.0
is a perfect fit; that is, all points lie exactly on the curve).
View standard Line of best fit Curve type R2 value (1.0 equals perfect fit)
curve equation

Standard curve

Click row to
Selected data point select sample
and update
graph; select
more rows to
overlay spectra
for up to five
samples.

Orange dots indicate Pink dots indicate Protein Absorbance

sample data points standard data points concentration at 650 nm
Standard curve view

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5 Protein Applications
Measure Protein Lowry

Protein Lowry reported values

Reported values are shown in the Data table. Select which of the reported results is
shown in the data table by selecting from the data table options menu. Here are the
available reported values:

Date/time measured

Protein conc.
Number of standard replicates
Absorbance at 650 nm
Average absorbance at
650 nm for replicate
Baseline correction absorbance
standard measurements
Equation of the standard curve
Additional monitored wavelength

Related Topics
• Example standard curve
• Basic Instrument Operations

Settings for Protein Lowry Measurements

To show the Protein Lowry settings, click the Protein Lowry Setup Icon.

Note You can edit the Curve Type setting when measuring standards by
changing the list box at the top of the application measurement screen. You can
edit the concentration value for a standard from the application setup screen.
After the first sample measurement, these settings cannot be changed.

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 5 Protein Applications
Measure Protein Lowry

Setting Description
Curve Type Specify type of equation used to create standard curve from standard concentration
values. Available options:
– Linear: Draws the linear least squares line through all measured standards
(requires reference measurement and at least one standard)
– Interpolation: Draws a series of straight lines to connect all measured
standards (requires reference measurement and at least one standard)
– 2nd order polynomial: Draws the 2nd order least squares polynomial
using all measured standards (requires reference measurement and at least
two standards)
– 3rd order polynomial: Draws the 3rd order least squares polynomial using
all measured standards (requires reference measurement and at least three
standards)

Replicates Enter number of measurements of the reference or the same standard or sample
that are averaged together to produce its associated concentration value.

Note: Replicates setting cannot be changed after the first standard has been
measured.

Standards Enter actual concentration value of each standard.

Note: Concentration values can be entered in any order but the standards must be
measured in the order they were entered.

Related Topics
• Instrument Settings

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 5 Protein Applications
Measure Protein Pierce 660

Measure Protein Pierce 660

Measures total protein concentration of
unpurified protein samples using a
proprietary colorimetric detection
reagent.

Measure Total Protein

Reported Results

Settings

Detection Limits

Measure Total Protein Concentration

The Protein Pierce 660 assay uses a proprietary protein binding material as a
colorimetric detection reagent to determine total protein concentration in unpurified
protein samples. This application is suitable for protein solutions that contain high
concentrations of detergents, reducing agents and other commonly used reagents.
The Pierce 660 application measures absorbance at 660 nm and uses a standard
curve to calculate protein concentration (see Working with Standard Curves for more
information). A single-point baseline correction is applied.

Theory of Protein Pierce 660 assay

The Protein Pierce 660 assay is based on the binding of a proprietary dye-metal
complex to protein in acidic conditions that causes a shift in the dye’s absorption
maximum, which is measured at 660 nm. The dye-metal complex is reddish-brown
and changes to green upon protein binding. The color change is produced by
deprotonation of the dye at low pH facilitated by interactions with positively charged
amino acid groups in proteins. The dye interacts mainly with basic residues in
proteins such as histidine, arginine and lysine and to a lesser extent tyrosine,
tryptophan and phenylalanine. The reaction product is measured at 660 nm and
baseline-corrected using the absorbance value at 750 nm.

The color produced in the assay is stable and increases in proportion to a broad
range of increasing protein concentrations. An optional Ionic Detergent Compatibility
Reagent (IDCR) may be added to the assay reagent to increase compatibility with
high amounts of ionic detergents, including Laemmli SDS sample buffer with
bromophenol blue. The IDCR dissolves completely by thorough mixing and has no
effect on the assay. Pre-formulated kits of the protein binding material are available
from us or a local distributor. For information about IDCR, refer to the kit
manufacturer.

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5 Protein Applications
Measure Protein Pierce 660

Protein assay kits and protocols

Please refer to the NanoDrop website for up-to-date kits and protocols for the
NanoDrop Eight instrument. Follow the assay kit manufacturer’s recommendations
for all standards and samples (unknowns). Ensure each is subjected to the same
timing and temperature throughout the assay.

Protein standards for generating a standard curve may also be provided by the kit
manufacturer. Since the NanoDrop Eight pedestals can measure higher protein
concentrations than traditional cuvette-based spectrophotometers, you may need to
supply your own protein standards at higher concentrations than provided by the
manufacturer. For example, additional standards may be required to ensure the
standard curve covers the dynamic range of the assay and the expected range of
the unknown samples.

To measure Protein Pierce 660 standards and samples

NOTICE
• Do not use a squirt or spray bottle on or near the instrument as liquids will
flow into the instrument and may cause permanent damage.
• Do not use hydrofluoric acid (HF) on the pedestals. Fluoride ions will
permanently damage the quartz fiber optic cables.

Before you begin...

Before taking pedestal measurements with the NanoDrop Eight instrument, lift the
instrument arm and clean the upper and lower pedestals. At a minimum, wipe the
pedestals with a new laboratory wipe. For more information, see Cleaning the
Pedestals.

To measure Protein Pierce 660 standards and samples

1. From the Home screen, select the Proteins tab and then click
Protein Pierce 660.
2. Specify a curve type and number of replicates for each standard and enter the
concentration of each standard.
Tip: For this assay, we recommend setting Curve Type to “Linear”.
3. Measure blank:
– Pipette 2 µL reference solution onto lower pedestal and lower arm,
Reference solution should contain none of the standard protein stock; see
Working With Standard Curves for details.
– select Blank and wait for measurement to complete
– lift arm and clean all pedestals with a new laboratory wipe,

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 5 Protein Applications
Measure Protein Pierce 660

– Measure reference standard:

– pipette 2 µL reference solution onto pedestal. Reference solution should
contain none of the standard protein stock, see Working With Standard
Curves for details.
– lower arm to start measurement (or select Measure if Auto-Measure is off)
– lift arm and clean all pedestals with a new wipe.
– if Replicates setting is greater than 1, repeat measurement.
4. Measure remaining standards:
– pipette 2 µL standard 1 onto pedestal,
– lower arm to start measurement (or select Measure if Auto-Measure is off)
– lift arm and clean all pedestals with a new wipe,
– if Replicates setting is greater than 1, repeat measurement.
– repeat substeps above for each additional standard (when specified number
of standards and replicates have been measured, a message asks whether
to load more standards or begin measuring samples)
– if finished measuring standards, select Load Sample ID File or
Continue to measurements (select Standard Curve from the view
drop-down to view standard curve).
5. Measure samples:
– pipette 2 µL sample 1 onto pedestal,
– lower arm to start measurement (or select Measure if Auto-Measure is off)
– lift arm and clean all pedestals with new wipe,
– if Replicates setting is greater than 1, repeat measurement.
6. When you are finished measuring samples, select End Experiment .
7. Lift the arm and clean all pedestals with a new wipe.

Related Topics
• Working with standard curves
• Best practices for protein measurements
• Measure a Micro-Volume Sample
• Prepare Samples and Blanks
• Basic Instrument Operations

Thermo Scientific NanoDrop Eight User Guide 113

5 Protein Applications
Measure Protein Pierce 660

Protein Pierce 660 Reported Results

Protein Pierce 660 measurement screen

For each measured sample and standard, this application shows the visible
absorbance spectrum and a summary of the results. Select “Standard Curve” to
view the Standard curve. Below is an example of the measurement screen:

Application Sample names Protein Pierce 660 setup Run Blank

of displayed
Graph View; Select measurements Load Standard Measure sample
Standard Curve to
End experiment
view standard curve

Right click
graph area
to view
UV spectrum display
options.

Menu of
table
options;
click to
choose
which
columns
to report

Click row
to select
sample and
Sample name; Protein Absorbance data for update
select to edit concentration Standard replicates spectrum.

Note
• A baseline correction is performed at 750 nm (absorbance value at 750 nm is
subtracted from absorbance values at all wavelengths in sample spectrum).
• Micro-volume absorbance measurements are normalized to a 10.0 mm
pathlength equivalent.

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 5 Protein Applications
Measure Protein Pierce 660

Protein Pierce 660 standard curve screen

The standard curve screen shows graphically the relationship between the
measured standards, the calculated standard curve, and the measured absorbance
and calculated concentration for a selected sample. A horizontal line connects the
sample absorbance value on the Y-axis to the standard curve. A vertical line
connects that point to the sample concentration value on the X-axis.

The R2 value indicates how well the standard curve fits the standard data points (1.0
is a perfect fit; that is, all points lie exactly on the curve).
View standard Line of best fit Curve type R2 value (1.0 equals perfect fit)
curve equation

Selected data point

Standard curve

Click row to
select sample
and update
graph; select
more rows to
overlay spectra
for up to five
samples.

Orange dots indicate Pink dots indicate Protein Absorbance

sample data points standard data points concentration at 660 nm
Standard curve view

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5 Protein Applications
Measure Protein Pierce 660

Protein Pierce 660 reported values

Reported values are shown in the Data table. Select which of the reported results is
shown in the data table by selecting from the data table options menu. Here are the
available reported values:

Date/time measured

Protein conc.
Number of standard replicates
Absorbance at 660 nm

Average absorbance at
Baseline correction absorbance 660 nm for replicate
standard measurements
Equation of the standard curve
Additional monitored wavelength

Related Topics
• Example standard curve
• Basic Instrument Operations

Settings for Protein Pierce 660 Measurements

To show the Protein Pierce 660 settings, the Protein Pierce 660 Setup
Icon.
Note You can edit the Curve Type setting when measuring standards by
changing the list box at the top of the application measurement screen. You can
edit the concentration value for a standard from the application setup screen.
After the first sample measurement, these settings cannot be changed.

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 5 Protein Applications
Measure Protein Pierce 660

Setting Description
Curve Type Specify type of equation used to create standard curve from standard concentration
values. Available options:
– Linear: Draws the linear least squares line through all measured standards
(requires reference measurement and at least one standard)
– Interpolation: Draws a series of straight lines to connect all measured
standards (requires reference measurement and at least one standard)
– 2nd order polynomial: Draws the 2nd order least squares polynomial
using all measured standards (requires reference measurement and at least
two standards)
– 3rd order polynomial: Draws the 3rd order least squares polynomial using
all measured standards (requires reference measurement and at least three
standards)

Replicates Enter number of measurements of the reference or the same standard or sample
that are averaged together to produce its associated concentration value.

Note: Replicates setting cannot be changed after the first standard has been
measured.

Standards Enter actual concentration value of each standard.

Note: Concentration values can be entered in any order but the standards must be
measured in the order in which they were entered.

Related Topics
• Instrument Settings

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118 NanoDrop Eight User Guide Thermo Scientific

 6

Measure OD600

Measures the concentration of microbial

cell cultures in solution by measuring
scattered light at 600 nm.

Measure OD600

Reported Results

Settings

Calculations

Measure OD600
Use the OD600 application to monitor the growth rate of bacterial or other microbial
cell cultures by measuring the optical density (absorbance) of the culture in growth
media at 600 nm. The Beer-Lambert equation and a user-entered conversion factor
are used to correlate absorbance with concentration. Reported concentration values
can be used to identify the phase of cultured cell populations, e.g., log or
exponential and stationary.

The OD600 application reports cell concentration in cells/mL. A single-point

absorbance correction can be used. This application does not require a standard
curve.
Note Due to the amount of scattered light present in this assay, absorbance
readings are typically very low.

Theory of OD600 application

The OD600 application measures light transmission and uses that value to calculate
absorbance. In spectroscopy, transmitted light is defined as any light that is not
absorbed by, reflected from and scattered off a sample.

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6 Measure OD600

In the case of living cells, most of the incident light is transmitted through the sample
rather than scattered, reflected or absorbed. The amount of scattered light is low
and can vary from instrument to instrument. As a result, calculated absorbance
readings are typically very low.

The calculated absorbance values are used to determine the density of cells in
solution in cells/mL. The physical concepts and formulas that relate optical
properties of living cells to concentration include:
• Cells, which have a different index of refraction from the surrounding medium,
randomly reflect and scatter light out of the incident light path. The amount of
scattering is proportional to the density of cells in the sample.
• The Beer’s Law equation is used to relate absorbance to concentration. See
Calculations for OD600 Measurements for details.
• All measurements should be made on the same type of spectrophotometer and
method (i.e., pedestal vs. cuvette) as the amount of scattered light captured
varies based on the optical configuration. When using a different
spectrophotometer or method, calculate and apply a conversion factor to the
reported results. For example, to compare OD readings using the pedestal vs. a
cuvette, a conversion factor can be calculated as follows:
Conversion factor = Cuvette OD/Pedestal OD

Best practices for OD600 measurements

• Ensure the sample is within the instrument’s absorbance detection limits.
• Blank with the growth or culture media the cells of interest are suspended in.
• Run a blanking cycle to assess the absorbance contribution of your media
solution. If the media solution exhibits strong absorbance at or near the analysis
wavelength (600 nm), you may need to choose a different media solution or
application. See Choosing and Measuring a Blank for more information.
• Make dilutions as necessary to ensure sample cultures do not exceed the linear
dynamic range of the assay before the culture reaches the stationary phase. The
linear range depends largely on optical configuration. To determine the linear
range:
– Measure a series of dilutions using a young overnight culture (~16 hrs) of the
microbial strain
– Graph the OD600 measurements against the dilution factor
The upper detection limit is the measured OD600 value at which there ceases to
be a linear correlation between dilution factors and OD600 readings.

