Pharmaceutical Chemistry Practical (Biochemistry)
S. No Name of Date of Date of
Experiment Experiment submission
1. To prepare standard buffers 26-5-2024 30-5-2024
(citrate, phosphate, and
carbonate) and measure their
pH
2. To quantitatively estimate the 5-6-2024 10-6-2024
concentration of amino acids
using a standard ninhydrin
reaction method.
3. To isolate DNA and RNA 12-6-2024 19-6-2024
from biological samples (e.g.,
plant tissue, animal tissue, or
bacterial cells) using
appropriate extraction
techniques
4. Estimation of SGOT (Serum 21-6-2024 12-7-2024
Glutamic Oxaloacetic
Transaminase) and SGPT
(Serum Glutamic Pyruvic
Transaminase)
5. Separation of Lipids by Thin 19-7-2024 22-7-2024
Layer Chromatography
(TLC)
6. Separation of Amino Acids by 25-7-2024 29-7-2024
2D Paper Chromatography
and Gel Electrophoresis
7. Separation of Amino Acids by 30-7-2024 2-8-2024
Gel Electrophoresis
Pharmaceutical Chemistry Practical
Experiment No-1
Aim: To prepare standard buffers (citrate, phosphate, and carbonate) and measure their pH.
References: Principles of Biochemistry by Lehninger, Harper’s Biochemistry by Robert K.
Murry, Daryl K. Granner, and Victor W. Rodwell.
Material Required: - Citric acid, Sodium citrate, Monosodium phosphate (NaH₂PO₄), Disodium
phosphate (Na₂HPO₄), Sodium carbonate (Na₂CO₃), Sodium bicarbonate (NaHCO₃), pH meter
or pH paper, Distilled water, Volumetric flasks (100 mL and 1 L), Beakers, Stirring rods and
Analytical balance.
Theory:
Introduction:
Buffers are aqueous systems that resist changes in pH when small amounts of acid or base are
added. They are essential in biochemical experiments where maintaining a constant pH is
critical for the stability of biomolecules and reactions. Buffer solutions usually consist of a
weak acid and its conjugate base. This experiment focuses on the preparation of three standard
buffers: citrate, phosphate, and carbonate buffers, and the measurement of their pH values.
1. Citrate Buffer:
Citrate buffer is prepared using citric acid and sodium citrate. It is commonly used to maintain
a pH in the acidic range (3.0–6.2).
Citric acid (H₃C₆H₅O₇): A weak organic acid.
Sodium citrate (Na₃C₆H₅O₇): The conjugate base of citric acid.
2. Phosphate Buffer:
Phosphate buffers are made from combinations of monosodium phosphate and disodium
phosphate. They are ideal for maintaining a pH in the range of 6.0–8.0.
Monosodium phosphate (NaH₂PO₄): Acts as an acid.
Disodium phosphate (Na₂HPO₄): Acts as a base.
3. Carbonate Buffer:
Carbonate buffers are typically used for maintaining pH in the range of 9.2–10.8. It is made
from sodium carbonate and sodium bicarbonate.
Sodium carbonate (Na₂CO₃): A basic salt.
Sodium bicarbonate (NaHCO₃): The acidic form of the buffer system.
Procedure:
1. Citrate Buffer Preparation (pH ~3.0 to 6.2):
1. Weigh 1.92 g of citric acid and dissolve it in 100 mL of distilled water.
2. Weigh 2.94 g of sodium citrate and dissolve it in 100 mL of distilled water.
3. Mix both solutions in appropriate volumes to obtain the desired pH.
4. For pH 4.0, use 61.5 mL of citric acid and 38.5 mL of sodium citrate solution.
5. Adjust the final volume to 200 mL with distilled water.
2. Phosphate Buffer Preparation (pH ~6.0 to 8.0):
Prepare 0.2 M solutions of monosodium phosphate (NaH₂PO₄) and disodium phosphate
(Na₂HPO₄).
