Laboratory Manual
Biochemistry II
(PHL 224)
Dr/ Gamal A Gabr
Department of Pharmacology
College of Pharmacy
Salman bin Abdulaziz University
1435-1436 / 2014 – 2015
Biochemistry Laboratory Manual PHL 224
TABLE OF CONTENTS
i. Development of Skills
ii. Laboratory Safety
iii. General Laboratory Rules
iv. In Case of an Accident
v. Laboratory Reports
vi. Use of Micropipettes & Volumetric and Serological Pipettes
vii. Units, Amounts and Concentrations
S. No.
Experiment
1.
To perform Molisch test, Fehling’s test, Benedict’s test for
identification of sugars.
2.
A. To measure pH of tap water and distilled water using pH
paper strips and a pH meter.
B. To prepare phosphate, citrate and carbonate buffer & measure
their pH.
3.
To detect the presence of amino acids qualitatively by Ninhydrin
test, Xanthoproteic test, Hopkins-Cole Test, Millon’s test, Pauly’s
test, Ehrlich test, Nitroprusside test, lead acetate test and
Sakaguchi test.
4.
To carry out qualitative tests to detect lipids in the given sample
using Sudan III reagent.
5.
Estimation of glucose content by kit method and glucometer in
blood & urine.
6.
Enzymatic hydrolysis of starch by α & β amylase.
7.
Effects of temperature & pH on the activity of enzymes (catalase
from fresh potato).
8.
Estimation of SGOT & SGPT activity by kit method in blood &
urine.
9.
Estimation of LDH activity by kit method in blood & urine.
10.
Estimation of alkaline phosphatase activity by kit method in blood
& urine.
11.
Estimation of total cholesterol, HDL-Cholesterol & Triglycerides
content by kit method in blood & urine.
12.
To estimate protein in the given sample by Folin--Lowry’s
method.
13.
To estimate the Urea in blood.
14.
To test the given urine sample for the presence of ketone bodies.
Dr. Gamal Gabr
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2-3
3
3-4
4
5
6-10
Page No.
11-12
13-14
15-16
17-19
20-20
21-22
23-24
25-26
27-28
29-30
31-33
34-37
38-39
40-41
42-42
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Biochemistry Laboratory Manual PHL 224
DEVELOPMENT OF SKILLS
Following is the broad perspective of acquisition of intellectual and motor skills. Due
care is to be taken, that a student systematically studying the subject will acquire the
skills enlisted below.
A) Intellectual skills
1. To understand the concepts.
2. To understand the procedure for performance of experiments.
3. To interpret results of experiments.
4. To investigate and discriminate the various situations.
5. To acquire ability to plan and design the experiment.
B) Motor skills:
1. Handling and using correctly the instruments and equipments.
2. Measuring and recording accurately with the help of instruments/equipments
3. To follow systematic, hygienic and safe procedure of working.
4. To collect sample of blood & Urine.
LABORATORY SAFETY
For the welfare of fellow students, and for your own well-being, each student is
expected to follow a set of accepted laboratory precautions. In the lab:
1. Do not eat, drink, or apply cosmetics at any time.
2. Mouth pipetting is not permitted.
3. Wear protective eyeware at all times; chemical splash goggles must be worn
when working with solutions of strong acids or bases.
4. Do not sit on the lab benches.
5. Use chemicals with high vapor pressure only in the hood.
6. Handle and dispose of hazardous chemicals properly. Disposal containers are
provided.
7. When chemicals are spilled they should be wiped or swept up or both as soon as
possible. If the spillage is large, immediately notify an instructor. Sponge and
brushes are provided and should be used to keep your work area clean.
8. Be aware of objects that can burn or give electrical shocks.
9. Do not use open flames near flammable chemicals.
10. In general, be alert to possible hazards from chemicals, glassware, electrical
connections, and flammable solvents. Read labels and observe suggested
precautions.
11. Sweep up broken glassware as soon as possible and deposit it in the labeled
container set aside for this purpose.
