LECTURE NOTES

ANALYTICAL BIOCHEMISTRY
CHAPTER – 1
Spectrophotometry and Colorimetry
Learning Objectives
By the end of this section, you will be able to:
 To measure the absorbance of the sample at different wavelengths.
 To find out the unknown concentration of the sample.
 Verification of Beer-Lambert's Law.

SPECTROPHOTOMETER
Introduction: A spectrophotometer is a photometer that can measure the intensity of light as a
function of its wavelength. Single beam and double beam are the two major classes of
spectrophotometers. Linear range of absorption and spectral bandwidth measurement are the
important features of spectrophotometers.
In Single Beam Spectrophotometers, all the light passes through the sample. To measure the
intensity of the incident light the sample must be removed so that all the light can pass through.
This type of spectrometer is usually less expensive and less complicated. The single beam
instruments are optically simpler and more compact, znc can also have a larger dynamic range.
In a Double Beam Spectrophotometer, before it reaches the sample, the light source is split into
two separate beams. One beam passes through the sample and the second one is used for
reference. This gives an advantage because the reference reading and sample reading can take
place at the same time.

In transmission measurements, the spectrophotometer quantitatively compares the amount of

light passing through the reference and test sample. For reflectance, it compares the amount of
light reflecting from the test and reference sample solutions.

Many spectrophotometers must be calibrated before they start to analyse the sample and the
procedure for calibrating spectrophotometer is known as "zeroing." Calibration is done by using
the reference substance, and the absorbencies of all other substances are measured relative to the
reference substance. % transmissivity (the amount of light transmitted through the substance
relative to the initial substance) is displayed on the spectrophotometer.

The major sequence of events in spectrophotometry is as follows:

1. The light source shines through a monochromator.

2. An output wavelength is selected and beamed at the sample.
3. A fraction of the monochromatic light is transmitted through the sample and to the
photo-detector.

Single Beam Spectrophotometer:

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Spectrophotometry deals with visible light, near UV and near IR. To acquire the spectral
information quicker in IR spectrophotometers, which use a Fourier transform technique and is
called Fourier Transform Infrared (FTIR).

Different Types of Spectrophotometers:

A. Single Beam: In this type, all the light passes through the sample .To measure the intensity
of the incident light the sample must be removed so that all the light can pass through. This type
of spectrometer is usually less expensive and less complicated.

B. Double Beam: In this type, before it reaches the sample, the light source is split into two
separate beams. From these one passes through the sample and second one is used for reference.
This gives an advantage because the reference reading and sample reading can take place at the
same time..

C. Visible Light (400-700 nm): Visible spectrophotometers can use incandescent, halogen,
LED, or a combination of these sources and these spectrophotometers vary in accuracy. Plastic
and glass cuvettes can be used for visible light spectroscopy.

D. Ultraviolet Light: UV spectroscopy is used for fluids, and even solids. Cuvettes, only made
of quartz, are used for placing the samples.

E. Infrared Light: IR spectroscopy helps to study different structures of molecules and their
vibrations. Different chemical structures vibrate in different ways due to variation of energy
associated with each wave length. For example, mid-range and near infrared (higher energy)
infrared tends to cause rotational vibrations and harmonic vibrations respectively.

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. For this reason, Beer's Law can only be
applied when there is a linear relationship. Beer's Law is written as:

\(A = \epsilon{lc}\)

where

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 \(A\) is the measure of absorbance (no units),
 \(\epsilon\) is the molar extinction coefficient or molar absorptivity (or absorption coefficient),
 \(l\) is the path length, and
 \(c\) is the concentration.

The molar extinction coefficient is given as a constant and varies for each molecule. Since
absorbance does not carry any units, the units for \(\epsilon\) must cancel out the units of length
and concentration. As a result, \(\epsilon\) has the units: L·mol-1·cm-1. The path length is
measured in centimeters. Because a standard spectrometer uses a cuvette that is 1 cm in width,
\(l\) is always assumed to equal 1 cm. Since absorption, \(\epsilon\), and path length are known,
we can calculate the concentration \(c\) of the sample.

Applications of a Spectrophotometer:

1. It is directly used to measure light intensity at different wavelengths.

2. It is used to determine the unknown concentration of solution.
3. Spectrometers can be used to determine the equilibrium constant of a reaction involving ions.

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COLORIMETER
Introduction
A colorimeter is a device used in colorimetry. The word generally refers to the device that
measures the absorbance of particular wavelengths of light by a specific solution. This device is
most commonly used to determine the concentration of a known solute in a given solution by the
application of the Beer-Lambert law.
Principle of colorimetry
Colored solutions have the property of absorbing light of definite wave lengths. The amount of
light absorbed or transmitted by a colored solution is in accordance with the Beer-Lambert law.
Beer’s law- The intensity of the color is directly proportional to the concentration of the colored
particles in the solution.
Lambert’s law- The amount of light absorbed by a colored solution depends on the length of the
column or the depth of the depth of the liquid through which the light passes.
Equations
When a monochromatic light with an original intensity ‘Io’, passes through a solution that can
absorb radiant energy, Is will be less than the Io.
Some of the radiant energy is reflected back by the cell containing the solution, or absorbed by
the cell wall or the solvent.
The amount of radiation absorbed may be measured in a number of ways:
1. By measuring transmittance
2. by measuring absorbance
1. By measuring transmittance- The transmittance (T) is defined as-
T= Is/ Io
The ratio is expressed as percentage, thus
% T= 100 x Is/ Io
As the concentration of the compound increases, less light is transmitted.
%T varies inversely and logarithmically with the concentration.
2) By measuring absorbance- Absorbance measurement is convenient than transmittance.
Absorbance (A) or optical density is directly proportional to the concentration.
The relationship between absorbance and transmittance can be expressed as –
A = – log Is/ Io
= – log T
= log 1/T
To convert T to % T,
A = log 1/T x 100/100
= log (100)/%T
= log 100-log %T
= 2-log %T
Thus,

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A = 2-log %T
In other words, absorbance (Optical density) and Transmittance (T) are reciprocally related.
So, if all the light passes through a solution without any absorption, then absorbance is zero, and
percent transmittance is 100%. If all the light is absorbed, then percent transmittance is zero, and
absorption is infinite (Figure-1)

Figure 1: Transmittance and absorbance are reciprocally related.

