BIOCHEMISTRY LABORATORY
PROTEINS, ENZYMES, AND CHROMATOGRAPHY (3)
JAILOUISE A. PEREZ
BACHELOR OF SCIENCE IN NURSING
PROTEINS AND ENZYMES
Proteins
 are large, complex molecules that play many critical
roles in the body. They do most of the work in cells and
are required for the structure, function, and regulation
of the body's tissues and organs
 Amino acids are the building blocks of proteins.
Laboratory Techniques
Isoelectric Focusing of Casein
 Casein, like proteins, are made up of many hundreds
of individual amino acids. Each may have a positive or
a negative charge, depending on the pH of the system.
At some pH value, all the positive charges and all the
negative charges on the [casein] protein will be in
balance, so that the net charge on the protein will be
zero.
 Milk is a mixture of many types of proteins, most of
them present in very small amounts. Milk proteins are
classified into three main groups of proteins based on
their widely different behaviors and forms of existence.
They are caseins (80%), whey proteins and minor
proteins.
 Casein is a heterogeneous mixture of phosphorous
containing proteins in milk. Casein is present in milk as
calcium salt and calcium caseinate. It is a mixture of
alpha, beta, and kappa caseins to form a cluster called
micelle. These micelles are responsible for the white
opaque appearance of milk.
 That pH value is known as the isoelectric point (IEP) of
the protein and is generally the pH at which the protein
is least soluble. For casein, the IEP is approximately
4.6 and it is the pH value at which acid casein is
precipitated. In milk, which has a pH of about 6.6, the
casein micelles have a net negative charge and are
quite stable. During the addition of acid to milk, the
negative charges on the outer surface of the micelle
are neutralized (the phosphate groups are protonated),
and the neutral protein precipitates.
Materials
1. Raw milk - 100ml
2. 0.2N HCl - 50ml
3. Diethyl ether - 50ml
4. 50% Ethanol - 50ml
5. Filter paper strip
6. Beaker
7. Glass stirring rod
8. Centrifugation Device
*Alternative - use non-fat milk if centrifuge machine is
not applicable*
Procedure
 Step 1: Measure 100ml of milk in a measuring cylinder
and transfer 25ml of milk to four Oakridge centrifuge
tubes each.
 Step 2: Centrifuge the milk in a centrifuge at 4000rpm
at room temperature (25- 30o C) for 20 minutes. This is
done to remove the fats and lipids from the mixture.
 Step 3: After centrifugation, carefully remove the fats
and lipids from the surface of the milk with a spatula.
 Step 4: Then transfer the milk from all the tubes into a
beaker and add equal volume of distilled water and stir
well. Now check the pH.
 Step 5: Start adding 0.2N HCl drop by drop into the
milk mixture and stir well.
 Step 6: Note the PH at which precipitation (white curdy
substances) appears. The pH should be 4.6.
 Step 7: Take the curdy precipitate and allow it to
sediment.
 Step 8: Now decant the supernatant using a filter paper
and funnel and wash the precipitate with distilled water
to remove the salts, then wash with diethyl ether and
ethanol.
 Step 9: Dry the precipitate and take the weight of the
casein and record it.
Isolation of Gluten from Flour
Gluten is a combination of the natural proteins, glutenin
and gliadin, found in the flour. Gluten molecules are activated
when flour is moistened and then mixed. When this happens,
the glutens literally stretch out as the proteins form longer and
longer chains. Isolation of proteins is the process of separating
a type of protein from a complex mixture. As for gluten, it is
separated through its difference in solubility. Insoluble proteins
are easily isolated, removing other substances that are soluble
by washing.
Materials
 Flour (Types of Flour - have different gluten content)
 Tap water
 Cheesecloth (alternative - clean cloth)
 Bowl
Methods
 Gluten can be readily prepared by gently washing
dough under a stream of running water. This removes
the bulk of the soluble and particulate matter to leave a
proteinaceous mass that retains its cohesiveness on
stretching. Gluten comprises some 75% protein on a
dry weight basis, with most of the remainder being
starch and lipids.
 Through a process called centrifugation the major
constituents of the flour are separated. The starch and
other constituents dissolve, but the gluten, which is not
water soluble, does not. Once starch and gluten are
separated by centrifugation, the gluten is washed
thoroughly and dried.
Procedures
 Select the flour.
 Put the flour in the cheesecloth then make sure there
will be a bowl underneath it.
 Gently wash the flour under the stream of running
water.
 Knead to develop the proteins into gluten.
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 Rinse it until the dough become elastic.
Biuret Test
 Biuret is a chemical made by heating urea, which
causes two urea molecules to condense into one.
Biuret is named for the peptide bonds in the reagent
that produces a positive response in the test. For
substances (proteins and peptides) with two or more
peptide (CO-NH) bonds, it is used as a generic test.
 A Biuret test is a chemical test that determines whether
or not a sample contains a peptide link. It is based on
the biuret reaction, in which an alkaline copper sulfate-
treated peptide structure with at least two peptide
linkages creates a violet color. The colored
coordination complex is generated by the Cu2+ ion and
the peptide bond's carbonyl oxygen (>C=O) and amide
nitrogen (=NH).
 The solution changes color from blue to purple once
this complex has formed. The more purple the tint, the
more peptide-copper complexes are present. The color
intensity is proportional to the number of peptide bonds
present in the responding protein molecule, as well as
the number of protein molecules present in the reaction
system.
 A solution of sodium hydroxide (NaOH) or potassium
hydroxide (KOH), hydrated copper (II) sulfate, and
potassium sodium tartrate is used to make the Biuret
reagent. The alkaline medium is made out of sodium
hydroxide and potassium hydroxide, with potassium
sodium tartrate added to chelate and thereby stabilize
the cupric ions in the solution, or to keep their solubility
in alkaline solution.
Biuret Test Principle
 The copper (II) present in the reaction binds itself to
the nitrogen atoms that are present in the protein
peptides. Since this test is not greatly disturbed by the
presence of amino acids in the sample, it can be used
to gauge the concentration of proteins in whole tissue
samples. However, the samples of proteins that are
purified via ammonium sulfate ((NH4)2SO4)
precipitation are not ideal for this test since buffers like
ammonia interfere with it.
 Now, four nitrogen atoms donate lone pairs to form
coordinate covalent bonds with the cupric ion, resulting
in the formation of a chelate complex. This chelate
complex has the ability to absorb light with a
wavelength of 540nm, which imparts a purple colour to
it. Therefore, the formation of a purple coloured
complex indicates the presence of proteins in the
analyte.
Procedure
 Step 1: Take 3 clean and dry test tubes.
 Step 2: Add 1-2 ml of the test solution, egg albumin,
and deionized water in the respective test tubes.
 Step 3: Add 1-2 ml of Biuret reagent to all the test
tubes.
 Step 4: Shake well and allow the mixtures to stand for
5 minutes.
 Step 5: Observe for any color change.
Observation and Interpretation
 No color change, i.e., the solution remains blue
Proteins are absent (Negative Biuret Test)
 The solution turns from blue to deep purple Proteins
are present (Positive Biuret Test)
Application
1. It can be used to detect the amount of protein in the
urine.
2. Biuret reaction with protein is applicable to the
quantitative determination of total protein by
spectrophotometric analysis.
Ninhydrin Test
 The ninhydrin test is a chemical test which is utilized
and performed to detect the presence of ammonia and
whether a given analyte contains amines or α- amino
acids. Ninhydrin is most commonly used as a forensic
chemical to detect “fingerprints”.
 In order to execute this test, ninhydrin is being added
to the test solution of the analyte. Ninhydrin reacts with
the α-amino group of primary amino acids producing
‘Ruhemann’s purple’. The chromophore formed is the
same for all primary amino acids. The intensity of the
colour formed depends on the number and chemical
nature of the amino groups being analysed. The
optimum pH for the overall reaction is 5.5. Ruhemann’s
purple has a spectral maximum at 570 nm
 An amino group that belongs to a free amino acid goes
through a chemical reation with ninhydrin, which acts
as an oxidizing agent. The amino acid goes through
oxidative deamination that results to the freedom of
CO2, NH3, and an aldehyde alongside hydrindantin
(which is a reduced type of ninhydrin) when it is
presented to the ninhydrin,
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Materials
1. Test tubes
2. Test tube stand
3. Pipette
4. Water bath
5. Spectrophotometer
Reagents
1. Ninhydrin reagent
2. Standard solution
3. Sample solution
Procedure
 Step 1: First, a 2% solution of ninhydrin must be
prepared by dissolving
 0.2 grams of ninhydrin in 10ml of either ethanol or
acetone.
 Step 2: Now a 1% solution of the amino acid (analyte)
in distilled water must be prepared. A few drops of the
2% ninhydrin solution must be added to this solution.
 Step 3: The test tube must be kept in a warm water
bath for approximately 5 minutes.
 Step 4: The development of a deep blue/violet colour
indicates the presence of amino acids.
Results
 Positive Test
o Blue-purple and yellow pigment occurs on its
reaction products
o Indication: Positive result indicates that there
is a presence of amino acid in the sample.
 Negative Test
o No change of color and any other observable
variety within the test solution.
o Indication: Negative result indicates that the
sample lacks of amino acids.
 For ammonia, primary/secondary amines, and amino
acids, deep purple color is obtained.
 For hydroxyproline and proline, a yellow color is
obtained.
 For asparagine, brown color is obtained.
 If no color change is observed, the analyte does not
contain amino acids, amines, or ammonia.
Xanthoproteic Test
 Xanthoproteic test is considered a biochemical test that
determines the detection of amino acids containing
phenolic or indolic groups like phenylalanine, tyrosine,
and tryptophan (aromatic amino acids). The test will
give a positive result for the amino acids that contain
benzene rings or other different aromatic groups. It is a
qualitative test that provides information and
determines the presence and absence of amino acids.
Principle of Xantoproteic Test
The Xanthoproteic test relies on the very fact that
aromatic groups within the amino acids are nitrated by heating
with concentrated HNO3 to yield intensely yellow-colored nitro
derivatives. In addition to alkali, the residue turns orange
because of the formation of a salt of the tautomeric kind of nitro
compound. Benzene ring-containing amino acids like
phenylalanine don’t provide a positive test to the current test
because the phenyl group in phenylalanine is extremely stable,
which doesn’t react with nitric acid within the conditions of this
test. However, phenylalanine might provide a positive result
after an extended period of heating.
Materials
 Test tubes
 Test tube stand
 Pipettes
Procedure of Xanthoproteic Test
1. About 1 ml of the sample solution is taken in a test
tube. To this, the same amount of concentrated nitric
acid is added.
2. The test tube is allowed to cool down to room
temperature. If the sample is a protein solution, a white
precipitate might develop due to the denaturation of
proteins.
3. Then, 1 ml of 40% NaOH solution is added to the test
tube and observed for color change.
Results and Interpretation
 Positive result: The appearance of a dark yellow or
orange-colored solution represents a positive test. This
indicates the presence of aromatic groups in the
proteins and amino acids.
 Negative result: The absence of a dark yellow or
orange-colored solution represents a negative test.
This indicates the absence of aromatic groups in
proteins and amino acids.
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 2. Add 1 ml of 10% percent of acid mercuric nitrate
solution on.
3. Boil gently for 30 seconds.
4. Cool down.
5. Mix well (observe color formation)
6. Formation of pinkish red color indicates the presence
of hydoxy phenyl (phenol) group containing amino acid
tyrosine.