12. Never work alone.
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Biochemistry Laboratory Manual PHL 224
13. All solutions that you prepare must be labeled with date, your last name, and the
type and concentration of the reagent. Unmarked solutions will be disposed.
14. Wipe your lab bench with a damp sponge at the end of each lab period.
15. Be familiar with location and use of safety items — location of safety showers,
eyewash stations, fire blankets, fire extinguishers, medical kit.
GENERAL LABORATORY RULES
1. Always wear a lab coat before enter the lab.
2. The instructors will make every effort to keep equipment in good working order.
It is your responsibility to read instructions for the use of equipment. Do not turn
on an instrument until you have done this. Do not hesitate to ask questions of
your instructor after you have read the instructions. If any equipment
malfunction is noted, report this immediately to an instructor.
3. Used glassware should be rinsed with tap water and deposited carefully on the
designated cart after all tape has been removed. Ink writing on glassware need
not be removed.
4. Items stored in the cold room or deep freeze should be removed and disposed of
when they are no longer needed.
5. Clean up after yourself after using any piece of equipment. Be considerate of
others. All equipment should be left ready for immediate use by another student.
6. Please clean the lab after each class. Your instructor will advise you for saving or
disposing your solutions.
7. Keep your work area clean. Your grades may be deducted if you keep littering
your bench.
8. You must have a lab notebook for writing everything in black or blue ink (no
pencils). When correcting your writing, scratch the error with a single line and
write the correct one above or below the line.
9. Pay attention to the due date for each experiment. You will be penalized for late
submitting your lab report.
10. You should finish your own lab report. Do not copy old lab reports, or copy your
classmate’s report. Plagiarism is illegal and unethical. Any evidence of
plagiarism in this class will result in your failure in the class. Further disciplinary
actions will be taken upon your instructor’s decision.
IN CASE OF AN ACCIDENT
1. Report all injuries, even minor ones, to an instructor immediately.
2. In case of even minor laboratory accidents, you should go to Health Center for
treatment.
3. For any chemicals splashed in the eye, hold the eye open and flush immediately
with cold water by using the eye wash or the rubber hose attached to the water
faucet at the end of each laboratory bench. Flush for at least 5 minutes and call
for help from an instructor.
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Biochemistry Laboratory Manual PHL 224
4. For chemicals spilled on the skin or splashed into the mouth, again, flush with
large amounts of cold water for 5 or more minutes. Call for an instructor.
5. For severe bleeding, apply pressure and call for help from an instructor.
6. For burns, flush with cold water and contact an instructor.
Note: Injury to students in the laboratory is not covered by University insurance.
Students must provide their own insurance coverage.
LABORATORY REPORTS
You are required for submitting a lab report for each experiment. Reports are due after
one week from the date you have performed your experiment. There will be a 10%
grade deduction upon your late submission. I also strongly recommend you use a
graphic program to plot your graphs, in which all axes should be labeled and the
proper units are displayed.
Your lab reports should consist of the following sections:
1. The title of the laboratory report: this includes the title of your report, the course
name and number, and your name and the date.
2. Objective section: the object will be a brief description of your experiment.
3. Principle section: Describe basic principle of the experiment.
4. Materials required: this section should describe the materials used, their strength.
5. Experimental Procedure: Describe the method used.
6. Calculation section: Calculations are required to be shown and explained.
7. Results section: this section presents your results that were obtained in your
experiments.
8. Clinical significance: in this section, you should discuss your findings and
compare your results with the normal values and write clinical relevance.
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Biochemistry Laboratory Manual PHL 224
USE OF MICROPIPETTES, VOLUMETRIC AND SEROLOGICAL
PIPETTES
Micropipettes
The first portion of this lab involves becoming familiar with the use of micropipettes.