Lambert -Beer’s law
The mathematical expression at a given wavelength can be represented as follows-
OD = A = Ʃcl
Since,
OD = – log Is/ Io
(Absorption has no units, since it is a ratio)
Thus:
– log Is/ Io = cl
or Is/ Io = e Ʃcl
Where =Ʃ is a Constant- It is the molar extinction coefficient( Molar absorptivity) with units of L
mol-1 cm-1
C = Concentration of the colored substance, expressed in mol L-1
l = is the path length of the sample – that is, the path length of the cuvette in which the sample is
contained.
e- base of the natural logarithm.
Since Is/ Io is known as transmittance (T)
Thus,
T= e Ʃcl
Taking logarithm:
-log 10T =Ʃcl
As per equation:
-log T= A
Hence
A = OD = Ʃcl

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Since the thickness of the layer of solution is constant in the instrument, optical density is
proportional to the concentration.
When optical density is plotted against concentration “c”, a straight line passing through the
origin should be obtained, because the absorbance is directly proportional to the concentration.
(Figure-2)

Figure 2: Relationship of absorbance and concentration of a solute in a solution

The concentration of an unknown solution can be readily determined by measurement of its
absorbance and interpolation of its concentration from the graph of the standards.
When % T is plotted versus concentration, a curvilinear relationship is obtained.
The linear relationship between concentration and absorbance is both simple and straightforward,
which is why it is preferred to express the Beer-Lambert law using absorbance as a measure of
the absorption rather than %T.
Calculation of unknown concentration in the test sample
Since there is a linear relationship between absorbance and concentration, it is possible to
calculate the unknown concentration of a substance in the test sample by simple proportional
equation-
Absorbance of unknown Concentration of unknown
———————————- = ————————————–
Absorbance of standard Concentration of Standard
Absorption of unknown
Concentration of unknown = ————————————— x Concentration of Standard
Absorption of standard
Thus,
Concentration of Unknown (Test sample T)
OD of Test
= ————————– x Concentration of Standard
OD of standard
Some of the incident energy may be reflected by the cell containing the solution or absorbed by
the cell wall or the solvent. To eliminate these factors and to consider the absorption by the
compound, a blank solution or a reference solution having everything but the compound to be
measured is used.

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Thus The concentration of unknown can be expressed as-
Concentration of Unknown (Test sample T)
OD of Test – OD of Blank
= —————————————— x Concentration of Standard
OD of standard – OD of Blank
Deviations from Beer’s law are observed when a very large concentration of an unknown
substance is measured or when the incident light is not mono chromatic light.
Components of a photo colorimeter
1) Light source
The light source is usually a tungsten lamp for wavelength in the visible range (320-700 nm) and
a deuterium or hydrogen lamp for ultraviolet light (below 350 nm). Hydrogen lamp is usually
preferred to UV range.
2) Monochromators
This is for the selection of sufficiently narrow wave band. The monochromator consists of an
entrance slit to exclude unwanted, followed by absorption or interference filters, prisms or
diffraction grating for wave length selection. (Figure-3)

Figure 3: Components of a colorimeter.

The interference filters consist of thin layer of magnesium fluoride crystals with a
semitransparent coating of silver on each side. The interference filters have a bandpass of 5-8
nm. The band pass is defined as the width of the spectrum that will be isolated by a
monochromator. The choice of filter depends upon the final color of the solution formed.
Wave length (nm) Filter used/Color absorbed Color of solution
350-430 Violet Yellow Blue
430-475 Blue Yellow
475- 495 Green blue Orange
495-505 Blue green Red

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505-555 Green Purple
555-575 Yellow green Violet
575-600 Yellow Blue
600=650 Orange Green blue
650-700 Red Blue green
3) Lens
Instruments using filters as wavelength selectors require lenses to focus correctly the light from
the source through the filter and cuvette to the detector. In the ultraviolet range, quartz or fused
silica is essential because the glass does not transmit light efficiently at wave length shorter than
340 nm.
An exit slit at the end of monochromator allows only a narrow fraction of the spectrum of reach
the sample cuvette.
4) Sample cuvette
For accurate and precise reading, cuvette must be transparent, clean, devoid of any scratches.
The optical path of the cuvette is always 1 cm. Glass cuvettes are used for reading in the visible
light range while quartz or fused silica cuvettes are used for UV range.
5) Photosensitive detectors
These detectors contain a light-sensitive surface that releases electrons in number proportional to
the intensity of light on it, converting light energy into electrical energy. Different detectors used
are-
a) Barrier layer cells
b) Photosensitive tubes
c) Photomultiplier tubes
d) Photoconductive cells
6) Read out devices– The detector response can be measured by any of the following read out
devices-
a) Galvanometer
b) Ammeter
c) Recorder
d) Digital read out.
The signal may be transmitted to computer or print out device. Most modern instruments are of
direct reading type where the amplified detector signal operates a galvanometer.

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CHAPTER – 2
PHOTOMETRY

Introduction: Photometry is the science of light measurement. Light is measured in terms of its
perceived brightness to human eye. Photometry is different than other measurements of light
within the field of optics like radiometry, which is the science of measurement of
electromagnetic radiation including visible light. Radiant power is weighted by a luminosity
function in photometry. Also, luminous function models account for human sensitivity to
brightness.

1. FLAME PHOTOMETER:
Introduction
During 1980s Bowling Barnes, David Richardson, John Berry and Robert Hood developed an
instrument to measure the low concentrations of sodium and potassium in a solution. They
named this instrument as Flame photometer. The principle of flame photometer is based on the
measurement of the emitted light intensity when a metal is introduced into the flame. The
wavelength of the colour gives information about the element and the colour of the flame gives
information about the amount of the element present in the sample.
Flame photometry is one of the branches of atomic absorption spectroscopy. It is also known as
flame emission spectroscopy. Currently, it has become a necessary tool in the field of analytical
chemistry. Flame photometer can be used to determine the concentration of certain metal ions
like sodium, potassium, lithium, calcium and cesium etc. In flame photometer spectra the metal
ions are used in the form of atoms. The International Union of Pure and Applied Chemistry
(IUPAC) Committee on Spectroscopic Nomenclature has named this technique as flame atomic
emission spectrometry (FAES).
Principle of Flame photometer
The compounds of the alkali and alkaline earth metals (Group II) dissociate into atoms when
introduced into the flame. Some of these atoms further get excited to even higher levels. But
these atoms are not stable at higher levels.
Hence, these atoms emit radiations when returning back to the ground state. These radiations
generally lie in the visible region of the spectrum. Each of the alkali and alkaline earth metals has
a specific wavelength.
Element Emitted wavelength Flame color
Sodium 589 nm Yellow
Potassium 766 nm Violet
Barium 554 nm Lime green
Calcium 622 nm Orange
Lithium 670 nm Red

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For certain concentration ranges,
The intensity of the emission is directly proportional to the number of atoms returning to the
ground state. And the light emitted is in turn proportional to the concentration of the sample.