Millon’s Test

Results and Interpretation

Millon’s test is an analytical test used for the detection
of the amino acid tyrosine, which is the only amino acid
containing the phenol group. Millon’s test is a specific test for
tyrosine, but it is not a specific test for protein as it also detects
the phenolic group present in other compounds as well.
Therefore, while performing Millon’s test, it is essential that
other tests like the Biuret test and Ninhydrin test also be
performed. As many proteins consist of tyrosine, the test is
useful in the detection of such proteins. The test was discovered
by and named after the French Chemist Auguste Nicolas
Eugene Millon.
 A POSITIVE RESULT or outcome in the Millon's test is
shown by the development of a red or pink shaded
pigment that has arouse or accelerated. With this, it
has implied that is has a strong presence of tyrosine or
tyrosine containing protein.
 A NEGATIVE RESULT or outcome in the Millon’s test
is shown by the absence of any color shade that has
aroused or accelerated upon executing the experiment.
There is no visible change on its color. This implies
that there is a shortfall and lack of tyrosine or tyrosine
containing protein in the test solution.

Reaction Involve

Sakaguchi Test
 The Sakaguchi test is a colorimetric biochemical test
Millon's test is based on the principle of nitrification of for the detection and quantification of guanidinium
the phenol group in tyrosine, which then forms complexes with groups that is used as a qualitative test for arginine
heavy metals like mercury. The reagent used for the test is that is either free or in protein. The Sakaguchi test is
called Millon's reagent, and it consists of mercuric nitrate and an example of a color reaction or test used to identify
mercurous nitrate that is dissolved in concentrated nitric acid. amino acids or proteins.

Materials Sakaguchi Test Principle

Equipment
1. Test tube
2. Test tube stand
3. Pipettes
4. Water bath

Reagents
 10% precent mercuric nitrate in 10% percent nitric acid
(acid mercuric nitrate solution)
 Test solution: 1 % arginine, 1 % tyrosine, egg albumen, The arginine reacts with α – napththol and an oxidizing
phenol solution agent such as bromine water or sodium hypochlorite/sodium
hypobromite to give a red colored product.
Procedure
1. Take 1 ml of above solutions in different test tubes. Reagent

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 Sakaguchi reagent: 1% 1-naphthol in alcohol with a  The test is qualitative, but with the addition of urea,
few drops of a 10% sodium hypobromite solution in which stabilizes the colored result, it can be made
bromine water. quantitative.
 40 percent sodium hydroxide a case study (0.1 percent
of arginine or 0.1 percent of creatine) Limitations
 Because Sakaguchi's reaction is sluggish, quantitative
examination of the colored result is not possible with
this test.
 Similarly, the hypochlorite may damage some of the
guanidinium groups in the solution, causing problems
with testing findings.

Lead Acetate Test

Lead Acetate test (or Lead Sulfide test) is a
biochemical test for the detection of amino acids like cysteine
and cystine. The test is a specific test for the detection of amino
acids containing sulfur, S-H group in cysteine, and S-S group in
cystine. The test is mainly called lead acetate test as the
Required Materials reagent for the test is lead acetate. Even though the test is
 Tubes for testing specific for the detection of sulfur-containing amino acids,
 Stand for test tubes methionine doesn’t give a positive result in this test.
 Pipettes
Reaction Involved

The sulfur-containing amino acid such cysteine and

cysteine (sulfhydryl/thiol group) reacts with lead acetate under
Procedure alkaline conditions to form a brown precipitate. These sulfur-
 Step 1: In a test tube, 3 ml of the test solution is added, containing amino acids are degraded in strongly alkaline media
followed by 1 ml of 40 percent NaOH, which is properly to release sulfide ion (S2-) in the form of H2S (hydrogen
mixed. sulfide). The sulfide ions can react with lead (II) acetate to form
 Step 2: Then, in the same test tube, two drops of 1- a brownish-black precipitate.
naphthol are added and thoroughly mixed.
 Step 3: 4-5 drops of sodium hypobromite (10%) or Materials
bromine water are now added. 1. Test tube
 Step 4: Color development in the test tube is 2. Test tube stand
monitored. 3. Pipettes
Results
Reagents
 POSITIVE: The creation of red color indicates a
1. 2% lead acetate solution
positive result on the Sakaguchi's test. This suggests
2. 40% NaOH
that an arginine or guanidinium molecule is present.
3. Sample
 NEGATIVE: The absence of red color indicates a
negative result in Sakaguchi's test. This suggests that Methods
arginine or a guanidinium molecule is missing.
 In a test tube, 2 ml of the amino acid solution is taken.
To this, 2 ml of NaOH is added, and a few drops of
lead acetate.
 The solution is boiled for a minute. Once the test tube
cools down, observe the solution.
 The test tube is then observed for the formation of a
precipitate