Micropipettes accurately measure volumes ranging from less than 1 µl to greater than 1
ml, the types that you will use measure from 0.1-2.5 µl (P-2.5), 1-20 µl (P-20), 20-200 µl
(P-200) and 100-1000 µl (P-1000). The disposable tips that fit on the end of the
micropipettes are sometimes colored yellow and blue. The yellow tips (which are white
for our experiments but are sometimes colored yellow) are for volumes of 1- 200 µl and
fit on the ends of the P-20 and P-200 micropipettes but not the P-1000 micropipette. The
blue tips (which are white for our experiments but are sometimes colored blue) are for
volumes of 100-1000 µl and fit on the end of the P-1000 micropipette.
The following steps describe the use of the micropipette. Adjust the micropipette
volume to the required setting. Press the tip on firmly with a slight twisting motion to
ensure an airtight seal. The plunger on the top of the micropipette is then depressed to
the first point of resistance and while holding the micropipette vertical the tip is placed
just below the surface of the appropriate solution (1-2 mm for yellow and 2-4 mm for
blue). The plunger is then slowly released and the solution is sucked up into the pipette
tip, wait 2 seconds to ensure that the full volume has been drawn up into the tip. Once
the plunger has been released the tip is removed from the solution (any excess liquid
should be wiped from the side of the tip but keep the cloth away from the end) and
transferred to the receptacle where the tip is placed on the side of the receptacle and the
plunger is once again depressed to the point of first resistance, wait, then continue on
past the point of first resistance (overshoot). With the plunger fully depressed withdraw
the micropipette from the vessel allowing the tip to slide along the wall of the vessel.
Allow the plunger to return to the top position.
Normally the tip would then be ejected by pressing the tip ejector button to
prevent cross contamination of samples but for our purposes in this exercise it should
only be necessary a few times. Attention should be given to the speed and smoothness
during depression and release of the button, pressure on the button at the first stop,
immersion depths (if the depth is incorrect bubbles may get into the sample), and
minimal angle from the vertical axis.
Please try the above procedure a few times with each of the micropipettes using
water as the solution to gain practice. Once the technique has been mastered, take your
solution of water over to the balances and pipette out five times each of the following
volumes in any order. Change the tip only when you change the volume.
i.
5.0 µl with the 1-20 µl micropipette
ii.
20.0 µl with the 1-20 µl micropipette
iii.
20.0 µl with the 20-200 µl micropipette
iv.
100.0 µl with the 20-200 µl micropipette
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Biochemistry Laboratory Manual PHL 224
Volumetric and serological pipettes
Volumetric pipettes are calibrated to deliver a specific volume of solution at a certain
temperature and have a single line with no scale. With a rubber bulb (DO NOT USE
YOUR MOUTH) draw liquid to a level 2-3 cm above the line, wipe the tip with a
kimwipe (not necessary for your practices), touch the tip of the pipette on the inside
wall of the container from which it was filled. Release liquid until the meniscus is
directly on the fill line. Transfer the pipette to the inside of the second container and
release the liquid, when the flow stops wait 5-10 seconds then touch the tip to the side
of the vesicle. The pipette should then be removed from the container. The pipette is TD
(“to deliver”) and therefore any remaining liquid should not be blown out of the
pipette.
Fractional volumes are transferred with graduated pipettes, one example being a
serological pipette. The procedure is the same as the volumetric pipette except that the
solution starts at the desired amount and the pipette is blown out at the end.
Please try the above procedure a number of times with each of the pipettes using
water as the solution to gain practice. Once the technique has been mastered, take your
solution of water over to the balances and pipette out five times:
i. 10.000 ml with the volumetric pipette
ii. 10.000 ml with the serological pipette.
UNITS, AMOUNTS AND CONCENTRATIONS
Units and abbreviations
The common units, and their respective abbreviations, used in biomedical sciences are
based on the International System of Measurements (SI units): grams (g), meters (m) etc.
Often it is necessary to deal with large quantities e.g. 1500 g or, more commonly, very
small quantities, e.g. 0.0000015 g. In such cases, such numbers are either expressed by
use of powers of 10 (positive or negative) or by use of the appropriate prefix.