Parts of flame photometer

A simple flame photometer consists of the following basic components:
Source of flame: A Burner in the flame photometer is the source of flame. It can be maintained
in at a constant temperature. The temperature of the flame is one of the critical factors in flame
photometry.
Fuel-Oxidant mixture Temperature (°C)
Natural gas-Air 1700
Propane-Air 1800
Hydrogen-Air 2000
Hydrogen-Oxygen 2650
Acetylene-Air 2300
Acetylene-Oxyen 3200
Acetylene-Nitrous oxide 2700
Cyanogen-Oxygen 4800
Nebuliser: Nebuliser is used to send homogeneous solution into the flame at a balanced rate.
Optical system: The optical system consists of convex mirror and convex lens. The convex
mirror transmits the light emitted from the atoms. Convex mirror also helps to focus the
emissions to the lens. The lens helps to focus the light on a point or slit.
Simple colour filters: The reflections from the mirror pass through the slit and reach the filters.
Filters will isolate the wavelength to be measured from that of irrelevant emissions.
Photo-detector: The intensity of radiation emitted by the flame is measured by photo detector.
Here the emitted radiation is converted to an electrical signal with the help of photo detector.
These electrical signals are directly proportional to the intensity of light.

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Working procedure
 Both the standard stock solution and sample solution are prepared in fresh distilled water.
 The flame of the photometer is calibrated by adjusting the air and gas. Then the flame is
allowed to stabilize for about 5 min.
 Now the instrument is switched on and the lids of the filter chamber are opened to insert
appropriate colour filters.
 The readings of the galvanometer are adjusted to zero by spraying distilled water into the
flame.
 The sensitivity is adjusted by spraying the most concentrated standard working solution
into the flame. Now the full scale deflection of the galvanometer is recorded.
 Again distilled water is sprayed into the flame to attain constant readings of
galvanometer. Then the galvanometer is readjusted to zero.
 Now each of the standard working solutions is sprayed into the flame for three times and
the readings of galvanometer are recorded. After each spray, the apparatus must be
thoroughly washed.
 Finally sample solution is sprayed into the flame for three times and the readings of
galvanometer are recorded. After each spray, the apparatus must be thoroughly washed.

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 Calculate the mean of the galvanometer reading.
 Plot the graph of concentration against the galvanometer reading to find out the
concentration of the element in the sample.

 The solvent is first aspirated to obtain fine solid particles.

 These molecules in the solid particles are moved towards the flame to produce gaseous
atoms and ions.
 These ions absorb the energy from the flame get excited to high energy levels from the
ground state.
 But as these ions are unstable, they return back to ground state. While returning they emit
characteristic radiation.
 The intensity of emitted light is proportional to the concentration of the element.

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The oxidants in flame photometer are mainly air, oxygen or nitrous oxide. The temperature of
the flame depends on the ratio of fuel and oxidant.
The processes occurring during flame photometer analysis are summarized below:
Desolvation: Desolvation involves drying a sample in a solution. The metal particles in the
solvent are dehydrated by the flame and thus solvent is evaporated.
Vaporization: The metal particles in the sample are also dehydrated. This also led to the
evaporation of the solvent.
Atomization: Atomization is the separation of all atoms in a chemical substance. The metal ions
in the sample are reduced to metal atoms by the flame.
Excitation: The electrostatic force of attraction between the electrons and nucleus of the atom
helps them to absorb a particular amount of energy. The atoms then jump to the higher energy
state when excited.
Emission: Since the higher energy state is unstable the atoms jump back to the ground state or
low energy state to gain stability. This jumping of atoms emits radiation with characteristic
wavelength. The radiation is measured by the photo detector.

Scheibe-Lomakin equation
Scheibe-Lomakin equation describes intensity of light emitted with the help of following
formula:
I = k × cn
Where:
I = Intensity of emitted light
c = Concentration of the element
k = Proportionality constant
At the linear part of the calibration curve n~1,
then I = k × c. In other words, the intensity of emitted light is directly related to the concentration
of the sample.

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Applications of flame photometer

1. Flame photometer can be applied both for quantitative and qualitative analysis of
elements. The radiations emitted by the flame photometer are characteristic to particular
metal. Hence with the help of Flame photometer we can detect the presence of any
specific element in the given sample.
2. The presence of some group II elements is critical for soil health. We can determine the
presence of various alkali and alkaline earth metals in soil sample by conducting flame
test and then the soil can be supplied with specific fertiliser.
3. The concentrations of Na+ and K+ ions are very important in the human body for
conducting various metabolic functions. Their concentrations can be determined by
diluting and aspirating blood serum sample into the flame.
4. Soft drinks, fruit juices and alcoholic beverages can also be analysed by using flame
photometry to determine the concentrations of various metals and elements.

Advantages of flame photometer

1. The method of analysis is very simple and economical.

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2. It is quick, convenient, selective and sensitive analysis.
3. It is both and qualitative and quantitative in nature.
4. Even very low concentrations (parts per million/ppm to parts per billion/ppb range) of
metals in the sample can be determined.
5. This method compensates for any unexpected interfering material present in the sample
solution.
6. This method can be used to estimate elements which are rarely analysed.

Disadvantages of flame photometer

1. In spite of many advantages, this analysis technique has quite a few disadvantages:
2. The accurate concentration of the metal ion in the solution cannot be measured.
3. It cannot directly detect and determine the presence of inert gases.
4. Though this technique measures the total metal content present in the sample, it does not
provide the information about the molecular structure of the metal present in the sample.
5. Only liquid samples may be used. Also sample preparation becomes lengthy in some
cases.
6. Flame photometry cannot be used for the direct determination of each and every metal
atom. A number of metal atoms cannot be analysed by this method. The elements such as
carbon, hydrogen and halides cannot be detected due to their non-radiating nature.

2. ATOMIC ABSORPTION SPECTROSCOPY:

Principle of AAS
AAS quantitatively measures the concentrations of elements present in a liquid sample. It utilises
the principle that elements in the gas phase absorb light at very specific wavelengths which gives
the technique excellent specificity and detection limits.
The sample may be an aqueous or organic solution, indeed it may even be solid provided it can
be dissolved successfully. The liquid is drawn in to a flame where it is ionised in the gas phase.
Light of a specific wavelength appropriate to the element being analysed is shone through the
flame, the absorption is proportional to the concentration of the element. Quantification is
achieved by preparing standards of the element.
AAS Instrumentation:
The Atomic absorption spectroscopy has simple instrumentation. But, unlike other spectroscopy
methods, it has two additional requirements. These include a specially designed lamp to produce
light of a desired wavelength and a burner to prepare the sample for absorption of light radiation.
Additionally, the instrument also sprays the sample in the solution state over an atomizer
(burner). This leads to evaporation of the solvent and leaves a fine dry residue. This residue has
neutral atoms in the ground state.
The Instrumentation includes:
1. The atomizer (burner) to dry the sample and produce atoms.