Results

Application
 Sakaguchi's Assay is a biochemical test for detecting
arginine in proteins, either free or mixed.
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 POSITIVE: There would be a formation of black
precipitate.
 NEGATIVE: There would be no formation of black
precipitate.
Bradford Assay
Background
 In 1976, an American scientist named Marion M.
Bradford developed the highly known and widely used
Bradford protein assay. What Bradford created is a
procedure for determining the concentration of protein
in a solution in a fast, easy, and accurate way. It also
determines the protein content of cell fractions and
asses protein concentrations for gel electrophoresis.
 Bradford assay’s principle results in the color change
from brown to blue by the binding of protein molecules
to Coomassie dye under acidic conditions. This assay
method contributes to formation of the protein-dye
complex and strictly measures the presence of the
basic amino acid residues, arginine lysine, and
histidine. Also, in the Bradford assay, samples that are
out of range can be retested within minutes.
Use
The Bradford assay is used to calculate the
concentration of the total amount of protein in a given sample. It
utilizes standards to both assess the amount of protein in
samples and to subtract any background due to interfering
substances that can shift the ratios between the three forms of
the dye. Samples that have protein concentrations higher than
the concentrations in the linear range must therefore be diluted
and re-assayed to obtain a more accurate estimation of the
protein concentration.
Materials and Methods
1. Summarized Procedures
a. Dilute protein sample with buffer (or protein
standards)
a. To fit in the standard curve (linear curve)
b. Linear curve – forms line using different
data sets, data is in real world set ups
and maintain pattern- we attempt the
pattern to represent plots or trend, real
world set ups may give us scattered lines
b. Add Bradford reagent
c. Incubate for 5 minutes
d. Read absorbance using spectrophotometer
a. Spectrophotometer- equipment; light will
pass on the sample and will be measured
through its color of light. The color of light
in the electromagnetic spectrum has a
specific wavelength. The result of the
sample when mix to Bradford reagent is
blue, we use color wheel to determine its
contrary color which is orange. The
wavelength of orange color is around
600-650 nm. Bradford assay can be
measured in 595 nm.
b. Refer to the established standard curve
equation to estimate protein
concentration of sample.
2. Full materials & procedures (long version)
a. Kindly refer to this link
https://www.youtube.com/watch?v=vfY3mVOlGBU
Conclusion
 The Bradford assay is based on the dye name
Coomassie brilliant blue G-250 that binds to the side
chain of basic amino acid. The sulfonic acid groups of
the dye interact with the positive amino group of the
proteins found in the basic amino residues present in
lysine, arginine, and histidine; this interaction makes
the brown color of Coomassie blue G-250 into a blue
color.
 To determine the amount of concentration of a protein,
first is we need to examine and measure a solution
without any protein content, next is we need a
reference solution where a known protein
concentration is measured into 3 different
concentration sample of an increasing optical density
such as 2 and 4 microgram so that we can determine
an unknown protein sample by the use of
spectrophotometer and with extrapolation, we can
calculate the protein concentration of the unknown
protein sample.
ENZYME
Enzyme
 Proteins that operate as biological catalysts are known
as enzymes. Catalysts help to speed up chemical
reactions. Substrates are the molecules on which
enzymes can function, and the enzyme changes the
substrates into various molecules called products.
 Enzymes are essential in different functions like
respiration, digesting food, muscle, and nerve function.
But for this type of experiment, we are going to focus
on the food digestion.
Salivary Amylase Enzyme Assay
Salivary Amylase
 Food are made up of a large molecules like lipids,
carbohydrates, and proteins that are too big to move to
our blood, in regard with this we need to digest it into
smaller molecules. And this is where physically
processes like chewing and chemically processes by
mean of special proteins called enzymes takes place.
 There are different types of food molecules we need to
digest and those are the lipids, carbohydrates, and
proteins. For this type of digestion, we are going to
focus on carbohydrates.
 Carbohydrates types of food can be found on rice and
pasta. The simplest carbohydrates are sugars which
can be joined into a big chain called starch. These
complex carbohydrates can be digested by these
carbohydrates’ enzyme called amylase. Amylase is a
special type of carbohydrates that breaks down starch
into smaller molecules which can be broken down
further into glucose which is small enough to be moved
into the blood.
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 Amylase can be found on saliva, where carbohydrates
will first start to be broken down chemically.
Effects of Different Factors on Enzyme Activity
1. pH
a. pH affects the enzymes activity by affecting
the structure of the enzymes itself.
b. As enzyme changes in pH, the structures of
the enzyme changes by means that the
substrate and the enzymes cannot bind
completely because of there charges. Many
amino acids in an enzyme molecule carry a
charge and these charges contributes to the
folding of the enzyme molecule, its shape,
and shape of the active site.
c. Thus changing the pH will affect the charges
on the amino acid molecules, which can affect
the shape of the active sites.
d. There are some instances where pH can work
on different enzymes with their own optimum
pH. Here for example the optimum pH of the
salivary amylase is at 6.8 pH level starting
from 4.5, the optimum pH is the highest point
of activity of the enzymes. Here we can see
as the pH decreases from the optimum pH,
the rate of enzyme activity decreases, and as
pH increases from optimum pH, rate of
enzyme activity decreases.
Enzyme Optimum pH
Salivary Amylase 4.5 – 6.8
Stomach Protease (pepsin) 1.5 – 2.0
Pancreatic Protease (trypsin) 3.5 – 8.0
e. The symmetrical shape of this enzyme activity
shows that below the optimum pH, the
enzymes start to denature. And above the
optimum pH, the enzymes start to denature
as well. Denature of denaturation means that
the enzymes active site loses it specific 3D
shape like on the temperature the enzymes
break down, the enzymes cannot also bind to
it substrate.
2. Temperature
a. As the temperature increases, the rate of
enzyme activity also increases which gives
the enzyme a more chance to bind with its
substrate.
b. Optimum temperature - the highest rate of
enzyme activity (for human the optimum
temperature is at 37 degrees Celsius).
Beyond the optimum temperature the rate of
enzyme activity decreases. Because as the
temperature goes higher the chemical bond
(intra and intermolecular bonds) that hold the
enzymes molecules begin to break down or
denature because the enzyme molecules gain
even more kinetic energy. (To be more
specific the active site of the enzyme can be
destroyed and therefor the substrate can no
longer bind to the active site).
c. At low temperature enzyme activity is almost
zero as well, because the kinetic energy of
enzyme and substrate moves slower and the
chances for them to bind as enzyme substrate
complex are low, which therefore conclude
having a low temperature gives a low rate of
activity for enzyme.
3. Substrate Concentration
a. As the temperature increases, the rate of an
enzyme-catalyzed reaction increases as the
temperature increases. At low temperatures,
the rate increases again because the enzyme
becomes denatured and can no longer
function.
b. Increasing the temperature (which is a
measure of the thermal energy in the system),
therefore, increases the frequency of the
collisions and thus increases the rate at which
the product is formed.
c. As you increase the concentration of the
substrate, the enzyme activity increases as
well as the rate of the reaction. In addition, as
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 you increase the concentration of the enzyme,
the rate of the chemical reaction will increase.
d. At some point, the rate is going to level off, as
for inhibitor A at 2 Mm ( having the maximum
amount of product formed) which can be due
to a limited number of enzyme molecules that
can interact with the substrate, by the case
that there is a maximum rate upon which it
works where there's a certain limit where if
you keep an increase in the concentration of 1. When inhibitor A binds with
the substrate, the rate will not keep increasing the enzyme, it doesn’t
as well. produce any product not
e. The rate of enzyme activity proportionally like when substrate bind
increases with the increase of substrate with enzyme it does
concentration until it reaches a maximum produce a product.
point. With this there will be a higher chance Because the inhibitor A only
of collision between the enzyme and the mimicking or copying the
substrate. Beyond a certain concentration of substrate but it doesn’t
substrate. really produce any product.
f. As the concentration of the substrate 2. For example, with a
increases, the enzyme activity increases as constant of 50 mmol/L of
well as the rate of the reaction. In addition, as substrate concentration.
you decrease the concentration of the The inhibitor A are
enzyme, the rate of the chemical reaction will decreasing little by little as
decrease. concentration of the
g. At low substrate concentrations, the reaction inhibitor increasing, from
rate increases sharply. But as the substrate 41.09 mM from its optimum
concentration climbs, the reaction rate begins pH suddenly decreased to
to increase less and less until it comes to a 38.23 mM with just an
point where it plateaus into a flat line. The increase of 87.5 mM of
reason this happens is because the enzyme inhibitor concentration.
becomes saturated with substrate.