For pH 7.4 buffer, mix 39 mL of 0.2 M NaH₂PO₄ with 61 mL of 0.2 M Na₂HPO₄.
Adjust the volume to 100 mL with distilled water.
3. Carbonate Buffer Preparation (pH ~9.2 to 10.8):
Dissolve 1.06 g of sodium carbonate and 0.84 g of sodium bicarbonate in 100 mL of distilled
water to prepare a buffer with pH 9.2.
For a pH of 10.8, use only sodium carbonate (1.06 g in 100 mL of water).
4. Measurement of pH:
Calibrate the pH meter with standard buffer solutions (pH 4.0, 7.0, and 10.0).
Measure the pH of the prepared buffer solutions using the pH meter.
Record the pH values and compare them with the theoretical values.
Observations:
S. No Buffer Type Theoretical pH Measured pH
1. Citrate Buffer 3-6.2 4.8
2. Phosphate Buffer 5.8-7.4 6.2
3. Carbonate Buffer 9.2 -10.6 9
Results:
The standard buffers (citrate, phosphate, and carbonate) were successfully prepared and the pH
values were measured using a pH meter. The measured pH values were close to the theoretical
values, confirming the successful preparation of the buffers.
Precautions:
- Handle all chemicals with care, especially citric acid and sodium carbonate, which are slightly
corrosive.
- Calibrate the pH meter properly before measuring the pH of buffer solutions.
- Ensure proper mixing of solutions to avoid discrepancies in the pH measurements.
Experiment No-2
Aim: To quantitatively estimate the concentration of amino acids using a standard ninhydrin
reaction method.
References: Principles of Biochemistry, Lehninger, Biochemical Methods, Sadasivam and
Manickam.
Material Required: Amino acid sample (unknown concentration), Ninhydrin reagent, Glycine
(or leucine) standard, Distilled water, Sodium acetate buffer (pH 5.5), Test tubes,
Spectrophotometer, Water bath, Volumetric flasks (10 mL, 50 mL, 100 mL), Pipettes,
Analytical balance.
Principle: When amino acids react with ninhydrin under heat, they produce a purple-blue
coloured complex known as Ruhemann's purple. This colour intensity is directly proportional
to the concentration of amino acids present in the sample. By measuring the absorbance of the
coloured complex at 570 nm using a spectrophotometer, the concentration of amino acids can
be quantified by comparing it with a standard curve generated from known concentrations of a
standard amino acid solution (typically glycine or leucine).
Procedure:
1. Preparation of Standard Glycine Solution:
- Weigh 50 mg of glycine and dissolve it in 50 mL of distilled water to prepare a 1 mg/mL
standard glycine solution.
- Prepare a series of dilutions (e.g., 0.2 mg/mL, 0.4 mg/mL, 0.6 mg/mL, 0.8 mg/mL, and 1.0
mg/mL) by diluting the stock solution with distilled water.
2. Preparation of Ninhydrin Reagent:
Prepare the ninhydrin reagent by dissolving 0.1 g of ninhydrin in 100 mL of sodium acetate
buffer (pH 5.5). Stir until completely dissolved.
3. Quantitative Estimation:
-Take 1 mL of each glycine standard solution and 1 mL of the unknown amino acid solution
in separate test tubes.
- Add 1 mL of ninhydrin reagent to each test tube.
- Mix the solutions thoroughly and place the test tubes in a boiling water bath for 10 minutes.
- After heating, cool the test tubes to room temperature.
- Dilute the reaction mixture with 5 mL of distilled water to stop the reaction and stabilize
the colour.
- Measure the absorbance of the resulting purple-blue solution at 570 nm using a
spectrophotometer.
4. Construction of Standard Curve:
- Plot a graph of the absorbance readings (y-axis) against the concentrations of the standard
glycine solutions (x-axis).
- Use the standard curve to determine the concentration of the unknown amino acid solution
from its absorbance value.
Observation Table:
Calculations:
Using the standard curve, calculate the concentration of the unknown amino acid sample using
formula
A = epsilon c× l
C=A/0.9
Where - A is the absorbance (no units, since it's a logarithmic ratio).