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2. Sample container.
3. Fuel and oxidant to burn the sample by heat.
4. Hollow cathode lamp to produce light of the desired wavelength.
5. Detector to detect the absorption intensity.
6. Amplifier and data recorder.
The instrument is available as single and double beam instruments.
Light source: The light source should produce a narrow spectrum with little background noise.
Besides the light should be stable and have sufficient intensity.
Two types of light sources can be used based on the requirement.
1. Hollow cathode lamp: This is most widely used as a light source. Inside the lamp, the cathode
is coated with a metal of analyte to be analyzed. For instance, if magnesium is to be analyzed
from the sample, a cathode coated with magnesium is used.
Similarly, for all the other elements like Na, Ca, K, Zn, etc. analysis respective metal coated
cathodes are used in the lamp. The lamp is filled with an inert gas like argon or neon which is
ionized by an electric arc. The ions get attracted toward cathodes and strike it leading to
excitation of metal ions. This leads to the emission of radiation with a characteristic wavelength
of analyte metal.
The advantages of Hollow cathode lamp is that it provides radiation with a bandwidth of 0.001 to
0.01nm. Use of other methods like monochromators gives radiation with a bandwidth of 1nm.
So, these lamps give highly specific radiation.
The disadvantage of this hollow cathode lamp is that for every metal different cathode lamp has
to be employed.
2. Electrode-less discharge lamps: These lamps are less conventional in regular use but are
essential of determination of Arsenic and selenium. A bulb containing an element of interest
(with argon gas) is present in the lamp. This element is excited using microwave energy or radio
frequency energy
Sample container: This is a beaker-like a container of the sample which is placed below the
burner preferably. A capillary tube drains the sample to the tip of the burner.
The burner (atomizer): Here the sample from the capillary rises to the tip of the burner. Here it
is burned with the flame. This flame is produced by a fuel and oxidant combination. The sample
after evaporation leaves a fine residue of neutral atoms.
Fuel and oxidant: This is a very important part of the entire process to be remembered. If the
heat produced is not sufficient then the sample doesn’t form neutral atoms. If the heat of the
burner is more, the sample molecules may ionize instead of forming atoms. So both are
undesirable for experimentation. Hence a proper combination of fuels and oxidant are to be used
to produce recommended temperatures. Commonly used flues include propane, Hydrogen, and
acetylene and oxidants are mostly air or oxygen.
Fuel combinations chart:
Fuel combinations Flame Metals Analyzed

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temperature
Acetylene + Air 2550 degrees For most samples
Acetylene + Nitrous Aluminum (Al), Molybdenum (Mo), Silicon (Si), Titanium
2900 degrees
oxide (Ti)
Hydrogen + Air 2200 degrees Lead (Pb), Tin (Sn)
Monochromator: As discussed before, elements have specific absorption line. But some
elements also have secondary absorption lines. Further, there is also emission from the lamp and
the flame. Hence, we need to isolate the desired spectral line for the measurement of absorption.
To achieve this a monochromator which can filter and provide a resolution of <1nm is employed.
Detector: This part of the instrument detects the intensity of radiation absorbed by the elements.
The detector consists of a photomultiplier tube or simple photocell. The current or potential
recorded for the sample absorption is recorded in computer software and then analyzed.
Read device: This can be a display computer. It displays the absorbance at a specific
wavelength.
Atomic absorption spectroscopy procedure:
1. Based on the metal of analysis a suitable cathode lamp is selected.
2. The sample is dissolved in a polar solvent is placed in the container.
3. With the help of fuel and oxidant in the presence of a mixer, the sample solution is sprayed on
to the flame.
4. The neutral atoms in the flame absorb light radiation from the cathode lamp. The unabsorbed
radiation is recorded by the detector.
Atomic Absorption Spectroscopy applications:
1. Atomic spectroscopy is used for quantitative analysis of metal elements in water, soil, plant
material and ceramics.
2. In health care, it is used to analyze ionic metal elements in blood, saliva, urine samples. The
elements analyzed routinely include sodium, potassium, magnesium, calcium and zinc.
3. To determine heavy metals like iron, manganese, copper, zinc, mercury, lead, nickel, and in
urine and blood.
This analysis is essential in case of heavy metal poisoning. Since heavy metal poisoning is
mostly lethal a regular monitoring of poison levels in the patient blood are essential.
4. To determine metal elements like copper, nickel and zinc in the food industry.
5. To estimate Lead in petroleum products.
6. To determine metal concentrations in groundwater and bore well samplings before using for
drinking and irrigation.

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Chapter - 4
CHROMATOGRAPHY

The word Chromatography is derived from two Greek words –Chroma means – color and
graphein to write. Chromatography is the collective term for a family of laboratory techniques
for the separation of mixtures.
Chromatography, literally “color writing”, was used—and named— in the first decade of the
20th century, primarily for the separation of plant pigments such as chlorophyll.
Chromatography is a misnomer since it is no longer limited to the separation of the colored
substances.
Principle- Chromatography is based on the principle of the partition of the solute between two
phases/solvents. It usually consists of a mobile phase and a stationary phase. The mobile phase
usually refers to the mixture of the substances to be separated dissolved in a liquid or a gas. The
stationary phase is a porous solid matrix through which the sample contained in the mobile phase
percolates. The interaction between the mobile and the stationary phases result in the separation
of the compounds from the mixture. These interactions include the physico chemical principles
such as the adsorption, ion- exchange, molecular sieving and affinity
Classification-There are two ways to classify the methodology of chromatography-
A) Based on nature of interactions between the sample components and the stationary
phase,whereby the components are retarded to different degrees in their migration with the
mobile phase and are consequently separated from each other- Following are the different types
of chromatographic procedures-
1) Partition chromatography
2) Adsorption chromatography
3) Ion – Exchange chromatography
4) Gel filtration chromatography
5) Affinity chromatography
6) High performance liquid chromatography
B) Base on nature of stationary phase or mobile phase
It is of two types
1) Planar- It may be Paper or Thin layer
2) Column- it may be Gas or Liquid
Planar chromatography–
 Planar chromatography is a separation technique in which the stationary phase is
present as or on a plane.
 The plane can be a paper, serving as such or impregnated by a substance as the stationary
bed (paper chromatography) or a layer of solid particles spread on a support such as a
glass plate (thin layer chromatography).
 Different compounds in the sample mixture travel different distances according to how
strongly they interact with the stationary phase as compared to the mobile phase.
 The specific Retardation factor (Rf) of each chemical can be used to aid in the
identification of an unknown substance.
Column Chromatography