4. Presence of Inhibitors
a. Irreversible Inhibitors - it permanently
inactivates the enzyme, meaning even you
increase your substrate concentration it does ii. Non-Competitive Inhibitors - binds to
not reverse the inhibition process of this the enzyme at allosteric site or the
inhibitors that’s why it is not compatible to use site away from the active site. When
as a inhibitor. non-competitive inhibitors bind to the
b. Reversible Inhibitors - It binds to enzymes enzyme it changes the shapes of the
either on the active site or in the allosteric site active site.
and only binds temporarily.
i. Competitive Inhibitors (Inhibitor A) - it
competes with the substrate for the
same active site of the enzyme.

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 1. Unlike the competitive
inhibitors it can compete
and win against the inhibitor
by simply adding the
substrate concentration.
Here in non-competitive
inhibitor or inhibitor B
increasing the substrate
concentration will not
relieve the inhibition.
Conclusion
 Salivary amylase enzyme assay is a laboratory
technique focused on the investigation of the effects of
pH, incubation, temperature, and substrate
concentration on the activity of specific enzyme. It was
concluded that for the pH, pH affects the enzyme
activity by affecting the structure of the enzyme. For
temperature, at low temperature, enzyme activity is
almost zero as well, while, as the temperature
increases, the rate of enzyme activity also increases
which gives the enzyme more chance to bind with its
substrate. For the concentration of substrate, the rate
of enzyme activity proportionally increases with the
increase of substrate concentration until it reaches the
maximum point.
 There is also a factor that affects the enzyme activity
which is the inhibitor, we discuss the two types of
reversible inhibitor both competitive and non-
competitive, where competitive inhibitor competes with
the substrate for the same active site of the enzyme,
the competitive inhibitor doesn’t produce product when
it is bind to the enzyme, therefore decrease the
enzyme activity by simply mimicking or copying the
substrate. While, non-competitive inhibitor it binds to
the allosteric site of an enzyme where it changes the
shape of an enzyme and make it difficult for the
substrate to bind and catalyze with the enzyme.
ELISA
The ELISA (enzyme-linked immunosorbent assay)
technique is a plate-based assay for detecting and measuring
peptides, proteins, antibodies, and hormones. An antigen must
be immobilized on a solid surface before being complexed with
an antibody coupled to an enzyme in an ELISA. The activity of
the conjugated enzyme is measured by incubating it with a
substrate to yield a quantifiable result. A highly specific
antibody-antigen interaction is the most important part of the
detection approach.
How does ELISA work
The concepts of enzyme-linked immunosorbent tests
(ELISA) are essentially similar to those of other immunoassay
methods. Specific antibodies bind the target antigen in ELISAs,
and a detection device indicates the presence and amount of
antigen binding. The plate must be thoroughly coated with high-
affinity antibodies to maximize the assay's sensitivity and
precision — a procedure that Boster Bio has mastered.
Materials
1. Pipettes, washer system, ELISA plate reader:
Readers, washers and pipettes are available as
manual or automated systems. One of the main factors
affecting equipment selection is the number and types
of test samples being run.
o ELISA Readers: Readers need to have
appropriate filters (650 nm and 450 nm).
o Pipette: Are available as fixed as well as
adjustable volume as well as single channel
and multi-channel.
o Washing system: It can be a manual system
that washes one row or column at a time or
semi-automated systems that wash one strip
or plate at a time or fully automated systems
that can process multiple plates
2. Reagents needed for the testing – Concluded in the kit
(coated plates, sample diluents, controls, wash
concentrate, conjugate, substrate, stop solution)
o Coated plates: The 96-well plates are made of
polystyrene and are coated with either
inactivated antigen or antibody. The function
of the plate has to hold the immobilized
antigen or antibody. Antigen or antibodies
present in the sample will bind to the plate.
This coating acts as the binding site for the
antibodies or antigens in the sample.
o Controls: Negative and positive controls are
provided in each kit. The controls help to
normalize or standardize each plate. Controls
are also used to validate the assay and to
calculate sample results. Controls might be
pre-diluted and ready to use. (Please refer to
the kit for specific instructions).
o Conjugates: ELISA conjugates are enzyme
labeled antibodies that react specifically to
plate bound sample analytes. Unbound
conjugates are washed away after incubation
and before the addition of substrate.
o Wash Concentrate: It acts as a buffered
solution containing detergent to wash
unbound material from the plate. (Not all test
kits have wash concentrate; in that case
distilled water can be used for washing;
please refer to kit insert for specific
instructions)
o Stop solution: It stops the enzyme substrate
reaction and color development.
Role/Function of Each Step
1. Preparation of reagents and equipment
9
 7. Washing
a. Remove the samples, and wash 5 times with
washing solution.
2. Immobilization of antibody
a. Add diluted antibody to each well of a 96-well
ELISA plate. Seal the plate to prevent
evaporation, and allow it to incubate at 4°C for
15-18 hours to immobilize the antibody.

8. Addition of Detection Antibody

a. Dilute the detection antibody in sample
dilution buffer, and add 100 µL to each well.
b. Allow it to incubate at 37°C for 1 hour.
3. Washing
a. Remove the diluted antibody, and wash 3
times with washing solution.

9. Washing
a. After reaction, remove the detection antibody,
and wash 5 times with washing solution.
4. Blocking buffer
a. Add blocking buffer to each well, and allow it
to incubate at 37°C for 1 hour to reduce non-
specific binding of the target protein to the
well

10. Addition of enzyme-linked secondary antibody

5. Washing
a. Remove the blocking buffer, and wash 3
times with a washing solution.

11. Washing

6. Addition of Samples\
a. Dilute the samples with sample dilution buffer,
and add 100 µL of each sample to each well.
For the calibration curve, prepare a dilution 12. Addition of substrate solution
series of the standard on the same plate.
Allow it to incubate at 37°C for 1 hour.