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 6 Measure OD600

• Mix samples gently but thoroughly immediately before taking an aliquot for
measurement.
• For micro-volume measurements:
– Ensure pedestal surfaces are properly cleaned and conditioned.
– Avoid introducing bubbles when mixing and pipetting.
– Start the measurement promptly to avoid settling or evaporation.
– Follow best practices for micro-volume measurements.
– Use 2 µL sample volume. See Recommended Sample Volumes for more
information.
– For dilute samples that exhibit low absorbance at 600 nm, use an alternative
wavelength such as 400 nm to measure absorbance.

To measure OD600 samples

NOTICE
• Do not use a squirt or spray bottle on or near the instrument as liquids will
flow into the instrument and may cause permanent damage.
• Do not use hydrofluoric acid (HF) on the pedestals. Fluoride ions will
permanently damage the quartz fiber optic cables.

Before you begin...

Before taking pedestal measurements with the NanoDrop Eight instrument, lift the
instrument arm and clean the upper and lower pedestals. At a minimum, wipe the
pedestals with a new laboratory wipe. For more information, see Cleaning the
Pedestals.

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To measure an OD600 sample

1. From the Home screen, select the OD600 tab and select OD600.
2. Specify the cell number conversion factor and a additional monitored
wavelength or absorbance correction if desired.
3. Pipette 2 µL blanking solution (i.e., the media solution the cells of interest are
suspended in) onto the lower pedestal and lower the arm.
4. Select Blank and wait for the measurement to complete.
Tip: If Auto-Blank is On, the blank measurement starts automatically after you
lower the arm.
5. Lift the arm and clean all pedestals with a new laboratory wipe.
6. Pipette 2 µL sample solution onto the pedestal and lower the arm.
7. Start the sample measurement:
– If Auto-Measure is On, lower arm;
– if Auto-Measure is off, lower arm and tap Measure.
When the sample measurement is completed, the spectrum and reported
values are displayed (see the next section).
8. When you are finished measuring samples, select End Experiment.
9. Lift the arm and clean all pedestals with a new wipe.

Related Topics
• Measure a Micro-Volume Sample
• Prepare Samples and Blanks
• Basic Instrument Operations

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 6 Measure OD600

OD600 Reported Results

OD600 measurement screen

The measurement screen shows the UV spectrum and all selected reported values:

Application Sample names of OD600 Run Measure sample

displayed Setup Blank
measurements End experiment

Right click graph

area to view
UV spectrum display options.

Menu of table
options;
click to choose
which columns
to report

Click row to
select sample
and update
spectrum.

Absorbance correction Cell culture

Sample name; concentration
select to edit Additional monitored wavelength (A600 * Factor)
Cell number
Corrected absorbance at conversion
Measurement screen additional wavelength factor

Note Micro-volume absorbance measurements are normalized to a 10.0 mm

pathlength equivalent.

OD600 reported values

Reported values are shown in the Data table. Select which of the reported results is
shown in the data table by selecting from the data table options menu. Here are the
available reported values:

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6 Measure OD600

Date/time measured

Corrected abs. at 600 nm

Absorbance correction
Additional wavelength
Corrected absorbance at additional wavelength
Factor
Cell culture concentration (A600 * Factor)

Related Topics
• Basic Instrument Operations
• OD600 Calculations

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 6 Measure OD600

Settings for OD600 Measurements

To show the OD600 settings, click the OD600 Setup Icon .

Setting Available Options Description

Absorbance Absorbance value User-defined absorbance correction. Enter absorbance
correction between 0 and 300 A correction for displayed spectrum. This can be useful, for
example, to correct baseline offset caused by any
difference between the media solution used to blank the
instrument and media used to suspend the cell culture
sample, and because scattered light generally produces
an offset.

Absorbance correction value is subtracted from

absorbance values at all wavelengths in sample spectrum.
(All displayed absorbance values are corrected values.)

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Setting Available Options Description

Additional monitored Any wavelength User-defined wavelength. Enter an additional wavelength
wavelength () between 250 nm and to measure if desired (useful for dilute samples that exhibit
700 nm low absorbance at 600 nm).

If an alternative wavelength is specified, use this equation

to calculate cell concentration:

c = A() * factor()
where:

c = analyte concentration in cells/mL

A() = UV-visible absorbance at specified wavelength in

absorbance units (A)

factor() = 1/(() * b) in mL/cell-cm

where:
() = molar absorption coefficient (or extinction
coefficient) at specified wavelength
b = pathlength in cm (1.0 cm for the NanoDrop Eight
instruments)
Cell number Any number User-defined factor. Generally accepted factor for
conversion factor measured cell type, or one derived empirically using a
(108) solution of study cells at known concentration using the
same media.

Default value is 1x108 which is the generally accepted

factor for most bacterial cell suspensions such as E. coli.

Tip: The factor is wavelength specific for each cell type

and can be affected by the type of media used for the
measurements. Ideally, the factor should be determined
empirically using a solution of the study cells at a known
concentration using the same media.

Related Topics
• Instrument Settings

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 6 Measure OD600

Calculations for OD600 Measurements

Similar to the nucleic acid applications, the Measured Values
OD600 application uses a modification of the
Beer-Lambert equation to calculate sample A600 absorbance
concentration where the extinction coefficient
and pathlength are combined and referred to as Note: For micro-volume absorbance measurements,
a “factor.” the spectra are normalized to a 10 mm pathlength
equivalent.
The OD600 application offers a user-specified
factor, to be used in conjunction with Beer’s
• Cell culture absorbance values are measured at
Law to calculate sample concentration. If the
factor is known, enter the factor. Otherwise, use
600 nm using the normalized spectrum. If no
1x108, which is the generally accepted factor Absorbance Correction is specified, this is the
for most bacterial cell suspensions such as E. reported A600 value and the value used to calculate
coli. cell concentration.
• If an Absorbance Correction is specified, the
normalized and (absorbance) corrected absorbance
value at 600 nm is reported and used to calculate
cell concentration.

A() absorbance
• Normalized and (absorbance) corrected (if used)
absorbance value at any specified Additional
Monitored Wavelength () is also reported.
Calculated cell concentrations are based on the Sample Pathlength
absorbance value at 600 nm, the entered factor
and the sample pathlength. A single-point • For micro-volume measurements, the software
absorbance correction may be applied. selects the optimal pathlength (between 1.0 mm and
0.1 mm) based on sample absorbance at the
analysis wavelength.
• Displayed spectra and absorbance values are
normalized to a 10 mm pathlength equivalent.
Reported Values

Cell concentration. Reported in cells/mL.

Calculations are based on Beer-Lambert equation using
corrected A600 absorbance value.

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 7

Custom Applications
Use the NanoDrop Eight to perform UV-Vis measurements.

The UV-Vis application allows the instrument to function as a conventional

spectrophotometer. Up to 40 wavelengths from 190 nm to 850 nm can be
monitored and reported.
• Measure UV-Vis ..... page 130
• Measure Custom ..... page 135

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7 Custom Applications
Measure UV-Vis

Measure UV-Vis
Measures the absorbance of any
sample at up to 40 wavelengths
across the ultra-violet (UV) and visible
regions of the spectrum.

Measure UV-Vis

Reported Results

Settings

Detection Limits

Measure UV-Vis
The UV-Vis application allows the instrument to function as a conventional
spectrophotometer. Sample absorbance is displayed on the screen from 190 nm to
850 nm. Up to 40 wavelengths can be designated for absorbance monitoring and
inclusion in the report. Automatic pathlength adjustment and a single-point baseline
correction can also be used.

To make UV-Vis measurements

NOTICE
• Do not use a squirt or spray bottle on or near the instrument as liquids will
flow into the instrument and may cause permanent damage.
• Do not use hydrofluoric acid (HF) on the pedestals. Fluoride ions will
permanently damage the quartz fiber optic cables.

Before you begin...

Before taking pedestal measurements with the NanoDrop Eight instrument, lift the
instrument arm and clean the upper and lower pedestals. At a minimum, wipe the
pedestals with a new laboratory wipe. For more information, see Cleaning the
Pedestals.

To measure a sample using the UV-Vis application

1. From the Home screen, from the Custom tab, select UV-Vis.
2. Specify up to 40 wavelengths to monitor (or you can specify them later if desired)
and whether automated pathlength adjustment, analysis wavelength, and
baseline correction will be used.
3. Pipette 1–2 µL blanking solution onto the lower pedestal and lower the arm.

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 7 Custom Applications
Measure UV-Vis

4. Select Blank and wait for the measurement to complete.

Tip: If Auto-Blank is On, the blank measurement starts automatically after you
lower the arm.
5. Lift the arm and clean all pedestals with a new laboratory wipe, .
6. Pipette 1-2 µL sample solution onto the pedestal and lower the arm.
7. Start the sample measurement: If Auto-Measure is On, lower arm;
if Auto-Measure is off, lower arm and select Measure .

When the sample measurement is completed, the spectrum and reported values
are displayed (see the next section).
8. When you are finished measuring samples, select End Experiment .
9. Lift the arm and clean all pedestals with a new wipe.

Best practices for UV-Vis measurements

• Ensure the sample absorbance is within the instrument’s absorbance detection
limits.
• Blank with the same buffer solution used to re-suspend the analyte of interest.
The blanking solution should be a similar pH and ionic strength as the analyte
solution.
• Run a blanking cycle to assess the absorbance contribution of your buffer
solution. If the buffer exhibits strong absorbance at or near an analysis
wavelength, you may need to choose a different buffer or application. See
Choosing and Measuring a Blank for more information.
• For micro-volume measurements:
– Ensure pedestal surfaces are properly cleaned and conditioned.
– Ensure samples are homogeneous before taking a measurement. Avoid
introducing bubbles when mixing and pipetting.
– Follow best practices for micro-volume measurements.
– Use a 1-2 µL sample volume. See Recommended Sample Volumes for more
information.

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Measure UV-Vis

UV-Vis Reported Results

UV-Vis measurement screen

For each measured sample, the absorbance spectrum and results are shown:

Back to View history data Run blank Measure sample solution

Home
screen Load Sample ID file
End experiment
and export data

Select to toggle
Auto-Measure
Right click graph area to view display options. ON/OFF
(Auto-Measure
default is ON
Auto-Blank
default is OFF)

Baseline
correction
wavelength;
select to edit

Sample rows

Well plate Automated Sample names; Select to add

options pathlength select to edit absorbance at
setting user- defined
Wellplate view; wavelengths
toggle to view/ hide
well plate map Tips:
Click sample row to select sample and update spectrum
Shift-click multiple sample rows to overlay spectra
Click a sample and hover locations on spectra to view measurement values

Note Micro-volume absorbance measurements are normalized to a 10.0 mm

pathlength equivalent.

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Measure UV-Vis

UV-Vis reported values

The lower half of the measurement screen shows the reported values:

Well plate Automated Analytical Select to add Baseline correction

options pathlength wavelength absorbance at wavelength;
setting setting user- defined select to edit
wavelengths

Sample
rows

Wellplate view;
toggle to view/ hide Sample names;
well plate map select to edit
Tips:
Click sample row to select sample and update spectrum
Shift-click multiple sample rows to overlay spectra
Click a sample and hover locations on spectra to view measurement values

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Settings for UV-Vis Measurements

To show the UV-Vis settings, from the Home screen, from the Custom tab, select
UV-Vis.

Setting Available Options Description

Monitored Enter up to 40 User-defined wavelengths to be measured and
wavelengths wavelengths between reported at run time. Absorbance values for the first
190 nm and 850 nm three entered wavelengths are displayed in the
measurement screen. To see absorbance values for 8
monitored wavelengths, swipe left in the measurement
screen to show the Data table. To see all monitored
wavelengths, press and hold a sample row to show the
Sample Details screen (scroll up to display absorbance
values for any additional user-defined wavelengths).

Note: If Baseline Correction is selected, all displayed

absorbance values are the corrected values.
Analytical Any wavelength between This is the wavelength the software will use to determine
Wavelength 190 nm and 850 nm the pathlength selection.
Automated On or Off Optional automated pathlength selection. Allows
Pathlength (affects pedestal the software to use the optimal (shorter) pedestal
measurements only) pathlength for high concentration samples to help prevent
detector saturation (see Detection Limits for details).
• When selected, the shorter pathlength is used when
any wavelength has 10 mm equivalent absorbance
value of 12.5 or higher.
• When deselected, the pedestal pathlength is restricted
to 1.0 mm across all wavelengths.

Note: In either case, displayed absorbance values have

been normalized to a 10 mm pathlength equivalent.
Baseline On or off Optional user-defined baseline correction. Can
Correction be used to correct for any offset caused by light scattering
Enter baseline correction particulates by subtracting measured absorbance at
wavelength in nm or use specified baseline correction wavelength from absorbance
default value (750 nm) values at all wavelengths in sample spectrum. As a result,
absorbance of sample spectrum is zero at specified
baseline correction wavelength.