- ε is the molar absorptivity (or molar extinction coefficient), with units of (L \c. mol^ {-1} \c.
cm^ -1).
- l is the path length of the sample (usually in cm, typically 1 cm for standard cuvettes).
- c is the concentration of the solution (usually in mol/L).
S. Absorbance (570nm) AA(Glycine) Conc(mg/mL)
No (unknown)
1. 0.18 0.2
2. 0.36 0.4
3. 0.54 0.6
4. 0.72 0.8
5. 0.90 1.0
Results:
The concentration of the unknown amino acid solution was found to be 0.2,0.4,0.6,0.8,1.0
mg/mL using the ninhydrin method and compared its absorbance with a standard curve.
Precautions:
- Ensure accurate pipetting and proper mixing of solutions.
- Handle ninhydrin reagent with care as it can be an irritant.
- Calibrate the spectrophotometer before taking measurements.
- Maintain the water bath at a constant temperature for uniform results.
Experiment No-3
Aim: To isolate DNA and RNA from biological samples (e.g., plant tissue, animal tissue, or
bacterial cells) using appropriate extraction techniques.
References: Biochemistry by Stryer, Biochemistry by D. Satyanarayan and U. Chakrapani.
Material Required: - Biological sample (e.g., onion, banana, animal cells, bacteria), DNA
extraction buffer (Tris-HCl, EDTA, NaCl, SDS), RNA extraction buffer (Triazole or guanidine
isothiocyanate solution), Proteinase K, Chloroform, Phenol (for RNA extraction), Isopropanol,
Ethanol (70%), RNase-free water (for RNA), Distilled water, Centrifuge tubes, Mortar and
pestle (for plant tissues), Ice, Pipettes and tips, Microcentrifuge, Spectrophotometer (for
quantification).
Theory: DNA (Deoxyribonucleic Acid) and RNA (Ribonucleic Acid) are vital macromolecules
that store and transfer genetic information in all living organisms. The isolation of DNA and
RNA is essential for molecular biology experiments including PCR, cloning and gene
expression studies.
Procedure:
Part A: Isolation of DNA
1. Sample Preparation:
- Collect about 1-2 grams of the biological sample (plant or animal tissue).
- Grind the sample thoroughly in a mortar and pestle with a small amount of extraction buffer
to release the cells.
2. Lysis of Cells:
- Transfer the ground sample into a centrifuge tube.
- Add an appropriate amount of DNA extraction buffer (Tris-HCl, EDTA, SDS, NaCl).
- Mix gently and incubate for 10 minutes at 37°C to lyse the cells and release the DNA.
3. Digestion of Proteins:
- Add Proteinase K to digest the proteins that are associated with DNA.
- Incubate for 1 hour at 55°C.
4. DNA Precipitation:
- Add equal volumes of chloroform to the lysate to remove proteins and other impurities.
- Centrifuge the mixture at 12,000 rpm for 15 minutes at 4°C.
- Carefully transfer the aqueous phase to a new tube (the DNA is in this layer).
- Add cold isopropanol to the aqueous phase to precipitate the DNA.
- Mix gently and incubate on ice for 20 minutes.
5. DNA Collection:
- Centrifuge the tube at 12,000 rpm for 10 minutes to pellet the DNA.
- Remove the supernatant, and wash the DNA pellet with 70% ethanol.
- Allow the pellet to air-dry, then resuspend it in a small volume of distilled water or TE
buffer.
6. DNA Quantification:
- Measure the DNA concentration using a spectrophotometer at 260 nm.
Part B: Isolation of RNA
1. Sample Preparation:
- Collect a small amount of fresh biological sample (around 100 mg).
- Grind the sample in liquid nitrogen using a mortar and pestle.
2. RNA Lysis and Extraction:
- Transfer the ground sample into a tube and add an appropriate volume of Triazole reagent
or guanidine isothiocyanate solution.
