Instrument 4:

Spectrophotometer
 Spectrophotometer
 A device to measure how much a chemical
substance absorbs light by measuring the intensity
of light as a beam of light passes through sample
solution.
 The basic principle is that each compound
absorbs or transmits light over a certain range of
wavelength.
 This measurement can also be used to measure
the amount of a known chemical substance.
 The most useful devices of quantitative analysis in
various fields such as chemistry, physics,
biochemistry, material and chemical engineering
and clinical applications.
 Common terms
Spectroscopy
 The science of studying the interaction between matter and radiated
energy
Spectrometry
 Method used to acquire a quantitative measurement of the spectrum.
Spectrum
 Band of colours, as seen in a rainbow, produced by separation of the
components of light by their different degrees of refraction according to
wavelength. (refer figure 1 & figure 2)
Photometry
 the science of the measurement of light, in terms of its perceived
brightness to the human eye.
Spectrophotometer
 Instrument for measuring the intensity of light in a part of the spectrum,
especially as transmitted or emitted by particular substances.
Spectrum
Band of colours, as seen in a rainbow, produced by
separation of the components of light by their different
degrees of refraction according to wavelength.

Figure 1
Wavelength
The distance between successive crests of a wave, especially
points in a sound wave or electromagnetic wave.

Figure 2
 Spectrometry & Photometry

Spectrometry Photometry
Spectrometer: It produces a desired range of wavelength Photometer: After the desired range of
wavelength of light passes through the
of light. First a collimator (lens) transmits a straight beam of solution of a sample in cuvette, the
light (photons) that passes through a monochromator photometer detects the amount of
(prism) to split it into several component wavelengths photons that is absorbed and then
(spectrum). Then a wavelength selector (slit) transmits only sends a signal to a galvanometer or a
the desired wavelengths digital display
 Prism

an optical component with a An optical element with flat,

periodic structure that splits polished surfaces that refract light.
and diffracts light into At least two of the flat surfaces
several beams travelling in must have an angle between
different directions. them.
 Collimator?
 Monochromator?
 How spectrophotometer works?
Video linked
https://www.youtube.com/watch?v=pxC6F7bK8CU
Component Spectrophotometry
Spectrophotometer consists of three
main parts:

1. Emission source which produces the

spectrum (eg ?)
2. Optical system which collimates
and disperses the spectrum (eg?)
3. Detecting device to measure the
emitted lines intensities. (eg?)
 Cuvette

 A cuvette (French: cuvette = "little

vessel") is a small tube-like
container with straight sides and
a circular or square cross section.
 It is sealed at one end, and made
of a clear, transparent material
such as plastic, glass, or fused
quartz.
 Cuvettes
•If cuvettes are mismatched, measurement errors may
result.
•Below 330 nm, silica or quartz cuvettes are required.
•Standard volume is 3 ml but some are designed for 0.1 to 1.0
ml.
•UV Spectrophotometer
Quartz (crystalline silica)

• Visible Spectrophotometer
Glass
 Colour intensity
 many solutions have distinctive colours
 absorb/transmit different wavelengths of visible light

absorbs: Red, Green

transmits: Blue

 intensity of colour is an indication of the solution’s concentration

 more concentrated = darker colour
Spectrophotometer vs
electromagnetic spectrum

 Spectrophotometer measure the light intensity of the samples.

 Why this can be measure?
Light is a wave of alternating electric and magnetic fields
known as the electromagnetic spectrum.
 What is electromagnetic spectrum?
Entire range of light that exists (Figure 3)
The electromagnetic spectrum
Can our eyes capture all the electromagnetic spectrum?

Figure 3
Electromagnetic Spectrum

400 700
nm nm
UV-Vis and IR
Spectrophotometer
 Depending on the range of wavelength of light
source, it can be classified into two different
types:

 UV-visible spectrophotometer: uses light over the

ultraviolet range (185 - 400 nm) and visible range
(400 - 700 nm) of electromagnetic radiation
spectrum.
 IR spectrophotometer: uses light over the infrared
range (700 - 15000 nm) of electromagnetic
radiation spectrum.
Q&A

1. In what way is visible light similar to ultraviolet?

2. What we called the entire range of EM waves?
3. What are the 7 EM waves in order from longest to
shortest wavelength?
4. What are the waves used for broadcasting radio
signals called?
5. What kind of light can some animals like bee and
dogs see that you cannot?
 Question & Answer

The passing of light through a sample?

