King Abdulaziz University
Faculty of Sciences
Department of Biochemistry
Girls Section
Analytical Biochemistry Lab
BIOC 343
Edited and Organized by a Collaborative Work with Lecturers
Mona Badahdah, Wedam Alghazzawi, and Sherin Bakhash
Table of Contents
Lab #
1
Experiment
Page #
Separation methods
3
Separation of amino acids by two-dimensional chromatography by Thin
Layer Chromatography (TLC) - Part I
11
The separation of amino acids by two-dimensional chromatography by Thin Layer
Chromatography (TLC) - Part II
11
2
Separation and identification of carotenoids pigments in tomatoes and carrots by column
chromatography
14
Part I: Dehydration and Extraction
3
Separation and identification of carotenoids pigments in tomatoes and carrots by column
chromatography:
Part II: Column chromatography
Part III: TLC analysis of carotenoids
16
21
Separation and identification of carotenoids pigments in tomatoes and carrots by column
Chromatography:
Part IV: UV/Vis spectroscopy
21
Determination of wavelength of maximum absorbance
26
Separation of a mixture of blue dextran and cobalt chloride molecules
30
A- Separation of a mixture of starch and maltose
34
B- Separation of a mixture of Starch and Glucose
39
7
Determination of the isoelectric point of glycine
40
8
Effect of salt on extractability of proteins
45
9
Chemical solutions
48
10
Gel electrophoresis of serum proteins.
55
11
Enzyme linked Immuno Sorbent Assay- ELISA
58
4
5
6
2
Principle:
Analytical biochemistry involves the use of laboratory techniques to determine the
composition of biological samples. It includes a qualitative analysis or a quantitative analysis.
A qualitative analysis indicates what is present in the sample. Chromatographic and
electrophoretic methods are an example of such analysis. A quantitative analysis indicates the
amount of a particular substances present in the sample such as spectrophotometric and
titrimetric methods.
The separation of molecules from biological materials is an important part of biochemical
work and often involves the isolation of one molecular species from a mixture of compounds
with very similar properties. Because of this, the usual methods of organic chemistry are
inadequate, and to be able to choose a suitable analytical technique it is essential to know
something about the chemical and the physical properties of the test substances. Generally,
the use of extreme pH, temperature, organic solvents, and oxidizing and reducing agents
should be avoided when dealing with molecules separated from living matter. These factors
lead to loss of the biological activity through denaturation.
In this lab manual, the experimental techniques employ mild conditions and utilize
differences in the basic physical properties of the molecules such as their size, shape, mass,
charge, solubility, and adsorption properties. The techniques involve different degrees of
interaction between three components:
1) Solute (sample to be separated from mixture).
2) Mobile phase (solvent) is the phase that moves through or over stationary phase carrying
the sample with it or it is a carrier sample.
3) Stationary phase (solid phase) - support material.
3
The basis of separation methods in biochemistry involving three components
Solvent
Solid phase
Usually contain a paper,
thin layer, column, gel,
or membrane.
Solute
(Molecules to be separated)
Table 1: common separation methods
Technique
Dialysis
Gel-filtration
Adsorption chromatography
Partition chromatography
Ion exchange chromatography
Polyacrylamide-gel
electrophoresis
Centrifugation
Solute properties
Solid phase
Solvent
Size and shape
Semi-permeable membrane Water
Size and shape
Hydrated gel
Usually aqueous
Absorbent usually inorganic
Adsorption
Non-polar
material
Mixture of polar and
Solubility
Inert support
non-polar solvents
Matrix containing ionized
Ionization
Aqueous buffer
groups
Charge and size
Inert support with pores
Size and shape
NB: Being in the laboratory you must:
1. Wear safety glasses and protective gloves.
2. Clean the bench and dispose of the waste chemicals.
3. Submit a lab report the following week to report your findings.
4
Aqueous buffer
Introduction
Chromatography, Color writing. "Chroma" is a Greek roots prefix for color and "graphy" is a
Greek roots suffix for writing. It is a separation technique based on the different interactions
of the compounds with the two phases, a stationary phase and a mobile phase, as the
compounds travel through the supporting medium. It is used to analyze, identify, purify and
quantify the compounds.




Mixture
Purify
Identify
Quantify
Analyze
Components
Chromatography is the physical separation of a mixture into its individual components on the
basis of their charge, size, shape, and their solubility. The mixture is separated by passing it
over the stationary phase (an insoluble material) to which the substances stick to varying
degrees. Substances that stick tightly to the stationary phase move very slowly, while those
that stick loosely or do not stick at all move rapidly. The solvent carrying the solutes over the
stationary phase is called the mobile phase.
Chromatography can be an analytical method, in which the investigator learns the number
and nature of the components in a very small amount of a mixture, but does not actually
isolate them. A common analytical method is silica-gel thin-layer chromatography. Or it
can be a preparative method, in which the investigator uses a large quantity of the mixture to
obtain useable amounts of each component. A common preparative method involving the
same phases is silica-gel column chromatography.
All chromatographic systems need
 Stationary phase (a solid or a liquid supported on a solid)
 Mobile phase (a liquid or a gas)
 Sample molecules (mixture to be separated)
5
Choice of Solvent
It is depend on the mixture investigated. If the compounds move close to the solvent front in
solvent A then they are too soluble, while if they are crowded around the origin in solvent B
then they are not sufficiently soluble. Therefore, a suitable solvent would be an appropriate
mixture of both solvent A & solvent B, so that the Rf values of the components of the
mixture are spread across the length of the paper.
The most common solvents are hydrocarbons and arranged in order of decreasing in their
polarity as the following:
Water > Methanol > Ethanol (very polar)> Propanol > Acetone (moderately polar) > Ethyl
Acetate > Diethyl Ether > Chloroform > Dichloromethane > Benzene > Carbon Tetrachloride
> Cyclohexane > Hexane (very non-polar) > Petroleum ether.
The pH may also be important in a particular separation because many solvents contain acetic
acid or ammonia to create a strongly acidic or basic environment.
Applications of chromatography

In pharmaceutical industry (penicillin and other antibiotics).

In separation of proteins into amino acids.

In crime scene investigation for DNA and RNA sequencing.

In many scientific studies to identify unknown organic and inorganic compounds.