Thus: 1500 g = 1.5 x 103 g = 1.5 kg (kilograms)
And 0.0000015 g = 1.5 x 10-6 g = 1.5 µg (micrograms)
Note also that biomedical scientists normally express volumes and concentrations in
terms of liters rather than in cubic measurements:
e.g. 1 liter, rather than 1 dm3
1 milliliter (1 ml) rather than 1 cm3
A concentration of 1 milligram per liter is also still commonly expressed in the form 1
mg/l rather than 1 mg.l-1 or 1 mg.dm-3.
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Biochemistry Laboratory Manual PHL 224
The commonly used prefixes are:
Prefix
Name
Which modifies an amount by
Examples
M
milli
1/1000th, i.e. by 10-3
mmol, mg, ml
µ
micro
1/1000 000th, i.e. by 10-6
µmol, µg, µl
N
nano
by 10-9
nmol, ng
P
Pico
by 10-12
pmol, pg
F
Femto
by 10-15
fmol, fg
K
Kilo
by 1000 times, i.e. by103
kg
M
mega
by 106
MPa
The prefixes centi (10-2) and deci (10-1) are only commonly used in specific cases e.g. cm
Concentrations
The determination of the concentration of a substance in a biological fluid is central to
many areas of medical and dental practice (e.g. electrolytes in serum or glucose in
urine). It is important, therefore, that concentrations are expressed in clear
unambiguous terms. There are several ways of doing this. The simplest is to express
the concentration as the weight or mass of the substance per unit volume:
e.g. 10 g/l or 20 mg/ml or 2 µg/ml
Another way is to express the concentration of a solution or mixture in terms of per cent
(%). This is a somewhat outdated method but you may still come across it, particularly
if you read the older medical literature, so you should know what it means. Note that
there are different types of % concentration:
% (v/v) (volume by volume)
% (w/v) (weight by volume)
% (w/w) (weight by weight)



A 1% (v/v) concentration is obtained by diluting 1 volume of a substance into 100
volumes (total) of solution, e.g. 1 ml ethanol diluted with water to a final volume of
100 ml gives a 1% (v/v) ethanol solution.
A 1% (w/v) concentration is obtained by dissolving 1 g of substance in a final
volume of 100 ml solution, e.g. 1 g glucose dissolved in water to a final volume of
100 ml solution gives a 1% (w/v) glucose solution.
A 1% (w/w) concentration is obtained by mixing 1 g of substance with something
else in a total weight of 100 g, e.g. 1% (w/w) salt in sand.
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Biochemistry Laboratory Manual PHL 224
Another old method of expressing concentration that you may still see (just look at the
side of a tube of toothpaste) is "parts per million (ppm)". One ppm is one part of
anything in one million parts of total material, e.g. 1 g of compound X in a million g
total, or 1 liter of Y in a million liters total.
Moles and molarity
By far the most important unit defining an amount of a biological substance is the mole,
with the corresponding concentration being the molarity. As far as possible, the
concentrations of specific compounds in serum, urine etc, are now expressed as
molarities in clinical laboratories.
One mole of a substance is the molecular weight of that substance expressed in
grams. Thus, the molecular weight of glucose is 180, so:
1 mole of glucose = 180 g
1 mmol glucose = 180 mg
1 µmol glucose =
180 µg
100 µmol glucose = 18,000 µg = 18 mg
Note the abbreviations: 1 mmol = 1 millimole; 5 µmol = 5 micromoles.
Concentrations in molarities are given by expressing the number of moles of the
substance present in a defined volume of solution:
A 1 molar (1 M) solution contains 1 mole per liter (1 mol/l)
A 1 millimolar (1 mM) solution contains 1 millimole per litre (1 mmol/l)
Note: mol and moles mean the same thing (an amount) and moles/litre (long winded
but correct) is a concentration and can be expressed as mol/l, or mol.l-1, or (best and
simplest of all) — M. Ensure you know the distinction between concentrations and
amounts.
So, if you dissolve 0.5 mol of a compound in one liter of solvent the
concentration of the compound is 0.5 mol/l, or 0.5 M. 100ml of the solution contains 0.05
mol.