A) % Transmittance
B) Transmittance
C) Nanometer
 Question & Answer

The manner in which a spectrophotometer reports the amount of

light that passes through a sample called ……..
A. Transmittance
B. % Transmittance
C. Absorbance
D. Ultraviolet
Spectrophotometer
 measures absorbance or transmittance of
light, as a function of wavelength

General procedure:
 sample is placed into cuvette a cuvette
 light of selected wavelength (λmax)
is passed through sample
 instrument measures the amount of light
absorbed by the sample
 Reference blank
 contains everything found in the sample
solution, except the substance being
analyzed
 calibrates the absorbance reading to
A=0.000

spectrophotometer
 • Io = Original intensity of light
• It = Intensity of transmitted light

Monochromator separates and Two ways of Reporting:

transmits a particular wavelength 1. Transmission, T = It/Io
(colour) of light
or %T = It/Io × 100%

2. Absorbance, A = -log(%T)
Absorbance and
wavelength
 Choosing a Wavelength
 use wavelength at which most light is absorbed (λmax)

λmax

a) What is λmax for this

compound?
b) What colour light would
you use to analyze this
compound?
c) What colour does this
solution appear to the
observer?

Absorption spectrum for a sample

Beer-Lambert Law
 Beer-Lambert Law (also known as Beer's Law)
states that there is a linear relationship between
the absorbance and the concentration of a
sample. Beer's Law can be applied when there is
a linear relationship. Beer's Law is written as:

(or molar extinction

coefficient)
Transmittance

 Transmittance is the fraction of light that

passes through the sample. This can be
calculated using the equation:
Absorbance
 Absorbance stands for the amount of photons that is
absorbed.
What does this tell us?
 There is a direct relationship between
absorbance and concentration
 Empirical evidence: prepare solutions of
known concentration and analyze them at
λmax: plot of absorbance vs. concentration
is linear.
 Since A is a linear function of c,

equation of line: y = mx + b
A = εLc

…slope of best-fit line = εL

 A=εLc
Just use the Beer’s Law equation!!
 seems simple, but ε is usually not readily available

Example 3: The molar absorptivity of ethanal in

hexane at its λmax is 15 L•cm-1•mol-1. Calculate the
concentration of ethanal in a solution that has an
absorbance of 0.652 with a path length of 1.2 cm.

ε=?
l =?
A =?
c= A/ εl
Calculation 1
 Guanosine has a maximum absorbance of 275
nm. ϵ275 = 8400 L mol-1 cm-1, and the path length
is 1 cm. Using a spectrophotometer, you find the
that A275=0.70. What is the concentration of
guanosine?
 Answer:
ϵ275 =?
A275=?
I=?
c=?
Lambert law =
Calculation 2

 Using a cuvette with a length of 1 cm, you

measured the absorbance of a solution with a
concentration of 0.05 mol/L. The absorbance at a
wavelength of 280 nm was 1.5. What is the molar
absorptivity of this solution?
 Answer:
Calculation 3

 There is a substance in a solution (4 mol/liter). The

length of cuvette is 2 cm and only 50% of the
certain light beam is transmitted. Find the value of
absorption coefficient.
 Answer:
Calculation 4
Information given:
 Concentration of compound M = 8 g/liter
 Cuvette length = 2 cm
 The value of molar extinction coefficient for
compound M = 0.0376
Based on information above, how much is the
percentage beam of light is transmitted through
compound M?
Answer:
Calculation 5
 The absorption coefficient of a glycogen-iodine complex is
0.20 at light of 450 nm. What is the concentration when the
transmission is 40 % in a cuvette of 2 cm?