In food and vegetables contained tiny amounts of pesticides and herbicides to check
dyes.
Types of Chromatography
There are many forms of chromatography, but all forms work on the same principle:
1. Partition Chromatography
2. Adsorption Chromatography
3. Gel Chromatography
4. Ion Exchange Chromatography uses a charged stationary phase to separate charged
compounds including amino acids, peptides, and proteins. The stationary phase is an
ion exchange resin that carries charged functional groups which interact with
oppositely charged groups of the compound to be retained.
6
Theory of partition chromatography
Partition chromatography is the distribution of similar solutes between two immiscible liquid
phases. The solute will distribute itself between the two phases according to its solubility in
each phase, this is called partitioning. It is mainly used for separation of molecules of small
molecular weight. In partition chromatography, one solvent usually water molecules bound as
a film on filter paper (inert support). Partitioning occurs between the bound water which is
the stationary phase and the solvent which is the mobile phase.
Paper chromatography
Paper chromatography is an analytical technique for separating and identifying both colored
(e.g. pigments) and colorless (e.g. amino acids) mixtures.
In paper chromatography, a filter paper is made of purified cellulose, which is a poly-hydroxy
compound of high molecular weight, makes an ideal support medium (the stationary phase)
where water is absorbed and retained strongly to the cellulose fibers and forms the stationary
hydrophilic phase. You can think of paper as being cellulose fibers with a very thin layer of
water molecules bound to the surface. The stationary phase is more polar than the mobile
organic phase.
Cellulose: a polymer of glucose has - OH groups.
7
Development
A. Ascending paper chromatography
Solvent running up the paper or TLC by capillary action. It is most employed
and has the advantage that separation can be carried out in two dimensions.
B. Descending paper chromatography
Solvent
In descending paper chromatography, the chromatogram is held vertically, and
the spot of the sample is drooped on the top of the chromatogram.
Solvent drips off the bottom of the paper by gravity. It is convenient for
compound which has similar Rf values. Descending chromatography is faster
because gravity helps the solvent flow but it’s difficult to set the apparatus.
Detection of spots
Most biological compounds are colorless and can be visualized on the "chromatogram" by:
1. Spraying the paper by specific reagents, as Ninhydrin.
2. Dipping in a solution of the reagent in a volatile solvent, as Iodine vapors.
3. Fluorescence compounds can be visualized with ultraviolet light.
4. Radioactive spots can be located with a detector, or the chromatogram can be pressed
against X-ray film.
Retention factor (Rf)
Different compounds should move different distances on the plate. Some compounds in a
mixture travel as the solvent moves; some stay much closer to the base line. The ratio of the
two distances is calculated. This ratio is called the retention fraction Rf. Individual compound
in a mixture can be identified by its Rf value when compared to one or more standards under
absolutely identical conditions to that of the test compound. If one of the Rf values of a
mixture matches the Rf value of the standard, this suggest the presence of the standard
compound in the mixture. When comparing two different compounds run under identical
conditions, the compound with the larger Rf is less polar because it interacts less strongly
with the polar adsorbent on the TLC.
Specifically, the retention fraction is defined as the fractional distance the spot moves
compared to the distance travelled by the solvent front. An Rf value will always be in the
range 0 to 1; if the compound moves, it can only move in the direction of the solvent flow,
8
and cannot move faster than the solvent. The Rf is constant for a particular compound; as
long as you keep everything else constant - the solvent system, the stationary phase,
temperature, amount of material spotted, pH. Spots with the same Rf values within
experimental error and the same appearance are likely to be the same compound.
For each compound the formula of Rf is:
Rf = Distance from baseline to spot, x_
Distance from baseline to solvent, y
=
The Rf is calculated as below:
Solvent front
New position of compound
3.0 cm 4.5 cm
Rf= 3.0 = 0.67
4.5
Origin
Theory of Adsorption chromatography
Adsorption chromatography is oldest form of chromatography since 1903. It utilizes a mobile
liquid or gaseous phase that is adsorbed onto the surface of a stationary solid phase. It is
achieved by the difference in degree of adsorption of the compounds to stationary phase. The
equilibrium between the mobile and stationary phase account for the separation of different
solutes.
Thin Layer Chromatography (TLC)
Thin layer chromatography (TLC) is among the most useful tools for assaying
the purity of organic compounds. Many compounds with varying functional
groups may be used as the stationary phase and several types of interactions
can aid in developing the desired separation (i.e. Van der Waals forces,
electrostatic interactions, hydrogen bonding, etc.).
The stationary phase consisting of very thin layer of adsorbent material,
usually the very polar silica gel (oxides of silicon) on a glass or plastic plate, aluminum
9
oxide, or cellulose immobilized onto a flat, inert carrier sheet. The surface of alumina is more
polar than that of silica gel. The mobile phase is a solvent (or mixture of solvents) that is less
polar than silica gel. TLC has advantages of faster runs, better separations, and can choice
between different stationary phases.
Both techniques, the adsorption and the partition chromatography, are based on the polarity
of the components of the mixture.
Non-polar compounds will be less strongly attracted to stationary solid phase and will spend
more time in the moving phase and will move faster and will appear closer to the top. They
will have relatively high Rf values.
Polar compounds will be more strongly attracted by hydrogen bonding or dipole-dipole
attractions to the stationary solid phase and will spend less time in the moving phase and they
aren't going to travel very fast up the stationary phase and appear near to the bottom. They
will have relatively low Rf values.
Solvent front
Component A
More polar
Component B
Less polar
Origin
10
The separation of amino acids by two-dimensional chromatography
by Thin Layer chromatography
Principle
Two-dimensional paper chromatography (Two-way paper chromatography) involves using
two solvents and rotating the paper 90° in between. This useful for separating complex
mixtures of similar compounds for example amino acids.
Ninhydrin is a test for proteins, peptones, peptides and α - amino acids in which it reacts with
such compounds to give blue to violet to red color. The blue color changes to a purple when
viewed in artificial light. Other compounds also react if present, and these include primary
and secondary aliphatic amines and some non-aromatic heterocyclic nitrogen compounds.
The imino acids, proline and hydroxyl proline, react to give a yellow color.
11
Materials and chemicals:
 Chromatography glass jar with lid
 Chromatography paper or filter paper: Whatman No.1,
(10 cm x 10 cm)
 Capillary tubes or pasteur pipettes
 Hairdryer or drying oven at 105°C
 Solvent 1
 For paper, n-butanol: glacial acetic acid: D.W. [4:1:5 v/v]
 For TLC, ethanol: water [7:3 v/v]
 Solvent 2
 For paper, phenol: D.W. [4:1 v/v].
 Add 125 ml of water to a 500 gm bottle of phenol and allow standing overnight. Just
before use, add a few drops of 0.88 ammonia to the solvent and mix well.
 For TLC, n-butanol: acetic acid: D.W. [8:2:2 v/v]). The solvent should be made up
fresh on the day.
 Ninhydrin reagent (Dissolve 0.2 gm in 100 ml of aceton just before use).
 Mixture of five amino acids (alanine, aspartic acid, histidine HCl, leucine and
proline). Prepare small volumes of 10 gm/liter solutions in 1 molar HCl (IRRITANT),
a drop of acid are needed to bring the compound into solution.
Procedure
1. Select five amino acids whose Rf values differ widely.
2. Obtain paper or TLC plate (10X10) and place it onto a paper towel. Be
careful to touch the paper or TLC sheet only on the edges, without
touching your fingers to the surface.
3. Using a pencil, draw a faint line (on the powdery side of the TLC),
about 1 cm from the bottom and about 1 cm from top of both paper and
TLC (Solvent front). The line should be parallel to the bottom edge. Do
not allow the pencil to dig into the coating on the plate at all. Then faintly draw one small
hash mark, about 2 cm towards the left corner.
4. Spot 10-20 ul of a single spot of amino acids mixture onto the paper or
the plate (up to a total of 3 times). To do this, dip one end of the
capillary tube into the top of the solution so that only the clear portion of
the liquid enters the capillary tube. Then lightly and quickly touch the
capillary tube to the surface of the paper or the TLC plate. Try to keep
the spot as small possible, preferably around 1mm, Dry the spot in a
current of air.
5. Use forceps to place the paper or TLC into the jar. Make sure that when the TLC plate is
placed in the solvent bath, the solvent does not immerse the spot of the sample mixture.
12
The solvent is below the spot, but then moves up across the spot, carrying the components
of the sample mixture up the plate at different rates. If the spot is immersed in the solvent
at the beginning, it will simply dissolve in the solvent and not rise up the plate.
6. Position the paper or the plate so that it is not touching the sides of the jar, and make sure
it does not curve or buckle in the jar. Covering the jar makes the atmosphere in the jar
saturated with solvent vapor. Saturating the atmosphere in the jar with vapor stops the
solvent from evaporating as it rises up the paper or the TLC plate.
7. As the solvent slowly travels up the paper sheet or TLC plate, the different components of
the mixture travel at different rates. As the solvent migrate up the paper or the TLC plate,
do not disturb the jar.
8. Watch the paper sheet or the TLC plate carefully. Once the solvent migrates almost to the
top of the plate use forceps to remove it and let the solvent evaporate from it in the hood.
9. Arrange the frame so that the second edge to which the spot was adjacent now dips in the
second solvent. Cover the jar with the lid. Once the solvent migrates almost to 1the top of
the plate use forceps to remove the plate and immediately mark the solvent front with a
pencil, before the solvent evaporates.
10. Dry the paper or the TLC plate by hairdryer. Rapidly spray the paper or the TLC plate
through the Ninhydrin reagent and allow the acetone to evaporate. Develop the colors by
heating at 105° C for 2-3 minute or using a hairdryer. The outcome of chromatography
experiment is a chromatogram.
11. Carefully circle the spots with a pencil and mark the center of each spot. Place your name
on the front or back of your TLC plate in the result sheet.
13
Introduction
Carotenoids are organic, fat-soluble pigments that are naturally occurring in plants and some
other photosynthetic organisms, called terpenes. These compounds are responsible for the
red, yellow, and orange color of fruits and vegetables. There are over 600 different
carotenoids known. Two classes of carotenoids are known xanthophylls and carotenes. Since
animals are incapable of synthesizing carotenoids, they must obtain them through their diet.
The two commonest carotenoids are lycopene, a red pigment found in tomato and betacarotene, a yellow pigment found in carrots. Other pigments also occur in the
leaves of plants but they are not obvious because their colors masked by the
chlorophylls in live, healthy leaves. These pigments become visible in the fall
when the leaf dies and the chlorophyll rapidly decompose.
Carotenes act as a precursor of vitamin A. Beta-carotene cleaves to form two molecules of
vitamin A when it is ingested. Vitamin A plays an important role in vision. Beta-carotene is
also a powerful antioxidant, and has been shown to help guard against cancer and heart
disease.
Lycopene is an antioxidant stop free radical production, and it may lower the risk of prostate
cancer. The best food source of lycopene is processed tomato products such as ketchup,
tomato paste, and tomato juice.
14
Separation and identification of carotenoids pigments in tomatoes and
carrots by column chromatography
Principle
This experiment performed to try and tentatively identify and isolate lycopene and betacarotene from two foods rich in them, tomato paste and carrots using refluxing, column
chromatography and thin layer chromatography. Analyze the fractions by thin-layer
chromatography to determine if the fraction contains more than one component. Since both
lycopene and β-carotene are colored pigments that absorb light in the UV and visible range,
they can be identified from their maximum wavelength.
PART I: Dehydration and Extraction
In this part you will extract pigments from tomato paste and carrots, using ethanol and
chloroform as organic solvents. The experiment should be performed under a portable fume
cupboard giving all-round visibility.
Materials and chemicals