Prefix notation
The value of the prefix notation can now be seen as it allows rapid mental calculations
to be performed (after much practice!). The following concentrations are all the same:
0.5 M, 0.5 mol/l, 0.5 mmol/ml, 0.5 µmol/µl, 0.5 pmol/pl
You see that by scaling both the units (amount and volume) in the concentration up or
down by a factor of 1000, the value of the concentration remains the same. If you only
scale one of the terms (amount or volume), then you can express the same concentration
in yet more ways:
0.5 mol/l = 0.5 mmol/ml = 500 µmol/ml = 500,000 pmol/µl
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Biochemistry Laboratory Manual PHL 224
Now, let’s say you wanted to determine the amount of cholesterol in the blood of a
newborn infant. You know it will be around 5 mM. The detection limit of the method
you are going to use is about 20 nmol and you can only take 20 µl of blood. Will this be
enough?
5 mM cholesterol contains 5 mmol/l, or 5 µmol/ml, or 5 nmol/µl.
It is easy to see now that 20µl contains 100 nmol of cholesterol - ENOUGH.
Examples
 How many µmol are dissolved in 2 l of a 20 mM solution?
20 mM = 20 mmol/l, so 2 l contain 40 mmol. 40 mmol = 40,000 µmol.

The molecular weight of NaCl is 58. How many mg are in 50 µmol of NaCl?
1 mol of NaCl is 58 g, so 1 µmol is 58 µg, so 50 µmol is 2,900 µg = 2.9 mg.

What is the molarity of a 1% (w/v) solution of glucose? (molecular weight = 180)
1% (w/v) contains 1 g in 100 ml and, therefore, 10 g in 1 liter.
A 1 M solution of glucose contains 180 g/l, so 10 g/l represents a molarity of 10/180
= 0.056 M (or 56 mM).
Dilutions
This is another source of great confusion. Most experiments require you to make
dilutions of reagents, either before use or as a consequence of the actual assay. When
you are asked to make a ten-fold dilution of a reagent, the objective is to produce a
solution that has a reagent concentration one-tenth of the original. It follows that the
molecules of reagent that occupied a volume of "x" before must now occupy a volume
of “10x". This is obtained by adding to one volume of reagent to nine volumes of
diluent (sometimes referred to as a “1 plus 9” dilution or, more commonly, a "1 in 10"
dilution).
When a solution of known concentration is diluted, it is obvious that the
concentration will fall. Less obvious is the amount by which it falls! Take a typical
example: A solution of a compound is maintained as a stock solution at 5 mM. What is the final
concentration of the compond in which 0.15 ml of stock solution is mixed with 0.4 ml of buffer
and 0.2 ml of water?
There are many ways to derive this answer. Here are a few different approaches — all
equally acceptable. Take the one that seems most logical to you!
i. The final volume is 0.75 ml, therefore the concentration of the substrate will
be 0.15/ 0.75 x 5 mM = 1 mM
ii. 5 mM is 5 mmol/l, or 5 µmol/ml. Therefore 0.15 ml of stock solution contains
0.15 x 5 = 0.75 µmol. This is expanded into a volume of 0.75 ml and therefore
there are now 0.75 µmol/0.75 ml, or 1 µmol/ml, or 1 mmol/l, or 1 mM
iii. The formula for a dilution is v1 x c1 = v2 x c2 (where c1 and c2 are the
concentrations before and after dilution, and v1 and v2 are the volumes
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Biochemistry Laboratory Manual PHL 224
before and after dilution). Therefore, c2 = v1/v2 x c1, which works out to be
c2 = 0.15 / 0.75 x 5 mM , i.e. 1 mM
Note that the last and first are identical, except that the latter defines the problem as a
formula. It may be valuable for dilutions when a solution of known concentration must
be diluted to a new concentration. The formula will give the new volume, but
remember that part of that volume will come from the sample before dilution! Method
2 seems tortuous with this example, but it suits some to work this way.
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Biochemistry Laboratory Manual PHL 224
Experiment No. 1
Objective: To perform Molisch test, Fehling’s test, Benedict’s test for identification of
sugars.