10

13. Addition of stop solution
14. Measuring using plate reader
4 Types of Elisa
1. Direct Elisa - An antigen or sample is directly
immobilized on the plate in a direct ELISA, and a
conjugated detection antibody binds to the target
protein. After that, the substrate is added, resulting in a
signal proportional to the amount of analyte in the
sample. Direct ELISAs are less specific than sandwich
ELISAs since just one antibody is utilized.
a. Advantages
i. Quick because only one antibody
and fewer steps are used.
ii. Cross-reactivity of secondary
antibody is eliminated.
iii. Fast and simple protocol
b. Disadvantages
i. Cell Smear: Adhere non-adherent
cells on coverslip with chemical bond
ii. Immunoreactivity of the primary
antibody might be adversely affected
by labeling with enzymes or tags.
iii. Labeling primary antibodies for each
specific ELISA system is time-
consuming and expensive.
iv. No flexibility in choice of primary
antibody label from one experiment
to another.
v. Minimal signal amplification.
2. Indirect Elisa - An indirect ELISA is similar to a direct
ELISA in that it uses a plate to immobilize an antigen,
but it also contains an amplification detection step.
First, a primary detection antibody that is unconjugated
is introduced and binds to the specific antigen. After
that, a conjugated secondary antibody directed against
the main antibody's host species is added. The amount
of antigen bound in the well is thus proportional to the
signal produced by the substrate.
a. Advantages
i. A wide variety of labeled secondary
antibodies are available
commercially.
ii. Versatile because many primary
antibodies can be made in one
species and the same labeled
secondary antibody can be used for
detection.
iii. Maximum immunoreactivity of the
primary antibody is retained because
it is not labeled.
iv. Sensitivity is increased because
each primary antibody contains
several epitopes that can be bound
by the labeled secondary antibody,
allowing for signal amplification.
b. Disadvantages
i. Cell Smear: Adhere non-adherent
cells on coverslip with chemical bond
ii. Cross-reactivity might occur with the
secondary antibody, resulting in
nonspecific signals.
iii. An extra incubation step is required
in the procedure.
3. Sandwich Elisa - The most frequent type of ELISA is a
sandwich ELISA. Matching antibody pairs are two
specific antibodies that are used to sandwich the
antigen. A microplate is covered with a capture
antibody, the sample is added, and the protein of
interest binds and immobilizes on the plate. The next
step is to add a conjugated-detection antibody, which
binds to an extra epitope on the target protein.
Substrate is added, and a signal proportional to the
amount of analyte in the sample is produced.
Sandwich ELISAs are very selective since they require
two antibodies to bind to the protein of interest.
a. Advantages
i. High specificity: the antigen/analyte
is specifically captured and detected
ii. Suitable for complex (or
crude/impure) samples: the antigen
does not require purification prior to
measurement
iii. Flexibility and sensitivity: both direct
or indirect detection methods can be
used
b. Disadvantages
i. Longer protocol
ii. Challenging to develop
4. Competitive Elisa - When the protein of interest is too
tiny to sandwich with two antibodies successfully,
11
 competitive ELISAs are routinely utilized. A capture
antibody is put on a microplate, similar to a sandwich
ELISA. A conjugated antigen is utilized instead of a
conjugated detection antibody to complete the binding
with the antigen present in the sample. The less
conjugated antigen in the sample, the less it will bind to
the capture antibody. When the substrate is
introduced, the resulting signal is inversely proportional
to the amount of protein in the sample.
a. Advantages
i. Ability to quantitate small molecules
b. Disadvantages
i. Less specific since you are only
using 1 antibody
ii. Requires a conjugated antigen
Conclusion
ELISA or Enzyme-Linked Immunosorbent Assay, also
known as Enzyme Immunoassay (EIA), is a biochemical
technique used to detect and quantify compounds like antigens,
antibodies, and hormones. The amount of compound in the
sample corresponds to the amount of substrate converted to
colored product by enzyme-linked antibodies. It is useful to
diagnose HIV, rotavirus, syphilis, Zika virus, and many others.
There are four types of ELISA, namely, Direct ELISA, Indirect
ELISA, Sandwich ELISA and Competitive ELISA. The major
difference between Direct and Indirect ELISA is that only one
antibody is used in Direct ELISA, while Indirect ELISA requires
two antibodies. In both Direct and Indirect ELISA, it is the
antigen that is immobilized to the plate, meanwhile in Sandwich
ELISA, it is the antibody that is immobilized to the plate, and this
antibody is called a capture antibody. Meanwhile, Competitive
ELISA is a type of ELISA wherein antigens compete with each
other for the binding with antibodies. The lower the intensity of
color in Competitive ELISA means there is a higher amount of
targeted antigen. The steps of this assay could be summarized
into 6 steps, which are:
1. Antibody coating of wells
2. Protein capture
3. Antibody detection
4. Addition of enzyme linked secondary antibody
5. Addition of substrate solution
6. Analysis
Summary
To conclude, the three experiments exhibit the
activities of proteins. Bradford Assay is focused on measuring
the number of proteins then Salivary Amylase Enzyme Assay
explains that proteins are controlled by other factors such as pH
level, temperature, substrate concentration, and inhibition, while
Elisa detects a specific protein.
CHROMATOGRAPHY
Chromatography
 Greek chroma meaning ‘color’; graphein meaning
‘writing’
 Physical process where the solutes of a sample
mixture are separated as a result of their differential
distribution between stationary and mobile phase
 Mikhail Tsvet- Russian-Italian botanist; Father of
Chromatography; Credited for the development of
Chromatography; Produced a colorful separation of
plant pigments through a column of calcium carbonate
Principles of Chromatography
 Usually based on the principle of partition of solute
between two phases
 Mobile Phase/Eluent- refers to the mixture of
substances to be separated dissolved in a liquid or a
gas; Stationary Phase- porous solid matrix through
which the sample contained in a mobile phase
percolate
 Column
o usually contains the stationary phase
o either packed or open tubular
o Packed Columns (filled with particles of the
stationary phase)
o the stationary phase is coated on the inside of
the column in open tubular columns
 Analyte- substance to be separated during
chromatography
 Eluate- solvent leaving column; Eluite- sample leaving
column
 Chromatogram
o graphical representation of detector response,
concentration of analyte in the effluent, other
quantity used as a measure of effluent
concentration
o the retention time or volume is when a solute
exits the injector and passes through the
column and the detector
o data represented by the chromatogram are
used to help identify and quantify
solute/solutes
o eluting solutes are displayed graphically as a
series of peaks, usually referred to as
Chromatographic Peaks (width, height, area)
 Molecules more soluble in mobile phase moves fast;
Molecules less soluble in mobile phase, takes longer
time to move
12
 1.
2.
3.
4.
Classification of Chromatographic Methods
 Based on the shape of chromatographic bed (e.g.
Planar and Column Chromatography)
5.
 Based on the physical state of mobile and stationary
phase (e.g. Gas and Liquid Chromatography)
 Based on the mechanism of separation (e.g. Ion-
Exchange Chromatography, Partition, Affinity and
Adsorption Chromatography)
1.
Paper Chromatography 2.
 A technique of separating dissolved chemical 3.
substances by their different migration rates across the 4.
sheets of paper. It is an inexpensive but powerful 5.
analytical tool that uses very small quantities of 6.
material. Paper chromatography was discovered by 7.
Synge and Martin in the year 1943. 8.
 It is necessary for the different chemicals in the
solution to have different properties such as molecule
size or a different ability to dissolve in a solvent. The 1.
stationary phase will absorb or slow down different
components of the tested solution to different degrees
creating layers as the components of the solution are
separated. Chromatography was invented by the
Russian botanist, Mikhail Tsvet. Chemists use this
process to identify unknown substances by separating 2.
them into the different molecules that make them up.
3.
4.
5.
6.
Mechanism of Separation
The separation of the component is called partition  This is a developed CHROMATOGRAM (the output of
chromatography where the substance is divided between
phases: the mobile phase which passes through the paper, and
the stationary phase which held in the pores of the filter paper.
The compounds in the mixture separate themselves based on
the differences in their affinity towards stationary and mobile
phase solvents under the capillary action of pores in the paper.
Types of Paper Chromatography
Ascending Paper Chromatography – The techniques
go with its name as the solvent moves in an upward
direction.
Descending Paper Chromatography – The movement
of the flow of solvent due to gravitational pull and
capillary action is downwards, hence the name
descending paper chromatography.
Ascending – Descending Paper Chromatography – In
this version of paper chromatography, movement of
solvent occurs in two directions after a particular point.
Initially, the solvent travels upwards on the paper which
is folded over a rod and after crossing the rod it
continues with its travel in the downward direction.
Radial or Circular Paper Chromatography – The
sample is deposited at the center of the circular filter
paper. Once the spot is dried, the filter paper is tied
horizontally on a Petri dish which contains the solvent.
Two-Dimensional Paper Chromatography –
Substances which have the same rf values can be
resolved with the help of two-dimensional paper
chromatography.
Materials
Water soluble pens/markers of different colors
Strips of paper towel
Water
Rubbing Alcohol
Nail polish remover
Straw/Pencil/Pen
Cups (transparent)
Tape
Procedure
Selecting a suitable type of development: It is decided
based on the complexity of the solvent, paper, mixture,
etc. Usually, ascending type or radial paper
chromatography is used as they are easy to perform.
Also, it is easy to handle, the chromatogram obtained
is faster and the process is less time-consuming.
Selecting a suitable filter paper: Selection of filter paper
is done based on the size of the pores and the sample
quality.
Prepare the sample: Sample preparation includes the
dissolution of the sample in a suitable solvent (inert
with the sample under analysis) used in making the
mobile phase.
Spot the sample on the paper: Samples should be
spotted at a proper position on the paper by using a
capillary tube.
Chromatogram development: Chromatogram
development is spotted by immersing the paper in the
mobile phase. Due to the capillary action of paper, the
mobile phase moves over the sample on the paper.
Paper drying and compound detection: Once the
chromatogram is developed, the paper is dried using
an air drier. Also, detecting solution can be sprayed on
the chromatogram developed paper and dried to
identify the sample chromatogram spots.
Analysis of Result
a chromatography run).
13