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Measure Custom

Measure Custom
Runs a custom measurement method
created using NanoDrop Eight software.

Measure Custom Method

Delete Custom Method

Reported Results

Measure using a Custom Method

Use the Custom application to run a user-defined method created using the
NanoDrop Eight software. For more information, see “Create Custom Method” on
page 138.

Custom methods can only be created on a personal computer running the

NanoDrop Eight software.

To measure using a custom method

NOTICE
• Do not use a squirt or spray bottle on or near the instrument as liquids will
flow into the instrument and may cause permanent damage.
• Do not use hydrofluoric acid (HF) on the pedestals. Fluoride ions will
permanently damage the quartz fiber optic cables.

Before you begin...

Before taking measurements with the NanoDrop Eight instrument, lift the instrument
arm and clean the upper and lower pedestals. At a minimum, wipe the pedestals
with a new laboratory wipe. For more information, see Cleaning the Pedestals.

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Measure Custom

To measure a sample using a custom method

1. From the Home screen, from the Custom tab, select Custom Methods.

Custom
Tab

Custom
Methods
Icon

2. In the method selection pane, select the method to run.

Information about the selected method appears in the method details pane.
3. Select .
4. Follow the on-screen instructions to measure a sample.

Delete Custom Method

– From Home screen, from the Custom tab, select Custom Methods.
– In Select Method box, select a method to delete

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 7 Custom Applications
Measure Custom

– From the drop-down menu select Delete

Custom Method Reported Results

Custom method measurement screen

For each measured sample, this application shows the absorbance spectrum and
results details. Here is an example:

Measure sample
Method Name View history data
Run Blank End experiment
Load Sample ID file

Auto measure
UV spectrum

Right click graph

area to view display
options.
Analyte
concentration
Menu of table
options;
click to choose
which columns to
report

Wellplate view; Sample name; Formula results

toggle to view/ hide select to edit
well plate map Well plate Date/time Click row to select sample
options measured and update spectrum.

Custom method reported values

• Result name
• Measurement Range
• Analysis wavelength correction

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Measure Custom

• Factor or Extinction coefficient

• Standards (Standard curve methods only)
• Baseline correction

Manage Custom Methods

The NanoDrop Eight software is your tool for creating and managing custom
methods, which contain user-defined settings that can be used to acquire data with
the instrument. Custom methods can be made with or without standards.

Create Custom Method

Create method to be used for sample measurements with user-defined settings.

Create new custom method

– From the NanoDrop Home screen, select the Custom tab and select
Custom Methods

Custom
Tab

Custom
Methods
Icon

– In the Manage Custom Methods screen, select and

choose one of the following:
– Formula (if your method will not have standards)
– Standard Curve (if your method will have standards)
– In the setup window, enter Method Name

– Enter detailed Description of method, if desired

– Specify how to calculate and report the method results:
– if method does not have standards, specify factor or extinction
coefficient of analyte (enter “1” to report absorbance measurements
only)

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Measure Custom

– if method has standards, enter name and concentration of each

standard and select the curve fit type.

Alternatively, load a standard curve.

– Enter or choose remaining custom settings as needed

– Choose Save

Note Any errors in the method will be listed in red text at the bottom of
the method editor screen. Errors must be addressed before the method
can be saved.

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Measure Custom

Load standard curve

– Select to open the Load Standard Setup window.

– Select a standard curve and click Load

– The measurement screen opens with the standard curve loaded. You can
begin measuring samples

View or edit custom method

– Select Custom Method (existing methods are listed in Select Method box
along with their type (formula or standards) and Description
– From the Custom Method Management screen, select the method you
would like to edit from the list of loaded methods.
– From the drop-down menu select Edit

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Measure Custom

– View and adjust the method settings as desired

– Select Save

Custom method settings

These settings are available for creating custom methods.

Setting Available Options

Result name Enter descriptive name for calculated concentration result (for example, “MTT
Assay”) and use adjacent drop down list to select appropriate unit. Result
name appears as column heading for reported concentration value.
Measurement range Select spectral range in which method will acquire data.
Available options:
• Ultra-violet only (190 nm - 350 nm)
• Visible only (350 nm - 850 nm)
• Ultra-violet and visible (190 nm - 850 nm)
• Custom (specify starting and ending point in nanometers)

Notes:
• If a Baseline correction and/or Analysis wavelength correction are used,
make sure your selected spectral range includes your specified baseline
correction and/or analysis correction wavelength.
• For micro-volume absorbance measurements, the spectra are normalized
to a 10 mm pathlength equivalent.
Analysis wavelength Use this option to specify absorbance correction at analysis wavelength only.
correction Available options:
• None. No correction at analysis wavelength.
• Single point. Enter wavelength for analysis correction. (Absorbance
value at specified analysis correction wavelength is subtracted from
absorbance value at analysis wavelength. Corrected value is used to
calculate sample concentration.)
• Sloping baseline. Enter two wavelengths that define sloping baseline
for analysis correction. (Absorbance value of sloping baseline at analysis
wavelength is subtracted from absorbance value at analysis wavelength.
Corrected value is used to calculate sample concentration.)

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Setting Available Options

Factor or Extinction coeffi- Specify whether to use factor or extinction coefficient to calculate concentra-
cient at 1 cm pathlength tion result:
(Formula methods only)
• User-defined factor. Enter factor for 1 cm pathlength and use
adjacent drop down list to select appropriate unit. Equation below
shows how factor is used to calculate sample concentration:

c = (A * f) / b
where:
c = analyte concentration
A = absorbance in absorbance units (A)
f = factor (typically 1/, where  = wavelength-dependent molar
absorptivity coefficient, or extinction coefficient)
b = pathlength in cm (determined at measurement time, then normalized
to 10 mm (1 cm) pathlength equivalent)
• Extinction coefficient and molecular weight. Enter extinction
coefficient for 1 cm pathlength and use adjacent drop down list to
select appropriate unit. Equation below shows how extinction coefficient
is used to calculate sample concentration:

c = A / ( * b)
where:
c = analyte concentration
A = absorbance in absorbance units (A)
 = wavelength-dependent molar absorptivity coefficient (or extinction
coefficient)
b = pathlength in cm (determined at measurement time, then normalized
to 10 mm (1 cm) pathlength equivalent)
Notes:
• Refer to product literature for information about factors and extinction
coefficients for specific materials.
• To set up a method that reports absorbance measurements only, select
Factor or Extinction Coefficient with the factor or extinction coefficient set
to “1”.
• If specified unit for factor or extinction coefficient is based on mass (such
as mg/mL) and specified unit for calculated result is based on molarity
(such as pmol/µL) or vice versa, enter molecular weight and use
adjacent drop down list to select appropriate unit.

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Setting Available Options

Standards (Standard Define the standards:
curve methods only)
• Enter name and analyte concentration of each standard and a reference,
if desired:
– Depending on the Curve Type setting, a standard curve can be
generated using two or more standards. (The software allows a
reference and up to 7 standards.)
– All reference and standards solutions should be in the same
buffer used to resuspend the samples plus the same volume of
reagent added to the samples.
– First standard can be a reference measurement. The reference
solution should contain none of the analyte of interest. (The reference
measurement is not the same as a blank measurement.)
– Concentration values for standards can be entered in any
order but the standards must be measured in the order in which they
were entered; however, best practice dictates that standards be
measured from the lowest concentration of the standard analyte
stock to the highest.
– Concentration range of the standards must cover the
dynamic range of the assay and the expected range of the unknown
samples. Sample analyte concentrations are not extrapolated beyond
the concentration of the highest standard.
• Select curve fit type.

Specify type of equation used to create standard curve from standard

concentration values. Available options:
– Linear: Draws the linear least squares line through all measured
standards (requires reference measurement and at least one
standard)
– Interpolation: Draws a series of straight lines to connect all measured
standards (requires reference measurement ans at least one
standard)
– 2nd order polynomial: Draws the 2nd order least squares polynomial
using all measured standards (requires reference measurement and at
least standards)
– 3rd order polynomial: Draws the 3rd order least squares polynomial
using all measured standards (requires reference measurement and at
least three standards)

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Setting Available Options

Analysis wavelength Monitor absorbance at specified wavelength (enter the wavelength in nano-
(Standard curve methods meters).
only)
Note: The specified wavelength must fall within the selected measurement
range.

The measurement results or the concentration will be calculated automatically

using the absorbance value at the specified wavelength and applying the
selected method type (factor or standard curve).
Baseline correction Select this option to correct offset caused by light scattering particulates by
subtracting the absorbance at a specified baseline point. Then specify
wavelength for baseline correction.

Note: Software subtracts absorbance value at specified baseline correction

wavelength from absorbance values at all wavelengths in sample spectrum.
As a result, absorbance of sample spectrum is zero at specified baseline
correction wavelength.
Automated pathlength Affects micro-volume measurements only.
• When Automated Pathlength is selected, software selects the optimal
pathlength (between 1.0 mm and 0.1 mm) based on sample absorbance
at the analysis wavelength. For example, when sample absorbance at the
analysis wavelength is less than or equal to 12.5 (10 mm pathlength
equivalent), the optimal longer pathlength is used. When sample
absorbance is greater than 12.5, the optimal shorter pathlength is used.
Recommended for samples that are highly absorbing at the analysis
wavelength. (This option may cause reduced sensitivity when the sample
spectra have a large absorbance peak that is not at the analysis
wavelength.)
Note: When the analysis wavelength is between 190 nm and 219 nm,
the optimal longer pathlength is used when sample absorbance is less
than or equal to 10 (10 mm pathlength equivalent), and the optimal
shorter pathlength is used when sample absorbance is greater than 10.
• When Automated Pathlength is deselected, the software uses a 1 mm
pathlength regardless of the sample absorbance. This can cause detector
saturation (resulting in jagged peaks) for highly absorbing samples (e.g.,
~15 A at 10 mm pathlength equivalent).

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Setting Available Options

Formula table (optional) Use the Formula table to specify additional reported results, such as a purity
ratio, for each sample.

Available options:
• Predefined. Select from a list of predefined formulas, which can be
used as is or edited, and choose Add. The predefined formula is listed in
the Formula Table.
• Add. Create formula for current method. Available options:
• Formula Name. Enter a name for the formula. After a
measurement, the name is reported in Data Table and Sample Details
screens.
• Formula. Enter valid formula (see below for rules and examples).
After a measurement, the measured or calculated value is reported in
Data Table and Sample Details screens.
• Unit. Enter unit for reported result. After a measurement, the unit is
reported in Data Table and Sample Details screens.
• Edit. Edit selected formula for current method.
• Delete. Delete selected formula from current method.
Formula rules Custom formulas can include the following operators and functions:
• Path(). Returns sample pathlength in cm.
• A(nm). Returns sample absorbance at specified wavelength (for
example, enter A(650) to add the measured absorbance at 650 nm to
your equation).
• Operators: + (add), - (subtract), * (multiply), / (divide).
• Functions: Log(x), Pow(x,y).

Notes: Follow these additional rules for all languages:

• Use period “.” decimal separators for floating point and double-floating
point numbers.
• Use comma “,” list separators (for example, “POW(2,8)”).
• Do not use comma “,” group separators for large numbers (for example,
enter 1000 rather than 1,000).

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Copy Custom Method

To create a custom method that is similar to an existing one, open the existing
method, make your changes, then select Save As and enter a new name.

Copy custom method

– From the Custom Methods screen, select a custom method
– From the drop-down menu choose Edit
– Enter new Method name and Description
– Select Save As
– Enter a filename for the method and click Save

You can now select the saved method and edit the Description and settings.

Run Custom Method

To run a custom method, first create or import a custom method.

Run custom method

– From the Custom Methods screen, select a custom method
– Select .
– Follow the on-screen instructions to measure a sample.

Export Custom Method

Export a custom method in order to run it and store the measurement results on
another PC for use with another NanoDrop Eight instrument.
– From the Custom Methods screen, select a custom method
– From the drop-down menu , choose Export (if method is invalid, an
error message is displayed; errors must be fixed before method can be
exported)
– Choose Save (method is exported to method file (*.method filename
extension) in proprietary format)

Import custom method

You can import an existing custom method file to edit the method settings.
– From the Custom Methods screen, choose Import
– Locate and select “.method” file
– Choose Open (imported method is added to end of Select Method list)

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Measure Custom

Edit custom method

Edit a custom method in order to change the method settings.
– From Custom Methods screen, select a custom method from the list of
available methods
– From the drop-down menu , choose Edit
– Edit method settings as desired
– Choose Save

Delete custom method

– From Custom Methods screen, select a custom method from the list of
available methods
– From the drop-down menu , choose Delete
– After the confirmation message, choose Yes

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 8

Learning Center

Contents
• Micro-Volume Sampling—How it Works 150
• Set Up the Instrument 152
• Measure a Micro-Volume Sample 154
• Sample Modes 158
• Prepare Samples and Blanks 158
• Basic Instrument Operations 164
• Acclaro Sample Intelligence 189
• Instrument Settings 195
• Measurement Screen Display Options 198

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Micro-Volume Sampling—How it Works

Micro-Volume Sampling—How it Works

Surface Tension

Absorbance Spectrum

Sample Absorbance

Sample Concentration

Baseline Correction

Surface Tension
The NanoDrop Eight spectrophotometer uses surface
tension to hold a small volume of sample between
two pedestals (upper and lower). The patented
sample retention system enables the measurement of
highly concentrated samples without the need for
dilutions.