- Vortex briefly to mix.
- Incubate the tube for 5 minutes at room temperature to allow complete cell lysis.
3. Phase Separation:
- Add chloroform (0.2 mL for every 1 mL of TRIZOL reagent used).
- Shake the tube vigorously for 15 seconds, and incubate at room temperature for 3 minutes.
- Centrifuge the mixture at 12,000 rpm for 15 minutes at 4°C.
- Carefully collect the upper aqueous phase (contains RNA) without disturbing the interface.
4. RNA Precipitation:
- Add an equal volume of isopropanol to the aqueous phase to precipitate RNA.
- Incubate on ice for 10 minutes, and centrifuge at 12,000 rpm for 10 minutes.
5. RNA Collection and Wash:
- Discard the supernatant, and wash the RNA pellet with 75% ethanol.
- Centrifuge briefly, and discard the ethanol wash.
- Air-dry the RNA pellet, then dissolve it in RNase-free water.
6. RNA Quantification:
- Measure the RNA concentration using a spectrophotometer at 260 nm.
Observations:
DNA: A white, stringy precipitate should be observed after the addition of isopropanol,
indicating the presence of DNA.
RNA: After isolation a clear or slightly cloudy solution should be observed.
Result: The DNA and RNA were successfully isolated from the biological sample. The purity
and concentration of nucleic acids were measured using a spectrophotometer. The
concentration of DNA for 1gm of sample was 25µg/ml and RNA was 0.025µg/ml.
Precautions:
1. Use RNase-free materials and gloves while handling RNA to avoid degradation.
2. Work quickly and efficiently to prevent contamination.
3. Ensure proper pH of the buffers to avoid DNA or RNA degradation.
4. Perform all steps involving RNA on ice to preserve its integrity.
Experiment No-4
Aim: Estimation of SGOT (Serum Glutamic Oxaloacetic Transaminase) and SGPT (Serum
Glutamic Pyruvic Transaminase).
References: Biochemistry by Stryer, Biochemistry by D. Satyanarayan and U. Chakrapani.
Material Required: Reagents: Serum sample, SGOT (AST) and SGPT (ALT) reagent kits
(commercially available), Phosphate buffer (pH 7.4), α-Ketoglutarate, L-aspartate (for SGOT)
and L-alanine (for SGPT), NADH (Nicotinamide adenine dinucleotide reduced form), LDH
(Lactate dehydrogenase). Apparatus: Test tubes, Micropipettes, Spectrophotometer, Incubator
or water bath, Timer.
Objective: To estimate the levels of SGOT (AST) and SGPT (ALT) in a given serum sample,
which are indicative of liver function.
Principle:
SGOT (AST) and SGPT (ALT) are enzymes present in various tissues, especially the liver.
When liver cells are damaged, these enzymes are released into the bloodstream. Elevated levels
of these enzymes indicate liver damage or disease.
The estimation of SGOT and SGPT is based on the following reactions:
- SGOT (AST) catalyses the conversion of L-aspartate and α-ketoglutarate into oxaloacetate
and L-glutamate.
-SGPT (ALT) catalyses the conversion of L-alanine and α-ketoglutarate into pyruvate and L-
glutamate.
In both reactions the products formed can be detected and quantified by a coupled enzymatic
reaction that produces a measurable colour change when measured spectrophotometrically.
Procedure:1. Preparation of Reaction Mixture:
a. Label test tubes for SGOT and SGPT tests.
b. For the SGOT test, add 0.5 mL of SGOT reagent (containing L-aspartate and α-
ketoglutarate) to the test tube.
c. For the SGPT test, add 0.5 mL of SGPT reagent (containing L-alanine and α-ketoglutarate)
to the test tube.
2. Addition of Serum Sample:
a. Add 0.1 mL of the serum sample to the respective test tubes (both SGOT and SGPT).
3. Incubation:
a. Incubate the test tubes at 37°C for 30 minutes.