 As the solvent slowly travels up the paper, the different
rates and mixtures are separated into different colored
layers/spots. These are the pigments contained in the
marker. The distance traveled by the solvent and the
pigments along the paper from the origin line should be
measured.
 Ink is a solution containing several different molecules.
These different molecules have different characteristics
such as size and solubility. Solubility is a molecule's
ability to dissolve in a particular solvent such as
alcohol, water, or nail polish remover. Because of their
different characteristics, each molecule travels at a
different speed when pulled along the piece of paper
toweling by the solvent. The lightest particles, which
are not necessarily the lightest colored particles, move
more quickly and a greater distance than the heavier
particles. Thus, all the pigments that make up an ink
sample are separated out.
Retention Factor (Rf Value)
It is the distance travelled relative to the solvent. The
Rf value indicate how soluble the pigments are in the solvent by
measuring how high these pigments traveled through the paper.
Each separated pigments or the components should have its
own measurement of the Rf value.
Application
 In the analysis of different classes of compounds
namely:
o Amino acids and organic acids
o Alkaloids
o Polysaccharides
o Proteins and peptides
o Natural and artificial pigments
o Inorganic cations
o Plant extracts
 Uses of paper chromatography are not confined to any
field. Some necessary areas include:
o Reaction monitoring – The progress of the
reaction can be estimated by developing the
chromatogram over different time intervals by
spotting the reactants.
o Isolation & Purification – This technique is
useful in the purification and isolation of
components of mixtures. Here, the separated
components on the paper are cut, dissolved in
suitable solvents, and using spectroscopic
methods, their absorption is characterized at
specific wavelengths.
o Foods – Analysis of food colors in synthetic
drinks and beverages, ice creams, sweets,
etc. Only edible colors are permitted for use;
therefore, identification and quantification are
of utmost importance.
o Forensics – Provides a basis for identification
and comparison against reference standards
for drugs and their metabolites. Paper
chromatography offers a vital role in the viable
analysis of samples that are available in
milligrams or microliter quantities.
o Pharmaceuticals – Provides information
related to the development of new drugs
molecules, reaction completion and progress
of manufacturing processes. This process is
cost-effective and hence used as an
alternative method in monitoring the active
ingredients present in the drug forms. Paper
chromatography is also applicable in color
identifications of pharmaceutical formulations.
Thin Layer Chromatography
 It is one of the chromatographic methods used for
separating non-volatile substances.
 The experiment is carried out on a thin layer of
adsorbent material covered by a sheet of aluminum
foil, plastic, or glass.
 Aluminium oxide, cellulose, or silica gel are the most
common materials utilized. This liquid phase contains
the solution that should be separated and dissolved as
an appropriate solvent and composed up to the plate
by capillary action dividing the solution based on the
compound's polarity.
Materials
 Pencil
 TLC plates
 Staining agent (Phosphomolybdic Acid (PMA) Stain)
 Capillary tube
 Developing Chamber
 UV light
Mechanism of Separation
TLC consist of a stationary and mobile phase. In this
procedure the mobile phase move up to the stationary phase.
When the solvent separated in the procedure it compose the
two polar compound. First sample which is the polar compound,
it is shown how the first substance strongly connection to the
silica gel in the stationary phase. That made first substance
slowly move in the stationary phase which made it sticky. On
the other hand the non polar compound has a weakly
connection to the silica on the stationary phase but it is visible
how rapidly move on the stationary phase up to the solvent
point.
Procedure
 Step 1: PREPARE THE DEVELOPING CONTAINER-
Fill the solvent in the developing chamber and cover it
for a while.
 Step 2: PREPARE THE TLC PLATE- Using a pencil
draw a line on the lower part of TLC plates.
14
 Step 3: SPOT THE TLC PLATE- Spot the plate by
using a capillary tube. After that press 3 times firmly to
the plate in order to deposited the solution.
 Step 4: DEVELOP THE PLATE- Get the TLC
developing chamber and put the TLC plate inside.
Cover it for a while then after a few minutes the solvent
will go up to the TLC plates. In this phase where the
mobile phase which is the solvent that we put in the
TLC developing chamber moves forward to the
stationary phase which is the TLC plate. It does it by
the capillary action, the two components will move up
the plate at different rates. Two components will
interact of hydroxyl group in the silica. First polar
compounds that has strongly absorbed to the
stationary phase that cause slow movement while the
Second is non-polar compound that move quickly
cause of weak interaction in the silica.
 Step 5: VISUALIZE THE SPOTS- Once the solvent
slightly move up, remove it before the solvent hits the
top of the plate. Then mark the solvent line with a
pencil. Let the TLC plate dry. When the spots are not
visible use UV light and trace all the spots results in the
experiments.
Analysis
 Sample #1: In solvent 1 where the 75% of a polar
solvent use it will move up quickly while the less polar
in the solvent two move slowly to the solvent point. It is
also show that the red spot on the stationary phase
move slowly due to the strongly absorbed on the silica
gel and the blue spot which is the less polar molecule
has weakly absorbed on the silica gel that cause blue
spots move rapidly than to the red spots.
 Sample #2: If the both sample solvent has same polar,
it will still the same results that shown on the TLC
plate.
 Sample #3: In the solvent #1 where 100% of non-polar
used, there no move and it will stay on the starting line.
While the solvent #2 used 100% polar both of the two
spots moves fast to the solvent line point. There will be
no sticky to the polar silica gel.
Layer VS Paper Chromatography
 Time saving- Thin layer chromatography experiment
takes 3-4 hours while the paper chromatography takes
13-16 hours
 Rigid support- In paper chromatography use support of
cellulose paper which is extremely flexible. On the
other hand TLC is use support of on rigid plastic,
mental or glass. In terms of fast development TLC has
more advantage than to Paper chromatography due to
the type of support that used in experiment. With the
use of proper rigidity, diffusion will be going less and
the formation of well-defined spots.
 Heating- Thin layer chromatography can be heated if it
is necessary for developing spots. While the sheets of
paper are not capable of that procedure.
 Choice of support- TLC has various of support
substance choices such as adsobernt, liquid coated
and fluorescence induces.However the paper
chromatography was limited in the choice of different
kind of paper.
Application
 Phytohemistry - used for plant chemistry, biochemistry
and molecular biology o Biological activities of plant
compounds. It can be used on thin layer
chromatography to recognize the variation of plant
compounds such as anti-oxidative, antibacterial, or
antifungal. In the process to test the antioxidants on
the TLC plates can be sprayed with 2,2- diphenhyl-
picrylhydrazyl (DPPH) and methanol. It will turns out
this compound have free radical like deep purple color.
After the reaction with the antioxidants, it turns out to
yellow. By examine the presence of anti-bacterial or
anti-fungal compounds, TLC plate can be incubated
with microorganism. It can monitor the inhibited growth
of microorganism.
 Food and Cosmetic Industry - to distinguish all the
measurements of colors, ingredients, preservatives,
and sweetening agents in food and cosmetic products.
 Pharmaceutical Industry
o It can control and monitor all the substances
in the testing of pharmaceutical formulations.
o One of the example is identifying drugs in
body fluids. It can be used in Thin layer
chromatography of forensic studies, such as
urine and blood test that can identify the
presence of the drugs on the body.
 Biochemical Analysis - In this Thin layer
chromatography examperiment which often used this