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 Column chromatography is a separation technique in which the stationary bed is within a
tube.
 The particles of the solid stationary phase or the support coated with a liquid stationary
phase may fill the whole inside volume of the tube (packed column) or be concentrated
on or along the inside tube wall leaving an open, unrestricted path for the mobile phase in
the middle part of the tube (open tubular column).
 Differences in rates of movement through the medium are calculated to different
retention times of the sample.
1. Partition Chromatography
This is more commonly used for the separation of mixture of amino acids and peptides. The
molecules of a mixture get partitioned between the stationary and the mobile phase depending on
the relative affinity of each one of the phases.
a) Paper chromatography- Paper chromatography is an analytical technique for separating
and identifying mixtures that are or can be colored, especially pigments. This method has been
largely replaced by thin layer chromatography, however it is still a powerful technique. It is a
liquid- liquid partition chromatography. The stationary phase is water held on a solid support of
filter paper (cellulose). The mobile phase is a mixture of immiscible solvents which are mixtures
of water, a non polar solvent and an acid or base e.g. Butanol, acetic acid water or phenol-water-
ammonia.
Technique(Figure-1)
 A small concentrated spot of solution that contains the sample of the solute is applied to a
strip of chromatography paper about two centimeters away from the base of the plate.
 This sample is absorbed onto the paper and may form interactions with it.
 Any substance that reacts or bonds with the paper cannot be measured using this
technique.
 The paper is then dipped in to a suitable solvent, such as ethanol or water, taking care that
the spot is above the surface of the solvent, and placed in a sealed container.
 The solvent moves up the paper by capillary action, which occurs as a result of the
attraction of the solvent molecules to the paper. (Figure-1)
 As the solvent rises through the paper it meets and dissolves the sample mixture, which
will then travel up the paper with the solvent solute sample.
 Different compounds in the sample mixture travel at different rates due to differences in
solubility in the solvent, and due to differences in their attraction to the fibers in the
paper.
 Paper chromatography takes anywhere from several minutes to several hours.
 In some cases, paper chromatography does not separate pigments completely; this occurs
when two substances appear to have the same values in a particular solvent.
 In these cases, two-way chromatography is used to separate the multiple-pigment spots.
Ascending Chromatography
In this method, the solvent is in pool at the bottom of the vessel in which the paper is supported.
It rises up the paper by capillary action against the force of gravity (Figure-1).
Descending Chromatography
In this method, the solvent is kept in a trough at the top of the chamber and is allowed to flow
down the paper. The liquid moves down by capillary action as well as by the gravitational force.

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In this case, the flow is more rapid as compared to the ascending method. Because of this rapid
speed, the chromatography is completed in a comparatively shorter time.
Analysis– After development, the spots corresponding to different compounds may be located
by their color, ultraviolet light, ninhydrin or by treatment with iodine vapors. The paper
remaining after the experiment is known as the Chromatogram.
Composition of Filter Paper
The original work in paper chromatography was carried on whatman no.1 filter paper. These
days paper for chromatography is made from cotton cellulose.
Rƒ value
The components which have been separated differ in their retention factor i.e Ratio of distance
traveled from the spot or origin by the solute component to that of the distance traveled from the
spot or origin by the solvent. Retention Factor can never be greater than one.
Rf, – Distance traveled by sample/ distance traveled by solvent.
The final chromatogram can be compared with other known mixture chromatograms to identify
sample mixes using the Rf value in an experiment. The retention values found can be compared
to known values, and from that conclusions can be drawn.
Rƒ values are usually expressed as a fraction of two decimal places.
If Rƒ value of a solution is zero, the solute remains in the stationary phase and thus it is
immobile. If Rƒ value = 1 then the solute has no affinity for the stationary phase and travels with
the solvent front.
Two dimensionalchromatography– Sometimes, it is difficult to separate a complex mixture of
substances by a single run with one solvent system. In such a case, a second run is carried out by
a different solvent system, in a direction perpendicular to the first run. This is referred to as-
two dimensional chromatography which enhances the separation of a mixture in to individual
components. (Figure-3)

Importance-
1) Paper chromatography is a very easy, simple, rapid and highly efficient method of separation.
2) It can be applied even in microgram quantities of the sample.
3)It can also be used for the separation of a wide variety of materials like amino acids,
oligopeptides, sugars, oligosaccharides, glycosides, purines and pyrimidines, steroids, vitamins
and some alkaloids like penicillin, tetracyclin and streptomycin.
4) It is not preferred for separating proteins because they are not soluble in many of the solvent
systems and are also denatured by them. Paper chromatography is inferior to thin layer
chromatography in resolving power.
b) Thin layer chromatography (TLC) is a chromatographic technique used to separate
mixtures. It involves a stationary phase consisting of a thin layer of adsorbent material, usually
silica gel, aluminum oxide, or cellulose immobilized onto a flat, inert carrier sheet. A liquid
phase consisting of the solution to be separated is then dissolved in an appropriate solvent and is
drawn up the plate via capillary action, separating the experimental solution based on the polarity
of the components of the compound in question. .
Importance of TLC– Its wide range of uses include
 determination of the pigments a plant contains
 detection of pesticides or insecticides in food
 identifying compounds present in a given substance

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 monitoring organic reaction
Technique
The process is similar to paper chromatography with the advantage of faster runs, better
separations, and the choice between different stationary phases. Because of its simplicity and
speed TLC is often used for monitoring chemical reactions and for the qualitative analysis of
reaction products.
A small spot of solution containing the sample is applied to a plate, about one centimeter from
the base. The plate is then dipped in to a suitable solvent, such as hexane or ethyl acetate, and
placed in a sealed container. The solvent moves up the plate by capillary action and meets the
sample mixture, which is dissolved and is carried up the plate by the solvent. Different
compounds in the sample mixture travel at different rates due to the differences in their attraction
to the stationary phase, and because of differences in solubility in the solvent.(Figure-4).
Separation of compounds is based on the competition of the solute and the mobile phase for
binding places on the stationary phase. For instance, if normal phase silica gel is used as the
stationary phase it can be considered polar. Given two compounds which differ in polarity, the
most polar compound has a stronger interaction with the silica and is therefore more capable to
dispel the mobile phase from the binding places. Consequently, the less polar compound moves
higher up the plate (resulting in a higher Rf value).

