CLS 571 Fall 2022-23

High Performance
Liquid Chromatography (HPLC) - 1

American University of Science and Technology

Nina Esipenko, Ph.D.

1
 Introduction
HPLC is a chromatographic technique used for separation,
purification and characterization of compounds. It relies on
pumps to pass a pressurized liquid solvent containing the
sample mixture through a column filled with a solid adsorbent
material.
Separation of the sample into its constituent parts occurs based
on the difference in the relative affinities of different molecules
for the mobile phase and the stationary phase used in the
separation.

2
 Introduction
Introduction
Qualitative HPLC: Separation and Identification of the sample composition
(identification of the compounds using reference standards ) using analytical column
(µg)
Quantitative HPLC: Separation and Quantification of the sample components
using analytical (µg) (using calibration curve), semi-preparative (mg) or
preparative columns (g).

Strengths:
 Precise
 Unlike GC, no heat is required (i.e. less sample degradation)
 Various types of columns (analytical, preparatory and semi
preparatory)
 Sensitive (traces of the analytes can be detected)
 Automated
Limitations:
 Requires large amount of organic solvents
 Requires specific detectors that monitor compounds that lack
3
a chromophore
 Instrumentation

tR
Peak

Signal
h
A

Retention Time

Controller /
Data processing

Degasser

Waste or
Fraction collector

Solvent reservoirs
 Instrumentation
1. Solvent reservoirs.
contain the solvents used to carry the sample
through the system. The solvent should be filtered
with an inlet solvent filter to remove any particles
that could potentially damage the system's
sensitive components.
 Instrumentation
2. Degasser.
HPLC degassing of solvents, is very essential for proper operation and
retrieval of uniform data.
Degassing removes entrapped air bubbles from the solvents. Improper
degassing leads to many problems like fluctuation in pressure,
baseline fluctuation, decrease in sensitivity, improper peaks, improper
detection, clogging of column etc.
Vacuum chamber

To Pump

The most common type –

Vacuum chamber type degasser
 Instrumentation
3. Pumps.
The role of the pump is to force a liquid (called the mobile phase)
through the liquid chromatograph at a specific flow rate (controlled
flow rate), expressed in milliliters per min (mL/min).
• Normal flow rates in HPLC are in the 1 to 2 mL/min range
Analytical 0.1 - 1 mL/min
Semi-preparative 0.5 – 2 mL/ min
Preparative 2 – 10 mL/min
• Typical pumps can reach pressures in the range of 6000-9000
psi (400- to 600-bar).
• During the chromatographic experiment, a pump can deliver a
constant mobile phase composition (isocratic) or a changing
mobile phase composition (gradient).
 Instrumentation

Mobile Phase

Pump head
Motor & Cam

Check valve

Plunger
Plunger seal
Mobile Phase
 Instrumentation
Isocratic mode - delivers constant mobile phase composition
(composition remains constant with time)
• solvent must be pre-mixed
• lowest cost pump
• best for simple separations

Gradient mode - delivers variable mobile phase composition

• best for the analysis of
complex samples
mobile phase solvent (“B”)
composition increases with
time:
 Instrumentation
 Binary gradient pump – delivers two solvents

 Quaternary gradient pump – delivers four solvents

 Instrumentation
4. Injector.
The injector serves to introduce the liquid sample into the
flow stream of the mobile phase.

• Typical sample volumes are 5 µL – 10 mL (depending on the

type of the column)
• The injector must be able to withstand the high pressures of
the liquid system.
• An auto-sampler (auto-injector) is the automatic version for
when the user has many samples to analyze or when
manual injection is not practical.
 Instrumentation
Manual Injector:
1. User manually loads sample into the sample loop of the injector
using a syringe LOAD position
2. and then turns the handle to inject sample into the flowing mobile
phase INJECT position
 Instrumentation

from Pump to Column from Pump to Column

Sample Loop Sample Loop

LOAD INJECT
 Instrumentation

from Pump

to Column
LOAD
from Pump

to Column
INJECT
 Instrumentation
Autosampler:
1. User loads vials filled with sample solution into the autosampler
tray (100 samples)
2. and the autosampler automatically
 measures the appropriate sample volume
 injects the sample,
 then flushes the injector to be ready for the next sample, etc.,
until all sample vials are processed …
… for unattended automatic operation
 Instrumentation