to identify, differentiate and characters the compound
and metabolites in the following:
o Blood
o Body fluids
o Urine
o Serum
Column Chromatography
15
  Column chromatography is a method in chemistry that
is used to isolate a single chemical component from a
mixture that has been dissolved in a fluid.
 It separates substances via differential adsorption of
compounds to the adsorbent as the compounds pass
along the column at various speeds, allowing them to
be separated into fractions.
 This procedure can be employed on a small or big
scale to purify materials for use in future investigations.
This technique is an example of the type of
chromatography used by researchers at Cambridge
University.
Ion Exchange Chromatography
 Ion exchange chromatography (or ion chromatography)
is a process that allows the separation of ions and
polar molecules based on their affinity to ion
exchangers.
 It offers a huge sample-handling capacity, a broader
application (particularly to proteins and enzymes), a
low cost, great resolving ability, and eases scale-up
and automation, making it one of the most flexible and
commonly used liquid chromatography methods. In this
process two types of exchangers i.e., cationic and
anionic exchangers can be used.
o Cationic exchangers possess negatively
charged group, and these will attract
positively charged cations. These exchangers
are also called “Acidic ion exchange”
materials, because their negative charges
result from the ionization of acidic group.
o Anionic exchangers have positively charged
groups that will attract negatively charged
anions. These are also called “Basic ion
exchange” materials.
Materials
Pump, injector, column, suppressor, detector, and recorder or
data system are examples of typical IC equipment.
1. Pump - The IC pump is regarded as one of the most
critical components in the system, since it must deliver
a consistent flow of eluent through the IC injector,
column, and detector.
2. Injector - A sample introduction can be done in a
variety of ways. The most basic approach is to utilize
an injection valve. Liquid samples can be injected
directly, whereas solid samples merely need to be
dissolved in a suitable solvent. Injectors should be able
to inject liquid samples ranging in volume from 0.1 to
100 ml with good repeatability and under high
pressure.
3. Columns - The column material may be stainless steel,
titanium, glass, or an inert plastic such as PEEK
(Polyether ether ketone), depending on its eventual
usage and region of application. Depending on
whether it is to be used for standard analytical
purposes, microanalysis, high speed analyses, or
preparative work, the column's width can range from
around 2mm to 5 cm and its length can range from 3
cm to 50 cm. The guard column is located ahead of the
dividing column. This acts as a protective element,
extending the separation column's life and
effectiveness. They are reliable columns that filter or
eliminate particles that block the separation column.
4. Suppressor - The suppressor suppresses the
background conductivity of the chemicals used to elute
samples from the ion-exchange column, improving the
conductivity measurement of the ions under test. IC
suppressors are membrane-based devices that are
meant to convert ionic eluent to water in order to
increase sensitivity.
5. Detectors - Electrical conductivity detectors are widely
used.
6. System of data - A pre-programmed computer
integrator may suffice in routine analysis when no
automation is required. A more sophisticated device,
such as a data station or minicomputer, is required for
greater control levels.
Mechanism of Separation
 The stationary phase in this method is an insoluble
porous resinous substance containing fixed charge-
carrying groups. These groups are loosely complexed
with counter-ions of opposite charge.
 The reversible exchange of these ions occurs when a
liquid mobile phase containing ionised or partly ionised
molecules of the same charge as the counter-ions
passes through the system.
 The pace of migration and hence the degree of
separation between the different solute species is
determined by the degree of affinity between the
stationary phase and feed ions.
 The most common form of stationary phase is a
synthetic copolymer of styrene and divinyl benzene
(DVB), which is manufactured as micrometer-sized
beads. Controlling the amount of DVB applied carefully
determines the degree of cross-linking and, as a result,
the porosity of the resinous structure.
 Big pores in low-cross-linking resins allow large ions to
diffuse into the resin beads and permit fast ion
exchange. Highly cross-linked resins feature holes that
are comparable in size to tiny ions.
 The selection of a certain resin will be heavily
influenced by the application. Chemical modifications
of the resin can be used to introduce cation (+) or
anion (-) exchange characteristics.
 In industrial operations, ion exchange chromatography
is widely used. This technology is used to separate
transition metals, remove trace metals from industrial
effluents, and purify a variety of organic chemicals and
medicines. When compared to other forms of
stationary phase, the resin matrix is generally very
affordable. Although ion exchange chromatography is
the most extensively used large-scale chromatographic
method, it is confined to ionisable, water-soluble
compounds.
Procedure
16
Key steps in the ion exchange chromatography procedure are
listed below:
 Ion exchange separations are carried out mainly in
columns packed with an ion-exchanger.
 These ionic exchangers are commercially available.
They are made up of styrene and divinyl benzene.
Example. DEAE-cellulose is an anionic exchanger,
CM-cellulose is a cationic exchanger.
 The choice of the exchanger depends upon the charge
of particle to be separated. To separate anions
“Anionic exchanger” is used, to separate cations
“Cationic exchanger” is used.
 First the column is filled with ion exchanger then the
sample is applied followed by the buffer. The tris-
buffer, pyridine buffer, acetate buffer, citrate and
phosphate buffers are widely used.
 The particles which have high affinity for ion exchanger
will come down the column along with buffers.
 In next step using corresponding buffer separates the
tightly bound particles.
 Then these particles are analyzed spectroscopically.
Analysis
Fluoride, chloride, nitrate, nitrite, and sulfate
concentrations may be measured using ion chromatographs.
They may also determine the concentrations of important
cations such as lithium, sodium, ammonium, potassium,
calcium, and magnesium. For water chemistry analysis, ion
chromatography is utilized.
 The ion exchangers basically contain charged
groups covalently linked to the surface of an
insoluble matrix.
 The charged groups of the matrix can be positively
or negatively charged.
 When suspended in an aqueous solution, the
charged groups of the matrix will be surrounded by
ions of the opposite charge.
 In this “ion cloud”, ions can be reversibly
exchanged without changing the nature and the
properties of the matrix.
Significance/Application
Ion exchange chromatography is the most extensively
used technology for separating and purifying charged
biomolecules such as polypeptides, proteins, polynucleotides,
and nucleic acids. The major reasons for its success as a
separation method are its wide application, high capacity and
simplicity, and excellent resolution. Ion exchange
chromatography is widely utilized in a variety of industrial
applications, including the following:
 Separation and Purification of blood
components such as albumin, recombinant
growth factors and enzymes.
 Biotechnology - Analytical applications such
as quality control and process monitoring
 Food and clinical research - to study wheat
varieties and the correlation of proteinuria with
different renal diseases.
 Fermentation - Cation exchange resins are
used to monitor the fermentation process
during ß-galactosidase production.
Size Exclusion Chromatography (SEC)
 Size-exclusion chromatography (SEC) is a phase can not diffuse into pores accordingly and will
chromatographic technique used for separating
substances according to their molecular size, or more
correctly, hydrodynamic volume.
 It is often described as a chromatographic-based
method that developed in 1955 and has been used to
fractionate molecules based on their hydrodynamic
dimensions.
 Further, according to ChemBam, it is a method where
separation of different compounds occurs according to