Analysis
As the chemicals being separated may be colorless, several methods exist to visualize the spots:
 Often a small amount of a fluorescent compound, usually manganese-activated zinc
silicate, is added to the adsorbent that allows the visualization of spots under a blacklight
(UV254)..
 Iodine vapors are a general unspecific color reagent
 Ninhydrin is used for amino acids and proteins
 Sulphuric acid is used for phospholipids
Once visible, the Rf value , or Retention factor, of each spot can be determined .These values
depend on the solvent used, and the type of TLC plate, and are not physical constants. The Rf
value for compounds (amino acids, peptides ,sugars, fatty acids, phospholipids etc.) for
commonly used solvent systems have been calculated and available for comparison.TLC can
also be used for two dimensional chromatography using the same plate with two solvent
systems, as in the case of paper chromatography.
Advantages of TLC over paper chromatography-
1) In case of paper chromatography , it takes 14-16 hrs for the separation of the components, but
in TLC, It takes only 3-4 hrs.
2) TLC has the advantage that the corrosive reagents like sulphuric acid can also be used which
pose a limitation for the paper chromatography.
3) It is easier to separate and visualize the components by this method.
4) It has the capacity to analyze multiple samples in a single run.
5) It is relatively a low cost.
2)Adsorption chromatography
 In this technique the separation is based on differences in adsorption at the surface of the
solid stationary medium.

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 The adsorbents such as silica gel, charcoal powder and calcium hydroxyapatite are
packed in to a column in a glass tube.
 This serves as the stationary phase. The sample mixture in a solvent is loaded on this
column.
 The individual components get differentially adsorbed on to the adsorbent (Figure-5).
 The elution is carried out by a buffer system (mobile phase).
 The most weakly held fraction moves fastest, followed by others, according to the order
of tightness in adsorption.
 The individual compounds come out of the column at different rates which may be
separately collected and identified

3)Ion – Exchange chromatography

 Ion-exchange chromatography (or ion chromatography) is a process that allows the
separation of ions and polar molecules based on the charge properties of the molecules
(Figure-6).
 It can be used for almost any kind of charged molecule including large proteins, small
nucleotides and amino acids.
 The solution to be injected is usually called a sample, and the individually separated
components are called analytes. It is often used in protein purification, water analysis,
and quality control.
 Ion exchange chromatography retains analyte molecules based on coulombic (ionic)
interactions.
 The stationary phase surface displays ionic functional groups that interact with analyte
ions of opposite charge. This type of chromatography is further subdivided into cation
exchange chromatography and anion exchange chromatography. The ionic compound
consisting of the cationic species and the anionic species can be retained by the
stationary phase.

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Figure-6 -showing ion exchange chromatography

Cation exchange chromatography retains positively charged cations because the stationary phase
displays a negatively charged functional group.
Cation Exchange resins- Polysterene sulfonate resins, CM- Sephadex gel, CM – cellulose,
These bear acidic groups and immobilize cations from adjacent solutions.
Anion exchange chromatography retains anions using positively charged functional group.
Anion exchangers- DEAE cellulose, Trimethyl amino polysterene, DEAE- sephadex. All these
bear basic groups ionizing into fixed positions and immobilize anions from neighboring
solutions.
The ionic groups in cation exchange resins are sulphonic and carboxyl groups, while the anion
exchange resins have a quaternary nitrogen.
4) Gel filtration chromatography or Molecular Sieve chromatography-
 This is extremely useful in separating ribosomes, viruses, nucleic acids and proteins
depending on their particle sizes and shapes.
 In Gel filtration chromatography, the separation of the particles is based on their size,
shape and molecular weight.
 This technique is also referred to as molecular exclusion chromatography.
 The apparatus consists of a column packed with sponge like gel beads( usually cross-
linked polysaccharides) containing pores.
 The gels serve as molecular sieves for the separation of smaller and bigger molecules
 The solution mixture containing molecules of different sizes( say protein) is applied to
the column and eluted with a buffer. (Figure-7).
 The larger molecules cannot pass through the pores of a gel and therefore move faster.

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 On the other hand, the smaller molecules enter the gel beads and are left behind which
come out slowly.
 By selecting the gel beads of different porosity, the molecules can be separated.
 The commercially available gels include (G-10, G-25,G-100), Bio gel(P-10,P-30,P-100)
and Sepharose(6B,4B,2B).
 The gel filtration chromatography can be used for an approximate determination of
molecular weights.
 This is done by using a calibrated column with substances of known molecular weights.

Figure-7- showing mechanism of molecular sieve chromatography.

5) Affinity chromatography
 The principle of affinity chromatography is based on the property of specific and non-
covalent binding of proteins to other molecules, referred to as substrates or cofactors.
 The technique involves the use of ligands covalently attached to an inert and porous
matrix in a column.
 The immobilized ligands act as moleculal hooks to selectively pick up the desired protein
while the remaining proteins pass through the column.
 The desired protein captured by the ligand, can be eluted by using free ligand molecules.
 Alternatively, some reagents that can break protein ligand interactions can also be
employed for the separation (Figure-8)
 Affinity chromatography is useful for the purification of enzymes, vitamins, nucleic
acids, drugs , hormone receptors, antibodies etc.
 For example NAD is used to purify dehydrogenases. By using antibodies, antigens can be
easily separated.
 Conversely, antibodies can be purified by passing through a column containing the
antigen.
 It is also widely used for the estimation of glycated Hb. Normal Hb does not bind and
comes out first, while glycated Hb binds with the Boronic acid used as a ligand. Sorbitol
is then added to elute the glycated Hb which can be quantitated then.

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Figure-8- Showing the mechanism of Affinity chromatography

6) High performance liquid chromatography
 In general chromatographic techniques are slow and time consuming.
 The separation can be greatly improved by applying high pressure in the range of 5000-
10,000 pounds per square inch, hence this technique is also referred to as high pressure
liquid chromatography.
 HPLC requires the use of non compressible resin materials and strong metal columns.
 The eluents of the columns are detected by methods such as UVabsorption and
fluorescence.
 It can be applied in the form of partition, adsorption, ion exchange or molecular sieve
chromatography .
 The stationary phase consists of an immobilized thin layer of a liquid on the micro glass
or plastic beads, tightly packed in to a narrow column. (Figure-9)
 The mobile phase consists of a buffered solvent system which is passed under high
pressure through the column for eluting the solutes of the sample.

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Figure-9- Showing the apparatus for high performance liquid chromatography

Due to rapidity in action it is used for assaying amino acids, peptides, proteins, carbohydrates,
lipids, nucleic acids and related compounds, vitamins, hormones, metabolites and drugs such as
antiarrytmics, antibiotics, antiepileptics,analgesics, bronchial smooth muscle relaxants and anti-
depressants.
6) Gas liquid chromatography-
 This is the method of choice for the separation of volatile substances or volatile
derivatives of certain in volatile substances.
 In GLC. the stationary phase is an inert solid material(diatomaceous earth or powdered
firebrick), impregnated with a non volatile liquid(silicon or polyethylene glycol).
 This is packed in a narrow column and maintained at high temperature (around 200
degree C).
 A mixture of volatile material is injected in to the column along with the mobile phase,
which is an inert gas (argon, helium or nitrogen).
 The separation of the volatile material is based on the partition of the components
between the mobile phase (gas) and stationary phase( liquid), hence the name gas liquid
chromatography. (Figure-10)
 The separated compounds can be identified and quantitated by a detector. Gas liquid
chromatography is sensitive, rapid and reliable.
 It is frequently used for the quantitative estimations of biological materials such as
lipids, drugs and vitamins.