A fiber optic cable embedded in the upper pedestal

leads to a xenon light source. A second cable
embedded in the lower pedestal leads to a detector.
When the instrument arm is down, the sample forms
a liquid column, essentially bridging the gap between
the two fiber optic cables.

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Absorbance Spectrum
The light passes through the liquid column to the
detector, which generates a spectrum of absorbance
versus wavelength. The spectrum shows the amount
of light absorbed by the molecules of the sample at
each measured wavelength.

Note: To prevent evaporation, which affects

measurement accuracy, close the arm quickly after
you finish loading a sample or blank.

The example at the left shows a typical absorbance

spectrum taken of a nucleic acid sample. The
spectrum is measured from 190 nm to 850 nm. The
displayed range may vary for each application.
Sample Absorbance
When the instrument is blanked, a reference
intensity sample
Absorbance = – log -------------------------------------- spectrum is taken of the blanking solution and stored
intensity blank in memory. For each sample measurement, the
sample intensities along with the blank intensities are
used to calculate the total absorbance of the sample
according to the equation at the left.
Beer-Lambert equation Sample Concentration

A = bc The Beer-Lambert equation (Beer’s law) shown at the

left is used to correlate sample absorbance with
where: concentration.

A = absorbance in absorbance units (A) The pathlength is the distance between the two
pedestals, which varies in real time during each
ε = wavelength-dependent molar absorptivity measurement. This auto-ranging pathlength
coefficient (or extinction coefficient) in liter/mol-cm technique produces accurate concentration results
b = pathlength in cm over a wide dynamic range.

c = analyte concentration in moles/liter or molarity (M)

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Set Up the Instrument

Baseline Correction
For some applications, the instrument can be set up
to apply a baseline correction to each measurement
to minimize any offset caused by light scattering
particulates in the sample spectra. The correction
subtracts the absorbance value at a reference
wavelength that is close to zero from the absorbance
value at each wavelength across the spectrum,
essentially “anchoring” the spectrum to zero
absorbance units at the reference wavelength.

Set Up the Instrument

USB-B

Power

Power
on/off

Connect Power
CAUTION Avoid shock hazard. Each wall outlet used must be equipped with a
ground. The ground must be a noncurrent-carrying wire connected to earth
ground at the main distribution box.

Connect the provided power cord to a grounded wall outlet. See “Power Cords” on
page 222 for more information.

Connect to a Computer
Connect the USB cable to the NanoDrop Eight and to an available USB port on
your PC.

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Operating Specifications
The instrument operates reliably when the room environment meets these
specifications:
• operating temperatures: 5 °C - 35 °C (41 °F - 95 °F)
• relative humidity (non-condensing): 20-80%

Locate the instrument away from air vents and exhaust fans to minimize evaporation.
Note If operating the instrument at the low end of the recommended humidity
range, use adequate sample volume to avoid evaporation.

After the instrument is installed, you can leave it turned on.

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Measure a Micro-Volume Sample

Measure a Micro-Volume Sample

The NanoDrop Eight spectrophotometer uses surface
tension to hold a small volume of sample between two
pedestals. The patented sample retention system enables
the measurement of highly concentrated samples without
the need for dilutions. See “Micro-Volume Sampling—How
it Works” on page 150 for details.

Supplies needed
• NanoDrop Eight spectrophotometer
• lint-free laboratory wipes
• calibrated precision pipettor
• sample material resuspended in appropriate buffer
solution (see Preparing Samples)
• pure buffer solution for blanking instrument (see
Choosing and Measuring a Blank.

Best practices for micro-volume measurements

Cleaning pedestals for daily operation
• Before first measurement, clean all pedestals with a new laboratory wipe.
• Run a blanking cycle to verify pedestals are clean.
• After each measurement, clean all pedestals with new wipe to prevent carryover.
• After each set of measurements, clean pedestals with DI H2O (see Clean
pedestals between users)
• Recondition pedestals periodically to maintain their hydrophobic property.

Pipetting Samples
• Position the instrument at an angle for optimal use of the pipette guide.
• Use calibrated precision pipettor (0–2 µL volume range) with well-fitting,
low-retention precision tips to apply sample material to instrument for
measurement. If using low accuracy (0-10 µL) pipettor, use 2 µL sample
volumes.

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• Filter tips are not recommended, as filter particulates can impact absorbance
measurements at 230 nm.
• Use recommended sample volumes to ensure proper liquid column formation.
• Use new tip for each blank and sample aliquot.
• Use new aliquot of sample for each measurement.
• When the measurement is complete, open the sampling arm and wipe the
samples from both the upper and lower pedestals using a soft laboratory wipe.

If solvents are used, make sure they are compatible with the pedestals. (see
“Compatible Solvents” in Hazardous Materials).

Recommended sample volumes

Application Sample Volume
Nucleic acid (aqueous solution) 1 µLa
Purified protein 2 µL
Other protein applications such as Bradford or BCA 2 µL
Microbial cell suspensions 2 µL
a
Use 2 µL for samples that contain materials that may reduce surface tension such as a surfactant.

To measure a micro-volume sample

NOTICE
• Do not use a squirt or spray bottle on or near the instrument as liquids will
flow into the instrument and may cause permanent damage.
• Do not use hydrofluoric acid (HF) on the pedestals. Fluoride ions will
permanently damage the quartz fiber optic cables.

1. From the Home screen, select an application from

one of the application categories, such as UV-Vis, or
Custom Methods.

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2. Lift the instrument arm and clean the upper and lower
pedestals with new laboratory wipe.

3. Measure a blank:
– Pipette 1–2 µL blanking solution onto the lower
pedestal and quickly lower the arm

– Click Blank and wait for the

measurement to complete
Tip: If Auto-Blank is On, blank measurement
starts automatically after you lower the arm.
– Lift the arm and clean all pedestals with a new
laboratory wipe

4. Measure the first sample:

– Pipette 1-2 µL sample solution onto the pedestal
and quickly lower the arm (see Recommended
Sample Volumes for more information)
– Start the sample measurement:
– if Auto-Measure is On, lower arm
– if Auto-Measure is off, lower arm and click

Measure
– When the sample measurement is completed, the
spectra and reported values are displayed.

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5. To measure another sample:

– Lift the arm
– Clean all pedestals with new wipe
– Load the next sample(s) and quickly lower the
arm
– Start the sample measurement
– Wait for the measurement to complete
The new spectrum replaces the previous one on the
spectral display and the new reported values replace
Select to end the previous ones in the table.
experiment

6. When you are finished measuring samples:

– Select the End Experiment icon (see previous
image)
– Enter an experiment name or leave the default
experiment name
– Select Save
– Lift the arm and clean all pedestals with a new
wipe
If finished with the instrument for the day, clean
Cancel to measure the pedestals with DI H2O (see Clean pedestals
Select Save to end between users)
more samples
and save experiment
Acquired data are automatically saved in an experiment
with the entered name. In the default configuration,
experiments are stored in a database according to
acquisition date, experiment name, application used and
any assigned labels (see Manage identifiers).

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Sample Modes

Sample Modes
The NanoDrop Eight can be used in either Single sample or an Eight sample mode.
Select the desired mode from the drop-down menu on the Home screen. In Single
Sample mode, measurements will only be taken using Channel A.

Module startup
Instrument self-test and preparation begins upon selection of an application after first
launching the software. The message 'Please wait - Initializing Spectrometer' will
appear. When this message disappears, the instrument will be ready for use and you
can prepare blanks. All data taken will automatically be logged in the appropriate
archive file.

Sample Plate Map

When running in Eight Sample mode, a plate map will display in the sample area.
This on-screen sample plate will populate with Sample ID information from an
imported sample ID file. You can also manually enter Sample ID or other identifying
information. The sample status color code displayed on the plate map is determined
by plate configuration at set up. See “Sample Position Illuminator” on page 174 for
details.

Prepare Samples and Blanks

Before making a sample measurement, a blank must be measured and stored. All
eight channels are blanked with each blanking command when using the 8 sample
mode. Only channel A is blanked for the single position mode.

Note: When using the 8 Sample mode, the software initiates each blank and
measurement cycle on the first position to be read. The user will, therefore, hear one
less position increment than expected. After making an initial blank measurement, a
straight line will appear on the individual graphs. Subsequent blanks will clear any
sample spectrum and again display straight baselines.

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Prepare Samples and Blanks

Preparing Samples
• Isolate and purify samples before measuring them with the instrument.
Commercial sample isolation kits are available for these purposes, or use an
in-house protocol. After purification, analyte of interest is typically dissolved in
aqueous buffer solution before it is measured.
Tip: Any molecule that absorbs light at analysis wavelength will contribute to
total absorbance value used to calculate sample concentration.
• Ensure final analyte concentration is within instrument’s absorbance detection
limits.
• For micro-volume measurements, gently (but thoroughly) vortex each sample
before taking a measurement.

Avoid introducing bubbles when mixing and pipetting.

Choosing and Measuring a Blank

The buffer used to resuspend a sample analyte can contribute absorbance. Blanking
minimizes any absorbance contribution due to the buffer components from the
sample measurement. The resulting sample spectrum represents the absorbance of
only the analyte of interest.
For best results:
• For most applications, blank with the same
buffer solution used to resuspend the analyte
of interest. The blanking solution should be a
similar pH and ionic strength as the analyte
solution. For details, see “To measure
samples” in the application used.
• Measure new blank before each set of
samples. It is not necessary to blank the
instrument before each sample
measurement unless the samples are
dissolved in different buffer solutions.
• It is recommended to measure a new blank
every 30 minutes.

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Prepare Samples and Blanks

• Run a blanking cycle to assess the suitability

of your blanking solution before using it to
perform sample measurements. For a quick
demonstration, watch the multimedia training
Evaluating a Blanking Solution for Suitability.
The resulting spectrum should vary no more
than 0.04 A (10 mm equivalent) across the
spectrum, especially at the analysis
wavelength as in the example at the right.
If the resulting spectrum is greater than
0.04 A around the analysis wavelength, that
buffer solution may interfere with the sample
analyses, especially for low concentration Good blanking buffer (measured abs < 0.04)
samples. See below for details.
Problems associated with blanking
• Residual sample was left on pedestal before
blank measurement was performed.
(Resulting sample spectra may exhibit
negative absorbance values, indicating blank
had more absorbance than sample in that
region of spectrum.)
• Blank measurement exhibits higher
absorbance than unknown sample at
analysis wavelength. (If buffer used as blank
differs in composition from that used to
resuspend sample, measurement results will
be incorrect.) Solution containing salt used to blank instrument results
in “mirror image” spectrum
• Sample was inadvertently used to blank
instrument. (Resulting sample spectra may
exhibit negative absorbance values or, in
some cases, resemble a mirror image of a
typical pure nucleic acid or protein
spectrum.)

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Prepare Samples and Blanks

Solutions for blanking problems

• Thoroughly clean and/or recondition both
pedestals and then:
– rerun blanking cycle, or
– measure new blank using new aliquot of
appropriate buffer solution, then measure
new aliquot of unknown sample
• For most applications, blank with the same
buffer solution used to resuspend the analyte
of interest. The blanking solution should be a
similar pH and ionic strength as the analyte
solution. For details, see “To measure
samples” in the application used.

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Prepare Samples and Blanks

Run a Blanking Cycle

Run a blanking cycle to verify the following:
• instrument is operating normally (with flat baseline)
• pedestals are clean (i.e., no dried-down sample material on pedestals)
• absorbance contribution of buffer solution you plan to use for sample analyses

Supplies needed
• lint-free laboratory wipes
• calibrated precision pipettor (0–2 µL)
• buffer solution for evaluation

To run a blanking cycle

For quick demonstration, watch multimedia training Evaluating a Blanking Solution

for Suitability.
NOTICE
• Do not use a squirt or spray bottle on or near the instrument as liquids will
flow into the instrument and may cause permanent damage.
• Do not use hydrofluoric acid (HF) on the pedestals. Fluoride ions will
permanently damage the quartz fiber optic cables.

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Prepare Samples and Blanks

1. From the Home screen, select an

application name.
2. Lift the instrument arm and clean the
upper and lower pedestals with new
laboratory wipe.
3. Measure a water blank:
– Pipette exactly 1 µL deionized
water (DI H2O) onto the lower
pedestal and lower the arm.
– Select Blank and wait for the
measurement to complete.
– Lift the arm and clean all pedestals
with new laboratory wipe.
Example spectrum of buffer suitable
4. Measure the buffer solution: for Protein A280 protein quantification
– Pipette 1-2 µL buffer solution onto
the pedestal and lower the arm.
– Start the sample measurement:
– if Auto-Measure is On, lower
arm
– if Auto-Measure is off, lower
arm and select Measure
– Wait for measurement to complete.
The resulting spectrum should vary no
more than 0.04 A from the baseline at
the analysis wavelength.
If your spectrum does not meet these
criteria, repeat steps 2–4.
If spectrum is still outside Example spectrum of buffer unsuitable
specifications, see Solutions for for Protein A280 protein quantification
Blanking Problems.
5. When you are finished with the
blanking cycle, select
End Experiment.
6. Lift the arm and clean all pedestals
with a new wipe.