4. Enzyme Reaction:
a. After incubation, add 0.5 mL of NADH solution to the reaction mixture.
b. The enzymes catalyse the conversion of substrates, and the resulting NADH consumption
causes a decrease in absorbance, which is measured spectrophotometrically.
5. Measurement:
a. Measure the absorbance at 340 nm using a spectrophotometer.
b. The decrease in absorbance is proportional to the activity of SGOT and SGPT in the serum
sample.
6. Calculation:
The enzyme activity is calculated using the following formula:
{SGOT/SGPT activity (U/L) = Absorbance ×Molar extinction coefficient of NADH ÷Volume
factor
where Δ Absorbance is the change in absorbance over time and the molar extinction
coefficient of NADH is 6.22 at 340 nm.
Normal Reference Values:
SGOT (AST): 5-40 U/L
SGPT (ALT): 7-56 U/L
Result: The concentration of SGOT and SGPT were quantified and reported successfully.
Interpretation: - Elevated levels of SGOT and SGPT indicate liver damage or hepatocellular
injury.
- Conditions such as hepatitis, cirrhosis, liver cancer, and alcoholic liver disease can lead to
increased levels of these enzymes in the blood.
Precautions:
1. The serum sample should be fresh and free from haemolysis to avoid interference with
results.
2. Proper calibration of the spectrophotometer should be done for accurate absorbance
readings.
3. Handle reagents and samples with care to prevent contamination.
Experiment No.-5
Aim: Separation of Lipids by Thin Layer Chromatography (TLC).
References: Principles of Biochemistry by Lehninger, Harper’s Biochemistry by Robert K.
Murry, Daryl K. Granner, and Victor W. Rodwell.
Material Required: Reagents -Lipid sample (mixture of triglycerides, phospholipids,
cholesterol, fatty acids, etc.), Solvent system (chloroform, methanol, and water or
hexane/diethyl ether/acetic acid), TLC silica gel plate, Iodine chamber (or other lipid detection
reagents like phosphomolybdic acid, iodine vapor, or sulfuric acid spray), Capillary tubes
Apparatus: TLC chamber, TLC silica gel plates (20 x 20 cm or smaller), Developing solvent,
Micropipette or capillary tube, Pencil and ruler and UV lamp or Iodine chamber for
visualization.
Objective:
To separate and identify different types of lipids present in a sample using Thin Layer
Chromatography (TLC).
Principle:
Thin Layer Chromatography (TLC) is a technique used for the separation of non-volatile
compounds. It relies on the differential affinities of compounds towards the stationary phase
(TLC plate) and the mobile phase (solvent mixture). Lipids due to their varied structures and
polarities can be separated using TLC based on their interaction with the polar stationary phase
and the less polar mobile phase.
Lipids such as triglycerides, phospholipids, fatty acids, and cholesterol esters migrate at
different rates on the TLC plate, allowing for their separation and identification.
Procedure:
1. Preparation of the TLC Chamber:
- Take a TLC chamber and pour a small amount of the solvent system (e.g., chloroform:
methanol: water in 65:25:4 ratio) into the chamber. Ensure that the solvent level is below the
starting point of the TLC plate.
- Close the chamber with a lid to allow saturation with solvent vapours.
2. Preparation of the TLC Plate:
- Take a silica gel-coated TLC plate and, using a pencil, draw a straight line about 1-2 cm
from the bottom edge of the plate. This will be the baseline for spotting the sample.
- Using a capillary tube or micropipette, apply small spots of the lipid sample mixture onto
the baseline. Avoid applying too much sample as it may lead to poor separation.
3. Development of the TLC Plate:
- Carefully place the TLC plate vertically in the developing chamber, ensuring that the spots
are above the solvent level.
- Allow the solvent to rise up the plate by capillary action. When the solvent front has reached
about 80% of the height of the plate, remove the plate from the chamber.
- Immediately mark the solvent front with a pencil before it evaporates.
4. Drying and Visualization:
- Allow the plate to air dry completely.