From pump

to the column
waiste
Step 1 Step 2

Step 3
 Instrumentation
5. HPLC Column:
Within the Column is where separation occurs.
Key Point – Proper choice of column is critical for success in HPLC
Stainless steel columns packed with porous materials
 Instrumentation
Columns are packed with small diameter porous particles.
– The most popular sizes are: 5-μm, 3.5- μm and 1.8-μm
• Columns are packed using high-pressure to ensure that they are
stable during use (most users purchase pre-packed columns to
use in their liquid chromatographs)
• These porous particles in the column usually have a chemically
bonded phase on their surface which interacts with the sample
components to separate them from one another
 Instrumentation
Optional:
 Column Oven can control and maintain the constant
column temperature.

Constant temperature for solvent and column is required

to perform reproducible results.
 Pre-Column contains the filter element which prevents the
column damage
 Instrumentation
Tubing
Materials
• Stainless steel (SUS) most popular, gives high pressure capabilities
• PEEK (Polyether ether ketone) biocampatable and chemically inert to
most solvents
• Glass (mostly for biomolecules)
Size
O.D. (Outer Diameter)
– 1.6 mm
I.D. (Inner Diameter)
– 0.1 mm
– 0.3 mm
– 0.5 mm
– 0.8 mm etc.
 Instrumentation
Connectors

• Male nut (SUS)

Ferrule
Ferrule (SUS)
– Pressure: up to 40 MPa
Male nut

• Male nut (PEEK)

– can be connected
without any tools
– Pressure: up to 25 MPa Male nut (PEEK)
 Instrumentation
• Dead volume may cause problems such as poor peak
shape, separations and reproducibility.

Male nut Dead volume

Tube

Excellent connection Poor connection

Instrumentation
 Instrumentation
Off-line Degassing and Filtration

Aspirator Membrane filter

(Size: 0.45 um)

Aspirator

Ultrasonic cleaning unit

 Instrumentation
6. Detectors

 UV-Visible detectors
 Photodiode Array Detectors (PDA)

 Fluorescence detector

 Mass Spectroscopic detector (MS)

 Refractive Index detector (RI)

 Electrochemical detector

 Light scattering detector (ELSO)

Characteristics of HPLC detector
Detector performance characteristics:
 Sensitivity (LoD, LoQ)
 Selectivity
 Linearity
 Qualitative information
 Reliability
 Ease of use
 Universality
 LOD and LOQ
The limit of detection (LOD) for a detector can be characterized
by its signal to noise ratio (S/N) for an analyte under a given set of
conditions.
Peak

Noise

SD - standard deviation of the response

S - slope of the calibration curve
 Limit of detection (LOD) is a result of the whole chromatography system, not
only the detector performance

 The limit of quantification (LOQ) is the lowest concentration at which the

performance of a method or measurement system is acceptable for a
specified use (specific purpose) (LOQ = 10 SN/S )
 Signal to noise ratio
S/N = 2H / h

CV (%RSD) = 50/ (S/N)

this equation gives us an idea of what S/N is required for a desired method
precision.

For example, if the %RSD for a method's precision can be no larger than
2%, this means that S/N = 25 is required (this assumes that S/N is the
primary contribution to %RSD, which might or might not be true).