Condenser
Hot plate
Beaker
Filter paper
Separation funnel
Glass funnel
Round-bottom flask
Two Conical flask
Tomato paste or carrot (natural or baby food)
Ethanol (95 %)
Chloroform
Saturated sodium chloride solution
Anhydrous sodium sulfate
Procedure
1. In a small beaker place 3 gm of tomato paste, 9 gm carrots, and 30 ml
of 95 % absolute ethanol. Then stir the suspension for at least five
minutes with a glass rod. Ethanol is added to remove water from the
sample, since chloroform immiscible with water and will not
effectively extract the carotenoids until the water is removed.
15
2. Filter to separate ethanol from the plant sample through a small funnel. Press out all the
liquid of the semi-solid residue collected in the funnel by pressing it gently with the flat
side of a spatula and discard the solvent. All glassware from this
point must be dry.
3. Return the solid residue, with or without any adhering filter paper,
to a round-bottom flask, then add 15 ml of chloroform. The flask is
fitted with a condenser and refluxed at 40°C for 4 minutes. This will
extract the β-carotene and lycopene from the plant. The yellow or
red extract is separated from the solid residue by decantation, and
then the solid residue is returned to the flask.
4. Repeat the extraction procedure two more times with 15 ml
chloroform each time. Combine the organic solvent extracts in a flask and discard the
plants.
5. Pour the combined organic solvent extracts into a separation funnel;
add 20 ml of saturated sodium chloride solution (aid in layer
separation). Invert the funnel and shake vigorously for 2 minutes to
extract carotenoids. Release the pressure by opening the lid
occasionally.
6. Replace the funnel in the stand and remove the lid. Allow the layers
to separate and then slowly drain the colored lower layer
(Chloroform is more dense than water) through a funnel which has a
cotton with 4 spatula tips of anhydrous sodium sulfate on top of it.
This removes any water from the colored extract.
7. Evaporate the colored extract on a hot plate under the hood until
about 1 ml of
solvent remains in beaker. Do not allow the solvent to completely evaporate.
8. Warp the flask with foil to avoid light oxidation, label it with your name and place it in a
beaker. Store it in the freezer until the next lab.
PART II: Column chromatography
In this part you will use the preparative method of column chromatography to separate two
major fractions of pigments, the carotenoids from extract. Since the different components are
colored differently, the separation is easily followed visually. Many factors can affect the
separation. These include: adsorbent, polarity of the column or solvents, size of the column,
and the rate of flow.
16
Principle
Column chromatography (LSC) is one of the most useful methods in
biochemical work in separation and purification of both solids and liquids.
The theory of column chromatography is analogous to that of thin-layer
chromatography. In TLC, The solvent (eluent), instead of rising by
capillary action up a TLC, flows down through the column filled with
the adsorbent by gravity action or by the application of air pressure.
The stationary phase (adsorbent) is a thin layer of the most common
stationary phase, silica gel (SiO2) or alumina (Al2O3) on a glass, metal or plastic plate. In
column chromatography the same adsorbent is packed into a vertical glass column. The
mixture of substances in appropriate solvents are applied to the top of the column and passed
through the column, more or less like filtration.
The column is a piece of cylindrical glass (polyethylene) tubing with a stopcock attached at
the bottom of the column to control the flow of the solvent through the column, or a piece of
flexible tubing is often attached at the bottom of the column, and a screw clamp would be
placed on the flexible tubing at the bottom to stop or regulate the flow of the solvent.
Column chromatography can be small or big, according to the amount of material which
needs to be loaded onto the column. It is often convenient to use burette and Pasteur pipette
as a chromatography column.
Materials and chemicals
 Column chromatography
 Beakers
 Glass rod
 Conical flask
 Glass funnel
 Pasteur pipette
 Small vial
 Crude carotenoids (dried pigment)
 Petroleum ether (60-80°C)
 You will need a petroleum ether solution of carotene into the glass cuvettes. Do not use an
ordinary test tube for your measurement. Care should be taken not to drop the cuvettes.
17
Procedure
A. Preparation of the column
1. Wear gloves and goggles at all times.
2. Place a very small glass wool plug in the bottom of the column with a long glass rod, until
all entrapped air is forced out. The plug is just to keep the solid alumina from entering the
stopcock. The plug must not interfere with the flow of the solvent through the stopcock.
3. Clamp the column to a ring stand in vertical position.
4. Close the stopcock, and fill the column with 6 ml of the eluent.
5. Place a small beaker under the stopcock of the column to catch any solvent that may
accidentally drip out of the tip of your column.
B. Preparation of the slurry
Prepare slurry (mixture of a solvent and an un-dissolved solid) by pouring dry alumina slowly
into a beaker of double volume of petroleum ether (60 ml), a little at a time while swirling to
form thick but flowing slurry. The addition adsorbent to solvent should be followed strictly
because the adsorbent will solvate and liberate heat. If the solvent is added to the adsorbent, it
may boil away almost as far as it is added due to heat evolved, especially if ether or another
low boiling solvent is used.
C. Packing of column
1. Quickly and carefully pour the slurry (10-12 cm) into the top of a clean
and dry column with a glass funnel. Stir by glass rod and pour immediately
to maximize the amount of alumina that goes into the column instead of
remaining behind in the beaker. Gently tape the column constantly by
tubing or rubber stopper on the side during the pouring of slurry to help the alumina settle
uniformly, compact, and to remove air bubbles inside the column.
2. Place a beaker under the column outlet, open the screw clamp, and allow the solvent to
drain into it. Add more solvent as necessary. The solvent collected prior to
the sample can be re-used.
3. Use a Pasteur pipette to rinse any alumina that is sticking to the sides of
the column. Allow the alumina to settle while eluent continues to drip into
the beaker. Once the alumina has settled, carefully add about 1cm of sand
to the top of the column to prevent it from being disturbed when fresh solvent is added.
18
4. When finish packing, drain the excess solvent until it just reaches the top level of alumina
(about 0.5 ml). Close the screw clamp. Your column is now “packed”.
5. Never let the solvent drop below the top of the adsorbent to prevent the column from going
dry out as the column progresses. Cracks will form within the alumina column if it dries, and
compounds can fall down the cracks instead of partitioning between mobile and stationary
phases. The success of separation will be dependent on how well you pack the column.
D. Adding the sample (pigment)
1. The sample is first dissolved in a very small amount of the solvent. It is important to use a
minimum amount (1-1.5 ml) of solvent to dissolve the pigments. A thin horizontal band of
sample is best for an optimal separation. If the extract has dried out, re-dissolve it in 1ml of
the solvent.
2. Place a beaker under the column then open the clamp at the tip of the column allowing the
solvent to drain just to the top of the column 1 mm above the surface of the column.
3. Add about half (and save the other half for the thin layer chromatography later) of the
crude plant pigment extract drop wise to the top of the sand using a long Pasteur pipette being
careful not to disturb the top of the column. Allow the sample to adsorb onto the top of the
alumina before adding more eluting solvent.
4. Carefully add solvent to the top of the column. To avoid disturbing the top of the column,
it’s a good idea to carefully pipette an inch or two of solvent onto the column instead of
pouring solvent directly onto the column. If the sample is colored, and the fresh layer of
solvent acquires this color, add small amount of eluting solvent until the solvent above the
top of the column is colorless.
5. Fill the column to the top with solvent. The column is now ready to run.
6. The components of the sample (pigment) begin to move down the column and separate
according to their polarity. Often a series of increasingly polar solvent systems are used to
elute a column. A less-polar solvent is first used to elute a less-polar compound. Once the
less-polar compound is off the column, a more-polar solvent is added to the column to elute
the more-polar compound. The yellow beta-carotene (less polar) moves rapidly through the
column, while the red lycopene (more polar) moves slowly.
E. Sample collection
1. Continue adding solvent at the top. Begin collecting solvent until you
see the band of the yellow β-carotene is just above the stopcock of the
19
column. Change the collecting beaker and begin collecting the yellow fraction in vial.
2. Switch collection vial and allow the solvent to run down to just the top of the column and
then add a solvent mixture of 5:45 petroleum ether/chloroform to elute the orange/red
lycopene band.
3. Switch beaker again when there appears to be a color change and begin to collect the
orange band in another vial when it reaches the middle to the lower half of the column to
minimizes contamination of the lycopene with β-carotene. The process is discontinued when
the band(s) desired is (are) off the column. Save the collected fractions for TLC analysis and
UV-Vis spectrum.
4. If the compounds to be separated from column chromatography are colorless, small equal
size fractions of the eluent are collected sequentially in labeled vials and the composition of
each fraction is analyzed by thin layer chromatography.
Clamp
Petroleum ether
Tomato sample
Alumina
Glass wool
Stopcock
PART III: TLC analysis of sample fractions
In this part you will use the analytical method of thin layer chromatography to estimate the
number of pigments in the extract.
Applying the sample
Procedure
1. Using a capillary tubes, spot the solutions to be analyzed on the silica gel side of the TLC
plate along the pencil line. One spot should be from the yellow fraction β-carotene from the
column. One spot should be from the red fraction lycopene from the column. One spot should
20
be from the colored crude extract that was not ever put on the column. The spots should be
very small in diameter, but clearly visible. If they are not visible, place enough of each
sample on the plate.
3. Follow the steps as in experiment 1 page 14.
4. Immediately circle the visible spots with pencil since the colors may fade over time.
5. Estimate the presence of β-carotene and lycopene in the sample through the calculation of
the Rf values. β-carotene carried the highest Rf values. Lycopene had the lowest values.
PART IV: UV/Vis spectroscopy
In this part you will explain the colors of pigment fractions by measuring the absorption of
electromagnetic spectrum of each, using UV-visible spectrophotometer.
Lycopene and β-carotene pigments may be identified from their absorption spectra. They
absorb light in the 400-500 nm region of the visible spectrum. Make sure a blank is run first.
Record the spectrum of your sample from 400-500 nm. Determine the λmax of any peaks.
There are actually several absorption peaks in the electronic spectrum of lycopene, but the
peak of maximum intensity corresponds to a wavelength near 473 nm. β-carotene behaves
similarly on irradiation, but the peak of maximum absorption is found at shorter wavelengths,
around 448 nm. The exact position of maximum absorption depending somewhat on the
solvent.
β-carotene: λ max at 426,448,474 nm
Lycopene: λ max at 444,473,502 nm
The following spectrum reveals that the sample absorbs UV radiation strongly at wavelengths
below 240 nm, and absorbs visible light near 410 nm. A sample transmits light of the
wavelengths that it does not absorb. The sample absorbs light at the blue end of the visible
spectrum, allowing light of higher wavelengths (yellow, orange, red) to pass through and
reach the eye of the observer and it appears orange in color.
21
Introduction
Most biological compounds in water are colorless and undetectable to the human eye. To test
for their presence we must find a way to “see” them. Colormetry is "the measurement of
color" and a colorimetric method is "any technique used to evaluate an unknown color in
reference to known colors". Two commonly used types of color measurement equipment are
colorimeters and spectrophotometers. A colorimeter or spectrophotometer can be used to
measure the amount of colored light absorbed by a test sample that is itself colored or can be
reacted to produce a color in reference to a colorless sample (blank). The most important idea
in measurement of color is that the intensity of the color from the reaction is directly
proportional to the concentration of the substance being tested and the absorbance of the
substance is proportional to its concentration.
Light and Electromagnetic spectrum (EMS)
The full range of wavelengths of light is called the electromagnetic spectrum.
Electromagnetic spectrum extends from very low frequency radio wave, through microwaves,
infrared, visible, and ultraviolet light to x-rays and gamma rays.
Sunlight is a portion of the electromagnetic spectrum given off by the sun, particularly
infrared, visible, and ultraviolet.
White light, one type of electromagnetic energy, is a very small part of the electromagnetic
spectrum that the human eye can only see. It is composed of all colors of visible light (seven
different colors of rainbow): red, orange, yellow, green, blue, indigo, and violet. It is most
22
important energy source which travels through space as wave. It is the only energy we can
see it in the form of color.
White light decomposed into its component colors by a prism or grating produces the color
spectrum. Each color of these has its own wavelength ranging from 400–700 nm.
The reddish color is the longest wavelength. The greenish is the mid-size, while the violet
has the shortest wavelength. Humans are especially sensitive to wavelength around 655 nm
(red).
Infrared (IR), invisible long wavelength (above 700 nm) before red in the visible spectrum.
Infrared can be felt, but it cannot be seen by the naked eye. Everything emits infrared light.
Because of this, movement can be detected in the dark with infrared detectors.
Ultraviolet (UV), invisible short wavelength of light (below 400 nm) beyond violet in the
visible spectrum. Most are reflected by the ozone layer, but some do get into our atmosphere.
These rays damage unprotected skin and have been known to cause skin cancer.
Color
Color is the visual effect that is caused by the spectral composition of the light emitted,
transmitted, or reflected by objects. A color solution appears colored because molecules in
the solution absorb light of particular colors (wavelengths) and not others. The particular
wavelengths of light that a given solution absorbs determine the color we perceive. The
perceived color (not absorbed) has a complementary relationship with the color of the visible
light absorbed. The wavelengths of light that are not absorbed are transmitted or reflected
completely to our eyes. A color wheel can be used to predict the appearance of the sample. A
color
wheel,
illustrates
the
approximate
complementary
relationship between the wavelengths of light absorbed and the
wavelengths transmitted or reflected. A color has the greatest
absorbance in its complementary color range. In general, if one
color of light is absorbed, the sample will appear to be the color
on the wheel opposite to the one absorbed. For example,
23
Tomatoes appear to be Red because contain a carotenoid known as "Lycopene". Lycopene
absorbs most colors of the visible light that are not red and reflects mainly red back to the
human eye, thus tomato appears to be Red.
If something absorbs all the colors, it appears black, if it reflects everything, it appears white.