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Basic Instrument Operations

Basic Instrument Operations

• NanoDrop Eight Home Screen
• NanoDrop Eight Measurement Screens
• View History
• NanoDrop Eight General Operations

NanoDrop Eight Home Screen

These operations are available from the NanoDrop Eight Home screen.
Applications System Status

Application categories

Control options

SciVault History Instrument Diagnostics Instrument Help

Settings

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Applications
The NanoDrop Eight software offers several configurable applications, which gives
users full control of the measurement. See“Custom Applications” on page 129 for
detailed information about each available application.

System Status
Select the icon on the Home screen to open the system status box.

The available information is described below.

Serial number Instrument serial number

Status Current status of the instrument
Data storage Indicates location of database set where instrument is
currently storing data.
Firmware Version of instrument firmware installed
Build Number Current Software build
Software Version Version of instrument operating software installed

Control options
History: View data stored locally. Filter by date or application.
Performance: Performance verification process using PV-1 solution.
See “Performance Verification” on page 212
Intensity: Run an intensity check for the pedestal.
See “Intensity Check” on page 210.

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Settings: Set security server location and path if desired.

Help: View help

History
Select on the Home screen to view any data acquired earlier today, last
week, last month, last six months, last year or in a specific date range. See “View
History” on page 176 for more information about the History feature.

Instrument Settings
Select on the Home screen to access instrument settings for software
updates, Protein Editor, and more. See “Instrument Settings” on page 195 for
detailed information about all available instrument settings.

Instrument Diagnostics
Instrument diagnostics (Performance and Intensity check) should be run periodically
according to the recommended maintenance schedule. See “Instrument
Diagnostics” on page 209 for information about how to run the available instrument
diagnostics.

SciVault Software
The Thermo Scientific™ SciVault™ software is available as an optional add-on. This
companion software allows users to operate their NanoDrop Eight instrument in a
manner that complies with US FDA 21 CFR Part 11. When purchased, the SciVault
software is shipped to you on a USB stick and integrates directly into the NanoDrop
Eight software user interface. For more information, visit
thermofisher.com/nanodrop.

Help
Select to launch the user guide, email a question to tech support, or find the
software logs.

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NanoDrop Eight Measurement Screens

These operations are available from any measurement screen within an Application.
Application Import View measurement Measure blank End experiment and
type Sample ID history solution export data

Auto-
Measure
UV absorbance
spectrum for
selected samples

Table display
options

Click row to
select sample
and update
spectrum; select
more rows to
Wellplate view; Sample Sample names; overlay spectra.
toggle to view/ hide location click to edit
well plate map Measurement results; see
Applications for details
Well plate options

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Measurement Screen Display Options

When performing measurements, right-click the graph on the measurement screen
to bring up the following display options:
Display multiple samples in overlay.

Hover spectrum to see plot data

Display graph legend

Calculate peaks for specified range

Scale axes to fit spectra measurement

Select to manually enter x-axis range

Select to manually enter y-axis range

Display options- Right click graph to view

Find Peaks
Select Find Peaks to view calculated peaks for specified range. You can enter the
range by dragging the color-coded limit lines, or enter values into the fields at the top
of the spectrum. Found peaks for the defined range are listed in the table below the
spectrum.

Wavelength start Wavelength end Absorbance minimum

UV spectrum
Peak 1

Table of calculated
peaks for the
Maximum number of peaks specified range

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Sample Name
Click the Sample Name field in any measurement screen to edit the sample name.

In Single Sample mode, each sample is given a default base name “sample”
followed by the number sample in the sequence. For example, the first sample
would be named “Sample 1” followed by “Sample 2,” etc. You can edit the default
base name and overwrite any sample name.

In 8 Sample mode, each sample is automatically labeled by the sample location. Add
sample names by clicking the sample name field, or by importing Sample ID file.
Note If you edit the sample base name during an experiment when
Auto-Naming is selected, the assigned sample ID numbers restart.

Edit sample base name

After you measure a blank and before the first sample is measured:
– click Sample Name field
– enter new base name
– press Enter key

Edit sample name

– from Home screen, click to open History
– select experiment
– select name field of sample
– enter new sample name
– press Enter key

Measurement Results
The types of results that appear in the measurement screens depend on the
selected application. For details, see the reported results section of that application
in this guide:
Applications > [application group] > Measure [application name] > Reported
Results

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Absorbance Spectrum
For each measured sample, each application shows the UV or
UV-Visible absorbance spectrum and a summary of the results. The vertical axis
shows absorbance in absorbance units (A). The horizontal axis shows wavelength in
nm. Here is an example for a UV-Vis method.

Sample Pathlength
All applications display the sample pathlength along the spectrum’s vertical axis.
Micro-volume absorbance measurements are normalized to a 10.0 mm pathlength
equivalent.

Measurement Alerts
The Acclaro Sample Intelligence technology built into the NanoDrop Eight instrument
provides important features to help you assess sample integrity. click a Sample
Intelligence icon in the software to view its associated information.
contaminant analysis is available to help qualify a sample before use
in downstream applications

on-demand technical support is available for measurements that

are atypical or very low concentrations

Blank Button
Select Blank to measure a blank for the selected experiment.

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A blank must be measured before each group of similar samples. The blank solution
is typically the pure buffer that was used to resuspend the sample. For more
information, see Choosing and Measuring a Blank.

Measure Button
Select Measure to measure a sample for the selected experiment.

Samples must be properly isolated and prepared before they can be measured with
the instrument and the concentration must be within the instrument’s absorbance
detection limits. For more information, see Preparing Samples, Measure a
Micro-Volume Sample, and Absorbance Detection Limits.
Note The Measure button is enabled after a valid blank measurement is
completed.

Auto-Measure and Auto-Blank Options

Speed up sample analysis with the NanoDrop Eight Auto-Measure and Auto-Blank
features, which cause the instrument to start the measurement immediately after you
lower the instrument arm. These options eliminate the need for repetitive Measure or
Blank operations for large batches of samples.
Note Auto-Measure and Auto-Blank are available for micro-volume
measurements only.

Auto-Measure

To select or deselect Auto-Measure, from any sample measurement screen, click the
On or Off button at the right of the Measure button.

Auto-Blank

To select or deselect Auto-Blank, from any blank measurement screen, click the On
or Off button at the right of the Blank button.

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End Experiment Button

Select End Experiment when you are ready to name and save your
experiment, add a label to help you locate the experiment later or export the data.
Depending on the administrative settings, you may be prompted to sign the
experiment upon ending the experiment.

Note The End Experiment button is enabled after the first sample
measurement is completed.

After you select End Experiment, the End Experiment dialog box is displayed:

Click Save and you will have the option to Print or Export the experiment data.

Available options:

Experiment Name Enter a name for this group of measurements. The

measurement results are saved in the selected database
location using the entered experiment name.
Tags Enter a descriptive label to help you find this experiment
later or to associate it with another experiment (see
Manage identifiers on the instrument for details).

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Export Select an available location for exporting the

measurements in this experiment. Experiments can be
exported to a USB device connected to the PC, any USB
port, or to a network location.

Available export file formats:

• comma-separated values spreadsheet (.csv) file
• tab-separated values spreadsheet (.tsv) file
• NanoDrop Eight software (.n8db) file

The filename is the entered experiment name (see above).

The file is stored in a folder named “NanodropEight”
followed by the instrument serial number. (Use System
Status to view your instrument serial number.)

Cancel (Return To Close the End Experiment box and display the results for
Experiment) the most recent measurement. From there you can add
measurements to the current experiment and save it later.
Print button Print measurement results for current experiment
Save (End Experiment) End the experiment and save the measurement results
using the entered experiment name. The experiment is
saved in the selected database location.

Sample Details
Sample details are shown in the sample row in any measurement screen or
History. This information includes all available measurement results and associated
details for the selected sample. To add or remove results shown in the sample row,
click the menu in the upper right corner of the data table. Information about the
measured values displayed in Sample Details is provided in this Help system, under
the application used to acquire the data.
Note You can also edit the sample name by clicking in the name field of the
sample.

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Data Table
The data table for the current experiment is shown below the spectrum graph. The
data table contains the measurement results for all samples in the experiment. The
image below highlights the available features.

Well plate map Sample name; Measurement results; see Menu of

click to edit Applications for details options;
click to
Active channel indicator; click to click row to open
activate or inactivate sample select sample
locations to add to measurement
Data Table is in the bottom half of the measurement screen

Sample Position Illuminator

The NanoDrop Eight Sample Position Illuminator and accompanying software user
interface are designed to help users keep track of which samples have already been
measured and which samples should be measured next. There are three different
templates designed for different sample holding formats. You can choose between
96-well plate, 1.5 mL tubes, and 0.5 mL tubes by clicking the 3-dot menu above the
plate matrix user interface in the bottom left of the measurement acquisition screen.
• 96-well plate: allows users to keep track of sample measurements for samples
stored in a standard 96-well microtiter plate
• 1.5 mL tubes: allows users to keep track of samples measurements for samples
stored in the 1.5 mL-sized spaces in the NanoDrop Eight tube rack accessory
• 0.5 mL tubes: allows users to keep track of samples measurements for samples
stored in the 0.5 mL-sized spaces in the NanoDrop Eight tube rack accessory

NOTE: This feature works most efficiently when a user imports sample names from
a file.

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NanoDrop Eight
tube rack accessory

The wells/tubes which should be measured next are filled with bright orange in the
software user interface. They correspond to the wells/tubes illuminated by the green
LEDs on the top of the instrument. Wells/tubes that have sample IDs pre-populated
will be filled with light orange. Wells/tubes that have already been measured will
either be filled bright green (indicating no sample alerts) or with an A in a circle
(indicating an Acclaro alert is present for that sample).
Sample group selector

Show/hide well
plate map View options menu

Sample groups Active sample group

Measurement alert
Measurement alert;
Acclaro warning

Currently active
channel measurement
Sample channels completed with no
errors

Currently active
channel with sample
IDs populated

Dark green
indicates
Measurement Dark orange indicates
completed with inactive well with
no errors Well plate map shown in the measurement screen sample ID populated

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After a set of measurements is complete, users can manually advance the sample
names and well illumination by clicking the and arrows above the well plate
map in the bottom left. This will prepare the instrument software for the next set of
measurements. Alternatively, users can click the 3-dot menu above the well plate
map and enable Auto Advance. Auto Advance sets the software to automatically
advance the columns a few seconds after the last measurement is made.

The entire well plate map interface can be hidden by clicking on the plate icon .

Manual Sample ID Entry

Users can manually enter sample names using a keyboard or barcode scanner.
When sample names are entered manually, the user needs to activate each channel
they wish to measure. To activate/inactive all channels at once, click the circle in the
column header area next to Sample. Alternatively, users can activate/inactivate
individual channels by clicking the circles within each row.
Sample column header; click to
activate/inactivate all channels

Sample name; click to edit

Measurement
alert; click to
learn more

Click rows to select

individual channels
and view spectra

Channel status; click to

activate/inactivate individual
channels for measurement
Active channel indicator of the measurement screen

View History
Whether you collect one sample or many in a row, after you choose End Experiment,
the acquired data are automatically saved in an experiment with an experiment
name. In the default configuration, experiments are stored in the NanoDrop Eight
database according to acquisition date, experiment name, application used and any
assigned labels.

Use the History feature to open the database in order to view acquired spectra and
associated data from any experiment at any time.

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Open instrument database of measurement results

– to open NanoDrop Eight database, from the Home screen, select History.

History Menu
Available options:

Home Return to NanoDrop Eight Home screen

Import Import data from a USB flash drive or folder on your
computer or network hard drive
Export Export the experiment

Measurement Menu
When viewing measurement data from History, Click the menu in the upper right of
the measurement screen to access available menu options.

Export Export the experiment

Print Print selected measurement results
Tags Manage experiment tags
Details Experiment details such as Name, Application type,
Creation date, and number of measurements

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Search Experiment Database

Use the search feature in History to search the selected database for an experiment
or to change the time range or other search filters. The database is filtered using the
current settings in the Search box. Filters include time range, application type and
any user-defined labels (see Manage Identifiers for information about adding and
deleting labels). Here is an example:

Change a filter to display updated list Change application filter

of experiments Change date range

Search

Export Selected Experiments

Use Select in History to select experiments to be exported.

Export selected experiments

– Open History. You can filter or use the Search feature to find experiment

Search

– click the checkbox to select row or rows to be exported

– click Export

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– select one or more formats to export to (see “Export Selected Experiments”)

– enter or browse to an available export location USB drive, PC storage, or a

network location) and select Export

– after “Export Success” message, select OK

Delete Selected Experiments

Select experiments to be deleted.

Delete selected experiments

– Filter experiments as needed, use Search feature to find desired experiment
– Click the checkbox of the row or rows to select one or more experiments to
delete (click again to deselect an experiment)
– Click Delete and OK
Note Deleted data cannot be recovered.

Open Experiment and View Associated Data

Use History to locate and open any experiment to see the measurement data it
contains.