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showing apparatus for gas liquid chromatography.

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Chapter-5
ELECTROPHORESIS

Electrophoresis: is a technique used to separate and sometimes purify macromolecules -

especially proteins and nucleic acids - that differ in size, charge or conformation. As such, it is
one of the most widely-used techniques in biochemistry and molecular biology.

Electrophoresis is defined as the migration of charged ions in an electric field. In metal

conductors, electric current is carried by the movement of electrons, largely along the surface of
the metal. In solutions, the electric current flows between electrodes and is carried by ions. The
ions that migrate towards the anode, because of their anodic migration, are called “anions”. The
ions which will migrate to the cathode are called “cations”.

Principle: When charged molecules are placed in an electric field, they migrate toward either the
positive or negative pole according to their charge. In contrast to proteins, which can have either
a net positive or net negative charge, nucleic acids have a consistent negative charge imparted by
their phosphate backbone, and migrate toward the anode.

An ion placed in such an electric field will experience a force:

Where,
F = electrophoretic force
K = a constant
q = net charge on the protein (atomic charges/protein molecule)

This force will cause the protein to accelerate towards either the cathode or the anode, depending
on the sign of its charge. Of course there are other forces such as frictional force when ions move
in the electric field. The influence of them cannot be understood easily by a formula, so we omit
it.
Electrophoresis exploits the fact that different ions have different mobility in an electric field and
so can be separated by this way.

Proteins and nucleic acids are electrophoresed within a matrix or "gel". Most commonly,
the gel is cast in the shape of a thin slab, with wells for loading the sample. The gel is immersed
within an electrophoresis buffer that provides ions to carry a current and some type of buffer to
maintain the pH at a relatively constant value.

The gel itself is composed of either agarose or polyacrylamide, each of which has attributes
suitable to particular tasks.

Factors influencing Electrophoresis:

Movement of proteins depends on various aspects. Within the gel the molecules must pass
through as they are moving from one pole to another. The smaller molecules can weave in and

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out of the matrix of the gel with more ease, compared with larger molecules. As a general rule,
the molecules move rapid if it has more net charge, has a shape of ball and shorter diameter

1) The buffer pH
It will influence the direction and rapid of the protein migration.
Movement of proteins depends on various aspects; one of them is the charges on the proteins.
Proteins are sequence of amino acids that can be ionized depend on their acid or basic character.
The protein’s net electric charge is the sum of the electric charges found on the surface of the
molecule as a function of the environment.
The rate of migration will depend on the strength of their net surface charges: The protein that
carries more +ve charges will move towards the cathode at a faster rate. On the contrary, the
protein that carries more -ve charges will move towards the anode at a faster rate. In this regard,
proteins can be separated based on their electric charges.
Depending on the pH of the buffer, proteins in a sample will carry different charges. At the pI
(isoelectric point) of a specific protein, the protein molecule carries no net charge and does not
migrate in an electric field. At pH above the pI, the protein has a net negative charge and
migrates towards the anode. At pH below the pI the protein obtains a net positive charge on its
surface and migrates towards the cathode.

2) The buffer ionic strength

It influences the proportion of the current carried by the proteins
At low ionic strength the proteins will carry a relatively large proportion of the current and so
will have a relatively fast migration. At high ionic strength, most of the current will be carried by
the buffer ions and so the proteins will migrate relatively slowly. An analogy might be useful in
visualizing this effect of ionic strength. Imagine a bank where there are two counters – one for
deposits the anode) and one for withdrawals (= the cathode), with electrons being the money.
The ions may be considered as customers waiting to be served at either counter, which one can
visualize as being at opposite ends of the banking hall.

In electrophoresis, therefore, a low ionic strength is preferred as it increases the rate of migration
of proteins. A low ionic strength is also preferred as it gives a lower heat generation. Assuming a
constant voltage, if the ionic strength is increased, the electrical resistance decreases but the
current will increase. A high ionic strength buffer will therefore lead to greater heat generation,
and so a low ionic strength is preferred.

3) The voltage gradient

the rate of migration will depend on the voltage gradient: There is more voltage gradient in the
electric field, protein will move towards the anode (or the cathode) at a faster rate.

4) Electo-osmosis
Liquid’s relative move upon solid medium in an electric field is called electo-osmosis. In applied
electric field, electo-osmosis distorts the sample stream and limits the separation. For example,
Paper electrophoresis has poor resolution because of electo-osmosis. The surface of paper has -e,
so the buffer has +e derived from hydrogen ions because of electrostatic induction.
Then +e drive buffer to cathode in electric field,these flows distort the electrophoretic migration

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of sample by causing a varying residence time. Thus, sample will move more or less than
normal.

Types of Electrophoresis

Electrophoresis encompasses several related analytical techniques. Examples include:

 affinity electrophoresis - Affinity electrophoresis is a type of electrophoresis in which

particles are separated based on complex formation or biospecific interaction
 capillary electrophoresis - Capillary electrophoresis is a type of electrophoresis used to
separate ions depending mainly on the atomic radius, charge, and viscosity. As the name
suggests, this technique is commonly performed in a glass tube. It yields quick results
and a high resolution separation.
 gel electrophoresis - Gel electrophoresis is a widely used type of electrophoresis in
which molecules are separated by movement through a porous gel under the influence of
an electrical field. The two main gel materials are agarose and polyacrylamide. Gel
electrophoresis is used to separate nucleic acids (DNA and RNA), nucleic acid fragments,
and proteins.
 immunoelectrophoresis - Immunoelectrophoresis is the general name given to a variety
of electrophoretic techniques used to characterize and separate proteins based on their
reaction to antibodies.
 electroblotting - Electroblotting is a technique used to recover nucleic acids or proteins
following electrophoresis by transferring them onto a membrane. The polymers
polyvinylidene fluoride (PVDF) or nitrocellulose are commonly used. Once the specimen
has been recovered, it can be further analyzed using stains or probes. A western blot is
one form of electroblotting used to detect specific proteins using artificial antibodies.
 pulsed-field gel electrophoresis - Pulsed-field electrophoresis is used to separate
macromolecules, such as DNA, by periodically changing the direction of the electric field
applied to a gel matrix. The reason the electric field is changed is because traditional gel
electrophoresis is unable to efficiently separate very large molecules that all tend to
migrate together. Changing the direction of the electric field gives the molecules
additional directions to travel, so they have a path through the gel. The voltage is
generally switched between three directions: one running along the axis of the gel and
two at 60 degrees to either side. Although the process takes longer than traditional gel
electrophoresis, it's better at separating large pieces of DNA.
 isoelectric focusing - Isoelectric focusing (IEF or electrofocusing) is a form of
electrophoresis that separates molecules based on different isoelectric points. IEF is most
often performed on proteins because their electrical charge depends on pH.