Many substances appear to be colorless to the eye since they absorb only UV and IR.
Wavelength
Wavelength is the distance between two peaks of a wave which determines its color.
Commonly designated by the Greek letter Lambda (λ). Common units of measurement are
angstroms,
nanometers
(nm),
one
nanometer equals one billionth of a meter,
or microns. The choice of the correct
wavelength for testing is important. It is
interesting to note that the wavelength that
gives the most sensitivity for a test factor is the complementary color of the test sample. Light
of different colors has different wavelengths, as shown beside.
24
Determination of wavelength of maximum absorbance
Principle
The purpose of this experiment is to find the wavelength of maximum absorbance (Lambda
max, λmax) for any colored solution. It is the wavelength of light at which absorbance are
greatest. There may be more than one λmax for a compound. The value of λmax is characteristic
of sample and may be used as an aid in its identification. It is the preferred wavelength to use
for running a calibration curve. The absorbance over the full range of wavelengths constitutes
the absorption spectrum, a plot of absorbance (on the y-axis) as a function of wavelength
(on the x-axis). It is characterized by the wavelength λmax (the highest portion of the curve).
The shape of the spectrum and the wavelength of maximum absorbance are characteristic of
the chemical compound. The absorbance of compounds is also directly related to the
concentration of the sample. You will use a spectrophotometer to do UV-Vis spectroscopy on
the extract and the fractions that you collected during the column chromatography. You will
graph the data manually to generate absorption spectrum. From this curve you will determine
a λmax for the carotene.
Carotenoids absorb light with wavelengths of 430 nm and 550 nm and appear red, orange, or
yellow.
Material and chemicals
 Spectrophotometer
 Two Cuvettes
 Dropper
 Lycopene
 β – carotene
 Extract of the dye
Procedure
1. Obtain two cuvettes that have been cleaned inside and outside. One will serve as blank (or
reference) and should be filled with distilled water (or whatever solvent is used for the
colored compound). The blank must be used to adjust the spectrophotometer every time
the wavelength is changed. If you are working with a single wavelength, the blank should
be placed in the spectrophotometer periodically to check for drift.
Note: It is important to use matched cuvettes, to check this, place at least 2.0 ml of water
(or whatever liquid in the reference cuvette) to the other cuvette (called the sample
25
cuvette) and read the absorbance if it is identical to that of the first cuvette (within 0.003
absorbance units). If not, get two that read the same.
2. Rinse the sample cuvette with a small amount of colored solution, and then fill it 3/4 full.
3. Turn on the spectrophotometer and warm up 15 min. Make sure to set the correct
wavelength. Without a tube in the sample compartment set the mode to transmittance,
rotate the front left knob until the digital display reads 0.00%T.
4. Wipe the blank cuvette with a Kim wipe. Insert the blank into the sample compartment
making sure the line of the cuvette matches the mark on the sample holder, and then close
the lid. Push the mode button changing the readout to absorbance. Rotate the right knob
until the digital display reads 0.00.
5. Remove the blank, wipe the sample cuvette, and insert the colored solution.
6. Record the absorbance at 10 nm intervals from 400 nm to 700 nm. Remember to zero the
spectrophotometer with the blank each time you change the wavelength. The absorbencies
over the full range of wavelengths constitute the absorption spectrum.
7. Record the data in the table in your result sheet and answer the questions.
8. All of your data must be taken on the same instrument, at the same time. If you need to
change instrument, you must start over and take all of the data for the spectrum again.
26
Introduction:
Size Exclusion Chromatography (SEC), also called Gel Permeation Chromatography or
molecular sieve chromatography is a liquid chromatography through a column packed with a
swollen gel media for separating molecules according to molecular weight or sizes.
A gel of polysaccharides (dextran) is formulated into small beads that contain pores of
varying diameters up to a maximum size, allowing smaller molecules to pass through pores
and quickly exclude larger molecules. Gel filtration works opposite to filtration.
It is reliable and simple, little equipment is required, the procedures are straight forward and
good separation and yields are usually obtained.
27
Gel filtration can also be used to determine the molecular weight of a molecule. A calibration
curve of the elution volumes are plotted against the log molecular weight of the
corresponding molecules. A straight line is obtained. If the elution volume of the unknown
molecular weight is then found, it can be compared to the calibration curve and the molecular
weight determined.
28
Separation a mixture of blue dextran and cobalt chloride molecules
Principle
Sephadex is most commonly used for gel-filtration column. It is an inert, bead-formed, cross
- linked dextran polymer consisting of many glucose molecules. Sephadex beads are porous.
Molecules larger than largest pores cannot enter the gel and are eluted from the column first.
Smaller molecules enter the beads and are retard. Therefore, molecules are eluted in order of
decreasing size. Sephadex G-25 excludes all molecules with a molecular weight greater than
5000, thereby eluting them first.
In this lab, you will separate a mixture of blue dextran and cobalt chloride molecules through
a gel-filtration column.
Blue dextran is a high molecular weight glucose polymer (2,000,000 Daltons) larger than the
exclusion limit of the gel can’t get into the beads and therefore excluded from the gel (Kd=0),
and eluted when a volume of solvent equal to the void volume Vo leaves the column and (the
space between gel beads). Blue dextran is often used as a marker to measured void volume.
Cobalt chloride, with a low molecular weight is freely accessible to the gel particles (Kd=1),
and elute at a volume equal to the total volume of solvent Vt, or the sum of Vo and Vi,
internal volume, (space within the beads). Molecules of an intermediate molecular size will
be eluted in volumes between Vt and Vo.
Both molecules, Blue dextran & CoCl2.6H2O, are colored so the progress of the filtration can
be followed by observing the separation of the colored bands. The completed fraction is then
analyzed by measuring the extinction of each fraction at 625 nm, λmax of blue dextran and
510 nm, λmax of CoCl2.6H2O.
It is possible to calculate a partition coefficient Kd. For a solute Kd is the partition
coefficient, the extent to which the molecules can penetrate the pores in stationary phase,
which values ranging between 0 and 1.
Kd = Ve – Vo
Vi
If the Kd is greater than 1, then adsorption of the compound on the gel has occurred.
However, because it is difficult to measure Vi precisely, the equation may be modified to
determine available part of the resin (Kav).
29
Kav = Ve – Vo
Vt – Vo
Where Vt is the total volume of the column (Vt = πr².h).
In order to separate easily two solutes, their values for (Kav) must be significantly different
from each other.
Materials and chemicals
 Column chromatography
 Spectrophotometer
 Fifteen test tubes
 Glass rod
 Glass funnel
 Pasteur pipette
 Beaker
 Stand with burette clamp
 Sephadex G25
 Blue dextran in saline
 Cobalt chloride in saline
 Sodium chloride (0.9 %), (Saline)
Procedure
1. Number fifteen small test tubes 1-15. Arrange them in order on a rack.
2. Prepare the gel bead column:
a. Clamp the column vertically.
b. Column (About 12 cm in hight) is filled with semisolid (Swollen) gel beads of
Sephadex G-25 [Note: the Sephadex gel has already been soaking in the elution buffer
for 3 to 4 hours]. Be gentle; do not allow gaps or bubbles to form. It may help the
beads to settle by flow a small amount of saline through the column between additions
of beads.
c. Equilibrate the column with saline by passing about 10 ml of saline through the
column beads after it has completely settled.
d. Avoid stirring up the top of the column bead when adding saline or samples, as this
will give poor resolutions of the samples.
3. Carefully add the sample mixture containing blue dextran and CoCl2.6H2O to the top of
the column using a pasture pipette. Again, do not stir up the top of the gel. Allow saline
solution to drain down so there is only about 1 mm of buffer above the gel before layering
the sample mixture carefully on top of the gel. Turn off the stopcock to allow the mixture
30
to enter the gel beads. As soon as it does, continue to add saline solution to the top, filling
the space at the top of the column.
4. Collect the fractions, 3 ml per tube, beginning with tube number “1” Early fractions
contain large molecules while later fractions contain smaller ones. Check to see if there is
any color in each fraction. Be sure to collect all fractions.
5. Measure the absorbance spectrum of samples in order to identify each molecule (Blue
dextran at 625 nm, CoCl2.6H2O at 510 nm).
6. Clean up the column as follows: Gently invert and shake the beads into the recycle beaker
for further use. Be patient. Some beads will come out the end. Then you can add some
buffer solution and shake. The rest of the beads will eventually come out. Try slipping a
pipette bulb over the thin end of the tube and onto the body. Try squeezing the bulb to
back flush the beads out. Clean and return your pipette.
7. Wash your test tubes. Line the tubes back up for the rest separation.
31
Introduction
Amino acids are the building blocks of roteins. In order to understand acid-base properties of
proteins and their behavior as polyionic macromolecules, we will begin by investigating the
properties of their constituent amino acids. Since all amino acids contain at least one amino
and one carboxyl group, they are classified as amphoteric substances (meaning that they can
act as either an acid or as a base). Such a molecule reacts with acids as follows:
R
R
+
H3 N C COO
H
+
+
H3O
H3 N C COOH
H
Acid
Zwitterio
+
H2 O
+
H2 O
Cationic
And with bases as follows:
R
R
+
H3 N C COO
H
+
Zwitterio
OH
H2 N C CO O
H
Base
Anionic form
Since alpha-carboxyl and alpha-amino groups are weak acids and bases, respectively,
buffering action by these groups will occur.
The COOH group is acidic with a pKa value of 1.7-2.4. Thus at pH values below this, the
group exists as COOH while at higher pH values, the group exists as COO-. The NH2 group
is basic with a pKa of 9-10.5, so below this it exists as NH3+ while above this pH it exists as
NH2. At neutral pH values, both groups are ionized and the amino acid exists in a dipolar
form with no net charge. This form is called a zwitterion. The pH at which all the amino
acid molecules are in this form is the isoelectric point (pI), where:
pI
=
pK1 + pK2
2
32
Determination of the isoelectric point of glycine
Principle
In this experiment, you will identify an unknown amino acid through acid-base titration.
Titration curves of amino acids are very useful for identification. As in the example for
glycine shown above, a simple amino acid has two dissociation steps corresponding to loss of
H+ from the acidic carboxyl group at low pH followed by loss of H+ from the more basic
amino group at high pH. The pKa value for each dissociable group of an amino acid can be
determined from such a titration curve by extrapolating the midpoint of each buffering region
(the plateau) in the titration curve. The diagram also shows that there is a point in the curve
where the amino acid behaves as a "neutral" salt. At this pH, the amino acid is predominantly
a zwitterion with a net charge of zero. This point of the titration curve is the “isoelectric
point” (pI) and can be approximated as halfway between the two points of strongest buffering
capacity (the two pKa values). The isoelectric point (pI) can be estimated by:
PI = (pKa + pKb)
2
Where Ka and Kb are the dissociation constants of the carboxyl and amino groups,
respectively.
Proteins can be separated according to their isoelectric point in a process known as isoelectric
focusing. At a pH below the pI, proteins carry a net positive charge. Above the pI they carry a
net negative charge. The pH of an electrophoresis gel is determined by the buffer used for
33
that gel. If the pH of the buffer is above the pI of the protein being run, the protein will
migrate to the positive pole. If the pH of the buffer is below the pI of the protein being run,
the protein will migrate to the negative pole of the gel. If the protein is run with a buffer pH
that is equal to the pI, it will not migrate at all. This is also true for individual amino acids.
Due to a preponderance of weakly acid residues in almost all proteins, they are nearly all
negatively charged at neutral pH. At pI the solubility is often minimal and the protein will
accumulate and it will not migrate at all. This is also true for individual amino acids.
Materials and Chemicals
 pH meter and standard buffer solutions
 Magnetic stirrer and bar
 Graduated burette and holder
 Beaker (400 ml)
 Graduated cylinder (20 ml)
 Distilled water
 NaOH solution (0.5 N)
 H2SO4 solution (0.25 N)
 Amino acid (Glycine)
Procedure
1. Use the electronic magnetic stirrer device. Place a magnetic bar
into your 400 ml beaker, and turn on the stirrer so that the bar turns
at a moderate speed. Turn off the stirrer.
2. Place approximately 20 ml (know and record the actual volume) of
your selected amino acid solution into the beaker. Assemble the
titration stand, graduated burette, and pH meter so the electrode is
submerged into the amino acid solution and the burette is
positioned to drop solution into the beaker. Be sure the electrode is
clear of the magnetic stir bar.
3. Record the initial pH of the amino acid solution and the initial reading of your burette
(Which should be 0.0 ml)
4. Turn on the stirrer, check that the magnetic bar does not collide with the electrodes.
5. Add enough 0.5 N NaOH solution to the titration burette. It is recommended that you
allow the first portion of NaOH solution to run through the burette to clear the bottom
constriction from residual distilled water (Discard this volume).
6. Record the starting volume reading on the burette, and then carefully titrate the amino acid
solution by drop wise addition of the 0.5 N NaOH.
34
7. Record the volume addition of NaOH (think!!) and pH of the solution throughout the
titration until a pH > 12 is reached. You do not have to take a reading for each pH unit
shown, but try not to skip more than two units.
8. Repeat the titration (Step 5 to 7) with 0.25 N of H2SO4 until a pH 1.5 is reached.
9. Record the volume of added H2SO4 and pH of the solution throughout the titration until a
pH below 1.5 is reached.
10. Use care in adding the H2SO4 and NaOH, as the pH may change slowly or rapidly
depending on the pH and buffering range of the ionizable group(s). Have patience!!!
Near the equivalence point, a single drop may change the pH dramatically. It is strongly
recommended that you proceed drop by drop.
35
Introduction
The movement of molecules through a cell membrane is termed osmosis or diffusion. Such
movement is principally possible because nutritive molecules are smaller than membrane
micro pores. If the molecules are too large, no molecular transfer, or diffusion occur.
Through dialysis molecules will separated according to their size by use a semi permeable
membranes that transmit selectively taking into consideration the concentration differences of
the two solutions on either side of the membrane. This porous membrane selectively allows
smaller solutes to diffuse from a high concentration solution to a low concentration solution
across the semi-permeable membrane until equilibrium is reached while the larger molecules
are retaining. This process is affected by the variables of temperature, viscosity, and pressure
gradient across the membrane. Also it is affected by the solvent, pore size, and the nature of
the membrane. The rate of dialysis is greatest in distilled water.
Advantages of lab dialysis:

Very gentle conditions

Easy operation

Wide range of samples volume

Many membrane types & MWCO’s

Inexpensive materials

Disposable membranes & devices
36
Dialysis application:

Macromolecular purification

Protein concentration

Solute fractionation

Contaminant removal

pH change

Desalting

Buffer exchange

Binding studies

Electro-elution
Dialysis membrane materials
Synthetic and natural membranes are commonly used for diffusion or osmosis. In the
following experiment, cellophane dialysis tubing serves as an excellent representation of the
cell membrane. Cellophane is the most commonly used as dialysis material. It is a thin
transparent sheet made of regenerated cellulose. The regenerated cellulose is derived from
cotton. It has a symmetric pore structure. The membrane contains traces of sulpher
compounds, metal ions, and some enzymes.
Dialysis is best carried out with freshly prepared tubing since, once it is wet, it becomes very
susceptible to attack by micro-organisms. If it has to be stored, then the tubing is best kept
with a trace of benzoic acid in the solution.
Preparing of the dialysis tubing
1. A suitable size and length of dialysis tubing is selected (about 10 cm). Always handle the
bag with gloves.
2. Boil the bag for 30 min in alkaline EDTA (Na2Co3, 10 g/L: EDTA, 1mmol/L) to avoid the
loss of activity of molecules dialyzed and to make the dialysis sac soft. After boiling the
tubing is washed with distilled water.
3. Soak the bag in water. Never let the dialysis bag dry out once it has been wetted.
37
A- Separation of a mixture of starch and maltose
Principle
Starch consists of two large molecules: amylose of molecular weight 50,000 and amylopectin
of molecular weight 1,000,000, neither of which will pass through a dialysis membrane.
Salivary amylase converts the starch to maltose, molecular weight 360, which diffuses out the
membrane. A solution of our sample (starch and salivary amylase) is placed into a dialysis
bag such as cellophane and the bag is sealed by knotting it at both ends. The sealed bag is
then immersed in a relatively large volume of the solvent (distilled water). Maltose that is
smaller than the pores of membrane allow moving freely across out of the membrane into the
solvent. Starch is greater than the pore diameter and will retain inside the dialysis bag.
Materials and chemicals
 Test tube rack to hold 16 small test tubes
 Two beaker
 Magnetic stirrer and bar
 Cellophane tubing
 Dropper, Pipette (2 ml)
 Hot plate
 Salivary amylase (1 ml saliva diluted to 5 ml with distilled water)
 Iodine solution (5 mmol/L in 30 g/L Potassium iodide)
 Soluble starch (20 g/L)
 Sodium chloride solution (1g/L) buffered with 0.02 mol/L Sodium phosphate pH 6.8).
 Fehling’s solution.
Procedure
1. Label sixteen small test tubes 1-16. Number test tubes 1 S through 8 S - and 1 M through 8
M (“S” is for starch an M is for maltose). Arrange them in order on a rack.
2. In test tubes 1 M through 8 M place one ml or 20 drops of Fehling’s solution (Test for
maltose).
3. Place 10 drops of iodine solution into each test tube numbered 1 S through 8 S (Test for
starch).
4. Seal one end of the tubing with a string. Carefully fill the sac with the reaction mixture by
pipetting 2.5 ml starch, 0.5 ml phosphate buffer and then 0.5 ml salivary amylase.
5. At 0 min, quickly, pipette 1.5 ml of the reaction mixture (From inside the bag). Add 0.5 ml
to tube # 1 S and 1 ml to tube # 1 M. Heat test tube # 1 M in a boiling water bath for 5min.
observes result for starch identification (Blue color). Observe result for maltose
identification (Red ppt).
38
6. Tie the second end of the tubing. Immerse the tubing in the 400 ml beaker containing 100
ml of the distilled water, the solvent. Place the beaker on a stir plate and stir in the cold for
at least 90 min (it takes about 2 hours for dialysis to be completed). Stirring help in the
entry of the water inside the sac and help in getting out of small molecules out of the sac.
7. Pipette 1.5 ml from the solvent (outside) every 15 min interval during the 90 min. Examine
each fraction (7 fractions) for the presence of starch or maltose as in step 4.
8. At the end of the experiment, cut the dialysis sac, carefully remove the dialyzed from the
sac with a pipette and squeeze out as much as possible of whatever is left into the test
tubes # 7 S (0.5 ml) and # 7 M (1ml). Examine the dialyzed (Inside the sac) for the
presence of starch or maltose as in step 4.
Incomplete hydrolysis
Complete hydrolysis
Incomplete separation
39
B-Separation of a mixture of starch and glucose
Materials and chemicals
 Glucose Solution
 Starch Solution
 Iodine Solution
 Glucose Test Strips
 Dialysis Tubing
 String & Scissors
 400 ml Beaker
 10 ml Graduated Cylinder
Procedure
1. Fill the 400 ml beaker ¾ full with tap water. Add 10 drops of Iodine solution and stir well
(what the color of the water). Iodine will turn blue-black in the presence of starch.
2. Test the solution in the beaker for the presence of glucose by dipping a glucose test strip
into it. After 30 seconds, compare the color on the strip to the color chart on the side of
the bottle.
3. Remove the tubing from the water and rub one of the ends between your thumb and
pointer finger to open. Once open, submerge it in water again for about thirty seconds.
4. Seal one end of the tubing with a string. Carefully pour into the tubing 5 ml of starch
solution and 5 ml of glucose solution as well, using the 10 ml graduated cylinder. Tie the
second end of the tubing.
5. Immerse the tubing in the 400 ml beaker of the solvent and allow it to remain undisturbed
for 15-20 minutes. Remove the tubing from the beaker.
6. Retest the solution in the beaker and in the bag with a new glucose test strip.
7. Test the solution in the beaker and in the bag for the presence of starch by notice any
change in the color of both solutions.
8. Record your observations in the data table for Initial and final status information.
40
Sedimentation
The motion of molecules in solutions or particles in solutions in response to an external force
as gravity, centrifugal force or electric force. The particles sediment at different rates
depending on:

Properties of molecules (size, shape, density)

Properties of solvent or gradient material (density, viscosity, temperature)

Interaction between the solute molecules and the solvent gradient material.
Centrifugation
Centrifugation is one of the most important and widely applied techniques that used to
separate immiscible liquids and solids from liquids by artificially controlling the gravity of a
solution. Due to gravity many particles in solution will settle at the bottom of a container.
Other particles extremely small in size (very light) will not separate unless subjected to high
centrifugal force (measured as Xg gravity) provided by a centrifuge. By putting the solution
in a centrifuge and rotating particles at a high rate of speed. This creates a centrifugal force
and very light particles will fall out and form a pellet at the bottom of the tube.
Relative centrifugal force
Relative centrifugal force (RCF) is the measurement of the force applied to a sample within a
centrifuge. This can be calculated using the following calculation.
g = RCF = 0.00001118 r (rpm)2, where:
g = Relative Centrifuge Force
r = rotational radius (centimeters, cm)
rpm = rotating speed (revolutions per minute, r/min)
An RCF of 500Xg indicates that the centrifugal force applied is 500 times greater than earth
gravitational force.
Common example of the use of centrifugal force
1. In a washing machine, it is centrifugal force generated in the spin cycle.
2. To separate cream from whole milk.
3. To separate uranium 235, to produce nuclear energy.
41
Centrifuge
It is an apparatus that rotates at high speed and by centrifugal force
separates substances of different densities. The materials with the
highest density travel towards the bottom of the centrifuge tube at
a higher rate of speed than they would under the force of normal
gravity.
Centrifuge can be considered devices for increasing the effects of the
earth's gravitational pull to quicken the precipitate of substances to the
bottom. The material is placed in a centrifuge tube which is then placed
Supernatant
in rotor. It is separates into pellet (precipitate) and supernatant or
supernatant (The clarified liquid above the pellet). The pellet (denser
materials) is settled at bottom of the centrifuge tube. The supernatant
(liquid) is then either quickly decanted from the tube without disturbing
the precipitate, or withdrawn with a Pasteur pipette.
Types of centrifuges
The major distinguishing features between centrifuge types are speed and capacity. At least
there are five types of centrifuges:

Table top/clinical/bench top centrifuge or micro centrifuge (speed up to 12,000 to
13,000 rpm)

Low-speed centrifuge (maximum speed 5000 rpm)

High-speed centrifuge (speed up to about 25,000 rpm.

Ultracentrifuge (speed up to 70,000 rpm). Speeds up to 100,000 rpm are available on
typical modern versions. At such speeds, the centrifuge chamber must be evacuated
and refrigerated to counter the heat generated as a result of friction.

Geotechnical centrifuge (in soil study)
Types of rotors
There are three types of rotors, fixed angle, swinging-bucket, and vertical rotors. Fixedangle and swinging-bucket rotors are the most common styles for bench top, low speed, and
high-speed floor-model centrifuge applications. Vertical rotors are used primarily in
ultracentrifugation.
42
Effect of salt on extractability of proteins
Principle
The purpose of this lab is to know how proteins can be separated from a tissue homogenate
into different fractions by centrifugation. The process of centrifugation is based on the fact
that proteins have differences in size, shape and density. At each step of centrifugation, more
dense particles are separated from less dense particles. You will also need to measure the
concentration of protein in each fraction by taking a sample of the original homogenate and
the supernatants. The procedure you will use to determine protein concentration is the Biuret
method. It permits the determination of protein concentration by measuring the intensity of
the purple color at 540 nm.
Extraction of proteins from tissues
In this lab you will isolate proteins from liver tissue. In biological systems proteins exist in a
variety of forms; some are in free soluble form and some are bound to nucleic acids, lipid or
sugar. Each type has to be extracted differently.
Materials and chemicals
 Centrifuge
 Balance
 Spectrophotometer
 Water bath (37o C)
 Mortar and pestle
 Filter paper & Muslin
 Measuring cylinder, Glass funnel & Beaker
 Centrifuge tube ,Test tube & Cuvette
 Dropper & Pipette
 Cold distilled water
 Liver tissue
 Biuret reagent
 NaCl )0.2 M(, NaCl (1.0 M), &NaCl + triton X (1.0 M)
Procedure
A. Preparation of homogenate
Prepare a homogenate of a biological tissue (liver) as follows:
1. Weigh 5 gm of liver. Dry it by folding between folds of filter paper
sheet and drop it in mortar.
43
2. Using a scissors, mince it into small pieces. Add 10 ml of cold distilled water and grind
well by pestle until a uniform paste is obtained.
3. Filter the homogenate through 2 or 3 layers of muslin into the measuring cylinder to
remove any lumps that may be present. Rinse the mortar with 5 ml of cold distilled water
and combine it in the measuring cylinder with the original homogenate.
4. Complete the volume to 20 ml with cold distilled water (Label it as F0).
5. Pipette 0.1 ml of (F0) into test tube for Biuret assay.
B. Before carry the procedure

Remember to take a 0.1 ml samples of each fraction (0 to 4 ).

Do all the following procedure in the cold, keeping all solutions on ice.

If a refrigerated centrifuge is not available the centrifuge holders need to
precooled to keep them at or near the operating temperature.