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Open an experiment
– In History, if you don’t see the experiment you want to open, you can use the
Search feature to find the desired experiment
– click the experiment name to open the experiment

The History provides measurement data as spectral data and data tables, similar to
what you see after you complete a measurement.
Note The data shown are dependent upon the application used to measure the
samples (nucleic acids in these examples). For more information, see the
application details.

Spectral data—
After you open an experiment, the software shows the UV or UV-visible absorbance
spectrum and associated data for the selected experiment, much like it appears
during a measurement. The image below describes the available features.

Selected experiment Selected application Menu of options;

click to open

UV spectrum for
selected sample(s)

Click row to select sample and

display spectrum. Select multiple Measurement results; see
rows to overlay spectra Applications for details

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Data Table—
The lower area of the measurement history screen (shown above) shows the data
table for the current experiment. The data table contains the measurement results
for all samples in the experiment. The image below describes the available features.

Menu

Select the menu in the upper right corner from any Spectral Data or Data Table
screen to see the available menu options.
Home Return to NanoDrop Eight Home screen
Manage Identifiers Add or delete labels for selected experiment to make it
easier to find (see Manage identifiers on the instrument)
Export Export selected experiments
Print Print plot or data table for selected measurement results;
if no results are selected, prints all results in data table
Settings View or change instrument settings

NanoDrop Eight General Operations

These operations are available from any measurement screen or from the History.

Manage Identifiers
You can add one or more “tags” (i.e., labels or metadata tags) to an experiment to
make the experiment easier to find.

Use the History to add labels to experiments, assign existing labels, view assigned
labels and remove or delete labels on the instrument. You can filter the list of
experiments in the History based on one or more user-defined labels.

Label new experiment when you save it

– after the last sample has been measured, select End Experiment.

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– in End Experiment box, enter user defined labels in the Tags field

– click Save

Label experiment in History

– from the Home screen, select History
– Click the menu of the experiment row and select Tags
– in Manage Tags box, enter a tag as an Identifier
– Click Save to add the tag. Add additional tags as needed.
– Click OK

Remove a label
– from Home screen, select History
– Click the menu of the experiment row and select Tags
– in Manage Tags box, click the X for any tag you wish to delete
– Click OK

Edit Experiment Name

You can edit the experiment name when you save the experiment or afterwards from
the History.

Edit experiment name at end of experiment

– when finished measuring samples, select End Experiment

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– enter a name for this group of measurements in the Experiment Name box

– click Save

Edit experiment name from History

– from Home screen, open History
– use Search feature to find experiment
– Double click experiment to open experiment

– Click the Experiment Name field

– enter new experiment name
– the new name is saved automatically

Export Selected Experiments

You can export measurement data when you save the experiment or afterwards
from the History
Note Data exported during a save are still saved to a database (local or remote,
depending on the Data Storage setting.

Measurement data can be exported in three formats:

• as comma-separated values (.csv) files containing:
– whole spectrum data in columns
– report data in columns
– both report and spectrum data combined in columns
• as tab-separated values (.tsv) files containing:
– whole spectrum data in columns
– report data in columns
– both report and spectrum data combined in columns

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– whole spectrum data in single column for TQ Analyst

• as NanoDrop Eight software (.n8db) files containing spectra and measurement
results for each exported experiment

Use any spreadsheet or word processing application to open a CSV or TSV file. Here
is an example of several sample measurement results in CSV format:

Note The types of data exported are dependent upon the application used to
measure the samples (nucleic acids in this example). For more information, see
the application details.

Data can be exported to any file path connected to the PC or to a network location.
If you select multiple experiments for export, each exported experiment has a
corresponding file. The filenames are the same as the experiment names. The files
are stored in a folder named “NanodropEight” followed by the instrument serial
number. (Use System Status to view your instrument serial number.)

Export data at end of experiment

– when finished measuring samples, click End Experiment
– from End Experiment box, enter Tags and edit experiment name if desired,
and clock Save
– Once the experiment is saved, you can click Export

– select one or more formats to export to (see above for details)

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– Set File Path: to an available export path

– click Export

Export data from History

– From Home screen, open History
– Click the checkbox for the experiment(s) you want to export. You can use the
Search feature to find experiment
– Click Export
– Select one or more formats to export to (see above for details)
– Set File Path: to an available export path and click Export

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– after “Export Success” message, click OK

Delete Selected Measurements

You can delete selected sample measurements from any experiment, or all the
measurements in the database.
Note Deleted data cannot be recovered.

Delete data from any measurement screen

– click a sample measurement row
– click

Delete data from History

– from Home screen, open History
– select row in History. You can filter experiments by date or type, or use
Search feature to find desired experiment
– click to select one or more experiments to export
– click Delete

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Print Selected Measurements

Connect a compatible printer to the PC to quickly print measurement results,
including spectral data, standard curves, data tables, sample details and diagnostic
results.

Print data from any measurement screen

– after you have measured a sample, display the measurement results to be
printed such as the spectral data, standard curve, data table or sample
details (see NanoDrop Eight Measurement Screens)
– from the options menu, select Print
– In Print Review, select the data you want to include in the data tables.

– choose Print to confirm

– in the Print Preview window, make sure the correct printer is selected and set
other print options as desired such as paper size and orientation (“Auto”
setting is recommended), margin and alignment to adjust the image in the
preview window
Note The software saves the print settings each time you print.

– choose Print
The selected measurement screen is printed for each selected measurement.

Print data from History

– from Home screen, open History
– click a row in History or use Search feature to find desired experiment
– click the experiment name to open the experiment
– click the menu in the upper right corner of the screen and choose Print

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– In Print Review, select the data you want to include in the data tables.

– choose Print to confirm

– in the Print Preview window, make sure the correct printer is selected and set
other print options as desired such as paper size and orientation (“Auto”
setting is recommended), margin and alignment to adjust the image in the
preview window
Note The software saves the print settings each time you print.

– choose Print
The selected measurement screen is printed for each selected measurement.

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Acclaro Sample Intelligence

Acclaro Sample Intelligence

The Thermo Scientific™ Acclaro™ Sample
Intelligence technology built into the
NanoDrop Eight instrument provides these
exclusive features to help you assess sample
integrity:

contaminant analysis to help qualify a

sample before use in downstream
applications

on-demand technical support for

measurements that are atypical or very low
concentration

Use these embedded resources to quickly troubleshoot possible problem

measurements and make informed decisions on whether to use, re-purify or take
other actions with an atypical sample result. The Sample Intelligence feature also
serves as a resource for further study and a learning tool for new or novice users.

Activate Detection
From the measurement screen, click the activate contaminant detection option.

When enabled, RNA Contaminant Detection/DNA Contaminant detection will apply

mathematical models to predict the amount of RNA contaminant in dsDNA or
dsDNA in RNA. These models are specific to the source of the nucleic acid. The
mouse icon is representative of all mammalian sources of nucleic acid. If you are
measuring nucleic acid from a source for which we do not have mathematical
model, leave the selections unchecked.

View Acclaro Sample Intelligence Information

Measurements that include a contaminant analysis or technical information are
flagged automatically (see examples below). Select the icon to review the associated
data or information.

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Sample alert information is Contaminant analysis is available

available for this measurement for this measurement

The icons appear in the Sample Plate Map, the data table, and in History (see
below).

The icons are active in all three places; the information remains with the data
indefinitely, even after it has been exported.

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Acclaro Sample Intelligence

Contaminant Analysis
For the dsDNA, RNA and Protein A280 applications, the NanoDrop Eight software
automatically initiates a spectral analysis for several known contaminants during the
measurement. Examples of known contaminants include:
• for dsDNA and RNA measurements:
– in the analysis region: protein and phenol
– detects presence of guanidine HCl and guanidinium isothiocyanate
– detects species-specific dsDNA contamination in the RNA application and
detects species-specific RNA contamination is in the dsDNA application
• for protein measurements:
– in the analysis region: nucleic acids and phenol

If contaminants are identified in a sample, the “Contaminant Analysis” icon appears

to the left of the measurement results.

Click the icon to view the contaminant analysis and associated information.

The following is an example of results from a nucleic acid contaminant analysis that
contains enough protein contaminant to affect the measurement results.

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Original nucleic acid spectrum

Corrected nucleic acid spectrum

Phenol spectrum

Original nucleic acid Out-of-range Phenol absorbance

concentration1 purity ratios value at 260 nm

Corrected nucleic acid concentration2 % coefficient of variation

1Based on total sample absorbance (sample plus contaminant)

2Based on corrected sample absorbance (sample minus contaminant)

Since phenols absorb light near the analysis wavelengths for nucleic acid (230 nm,
260 nm, and 280 nm), the presence of phenol in the nucleic acid sample shown
above has pushed the A260/A280 and A260/A230 ratios out of range and caused
the reported nucleic acid concentration to be higher than the real value. The
software identifies the impurity (phenol), and reports the following:
• baseline-corrected absorbance due to phenol (2.53) at the analysis wavelength
(260 nm)
• % coefficient of variation for the measurement result (uncertainty x
100/measurement result = 4.22%; a high %CV indicates the measurement result
is close to the instrument detection limit or there is an interfering component)
• original nucleic acid concentration (462.6 ng/µL), which is based on the total
baseline-corrected absorbance (sample plus contaminant) at the analysis
wavelength
• corrected nucleic acid concentration (286.4 ng/µL), which is based on the
corrected absorbance (sample minus contaminant) at the analysis wavelength

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Theory behind contaminant analysis

UV and UV-visible absorbance measurements are used to quantify nucleic acid and
protein samples at 260 nm and 280 nm, respectively. The analysis is based on the
fact that the total absorbance of a mixture solution at a given wavelength is the sum
of the absorbance values of each component in the mixture.

An ongoing challenge of this method is that a number of materials used in the

extraction process can absorb in various regions across the spectrum. When these
contaminants are present in a sample, they can interfere with the analysis by
artificially inflating the absorbance at the wavelength of interest, which causes the
analyte concentration to be overestimated.

Traditionally, purity ratios are used to detect the presence of contaminants that could
affect downstream applications. However, purity ratios do not always provide a
complete picture of possible contamination. When a purity ratio falls outside the
expected range, the spectral profile is often examined qualitatively.

Our Acclaro technology applies a quantitative approach to contaminant analysis.

Through sophisticated mathematical algorithms, Acclaro analyzes the spectral data
to identify probable contaminants in a sample and removes any contribution due to
the contaminant from the sample result. This results in a more accurate
concentration value of the analyte of interest and a more quantitative analysis of the
level of contamination.

Since the spectrum of a pure compound is unique to that compound, a mixture

spectrum of mostly known materials that have few interactions can be
mathematically broken down into its component spectra and the components
identified. The contaminant analysis algorithm uses a narrow spectral region
(220-285 nm) around the analysis wavelength (260 nm for nucleic acids, 280 nm for
proteins) to determine any absorbance contribution from possible known
contaminants (protein or nucleic acid, and phenol) that absorb in that region. The
entire spectrum is analyzed to determine the presence of other possible
contaminants such as guanidine HCl and/or guanidinium isothiocyanate, which are
common reagents used for nucleic acid purification.

Note Achieving consistent, high quality contaminant analysis results is

dependent on the quality of the measured sample spectra, which is dependent
on the maintenance status of the instrument. For more information, see
Maintenance Schedule.

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On-Demand Technical Support

For the dsDNA and Protein A280 applications, the NanoDrop Eight software
monitors all sample measurements for the presence of contaminants or other
anomalies that may affect the measurement. Examples of monitored characteristics
include:
• absorbance ratios, which indicate the presence of compounds that may interfere
with sample measurements (also referred to as “purity ratios”). For more
information, watch the multimedia training What is a Purity Ratio?.

If sample alert information is available, appears to the left of the measurement

results.

Click the icon to view the information.

Here are results from a nucleic acid analysis for which measured purity ratios are
above the expected value

Click the More Information icon for the next level of information

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Instrument Settings

Instrument Settings
From the Home screen, select Settings. Available options include System Settings,
Dye Editor, Protein Editor, and Preferences.

System Settings
These options are available:

Database The NanoDrop Eight uses a built-in database

Security Used when the optional Thermo Scientific™ SciVault™

software is installed. The server location is automatically
populated for single computer installations, but must be
Copy and Pasted for different computer installations.

Database Backup Set database backup and restore options such as file
& Restore path and data and backup schedule.

Language Select language for displaying NanoDrop Eight software.

Notice: Changing the language requires a software

restart.

Auto-Export Select a file path for exported files and enable/disable

Auto-export at the end of experiments

LIMS Integration Enable when importing data into LIMS software.

Support Incorporates a checksum into the exported file.

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Instrument Settings

Database Backup and Restore

You can backup and restore the measurement database to a specified location of
your choosing.

Configure database backup

From the Settings screen, in the Database Backup & Restore region, select
Configure.

The current backup location is displayed.

Click Browse to assign the backup location.

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Instrument Settings

You can configure a database backup as a one-time event, or as a periodic

scheduled backup. Specify the date and time of a one time backup, or toggle
Backup Schedule to then select the period of the backup from the drop-down
menu.

Select Back Up to save the database to the backup location.

Restore database from backup

To restore data from a previous backup event, from Database Maintenance, select
Restore and browse for the database backup you wish to use.