their size (hydrodynamic volume) measured by how
efficiently they penetrate the pores of the stationary
phase.
 It is usually applied to large molecules or
macromolecular complexes such as proteins and
industrial polymers. There are two basic types of size
exclusion chromatography.
 One is gel permeation chromatography (GPC), which
uses a hydrophobic column packing material and a
non-aqueous mobile phase (organic solvent) to
measure the molecular weight distribution of synthetic
polymers.
 The other is gel filtration chromatography (GFC), which
uses a hydrophilic packing material and an aqueous
mobile phase to separate and measure the molecular
weight distribution of molecules soluble in water, such
as polysaccharides and proteins.
Materials
As indicated by Mohammad (2020), coming up next
are the Instruments that are utilized in doing this investigation:
1. Stationary Phase/Gel - Gels that are usually utilized
incorporate cross-linked dextran, agarose,
polyacrylamide, poly acryloyl morphine, and
Polystyrenes. In addition, there are three sorts of gel
and these are the following:
a. Soft gel separation of proteins
i. E.g. dextran (Sephadex),
polyacrylamide gel
b. Semi-rigid gels separation of non-polar
polymers in non-polar solvents.
i. E.g. bio beats
c. Highly rigid gels and glasses separation of
polar solvents.
2. Mobile Phase/Buffer - the fluid used to disintegrate the
biomolecules to make the mobile phase is generally
known as a buffer. The combination of biomolecules
broken up in the cradle is known as the sample. 3.
3. Sample Preparation - The sample preparation should
be ready in weakened fixations (under 2 mg/ml). A
decent solvent can break up a sample to any extent in
the scope of temperatures. Tests with expansive sub-
atomic weight dissemination might require higher
focus. It is prescribed to filter the sample solution
before injecting it into the columns to dispose of
clogging and unnecessarily high-pressure problems.
4. Column Packing - The packing of a column depends
on either permeable silica or semi-rigid(highly cross-
connected) organic gels, much of the time copolymers
of styrene and divinylbenzene. The following are the
given degrees of gels that have different purposes:
5. Pumps - Pumps are utilized to keep a steady stream
rate during the whole chromatogram, this is vital in size
exclusion chromatography.
6. Chromatogram - The extent of retention relies upon the
size of the included particles relying upon the size of
the pores. Small molecules will enter all pores.
Intermediate molecules due to the speed of the mobile
be held less adequately. The initial peak contains a
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 barred solute. The final peak contains an included
solute.
7. Detectors = Detectors are utilized for measuring the
consequence of chromatograms. There are various
kinds of detectors, and these are the following:
a. Concentration Sensitive Detectors
b. Solute property detectors- UV absorption
detectors.
c. Bulk property detectors- Refractive index (R.I)
detectors.
d. Evaporation detectors- Evaporation light
scattering detectors (ELSD).
e. Molecular Weight-Sensitive Detectors
f. Light scattering detectors-
g. Low Angle Light Scattering (LALS) Detectors,
h. Multi-Angle Light Scattering (MALS)
Detectors.
i. Viscosity detectors- Differential viscometer.
Mechanism of Separation
Since Size exclusion chromatography (SEC) isolates
molecules dependent on their size by filtration through a gel, the
separation happens when particles of various sizes are included
or excluded from the pores inside the lattice. Small particles
diffuse into the pores and their flow through the segment is
impeded by their size, while huge particles don't enter the pores
and are eluted in the column's void volume. Thus, particles
separate depending on their size as they go through the section
and are eluted, arranged by diminishing molecular weight (MW).
Further, this partition is separated into two as per Bio-
Rad (n.d.).
 Desalting — A typical utilization of SEC is
for desalting protein or nucleic acid
samples. The particle of interest is eluted
in the void volume, while smaller
molecules are held in the gel pores. To
acquire the ideal separation, the gel
should have an exclusion limit
significantly smaller than the molecule of
interest
 Fractionation — Molecules of differing
molecular weights are isolated inside the
gel matrix. With this partition technique,
the particles of interest should fall inside
the fractionation scope of the gel
Procedure
The procedure in Size Exclusion Chromatography can
be traced using the steps given by Aryal (2019). Hence, the
following are the steps for conducting SEC.
1. Spherical particles of gel filtration medium are
stuffed into a column.
2. The sample is applied to the column.
3. Buffer (mobile phase) and samples travel through
the column.
4. Molecules diffuse all through the pores of the
matrix (additionally depicted as the partitioning of
the sample between the mobile phase and
stationary phase).
5. Smaller molecules move further into the matrix
thus staying longer on the section.
6. As the buffer goes constantly through the column,
molecules that are bigger than the pores of the
matrix can't diffuse into the pores and pass
through the column.
7. Smaller particles diffuse into the pores and are
postponed in their entry down the segment.
8. Separation happens at various stretches which are
trailed by recognition of parts
Analysis
A mixture of molecules broke up in liquid is applied to a
chromatography column which contains solid support in the
form of microscopic spheres and beads. The mass of beads
inside the column is regularly alluded to as the column bed. The
beads act as "traps" and function to channel small molecules
which become briefly caught inside the pores. Besides, bigger
molecules pass around or are excluded from the beads. They
can't or can only partially penetrate the pores, while smaller
molecules can access almost to all pores. In this manner, they'll
be eluted first, then again, average molecules elute later, and
small molecules will elute last from the column since they can
get to every one of the pores.
Application
As indicated by Pharmatutor/Pharma Infopedia
(2016) ,size exclusion chromatography has been utilized with
incredible accomplishment in the partition of the sugars,
polypeptides, proteins, fluids, pavements, butyl rubbers,
polyethylene, polystyrenes, silicon polymers, and others. Size
exclusion chromatography has been for the most part applied to
investigations of complex, biochemical or exceptionally
polymerized molecules. In any case, the fundamental
utilizations of the size exclusion chromatography are as per the
following:
a. Purification: The principal use of size exclusion
chromatography is in the cleaning of natural
macromolecules. Infections, proteins, catalysts,
chemicals, antibodies, nucleic acids, and
polysaccharides have all been isolated and
sanitized by the utilization of fitting gels or glass
granules.
b. Molecular Weight Determination: The profluent
volumes of globular proteins are to a great extent
controlled by their sub-atomic weight. It has been
shown, that over an impressive atomic weight
territory, the profluent volume is around a straight
capacity of the logarithm of the sub-atomic weight.
c. Solution Concentration: Solution of high molecular
weight substances can be concentrated by the
expansion of dry sephadex G-25 (coarse). Water
and low molecular weight substances stay in
arrangement. After ten minutes, the gel is
eliminated by centrifugation, leaving the high
molecular material in an answer whose fixation
has expanded however whose pH and ionic
strength are unaltered.
d. Desalting: By utilization of a section of sephadex
G-25, arrangements of high molecular weight
mixtures might be desalted. The high atomic
weight substances move with the void volume
while the low sub-atomic weight parts are
disseminated between the versatile stage and
subsequently move gradually.
e. Protein building studies: Exclusion
chromatography is one of various techniques
ordinarily used to concentrate on the reversible
restricting of a ligand to a macromolecular, for
example, proteins including receptor proteins. An
example of the protein/ligand blend is applied to a
section of an appropriate gel (for example G-25)
which has recently been equilibrated with an
answer of the ligand of the very focus as that in
the blend.
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