Carry out the procedure with the homogenate into two centrifuge tubes
according to the diagram in the next page.
44
45
Chemical solutions
Introduction
Chemical solutions is a homogeneous mixtures in which one substance (the solute) is
dissolved in another substance (the solvent), but not chemically combined. A solution may
exist in any phase. The three types of solution are:
1. Gaseous Solution includes gases or vapors dissolved in one another. Two or more
gases can form a solution. Air is an example of a gaseous solution.
2. Liquid Solution contains a liquid solvent in which gas, liquid, or solid is dissolved.
Water is the most common liquid solution. Many things can be dissolved in it. Table
salt is an example of a solid dissolved in a liquid. A liquid and a gas can also be
dissolved in a liquid solution.
3. Solid Solution is a mixture of solids spread equally throughout one another. Metal is
an example of a solid solution at room temperature.
Lab experiments often require preparation of chemical solutions in their procedure.
This may involve weighing a precise amount of dry material or measuring a precise
amount of liquid. Preparing solutions accurately will improve an experiment's safety
and chances for success.
We look at preparation of these chemical solutions by:
Weight (w/v): [Mass of solute (g) / Volume of solution (ml)]
Volume (v/v): [Volume of solute (ml) / Volume of solution (ml)]
Molar solutions (M): One of the most common ways to express the concentration of
the solution which is moles of solute / 1 liter of solution or gram-molecular masses
of solute / 1 liter of solution. Mole a fundamental unit of mass 1 mole is 6.02
x1023 molecules of that substance (Avogadro's number).
In this lab, you will deal with liquid solution and you will learn how to prepare it.
46
A- Preparation of liquid solutions
1. Preparation of solutions from solid materials
To prepare a solution of a specific concentration by dissolving a solid in a liquid,
you must know the concentration and total volume of the solution you want to make
(probably given to you) so that the specific amount of solute is measured.
In this lab, you will prepare the following solutions:
0.1 M Na2HPO4
0.1 M NaH2PO4
To prepare 0.1 M Na2HPO4 or 0.1 M NaH2PO4 in 500.0 ml, first make the following
calculation to obtain the required amount of each substance.
Calculation
Molarity (M) =
Molarity (M) =
No. of moles of solute
Volume of solution (L)
Weight (gm) X 1000
Volume (ml) X Molecular weight
Weight (gm) = Molarity (M) X Volume (ml) X Molecular weight
1000
Procedure
1. Take a 400 ml beaker and place it on the balance. Tare the balance (set it to zero). Weigh
out the required amount in gram of Na2HPO4 or NaH2PO4.
2. Add approx. 1/2 the volume of dH2O to the beaker and stir with a glass rod until the solid
is completely dissolved or stirring with magnetic stir.
3. Transfer the solution to 500 ml volumetric flask. Rinse out the beaker and the glass rod
with dH2O. Do this at least twice. Add water to just below the line on the volumetric
flask. Add the final drops with a dropper to ensure that the bottom of the meniscus is at
the fill mark. Put the lid on the flask and invert to mix the solution.
4. Label your solution with your name, the concentration, the date, and the contents, e.g.
0.1 M Na2HPO4.
47
2. Preparation of solutions from liquid materials
To prepare a solution of a specific concentration from a liquid, you must know the
concentration and total volume of the solution you want to make (probably given to you) so
that the specific volume of liquid is measured.
In this lab, you will prepare the following solutions
0.2 N HCl
0.05 N H2SO4
To prepare 0.2 N HCl or 0.05 N H2SO4 in 50.0 ml, first make the following calculation to
obtain the specific volume of liquids.
Note: When preparing a strong acid, always add acid to water (same for a strong base).
Calculation
Molarity (liquid) = Percentage x Specific gravity x 1000
Molecular weight
Volume = Molarity x Volume x Molecular weight
Specific gravity x Percentage x 1000
Normality (liquid) =Percentage x Specific gravity x 1000
Equivalent weight
Equivalent weight = Molecular weight
Valence
Volume = Normality x Volume x Equivalent weight
Specific gravity x Percentage x 1000
Procedure
1. Add approximately ¾ of the water from the wash bottle to a 50 ml volumetric flask.
2. Measure the specific volume of HCl or H2SO4 with a pipette then transfer it to the flask.
3. Put the lid on the flask and invert several times to mix the solution.
4. Add more water, if necessary, but do not go over the fill mark. Add the final drops with a
dropper to ensure that the bottom of the meniscus is at the fill mark. Put the lid on the
flask an again invert to mix the solution.
5. Label your solution with your name, the concentration, the date, and the contents, e.g. 0.1
HCl.
48
3. Preparation of buffer solutions
Buffer solutions are needed to control the pH of solutions in a narrow range for certain
reactions to be optimized. We have buffers in our blood; it is mainly HCO3-(aq) / CO2-(aq).
Without buffers, our blood pH would change too quickly. For example, without buffers,
imagine drinking orange juice (citric acid). The weak acid will lower our blood pH and will
lead into a lot of trouble. The enzymes in our body will not be optimal.
In this lab, you will prepare phosphate buffer. Phosphate buffer is one of the more common
buffers used for many molecular biology procedures and is made up of a mixture of
dihydrogen phosphate (NaH2PO4) and its conjugate base, hydrogen phosphate (Na2HPO4).
The dihydrogen phosphate acts as the weak acid because it donates an H+ to the OH- to form
water. The hydrogen phosphate will accept excess H+ to create dihydrogen phosphate. The
buffer is most commonly prepared at pH 7.
Measurement of pH
The pH reading of a solution is usually obtained by comparing unknown solutions to those
of known pH; there are several ways for doing this that described below:
1. By acid – base titration with a dye (pH indicators).
2. By using pH strips or pH papers (color patterns indicate pH).
3. By using pH meter (read the number)
The formal definition of pH is the negative logarithm of the hydrogen ion concentration:
pH = -log[H+]
Henderson-Hasselbalch equation or a buffer equation can be used for pH calculation of a
solution containing pair of acid and conjugate base.
pH = pKa + log ([A-]/[HA])
[A-] = molar concentration of a conjugate base (M)
[HA] = molar concentration of a weak acid (M)
pKa = the effective ionization constant of the acid
Buffer systems in the human body
There are three primary systems that regulate the H+ concentration in the body fluids to
prevent acidosis or alkalosis:
1) Carbonic Acid Buffering System (H2CO3 and HCO3) - Major ECF buffer
2) Phosphate Buffering System - Urinary buffer
3) Protein Buffer System - Major ICF buffer
49
Before making a buffer you must know the component of the buffer, desired pH, the
concentration and total volume of the buffer you want to make (probably given to you).
Procedure
1. Make up 0.1 M monobasic stock (NaH2PO4) and add dH2O to bring up to 100 ml.
2. Make up 0.1 M dibasic stock (Na2HPO4) and add dH2O to bring up to 100 ml.
3. To make 1 L (0.1 M) from sodium phosphate buffer, combine the following quantities
with different pH of each stock and add dH2O to bring up to 100 ml as described in table1.
4. Check the pH using a pH meter and adjust the pH as necessary using phosphoric acid or
sodium hydroxide.
5. Once you have reached the desired pH, add water to bring the total volume of phosphoric
acid buffer to 100 ml.
6. Label the volumetric flask. The label includes your name, chemical name of substance, the
pH, the concentration, and the date, e.g. 0.1 M phosphate buffer pH 6.8
Preparation of phosphate buffer
Vol. of NaH2PO4
(0.1 M)
10 ml
9 ml
8 ml
6 ml
11 ml
12 ml
14 ml
Vol. of Na2HPO4
(0.1 M)
10 ml
11 ml
12 ml
14 ml
9 ml
8 ml
6 ml
Calculated
pH
6.77
6.61
6.52
6.33
6.79
6.88
7.07
4. Dilution
Solutions are often prepared by dilutions of a concentrated solution, usually a laboratory
stock solution. Given the initial concentration of the stock solution, Ci, desired
concentration and volume of the diluted solution, its preparation is straightforward. The
following equation is useful to do calculations involving dilutions
Ci Vi (stock solution)
= Cf Vf (new solution)
Ci = initial concentration to be diluted (stock solution)
Vi = initial volume to be diluted (stock solution)
Cf = final concentration to prepare (new solution)
Vf = final volume to prepare (new solution)
50
The volume to be taken from the initial solution can be calculated as: Vi = CfVf / Ci
To make 200 mL of a 0.1 M HCl (new solution) from a solution of 1.0 M HCl (stock
solution), we need 20 ml of 1.0 M HCl.
Procedure
1. Measure 20 ml of 1.0 M HCl (stock solution) with a pipette then transfer it to a 200 ml
volumetric flask.
2. Add approximately ¾ of the water volume to the volumetric flask.
3. Put the lid on the flask and invert several times to mix the solution.
4. Add more water, if necessary, but do not go over the fill mark.
5. Add the final drops with a teat pipette to ensure that the bottom of the meniscus is at the
fill mark. Put the lid on the flask an again invert to mix the solution.
6. Label your solution with your name, the concentration, the date, and the contents, e.g. 0.1
HCl.
Note: When diluting a strong acid, always add acid to water (same for a strong base).
Meniscus
51
Principle
Electrophoresis is a technique used for the separation and sometimes purification of
macromolecules especially proteins and DNA, or RNA fragments that differ in size, charge
or conformation. It refers to the movement of charged particles (ions, molecules,
macromolecules) through a porous matrix under the influence of electric field. Particles will
move through the matrix at different rates, according to their charge, toward either the
negative pole (cathode) if positively charged or the positive pole (anode) if negatively
charged. Similar charge molecules will migrate according to their size. Smaller molecules
migrate faster through the gel matrix than the larger molecules.
The rate of migration through the electric field depends on the strength of the field, the net
charge, size and shape of the molecules, and the ionic strength, viscosity and temperature of
the buffer.
Many important biological molecules such as amino acids, peptides, proteins, nucleotides,
and nucleic acids, possess ionisable groups and, therefore, at any given pH, exist in solution
as charged species either as cations (+) or anions (-).
Materials and chemicals
A. Supporting medium
B. Buffer
C. Power supply: provides the regulated electric current
D. Detection and Quantification
E. Electrophoresis apparatus: tank, electrodes, tray, and combs.
A. Supporting medium:
1. Paper:
Whatman filter paper and Cellulose acetate paper
2. Gel:
Agarose gel is a linear polysaccharide extracted from seaweed
Starch gel: from the partial hydrolysis of potato starch.
52
Polyacrylamide gel: is a cross-linked polymer, sponge like structure made of
acrylamide.
B. Buffer composition (ionic strength, pH)
Commonly used buffer

TBE (Tris-borate-EDTA)

TAE (Tris-acetate-EDTA)

Barbital buffer and TE (Tris-EDTA) for protein
High ionic strength of the buffer increases sharp zones, but decreases the migration rate.
Function of buffer. Tris, chemical which helps maintain a consistent pH of the solution.

Carries the electric current

Determine the electric charge

Establish the pH
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. The buffer also will
generate greater heat.
C. Detection and Quantification
After the separation is complete, the molecules can be visualized by treating the gel with a
stain such as ethidium bromide, silver, or coomassie blue, which bind to the molecules but
not to the gel itself.
Bands in different lanes that end up at the same distance from the top contain molecules that
passed through the gel with the same speed, which usually means they are approximately the
same size. There are molecular weight sizes markers can be used to compare it with those of
the unknown to determine their size.
D. Determination of Molecular Weight
A linear relationship exists between the logarithm of the molecular weight of an SDSdenatured polypeptide, or native nucleic acid, and it’s Rf. The Rf is calculated as the ratio of
the distance migrated by the molecule to that migrated by a marker dye-front. A simple way
of determining relative molecular weight by electrophoresis (MW) is to plot a standard curve
of distance migrate vs log MW for known samples, and read the log MW of the unknown
sample after measuring distance migrated on the same gel.
53
Gel electrophoresis of serum proteins
Principle
Proteins can be separated by gel electrophoresis according to their size and electrical charge
by applying an electric current to them while they are in a gel. The current forces the
molecules through pores in a thin layer of gel, a firm jelly-like substance. The gel can be
made so that its pores are just the right dimensions for separating molecules within a specific
range of sizes and shapes. Smaller fragments usually travel further than large ones. Gel
electrophoresis can provide information about the molecular weights and charges of proteins,
the subunit structures of proteins, and the purity of a particular protein preparation. It is
relatively simple to use and it is highly reproducible. SDS-PAGE, Sodium dodecyle sulfatepolyacrylamide gel electrophoresis is the most commonly technique used for proteins.
To run this test, a gel is prepared with alternating wells and slots cut into it. The serum
sample is placed in the wells and electrophoresis is performed to separate the proteins. The
separated proteins are then stained with coomassie blue.
Serum proteins are separated into albumin and globulins (the total proteins of serum).
Gamma
Beta
Alpha 2
Alpha 1
Albumin
Globulins are divided into alpha-1, alpha-2, beta, and gamma globulins
The major components of serum proteins
54
Materials:
 Barbiturate buffer (0.07mol/liter, pH 8.6)
 Agarose gel (1 % w/v in the barbiturate buffer: heat the agarose at 90oC until the lumps
are dissolved, then cool to 55oC).
 Tracking dye (bromophenol blue, 0.1 g/liter)
 Electrophoresis equipment
 Human serum
 Coomassie blue (0.25 % w/v in methanol: acetic acid: water, 5: 1: 5)
 Destaining solution (methanol: acetic acid: water, 5: 1: 5)
Procedure:
1. Prepare 1% agarose solution, by measuring 1g agarose into a conical flask and add 100
ml barbiturate buffer.
2. Microwave or stir on a hot plate until agarose is dissolved and solution is clear.
3. Allow solution to cool to about 55oC before pouring.
4. Place comb in gel tray about 1 inch from one end of the tray and position the comb
vertically.
5. Pour the gel solution into tray to about half the comb to be filled. Allow the gel to
solidify about 20 minutes at room temperature.
6. Gently remove the comb, place tray in electrophoresis chamber and cover with the buffer
(the same buffer used to prepare the agarose) just until wells are submerged.
7. To prepare samples, add 1 μl of tracking dye for every 5 μl of serum. Mix well and then
load 5 μl of serum sample per well.
8. Run the samples at 90 volts until dye markers have migrated an appropriate distance.
9. Stain the gel in Coomassie blue for 10 minutes. Remove the excess stain with the
destaining solution.
55
Introduction
Also known as ELISA an abbreviation for enzyme-linked immunosorbent assay or enzyme
immunoassay (EIA). It is similar in principle to radioimmunoassay (RIA), but depends on an
enzyme rather than a radioactive label. It is widely used immunological method to detect the
presence of an antibody or an antigen in a sample such as serum or urine. The molecule as a
hormone or drug is detected by antibodies that have been made against it. An antibody reacts
with the concerned antigen in a highly specific manner to produce an Antigen-Antibody
complex.
ELISA may be run in a qualitative or quantitative format. Qualitative results provide a simple
positive or negative result for a sample. In quantitative ELISA, the amount of colored product
is proportional to the amount of enzyme-linked antibody that binds, which is directly related
to the amount of antibody that was present to bind antigen or antigen that was present to bind
antibody. If known amounts of antigen or antibody are added, a
standard curve, which is typically a serial dilution of the target,
can be constructed by plotting each standard optical density vs.
the standard concentrations on graph paper. The concentration
of each unknown antigen or antibody can then be extrapolated
and determined from the standard curve.
ELISA results are reported as a number using a spectrophotometer, spectrofluorometer, or
other optical device. This test is determining the "cut-off" point between a positive and
negative result.
Unknowns that generate a signal that is stronger than the known
sample are called "positive"; those that generate weaker signal are
called "negative."
Most commonly, ELISAs are performed in 96-well (or 384-well)
usually polystyrene microtiter plates, which will passively bind
96 - well microtiter plate
antibodies and proteins
56
Advantages of ELISA