Once you have selected your backup, click the Restore button.

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Measurement Screen Display Options

Preferences

Set your preferences for remembering certain settings when restarting the
application.

Dye Editor
See “Dye/chromophore editor” on page 32 for information on using the dye editor.

Protein Editor
See “Protein editor” on page 63 for information on using the protein editor.

Measurement Screen Display Options

When performing measurements using the PC Control software, right-click the
graph on the measurement screen to bring up the following display options:
Display multiple samples in overlay.

Hover spectrum to see plot data

Display graph legend

Calculate peaks for specified range

Scale axes to fit spectra measurement

Select to manually enter x-axis range

Select to manually enter y-axis range

Display options- Right click graph to view

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Measurement Screen Display Options

Find Peaks
Select Find Peaks to view calculated peaks for specified range. You can enter the
range by dragging the color-coded limit lines, or enter values into the fields at the top
of the spectrum. Found peaks for the defined range are listed in the table below the
spectrum.
Wavelength start Wavelength end Absorbance minimum

UV spectrum
Peak 1

Table of calculated
peaks for the
Maximum number of peaks specified range

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 9

Maintenance
• Maintenance Schedule 202
• Maintaining the Pedestals 203
• Decontaminating the Instrument 207
• Instrument Diagnostics 209

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Maintenance Schedule

Maintenance Schedule

Daily Maintenance
• Clean pedestals with deionized water

Periodic Maintenance
• Clean and recondition pedestals

Every 6 Months
• Clean and recondition pedestals
• Run Intensity Check
• Run Performance Verification

If you are experiencing an issue with your system,

refer to the troubleshooting information. If the issue
persists, contact us. If you are outside the U.S.A.
and Canada, please contact your local distributor.

If your instrument requires maintenance or repair,

contact us or your local distributor.

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Maintaining the Pedestals

Maintaining the Pedestals

The pedestals require periodic maintenance to maintain measurement integrity. Time
lines and procedures for cleaning and reconditioning the pedestals are provided
below.

Cleaning the Pedestals

To avoid carryover and cross contamination, clean the pedestals before the first
blank or sample measurement and at the end of each measurement. Additional
cleaning (see below) or reconditioning may be required for periodic maintenance.
Note
• Do not use hydrofluoric acid (HF) on the pedestals. Fluoride ions will
permanently damage the quartz fiber optic cables.
• To prevent damage from spills, keep containers of liquids away from the
instrument.
• Do not use a squirt or spray bottle on or near the instrument as liquids may
flow into the instrument and may cause permanent damage.
• Do not attempt to remove the diaphragm around the lower pedestals as it is
permanently affixed to the instrument.
• Do not allow HCl, alcohol, bleach, acetone or any other solvent to remain on
the diaphragm for more than one minute or it may loosen the seals. If the
diaphragm becomes loose, contact us.

Note Solutions containing detergent or isopropyl alcohol may uncondition the

pedestals. If these are required for sample analyses, follow immediately with 3–
5 µL DI H2O.

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Maintaining the Pedestals

Supplies needed
• lint-free laboratory wipes
• deionized water (DI H2O)
• for thorough cleaning: PR-1 kit

To clean the pedestals between

measurements

Lift the instrument arm and clean the

upper and lower pedestals with a new
laboratory wipe.

To clean the pedestals between

users
1. Lift the arm and clean all pedestals
with a new laboratory wipe.
2. Pipette 3 µL DI H2O onto the lower
pedestal.
3. Lower the arm and wait 2–3
minutes.
4. Lift the arm and clean all pedestals
with a new wipe.

Tip: When thorough cleaning is

required (for example, to remove dried
sample left on the pedestals), clean
and recondition the pedestals using
PR-1 compound. If you do not have
PR-1, you can also substitute 0.5M
HCl for the DI H2O in the procedure
above and follow with 3 uL DI H2O.

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Maintaining the Pedestals

Reconditioning the Pedestals

The pedestal surfaces may lose their “conditioned” properties over time, especially
after measurements with isopropyl alcohol or solutions that contain surfactants or
detergents such as the Bradford reagent. An unconditioned pedestal causes
droplets on the lower pedestal to “flatten out,” preventing proper formation of the
liquid column when the arm is lowered. The resulting spectrum may look “rough” or
“jagged.”

If samples flatten out on the pedestal (rather than “beading up” or forming a rounded
droplet) or the liquid column breaks during a measurement, recondition the
pedestals.

Unconditioned pedestal Properly conditioned pedestal

(droplet flattens out) (droplet beads up)

Supplies needed
• lint-free laboratory wipes
• PR-1 pedestal reconditioning kit (available from us or a local distributor)
• calibrated precision pipettor (0-2 µL)
• canned air

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Maintaining the Pedestals

To recondition the pedestals

1. Open the container of PR-1 compound
and use the provided applicator to remove
a pin-head sized amount of the
compound.
2. Apply a thin, even layer of reconditioning
compound to the surface of the upper and
lower pedestal.
Wait 30 seconds for the PR-1 compound
to dry.

3. Fold a clean laboratory wipe into quarters

and use it to vigorously buff the surface of
each pedestal.
Notice: Support the instrument arm with
one hand while you buff the upper
pedestal to avoid damaging the arm.
Tip: Black residue on the wipe is normal.
4. Repeat step 3 with a new folded wipe until
all residue is removed and the pedestals
buff clean.
5. Use canned air to remove any paper
residue from the pedestals.
6. Pipette 1 µL DI H2O onto the lower
pedestals.
The DI H2O should “bead up” or form a
rounded droplet.

Droplet “beads up” on properly

conditioned pedestal

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Decontaminating the Instrument

Tip The PR-1 pedestal reconditioning compound is the easiest way to

recondition the pedestals. If you don’t have a PR-1 kit, follow these steps:
1. Lift the instrument arm and pipette 3 µL 0.5M HCl onto the lower pedestals.
2. Lower the arm and wait 2–3 minutes.
3. Lift the arm and clean all pedestals with a new laboratory wipe.
4. Pipette 3 µL DI H2O onto the lower pedestals.
5. Lower the arm and wait 2–3 minutes.
6. Lift the arm and clean all pedestals with a new wipe.
NOTICE: Support the instrument arm with one hand while you buff the upper
pedestal to avoid damaging the arm.
7. Fold a clean laboratory wipe into quarters and use it to vigorously buff the
surface of each pedestal at least 50 times.
8. Use canned air to remove any paper residue from the pedestals.

Decontaminating the Instrument

Decontaminate the instrument after measurements with samples that contain
hazardous materials and before returning the instrument to us for maintenance or
repair.
Note If your instrument requires maintenance or repair, contact us or your local
distributor.

Note
• Do not use hydrofluoric acid (HF) on the pedestals. Fluoride ions will
permanently damage the quartz fiber optic cables.
• To prevent damage from spills, keep containers of liquids away from the
instrument.
• Do not use a squirt or spray bottle on or near the instrument as liquids may
flow into the instrument and may cause permanent damage.
• Do not attempt to remove the diaphragm around the lower pedestals as it is
permanently affixed to the instrument.
• Do not allow HCl, alcohol, bleach, acetone or any other solvent to remain on
the diaphragm for more than one minute or it may loosen the seals. If the
diaphragm becomes loose, contact us.
•

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Decontaminating the Instrument

Supplies needed
• lint-free laboratory wipes
• deionized water (DI H2O)
• 0.5% sodium hypochlorite solution (1:10 dilution of commercial bleach, freshly
prepared)
• pipettor

To decontaminate the pedestals

1. Lift the instrument arm and clean the upper
and lower pedestal with a new laboratory
wipe.
2. Pipette 2–3 µL diluted bleach solution (see
Supplies needed) onto the lower pedestal.
3. Lower the arm and wait 2–3 minutes.
4. Lift the arm and clean all pedestals with a
new wipe.
5. Pipette 3–5 µL DI H2O onto the lower
pedestal.
6. Lower the arm and wait 2–3 minutes.
7. Lift the arm and clean all pedestals with a
new wipe.

To decontaminate the instrument surfaces

1. Dampen a clean, soft cloth or laboratory
wipe with the diluted bleach solution (see
Supplies needed) and use it to gently wipe
the outside surfaces of the instrument.
2. Use a clean cloth or wipe dampened with
DI H2O to remove the bleach solution.

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Instrument Diagnostics

Instrument Diagnostics
Every 6 months, run the following performance and quality checks to verify
instrument operation.

Intensity Check 210

Performance Verification 212

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Instrument Diagnostics

Diagnostics can be performed using the NanoDrop Eight software. Intensity

Check and Performance Verification, are all accessible from the software
Home screen:

Figure 1. Control options

History: View data stored locally. Filter by date or application.

Performance: Performance verification process using PV-1 solution
Intensity: Run an intensity check for the pedestal
Settings: Set security server location and path if desired
Help: View help

Intensity Check
Run Intensity Check every 6 months to verify operation of the instrument’s internal
components. The test measures the intensity of light from the xenon source through
the instrument to verify that throughput, wavelength accuracy, and bias are within
specifications. The test is automatically performed using the pedestal paths.

Supplies needed
• lint-free laboratory wipes

To run intensity check

1. From the Home screen, select Intensity.
2. Lift the instrument arm and clean the upper and lower pedestal with a new
laboratory wipe.
3. Click Ready and lower the arm.
4. If Auto-Measure feature is OFF, click Measure to begin the measurement. If
Auto-Measure feature is ON, the measurement will begin automatically after
the arm is lowered.

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Here is an example of a typical intensity check result screen.

Channel table

5. To rerun the intensity check, select Run.

6. When finished, select End Experiment.
After the test is completed, the results are available from the History. See
Manage identifiers on the instrument for details.

To interpret intensity check results

The results will display the lamp intensity using the UV and Visible integration times in
two separate graphs (see image above).

Users can see the intensity graphs of a single channel by clicking a single row from
the Channel table or Shift + Click to highlight multiple rows (see image to the right).

A green check mark and banner will be displayed at the top if the UV intensity, Visible
intensity, bias, and maximum single offset are all within specification. If one or more
is not within specification, a message will be displayed at the top indicating what
parameter(s) failed. If any parameter has failed, clean the pedestals with deionized
water and then repeat the Intensity Check.

If a yellow triangle appears next to the Bias indicator, make sure the room is within
the temperature specifications for the instrument.

If the Intensity Check fails again, contact us.

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Instrument Diagnostics

Performance Verification
Run Performance Verification every 6 months to confirm pathlength accuracy is
within specifications.

A CHEM-PV-8 kit containing two vials of PV-1 {aqueous nicotinic acid (C6H5NO2),
potassium nitrate (KNO3)} is required to verify the performance of the Thermo
Scientific™ NanoDrop™ Eight microvolume UV-Vis spectrophotometer.

Supplies needed
• lint-free laboratory wipes
• deionized water (DI H2O)
• Calibrated Precision 8-channel Pipettor (0.5 - 10 µL)
• CHEM-PV-8 kit containing two ampoules of PV-1, two 8-well PCR strip tubes,
and transfer pipettes
• laboratory gloves
Note The PV-1 solution comes in a single-use ampoule. Before you open the
ampoule, shake it vigorously and then allow the liquid to collect in the bottom
portion of the ampoule. After the ampoule is opened, its contents must be used
within one hour. Pipette directly from the ampoule; do not transfer the solution.

Before you begin

First make sure the pedestals are properly conditioned. To test pedestal
conditioning, clean the upper and lower pedestal surfaces with a dry, lint free
laboratory wipe, then pipette 1.5 µL DI H2O onto the eight lower pedestal surfaces.
The droplets should “bead up” as shown below. If it does not, recondition the
pedestals. Remove the water sample from the upper and lower pedestal surfaces
with a dry laboratory wipe.
Droplets “bead up” on properly
conditioned pedestals

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Instrument Diagnostics

Sample Loading Hints

• Ensure the NanoDrop Eight instrument is not situated near an air vent or an
exhaust fan from a nearby instrument.
• Use a small-volume (0.5 - 10 µL) 8-channel pipettor to load the diH20 and the
PV-1 aliquots.

Discharge and Touch Sample Delivery Method Practice

It is very important to deliver the PV-1 aliquots to all eight pedestals in a single
motion using a multichannel pipettor. It is highly recommended that the steps below
be practiced using water before starting the calibration check procedure.
1. When the pipette tips are close to the measurement pedestals, discharge the
fluid and allow the drops to hang on the end of the tips.
2. Gently touch the droplets to the pedestals and allow them to be pulled off the
tips and onto the pedestals by surface tension.

Tip: It is important to use tips with a rigid structure to ensure the tips do not
splay or skew when delivering the samples.

Tip: It is suggested that this technique be practiced until consistent delivery to all
eight positions is routine and quick.

To run performance verification

1. Click on Performance at the bottom of the screen.
2. Enter the PV-1 lot number (found on the PV-1 ampoule label) into the
corresponding PV-1 Lot Number entry box.
3. Enter Target Absorbance Value #1 (found on PV-1 ampoule label) into
corresponding Target #1 Abs entry box.
4. Repeat step 2 for the Target #2 Abs entry box, using Target Absorbance
Value #2 found on PV-1 ampoule label.
Note Target Abs. values are lot specific and must be entered into the
correct, corresponding entry box.