- Visualize the separated lipid spots using appropriate detection methods:
- Iodine chamber: Place the plate in a chamber with iodine crystals. The lipids will appear
as yellow/brown spots.
- Phosphomolybdic acid or sulfuric acid spray: Spray the TLC plate with the reagent and
heat it to develop the spots.
5. Identification of Lipids:
- Measure the distance travelled by each spot (from the baseline) and the distance travelled
by the solvent front.
Calculation of the Retention Factor (Rf) for each lipid spot using the formula:
Rf = Distance travelled by the compound ÷Distance travelled by the solvent.
Compare the Rf values with standard lipid Rf values to identify the types of lipids.
S. No Different Lipids Rf value
1. Triglyceride 0.2-o.5
2. Phospholipid 0.3-0.7
3. Cholesterol 0.4-0.7
4. Fatty Acid 0.2-0.5
Result:
- The different lipids present in the sample were successfully separated using TLC. The Rf
values obtained from the spots can be compared with standard Rf values to identify the lipids
such as triglycerides, phospholipids, fatty acids, and cholesterol esters.
Precautions:
1. Avoid touching the silica gel surface of the TLC plate to prevent contamination.
2. Ensure that the sample spots are small and concentrated to avoid smearing and poor
resolution.
3. The solvent system should be freshly prepared, and the chamber should be saturated with
solvent vapours for proper development of the TLC plate.
Experiment No.-6
AIM: Separation of Amino Acids by 2D Paper Chromatography and Gel Electrophoresis.
References: Principles of Biochemistry by Lehninger, Harper’s Biochemistry by Robert K.
Murry, Daryl K. Granner, and Victor W. Rodwell.
Material Required: Reagent-Amino acid mixture, Solvents for 2D chromatography (e.g., n-
butanol, acetic acid, water for solvent 1; phenol for solvent 2), Ninhydrin reagent (for detection
of amino acids), Electrophoresis buffer (e.g., Tris-glycine buffer), Agarose or polyacrylamide
gel (depending on the technique), pH indicator paper (for adjusting buffer pH), Loading dye
(for electrophoresis), Staining solution for gel (e.g., Coomassie Brilliant Blue for protein/amino
acid detection)
Apparatus: Chromatography paper (Whatman No. 1 filter paper), Chromatography chamber,
Capillary tubes or micropipette, Ruler and pencil, Hot air oven or hair dryer, Gel
electrophoresis apparatus, Power supply for electrophoresis, UV or visible light source (for
visualization), Electrophoresis gel casting tray and comb, Gel staining and destaining trays
Objective: To separate and identify amino acids in a mixture using two-dimensional paper
chromatography and gel electrophoresis techniques.
Principle:
1. 2D Paper Chromatography:
In two-dimensional chromatography, separation occurs by subjecting a sample to two
different solvent systems in two orthogonal directions. The amino acids in the mixture move
based on their solubility in the mobile phase (solvent) and their affinity for the stationary phase
(paper). Different amino acids have different Rf values (retention factors) in different solvents,
allowing for their separation and identification.
2. Gel Electrophoresis:
Gel electrophoresis is a technique that separates molecules (in this case, amino acids) based
on their size and charge. When an electric field is applied, amino acids migrate through a gel
matrix toward either the anode or cathode depending on their charge at a given pH. The rate of
movement is influenced by their molecular weight and charge.
Procedure:
Part A: Separation of Amino Acids by 2D Paper Chromatography.
1. Preparation of Chromatography Paper:
- Take a square piece of chromatography paper (10 x 10 cm).
- Using a pencil, draw a faint pencil line 2 cm from one corner. This will be the origin for
spotting the amino acid mixture.
- Apply a small spot of the amino acid mixture onto the origin using a capillary tube. Allow
it to dry completely.
2. First Dimension Separation:
- Place the chromatography paper in the chromatography chamber with the first solvent
system (e.g., n-butanol: acetic acid: water in 4:1:5 ratio).
- Ensure the solvent level is below the origin line.
- Allow the solvent to rise until it is about 3/4 of the way up the paper.