Less costly and safest.

Easy visualization of results with high level of accuracy.

Highly specific and sensitive assay that can detect protein at the picomolar to
nanomolar range.

Easily automated for performance of large numbers of tests.

Require minimal reagents.

Qualitative detection or Quantitative measurement of either antigen or antibody.

Wells can be coated with antigens or antibodies.

Can be done by personnel with only minimal training.
Applications of ELISA

Analysis of hormones, vitamins, metabolites, and diagnostic markers.

Therapeutic drug monitoring.

Diagnostic procedures for detecting infection.
Requirements for ELISA test

Purified antigen (if you want to detect or quantify antibody).

Purified antibody (if you want to detect or quantify antigen).

Standard solutions (positive and negative controls).

Sample to be tested.

Microtiter plates: plastic trays with small wells in which the assay
is done.

Wash fluid (buffer).

Enzyme-labeled antibody and enzyme substrate.

ELISA reader (spectrophotometer) for quantitative measurements.
Enzyme labels
Enzyme labels are used to detect the binding of antigen-antibody complex. It should have
high specific reactivity. The reactivity should be retained after linking of the enzyme to the
antigen/antibody. It should be easily coupled to antigen-antibody complex and must stable.
Enzymes used in labelling should not be normally present in the patient samples. Examples
of enzyme labels are Horse radish peroxidase, Alkaline phosphatase, and Glucose oxidase.
57
Stages in ELISA
1. The adsorption of either antigen or antibody to the plastic solid phase (micro-titer
plate).
2. The addition of the test sample and subsequent reagents.
3. The incubation of reactants (formation of antigen-antibody complex).
4. The separation of bound and free reactants by washing.
5. The binding of enzyme-conjugated antibody or antigen to the target antigen or
antibody.
6. The addition of substrate (production of a visible signal).
7. The visual or spectrophotometric reading of the assay.
Types of ELISA assay
There are variations of the ELISA test, but the most basic type consists of an antibody
attached to a solid surface. The antibody has affinity for the substance of interest. Variation
of different types of ELISA assay depends upon the labeling and signal detection
methodology. ELISA used for detecting antibody-antigen complex by fixing either antigen or
antibody.
Substrate
Direct ELISA
In this ELISA method, the antigen is first adsorbed to the
plate followed by an enzyme- conjugated primary
E
Primary
antibody
antibody. The color or the signal produced as a result of
addition of substrate is proportional to antibodies in the
Antigen
Ag
Direct ELISA
sample.
Indirect ELIS
Substrate
This method differs than direct ELISA in that the primary
antibody
is
not
labeled.
An
enzyme-conjugated
secondary antibody, directed at the first antibody, is then
added. This format is used most often to determine
whether a specific antibodies present in the serum. The
color or the signal produced as a result of addition of
substrate is proportional to antibodies in the sample.
E
Secondary
antibody
conjugate
Primary
antibod
y
Antigen
Ag
Indirect
ELISA
58
Sandwich ELISA
Substrate
It measures the amount of antigen between two layers
of primary antibodies –the capture antibody and the
detection antibody, just like a sandwich. The capture
antibody is adsorbed to the plate. The antigens to be
measured must contain at least two antigenic sites,
since at least two antibodies bind to antigen. The
color or the signal produced as a result of addition of
substrate is proportional to antigen.
E
Secondary
antibody
Detection
antibody
Target
antigen
Ag
Capture
antibody
Sandwich ELISA
Competitive ELISA
It measures the amount of antigen in a sample. In this type of ELISA, the antigen is labeled
instead of the antibody. Unlabeled antigen from samples and the labeled antigen compete for
binding to the capture antibody. The color or the signal produced as a result of addition of
substrate is inversely proportional to antigens in the sample. For example, the absence of the
antigen in the sample will result in a dark color, whereas the presence of the antigen will
result in a light color or no color as the concentration of the antigen increases.
Substrate
Labele
Antigen
Target
antigen
Substrate
E
Ag
E
Ag
Ag
E
Ag
Capture
antibody
Competitive ELISA
59
E
Ag
Ag
E
Ag
Ag
References:
1. Plummer D.T., An introduction to Practical biochemistry, Tata McGraw – Hill.
2. http://www.columbia.edu/cu/biology/courses/c2005/hand04.html
3. http://www.morrisonlabs.com/ph_study_guide.htm
4. http://www.ph-meter.info/
5. Carlsberg Research Centre history page, http://www.crc.dk/history.shtml
6. Definitions of pH scales, standard reference values, measurement of pH, and related terminology
7. http://hyperphysics.phy-astr.gsu.edu/hbase/chemical/ph.html
8. http://www.virtualsciencefair.org/2005/wali5s0/public_html/pH_scale.htm
9. http://www.gcsechemistry.com/aa29.htm
10. http://www.bbc.co.uk/schools/gcsebitesize/chemistry/chemicalreactions/0acidsbasesrev2.shtml
11. http://www.seafriends.org.nz/dda/ph.htm
12. http://znk.ptfos.hr/dodatno/suradnja/ph%20meter.htm
13. http://www.chem.tamu.edu/class/majors/tutorialnotefiles/buffers.htm
14. Pictures are captured & drawn by KAU/Biochemistry department.
15. http://www.aai.org/committees/education/Curriculum/gel.htm
16. Plummer D.T., An introduction to Practical biochemistry, Tata McGraw – Hill.
17. David J Holme and Hazel Peck (1993) analytical biochemistry, second edition, Longman
Singapore, Singapore, pp. 143-145.
18. http://www.colby.edu/chemistry/CH367/laboratory/
19. http://www.unm.edu/~rrobergs/titration.htm
20. http://www.daviddarling.info/encyclopedia/G/glycine.html
21. http://www.hannainst.co.uk/acatalog/Getting_the_best_from_your_Colorimeter.html
22. http://dl.clackamas.edu/ch105-04/importan.htm
23. http://faculty.uca.edu/~march/bio1/scimethod/spectro.htm
24. http://abacus.bates.edu/~ganderso/biology/resources/spec20.html
25. http://www.biology.lsu.edu/introbio/tutorial/webtutorial/spectrophotometry.php
26. http://services.juniata.edu/ScienceInMotion/chem/labs/spec/maxwave.doc
27. http://www.diycalculator.com/sp-cvision.shtml
28. http://www.wfu.edu/academics/chemistry/courses/CC/index.htm
29. http://www.piercenet.com/browse.cfm?fldID=F88ADEC9-1B43-4585-922E-836FE09D8403
30. http://immunoassays.blogspot.com/
31. http://exploreable.wordpress.com/2011/05/25/elisa-enzyme-linked-immunosorbent-assay/
32. Holme DJ and Peck H, Analytical Biochemistry (2nd edition).
33. cellbiologyolm.stevegallik.org/node/74
34. http://ga.water.usgs.gov/edu/meniscus.html
60
Useful links:
 Buffer solution
 pH
 Amino acid titration curve and pKa values
 ph-meter
 Buffer solutions
 Solutions
 Phosphate buffer
 Buffer preparation
 Buffered solution
 ph_meter
 http://www.moafa.com/c3.htm
 Chromatography-online
 Chromatography menu
 Chromtutorial
 Biochemlab
 See these videos for TLC: Video 1 and Video 2
See this video for the differences between absorption and adsorption
 TLC and Column Chromatography part 1
TLC and Column Chromatography part 2
TLC and Column Chromatography part 3
TLC and Column Chromatography part 4
 The spectrophotometer: A demo and practice experiment
 Lycopene
 Carotenoids
 Column chromatography
 Column Chromatography Procedures
 Separatory Funnel Extraction Procrdure
 Principles of gel filtration chromatography
 http://www.hoeferinc.com/
 http://en.wikipedia.org/wiki/Gel_electrophoresis#cite_note-1
 http://www.mcb.uct.ac.za/sdspage.html
 http://www.spectrapor.com/dialysis/Fund.html
 www.membrane-mfpi.com/labdialy.html
 http://www.membrane-mfpi.com/t_instrs.html
 http://web.siumed.edu/~bbartholomew/course_material/protein_methods.htm
 http://highered.mcgrawhill.com/sites/0072556781/student_view0/chapter33/animation_quiz_1.html
 http://www.sumanasinc.com/webcontent/animations/content/ELISA.html
61