5. Once the target values have been entered, click Continue.

6. Add 55 µL of diH20 to each well of one of the 8-well strips provided. Do NOT
aliquot the PV-1 at this time.
7. Use an 8-channel pipettor to pipette a 1.5 ìL aliquot of diH2O onto each of the
lower pedestals, lower the arm, and click Blank.

Tip: It is important to use tips with a rigid structure to ensure the tips do not
splay or skew when delivering the samples.

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9 Maintenance
Instrument Diagnostics

8. Remove water from upper and lower pedestal surfaces using a clean, dry
laboratory wipe.
9. Ensure PV-1 solution is thoroughly mixed by vigorously shaking both ampoules.
Allow the solution to collect in the bottom portion of the ampoule, if needed
gently tap the ampoule.
10. Carefully snap off top portion of each ampoule using an ampoule cracker,
discard top along with ampoule cracker (use proper safety precautions for
disposal).
11. Use the provided transfer pipette to dispense the contents of both PV-1
ampoules into each well of the 8-well PCR strip tube.
12. Using an 8-channel pipettor, withdraw 1.5 µL of PV-1 solution from each of the
8- wells of the PCR strip tube and pipette onto each of the 8 lower pedestals.
Lower arm.
Note If Auto-Measure feature is OFF, click Measure to begin the
measurement. If Auto-Measure feature is ON, the measurement will begin
automatically after the arm is lowered.

13. After the measurement is complete, remove sample from both upper and lower
pedestals using a dry laboratory wipe.
14. Repeat steps 11 and 12 to measure five additional individual replicates of the
PV-1 solution.
a. Always use fresh pipette tips to remove each aliquot and use a fresh aliquot
of PV-1 for each measurement.
b. In between each measurement, remove PV-1 solution from all 16 pedestals,
upper and lower, using a clean, dry laboratory wipe.
15. After each measurement is complete, the individual results will be displayed on
screen and subsequently added to the existing results.
16. After six replicates have been measured, the Status columns for each
pathlength will indicate whether the channel passed the Performance
Verification.

To interpret performance verification results

1. Results will display for Pass, for Conditional Pass, and for Fail.
a. If results are not within specifications, repeat the procedure using 2 µL
aliquots of PV-1.
b. If results fail to meet specifications using 2 µL aliquots, contact support or
local distributor for assistance.
2. Click End Experiment when done.
a. Results can be exported and printed at this time or later from the History.

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 9 Maintenance
Instrument Diagnostics

b. The Experiment name can be changed at this time and Identifiers can be
added.
3. Click End Experiment when done.
4. To review results from a previous Performance Verification check, select History
from the Home screen and locate the Performance Verification check results
from the list of experiments.

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 10

Safety and Operating Precautions

Contents
• Operating Precautions 218
• Safety Information 219

Note Be sure that all persons operating this system read the safety manual first.

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10 Safety and Operating Precautions
Operating Precautions

Operating Precautions
CAUTION Do not remove the instrument cover. Removing the cover exposes
the operator to sharp edges and delicate fiber optic cables. The instrument
warranty is void if the cover has been removed.

NanoDrop Eight spectrophotometers are designed to operate indoors in an

environment that meets our specifications. For details, see the site preparation guide
for your instrument.

Follow these precautions to avoid damaging your NanoDrop spectrophotometer

during use:
• Use a grounded power cord appropriate for your electrical service. If the
supplied power cord is incompatible or if it becomes damaged, contact us.
• Do not remove the instrument cover.
• Use solvents that are compatible with the instrument (see Hazardous Materials)
• Do not use hydrofluoric acid (HF) on the pedestals. Fluoride ions will permanently
damage the quartz fiber optic cables.
• To prevent damage from spills, keep containers of liquids away from the
instrument.
• Do not use a squirt or spray bottle on or near the instrument as liquids may flow
into the instrument and may cause permanent damage.
• Do not attempt to remove the diaphragm around the lower pedestals as it is
permanently affixed to the instrument.
• Do not allow HCl, alcohol, bleach, acetone or any other solvent to remain on the
diaphragm for more than one minute or it may loosen the seals. If the diaphragm
becomes loose, contact us.

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 10 Safety and Operating Precautions
Safety Information

Safety Information
Before operating a NanoDrop Eight instrument, please read the safety information
and follow its recommendations for the system.

Safety and Special Notices

In many cases, safety information is displayed on the instrument itself. The symbol
indicates that there is additional safety information in the documentation and failure
to heed the safety precautions could result in injury.

WARNING Indicates a hazardous situation which, if not avoided, could result in

death or serious injury.

CAUTION Indicates a hazardous situation which, if not avoided, could result in

minor or moderate injury.

Note Follow instructions with this label to avoid damaging the system hardware
or losing data.

Note Contains helpful supplementary information.

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10 Safety and Operating Precautions
Safety Information

The following table lists some of the safety symbols and their indications that may
appear in the user documentation.

Symbols Indication
This is a mandatory action symbol. It is used to indicate that an
action shall be taken to avoid a hazard.

This is a prohibition symbol. The graphic in this symbol is used to

alert the user to actions that shall not be taken or shall be
stopped.

This is the general warning sign. Failure to heed the safety

precautions could result in personal injury.

Avoid shock hazard. If you see either of these symbols, there is a

risk of electrical shock in the vicinity. Only qualified persons shall
perform the related procedures.

Avoid fire hazard. Do not test flammable or explosive samples.

Read and follow the associated instructions carefully.

Avoid eye injury. If you see these symbols, there is a risk of

exposure to ultraviolet light, which can harm your eyes if safety
glasses are not worn.

Avoid Biohazard. This icon informs of a biological hazard in the

area. Read and follow the associated instructions carefully.

Avoid chemical burns. This symbol alerts you to possible skin

irritation. Wear gloves when handling toxic, carcinogenic,
mutagenic, or corrosive or irritant chemicals. Use approved
containers and proper procedures to dispose of waste.

Symbol Description
Alternating current
Earth terminal or ground
Direct current
Protective conductor terminal
Frame or chassis terminal
Fuse
Power on
Power off

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 10 Safety and Operating Precautions
Safety Information

When the System Arrives

WARNING Avoid personal injury. If this equipment is used in a manner not
specified in the accompanying documentation, the protection provided by the
equipment may be impaired.

CAUTION Avoid personal injury. Perform only those procedures described in

the documentation. If there are other problems, contact us. Any other service
must be performed by trained personnel.

CAUTION Avoid shock hazard. Do not remove the cover of the instrument. All
service to the instrument must be performed by trained personnel.

When the instrument arrives, check the exterior of the shipping box for signs of
damage. If damage is apparent, contact us or your local distributor for instructions.
• Move the shipping box to the installation location at least 24 hours before
installation.
Note
• Inside the shipping box, the instrument is sealed in a plastic bag to keep
the unit dry.
• Allow 24 hours for the instrument to reach room temperature before
opening the bag. If the bag is opened before the instrument reaches
room temperature, moisture could condense on the optical components
and cause permanent damage.

• Keep the instrument upright at all times.

The warranty will not cover:

• Damage due to improper moving techniques.
• Damage due to removing the sealed plastic bag before the instrument has come
to room temperature.
•

Note It is important to have all system utilities installed before the instrument
arrives. Utility installations must comply with all local building and safety
codes.

Lifting or Moving the Instrument

To avoid risk of injury, use proper lifting techniques when lifting or moving the
instrument or other system components.

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10 Safety and Operating Precautions
Safety Information

Electrical Requirements and Safety

Power supplied to the system must be from dedicated, uninterrupted sources.
Power must be free of voltage dropouts, transient spikes, frequency shifts, and other
line disturbances that impair reliable performance.

If you suspect power quality problems at your site, or if your system will be installed
in a heavy industrial environment, we recommend a power quality audit before
installation. Contact us or your local electrical authority for more information.
CAUTION Avoid shock hazard.
• Only a qualified person using the appropriate measuring device shall check
the line voltage, current and frequency.
• Only our trained and certified service representatives shall attempt to service
a component that carries this symbol.
• If a protective cover on a system component appears damaged, turn off the
system and secure it against any unintended operation. Always examine the
protective cover for transport stresses after shipping.
• Even after this instrument has been disconnected from all voltage sources,
capacitors may remain charged for up to 30 seconds and can cause an
electrical shock.
• Do not allow liquid to run over or into any surface where it may gain entry into
the instrument.
• Do not attempt to remove the cover of the instrument.

Grounding

CAUTION Avoid shock hazard. Each wall outlet used must be equipped with a
ground. The ground must be a noncurrent-carrying wire connected to earth
ground at the main distribution box.

Power Cords
Be sure to use an appropriate grounded power cord for your electrical service. If the
power cord received is not appropriate for the electrical system in your location, or if
the power cord becomes damaged, contact us.

Power Line Conditioning Accessories

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 10 Safety and Operating Precautions
Safety Information

A UPS reduces the probability of a system shutdown if power is lost elsewhere in the
building. Power line conditioners (which ensure that your service is free from sags,
surges or other line disturbances) also are available in the U.S.A. from us for 120 volt
operation. Line conditioners for 220 volt operation can be purchased locally. Contact
technical support for information about power conditioners and UPS.

Electrical Service Specifications

The following table lists the specifications for electrical service. Contact our service
representative in your area if you have questions about the requirements.
Requirements Specifications
Input current 5.0 A (max.)
Input voltage 100-240 VAC
Line frequency 50-60 Hz
Line disturbances Sags, surges or other line disturbances must
not exceed 10% of input voltage (even for a
half cycle).
Noise < 2 V (common mode)
< 20 V (normal mode)

Power Consumption

Generally, 50% more power should be available than the entire system (including
accessories) typically uses. Maximum power consumption and heat dissipation
specifications for the spectrometer and accessories are shown below. The values
are approximate.
Item Power Consumption Max. Heat Dissipation
instrument 60 W 205 Btu/hr

Fire Safety and Burn Hazards

Note Do not position the instrument so that it is difficult to operate the power
switch or access the power supply and power cord.

To avoid a burn injury and the risk of fire or explosion:

• Use caution when testing flammable or explosive samples (see the “Hazardous
Materials” section)
• Never block any of the vents on the instrument or its power supply
• Only use exact replacement power supplies from us

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10 Safety and Operating Precautions
Safety Information

Optical Safety
This instrument was designed with a protective housing to prevent user exposure to
ultraviolet light.

WARNING Avoid personal injury. Never look at the lamp while illuminated.

Hazardous Materials
Many standard spectroscopy methods are based on the use of solvents. Others
involve corrosive samples or pressurized samples in a gaseous state.

Volatile Solvents and Flammable Samples

CAUTION Avoid personal injury. Do not leave solvents or flammable samples

near the instrument. Be sure that the workspace is properly ventilated.

Compatible Solvents

Most solvents typically used in life science laboratories are compatible with the fiber
optic pedestals of all NanoDrop spectrophotometers. However, the high vapor
pressure properties of some solvents may not be conducive to small volume
measurements when using the pedestal for measurements on any of the NanoDrop
instruments. If you are measuring samples with high vapor pressures, use an
instrument with provision for measuring samples in cuvettes.

The following solvents are compatible for use on the pedestals of all NanoDrop
instruments.
Note Spillage of these solvents on surfaces other than the pedestals may
damage the instrument.

• methanol • ethanol • n-propanol

• isopropanol • butanol • acetone
• ether • chloroform • carbon tetrachloride
• DMSO • DMF • acetonitrile
• THF • toluene • hexane
• benzene • sodium hydroxide • sodium hypochlorite (bleach)
• dilute HCl • dilute HNO3 • dilute acetic acid

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Safety Information

It is recommended that all corrosive solvents be wiped from the pedestal

immediately upon completion of a measurement. It is also recommended that the
user end a series of measurements with a diH2O sample to ensure that solvents are
not inadvertently left on the pedestal.

The diaphragm around the pedestal of the NanoDrop Eight is permanently affixed to
the instrument. Do not attempt to remove the diaphragm or break the seal. Avoid
prolonged exposure of the diaphragm to HCl, alcohol, bleach, acetone or other
solvents as the adhesive securing the seal may be affected. If the seal comes loose
please contact us.
Note All forms of Hydrofluoric Acid (HF) are incompatible as the fluoride ion will
etch the fiber optic cable.

Biohazard or Radioactive Materials and Infectious Agents

Biological samples such as tissues, body fluids, infectious agents, and blood of
humans and other animals have the potential to transmit infectious diseases. Wear
appropriate protective equipment. Individuals should be trained according to
applicable regulatory and organization requirements before working with potentially
infectious materials. Follow your organization’s Biosafety Program protocols for
working with and/or handling potentially infectious materials.

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10 Safety and Operating Precautions
Safety Information

WARNING Reduce the risk associated with potentially infectious samples:

• Do not spill samples on any of the instrument components.
• If spill occurs, disinfect the external surfaces immediately following your
laboratory protocols.

Instruments, accessories, components or other associated materials should not be

disposed of and may not be returned to us or other accessory manufacturers if they
are contaminated with biohazard or radioactive materials, infectious agents, or any
other materials and/or conditions that could constitute a health or injury hazard to
employees. Contact us if you have questions about decontamination requirements.

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