 Answer
Uses of Spectrophotometry

Concentration of Both Inorganic

and Organic Molecules

Absorption Spectra

Kinetic Assays
Measure quantity
 Some bio-molecules have properties
which allow direct measurement
 proteins have aromatic amino acids
(280nm)
 Nucleic acids have unsaturated ring
structures (260nm)

 Other molecules have chemical

properties which can be used in indirect
measurement.
eg ;Growth rate of bacteria such as E. coli etc
UV SPECTROMETER APPLICATION
 180 nm to 340 nm
 Hydrogen Gas Lamp & Mercury Lamp
Procedure
 Dilute of each of your plasmid DNA
 Your blank will be sterile MilliQ-filtered water.
 Your wavelength is 260nm.
 Blank the UV-spectrophotometer with MilliQ
water.
 Careful, the Quartz cuvette is small, expensive,
and it breaks when dropped on the floor.
 Exchange the water with your diluted sample
 Read sample.
UV SPECTROMETER APPLICATION

Protein
Amino Acids (aromatic)
Pantothenic Acid
Glucose Determination
Enzyme Activity (Hexokinase)
GeneRay UV Photometer
 The GeneRay UV Photometer is
a powerful tool for the
molecular biologist enabling
rapid
 UV purity determination and
quantification of single-
stranded and double-stranded
DNA, ssRNA and
oligonucleotides,
 as well as bacterial growth
rates.
 Results can be obtained in
µg/ml µg/µl or pmol/ml
 Measures at 260, 280 and 600
nm
 No warm-up time
 Automatic calculation of
concentrations
 Virtually unlimited life time of
lamp
VISIBLE SPECTROPHOTOMETER APPLICATION

• 340 nm to 700 nm
• Tungsten Lamp
• Material detection like
 Vitamins like Niacin (Vit B3), Vit B12 &
Pyridoxine (Vit B6)
 Metal Determination (Fe)
 Fat-quality Determination (TBA)
 Enzyme Activity (glucose oxidase)
Common spectrophotometer
Precautions When Handling a
Spectrophotometer

 Allowing the lamps and electronics to warm up

 Using the correct wavelength
 Wiping fingerprints and spilt sample off the outside of the
cuvette before measuring.
 Avoid to make any scratch on the surface of cuvette.
 Carrying out the set-up procedure in the correct order
 Performing calibration checks after set up
 Closing the door to the cuvette compartment before
reading the result
 Cleaning up any spills inside the cuvette compartment
1

Turn on the spectrophotometer. Most spectrophotometers

need to warm up before they can give an accurate
reading. Turn on the machine and let it sit for at least 15
minutes before running any samples. However, this is still
depending on the unit/brand/model that you are using
2

Clean the cuvettes or test tubes. Rinse each cuvette

thoroughly with deionized water.
3

Load the proper volume of the sample into the cuvette.

Some cuvettes have a maximum volume of 1 milliliter (mL)
while test tubes may have a maximum volume of 5 mL. As
long as the laser producing the light is passing through the
liquid and not an empty part of the container, you will get
an accurate reading. If you are using a pipette to load your
samples, use a new tip for each sample to prevent cross-
4

Prepare a control solution. Known as a blank, the control

solution has only the chemical solvent in which the solute to
be analyzed is dissolved in. For example, if you had salt
dissolved in water, your blank would be just water. If you dye
the water red, the blank must also contain red water. The
blank is the same volume as the solution to be analyzed and
kept in the same kind of container.
5

Wipe the outside of the cuvette. Before placing the cuvette

into the spectrophotometer you want to make sure it is as
clean as possible to avoid interference from dirt or dust
particles. Using a lint free cloth, remove any water droplets or
dust that may be on the outside of the cuvette. Avoid from
making any scratch on the surface of cuvette.
6

Choose and set the wavelength of light to analyze the

sample with.
7

Calibrate the machine with the blank. Place the blank into
the cuvette holder and shut the lid.
8

Remove the blank and test the calibration.

9

Place the test sample, then make the reading

1
0

Calculate the transmittance and absorbance of the sample,

in order to determine the amount of species/compound
solution. Using current spectrophotometer models,
everything is calculated for you.
Care of Spectrophotometers

 The following rules should be followed when

using the spectrophotometers:
 Always use a Kimwipe to wipe the outside of the
tube before inserting it into the sample holder
 Never place anything on top of the machine
 Immediately wipe up any spills
 Always turn off the spec when you are finished
with it unless otherwise instructed
 Always remove the tube from the sample holder
when you are finished
Thank you