For bioanalytical methods, S/N = 2.5 would translate into %RSD ≈ 20%.
Repeatability: Closeness between the results of measurements of the
same sample carried out using following conditions:
The same measurement procedure
The same observer
The same measuring instrument used under the same conditions
The same location
Repetition over a short period of time

Reproducibility: Closeness between the results of measurements of the

same sample, where the measurements are carried out under changed
conditions such as:•
Principle or method of measurement
Observer
Measuring instrument
Location
Conditions of use
Time
 A replicates (batch) cannot be broken apart without loosing relevance
– differences should be minimal.
 Every batch should be accompanied by a positive and a negative control.
Positive control – produces a positive result
Negative control (blank) – produces no result

 Blank samples: analyses the solvent/matrix without the analyte and ensures that
glassware/instruments/equipment are free from contamination.
 Known samples/open control: uses a standard with known concentration in order
to validate the method and ensure accuracy.
 Calibration check: Different concentrations of an analyte are analyzed and a
calibration curve is plotted. This curve has to be validated regularly using a known
analyte concentration.
 Replicates: A subsample aliquot (taken from the original sample) analyzed to
determine the mean and standard deviation (%RSD).
 Duplicates: Two original analyte samples are obtained and analyzed.
 Spikes: A known concentration of an analyte is added to a sample matrix and then
analyzed to determine the percentage recovery. Often, deuterated compounds can
be used as spikes as they can be distinguished from the target analyte via MS
analysis.
 Instrumentation
Qualitative Information

Chlortoluron Atrazine
? ?

Take peak spectrum Take peak spectrum

(UV) (MS)

200

58

215
44
68 172

96 104 132 138158

60 80 100 120 140 160 180 200 220

Wavelength (nm) Mass/Charge

Necessity for more than one detector

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UV-signal

248/411
270/388
241/394

247/504
302/420
WL
WL

WL
Fluorescence

WL
WL
PAH's extracted from soil;
Flecainide in
Sup.LC-PAH 150x4.6mm; Serum
Solv.: H2O/CH3OH= 10:90

UV signal

FL signal

Therapeutic concentration: 1.8mg/l, 20ul injected

UV and fluorescence signal
 Detectors
Ultraviolet (UV) Absorption

An ultraviolet light beam is directed through a flow cell and a

sensor measures the light passing through the cell.
• If a compound elutes from the column that absorbs this light
energy, it will change the amount of light energy falling on the
sensor.
• The resulting change in this electrical signal is amplified and
directed to a recorder or data system.
• A UV spectrum is sometimes also obtained which may aid in
the identification of a compound or series of compounds.
 Detectors
UV/UV-VIS detector

Cell C : Concentration

Iin Iout

D2 / W Lamps
l

A = e·C·l = –log (Iout / Iin) A

(A : Absorbance)
C
 Detectors
UV/UV-VIS detector
• Lamp selection
– D2 (160-400 nm) / W (350-2500 nm) / OFF
• Wavelength
• Signal unit
– Volt / AU
 Detectors
PDA detector

Cell
Grating

D2 / W Lamps
1 nm / element

512…… photodiodes
 Detectors
PDA detector Spectrum

Chromatogram
Absorbance

Retention time
 Detectors
Fluorescence detector
Compared to UV-Vis detectors fluorescence detectors offer a higher sensitivity and
selectivity that allows to quantify and identify compounds and impurities in complex
matrices at extremely low concentration levels (trace level analysis).
• Fluorescence detectors sense only those substances that fluoresce
Excitation wavelength

+ hn1 *

* hn2 +
Emission wavelength
Excitation state
Quasi-excitation state
hn1
hn2
Fluorescence
Ground state
 Detectors
Refractive Index (RI) detector
The ability of a compound or solvent to deflect light
• The RI is a measure of molecule’s ability to deflect light in a flowing mobile
phase in a flow cell relative to a static mobile phase contained in a reference
flow cell.
• The amount of deflection is proportional to concentration.
• The RI detector is considered to be a universal detector but it is not very
sensitive.

Photodiode
Reference

W Lamp
Sample
 Detectors
Refractive Index (RI) detector

Reference cell
Reference cell
containing
containing
flowing solvent Sample cell
flowing solvent Sample cell
(mobile phase) containing only
(mobile phase) containing
mobile phase
mobile phase
with analyte
 Calculate the resolution of the compounds in red
 Calculate the retention factor for the last peak (the hold-up time (to) is
indicated by the vertical ‘tick mark’ on the baseline of the chromatogram).
 Calculate the plate number of the column (from the last peak eluted).
to = 1.64 min