- Remove the paper, mark the solvent front, and let it dry.
3. Second Dimension Separation:
- Rotate the chromatography paper 90° so that the previous separation runs horizontally.
- Place the paper in the chromatography chamber with the second solvent system (e.g.,
phenol: water in 4:1 ratio).
- Allow the solvent to rise again as in the first dimension.
- Remove the paper, mark the solvent front, and allow it to dry.
4. Visualization:
- Spray the dried paper with ninhydrin solution and heat the paper in an oven (or use a hair
dryer) until spots develop.
- Purple spots will appear at different positions on the paper, indicating the presence of amino
acids.
5. Calculation of Rf Values:
- Measure the distance travelled by each amino acid spot and the distance travelled by the
solvent front in both dimensions.
- Calculation of the retention factor (Rf) values for each amino acid using the
formula:
Rf = Distance travelled by the amino acid ÷Distance travelled by the solvent
S. No Name of Amino Acid Rf Value
1. Glycine 0.30-0.50
2. Alanine 0.20-0.45
3. Leucine 0.25-0.40
4. Lysin 0.15-0.35
5. Glutamic acid 0.15-0.35
Results: Different amino acids were separated based on their Rf values. The amino acids
appeared as purple spots on the chromatography paper each with a unique Rf value in both
dimensions.
Experiment No.-7
Aim: Separation of Amino Acids by Gel Electrophoresis.
Reference: Practical Biochemistry by R.C. Gupta and S. Bhargavan.
Material Required: Electrophoresis buffer (e.g., Tris-glycine buffer), Agarose or
polyacrylamide gel (depending on the technique), Loading dye (for electrophoresis), Staining
solution for gel (e.g., Coomassie Brilliant Blue for protein/amino acid detection)
Apparatus: Gel electrophoresis apparatus, Power supply for electrophoresis, UV or visible light
source (for visualization), Electrophoresis gel casting tray and comb, Gel staining and
destaining trays.
Theory: Gel electrophoresis is a technique that separates molecules (in this case, amino acids)
based on their size and charge. When an electric field is applied, amino acids migrate through
a gel matrix toward either the anode or cathode depending on their charge at a given pH. The
rate of movement is influenced by their molecular weight and charge.
Procedure:
1. Preparation of Gel:
- Prepare the agarose or polyacrylamide gel according to the manufacturer’s instructions.
- Pour the gel into the casting tray and insert the comb to create wells. Allow the gel to
solidify.
2. Preparation of Samples:
- Mix the amino acid samples with a small amount of loading dye (if applicable).
3. Running the Gel:
- Place the solidified gel in the electrophoresis chamber and fill the chamber with the
electrophoresis buffer (e.g., Tris-glycine buffer).
- Load the amino acid samples into the wells using a micropipette.
- Connect the electrophoresis chamber to a power supply and run the gel at the appropriate
voltage (e.g., 100V) for about 45 minutes to 1 hour.
4. Staining and Visualization:
- After the run, remove the gel and stain it using Coomassie Brilliant Blue or a suitable
protein/amino acid stain.
- Wash the gel in a destaining solution to remove excess stain.
- Amino acids will appear as bands in the gel.
5. Interpretation:
- The migration of amino acids will depend on their charge and size.
- Compare the migration distances of the separated amino acids with standard markers (if
available) to identify them.
Results: The amino acids migrated through the gel based on their charge at the chosen pH.
Bands were visualized after staining, indicating the separation of the amino acids in the
mixture.
Precautions:
1. Ensure that the chromatography paper is handled carefully to avoid contamination or
damage.
2. Avoid overloading the sample onto the chromatography paper or gel wells to prevent
smearing.
3. The electrophoresis gel must be run at the correct voltage to prevent overheating or poor
resolution.
4. Handle reagents like ninhydrin and Coomassie Brilliant Blue with care, as they may be toxic
or hazardous.
5. Ensure proper calibration of the electrophoresis apparatus to get accurate results.