PAPER ELECTROPHORESIS

Paper Electrophoresis is one of the zone electrophoresis. This is very important method in all
laboratories. In this article let us learn the details of the paper chromatography with suitable
notes. I have given the info about this in Notes.

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Principle:

“The charge carried by a molecule depends on the pH of the medium. Electrophoresis at low
voltage is not usually to separate low molecular weight compounds because of diffusion, but it is
easier to illustrate the relationship between charge and pH with amino acids than with proteins
(or) other macromolecules”.

Filter paper:
Paper of good quality should contain at least 95% α-cellulose and should have only a very slight
adsorption
capacity.

Apparatus:

 The equipment required for electrophoresis consists basically of two items, a

POWER PACK and an ELECTROPHORETIC CELL.
 Power pack provides a stabilized direct current & has controls for both voltage &
current out put, which have an out put of 0 to 500V and 0 to 150mA are available.
 The Electrophoretic cell contains the electrodes, buffer reservoirs, a support for
paper and a supporting transparent insulating cover. The electrodes are usually made of
platinum.
 The two arrangements of the filter strips are commonly used. The horizontal &
vertical arrangements. Both the arrangements are equally viable & the choice usually
depends upon personal preferences.

Sample application:

The sample may be applied as a spot (about 0.5cm in diameter) or as a narrow unifor
m streak.

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Special devices are available commercially for this purpose. The sample can be applied b
efore the paper has been equilibrated with buffer (or) after it.

Procedure:

After the sample has been applied to the paper and the paper has been equilibrated with the
buffer.
The current is switched on. Commonly used buffers are,

The device providing stable voltage (or) current is available. Frequent observation is necessary to
run electrophoretic apparatus. Overheating can be avoided by placing the entire equipmen
t in the cold
room. The process does not take longer than two hours. After 2 hours switched off the power and
paper is
removed. Once removed, the paper is dried in hot oven at 1100C.

Detection & Quantitative assay:

To identify the unknown electrophorogram, compare the Unknown electogram with standa
rd
electrogram under standard conditions.Individual compounds are usually identified by phys
ical properties by the following methods.

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i) Fluorescence:

a) Staining with “Ethidium bromide” and subsequent

visualization of the electrophoreticgram under UV light makes DNA & RNA fluoresce and
thus facilitates their detection.

b) Flourescamine staining is utilized for detecting amino acids, amino acid derivatives,
peptides & proteins.

c) DANSYl chloride may be used in place of fluorescamine. ii) UV absorption:

Proteins, Peptides & nucleic acids absorb in the range of 260 to 280nm, this property these can
be detect.

iii)) Staining:

iv) Detection of Enzymes in situ:

 If the component to be separated is an enzyme. Special techniques may be used

to detect it.
 The paper strip, which have separated enzyme, is impregnated with the
substrate for the enzyme desired to be separated.
 The paper strip is now placed in a suitable buffer along with electrophoret
ogram. The color bands will appear which indicates the position of enzyme.

v) Quantitative estimation:

The color density of the zone may be multiplied with the area of the zone and the resulting
value would be a rough estimate of the concentration of the component.

Applications:

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 Serum analysis for diagnostic purpose is routinely carried about by paper electrophoresis.
 Muscle proteins, egg white proteins, milk proteins & snake, insect venom analysis done
by this technique.

GEL ELECTROPHORESIS

Principles of gel electrophoresis

The gel electrophoresis technique exploits the difference in size and charge of different
molecules in a sample. The DNA or protein sample to be separated is loaded on to a porous gel
placed in an ionic buffer medium. On application of electric charge, each molecule having
different size and charge will move through the gel at different speeds.

The porous gel used in this technique acts as a molecular sieve that separates bigger molecules
from the smaller ones. Smaller molecules move faster across the gel while the bulkier ones are
left behind. The mobility of the particles is also controlled by their individual electric charge.
Two oppositely charged electrodes that are part of the system pull molecules of towards them on
the basis of their charge.

How does it work?

The gel used in gel electrophoresis is usually made of a material called agarose, which is a
gelatinous substance extracted from seaweed. This porous gel could be used to separate
macromolecules of many different sizes. The gel is submerged in a salt buffer solution in an
electrophoresis chamber. Tris-borate-EDTA (TBE) is commonly used as the buffer. Its main
function is to control the pH of the system. The chamber has two electrodes – one positive and
another negative - at its two ends.

Samples that need to be analyzed are then loaded into tiny wells in the gel with the help of a
pipette. Once loading is complete, an electrical current of 50–150 V is applied. Now, charged
molecules present in the sample start migrating through the gel towards the electrodes.
Negatively charged molecules move towards the positive electrode and positively charged
molecules migrate towards the negative electrode.

The speed at which each molecule travels through the gel is called its electrophoretic mobility
and is determined mainly by its net charge and size. Strongly charged molecules move faster
than weakly charged ones. Smaller molecules run faster leaving behind the larger ones. Thus,
strong charge and small size increases a molecule’s electrophoretic mobility, while weak charge
and large size decreases the mobility of a molecule. When all molecules in a sample are of the
same size, the separation will solely be based on their size.

Once the separation is complete, the gel is stained with a dye to reveal the separation bands.
Ethidium bromide is a fluorescent dye commonly used in gel electrophoresis. The gel is soaked

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in a diluted ethidium bromide solution and then placed on a UV transilluminator to visualize the
separation bands.

The bands are immediately examined or photographed for future reference, as they will diffuse
into the gel over time. The dye can also be loaded into the gel well in advance to track the
migration of the molecules as it happens.

Applications of gel electrophoresis

Gel electrophoresis is widely used in the molecular biology and biochemistry labs in areas such
as forensic science, conservational biology, and medicine.

Some key applications of the technique are listed below:

 In the separation of DNA fragments for DNA fingerprinting to investigate crime scenes
 To analyze results of polymerase chain reaction
 To analyze genes associated with a particular illness
 In DNA profiling for taxonomy studies to distinguish different species
 In paternity testing using DNA fingerprinting
 In the study of structure and function of proteins
 In the analysis of antibiotic resistance
 In blotting techniques for analysis of macromolecules
 In the study of evolutionary relationships by analyzing genetic similarity among populations or
species

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