Principle: Carbohydrates undergo dehydration reactions (loss of water) in the
presence of concentrated sulfuric acid. Pentoses and hexoses form five member oxygen
containing rings on dehydration. The five member ring, known as furfural, further
reacts with Molisch reagent to form colored compounds. Molisch test is used to detect
carbohydrates in several substances.
All monosaccharides and many disaccharides with free aldehyde groups can
reduce weak oxidizing agents like cupric ions (Cu2+ ion) in alkaline medium (Fehling's
or Benedict's reagents) to produce red or orange colored precipitate of cuprous ions.
These carbohydrates are called reducing sugars. Heating in a boiling water bath is
necessary for these reactions.
Materials required:
Glassware: Beaker, water bath, test tubes, graduated pipettes.
Chemicals: Molisch reagent (1% α-naphthol in alcohol), concentrated sulphuric acid,
distilled water, Fehling's solution A (7.93% copper sulphate in water), Fehling's solution
B (250g sodium hydroxide and 320g sodium potassium tartarate in 500 ml water),
Benedict's reagent (dissolve 173g sodium citrate and 100g anhydrous sodium carbonate
in about 800 ml water, separately dissolve 17.3g copper sulphate in 100ml water, mix
both the solution and make volume to 1000 ml with water).
Experimental Procedure:
S. No.
1.
2.
3.
Test
Molisch test: Mix 2ml of sugar
sample with 5 drops of
Molisch's Reagent in a test
tube. Add gently through the
side by tilting the tube, about
2 ml of concentrated H2SO4 so
as to form a bottom layer.
Solubility: Compound +
water
Observation
Inference
Violet / purple ring at Sugar present.
the junction of two
liquids
Mono and
disaccharides present
Insoluble
Polysaccharides
present
Fehling’s test: 2 ml of Yellow or brick red ppt Reducing sugar
Fehling's solution A + 2ml of is observed.
present
Fehling's solution B+ 2 ml of
Sugar solution Boil.
Dr. Gamal Gabr
Soluble
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Biochemistry Laboratory Manual PHL 224
4.
Benedict’s test: Take 5ml of Green, yellow, orange Reducing sugar
Benedict's qualitative reagent, or brick red ppt is present
add 8 drops of sugar solution. observed
Boil over a flame for 2 minutes
or place in boiling water bath
for 3 minutes. Allow to cool.
Results: On the basis of above observations the given sample was found to be sugar.
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Biochemistry Laboratory Manual PHL 224
Experiment No. 2 (A)
Objective: To measure pH of tap water and distilled water using pH paper strips and
a pH meter.
Principle: pH is the acidity or alkalinity of a substance. A pH of seven is neutral. Any
number lower than seven represents an acidic substance while any number higher than
seven represents an alkaline substance. There are substances which have the property of
changing their color when they come in contact with an acidic or basic environment.
These substances are called pH indicators. Usually, they are used as dissolved
substances, as for instance phenolphthalein and bromothymol blue. Often, to measure
the pH, special papers which have been soaked with indicators are used. These papers
change color when they are immersed in acidic or basic liquids. This is the case of the
well-known litmus paper. More recently, it has become possible to measure the pH with
electrical instruments like the pH meter.
The pH meter is an electronic instrument supplied with a special bulb which is
sensitive to the hydrogen ions which are present in the solution being tested. The signal
produced by the bulb is amplified and sent to a liquid-crystal or an analog meter
display. These instruments are much more precise and convenient to use than the
indicating papers.
Materials required:
1.
2.
3.
4.
5.
Tap water
Distilled water
pH meter
pH paper strips
Glassware
Experimental Procedure:
1. Measurement of pH by pH meter: The pH meter is put on and allowed to
warm and calibrate the probe using two standard solutions (pH 4, 7, and 10
buffers are recommended, dependant on the range). Calibration procedures vary
by instrument, so following the manufacturer's instructions is highly
recommended. BE SURE TO RINSE THE PROBE THOROUGHLY BETWEEN
BUFFERS USING DEIONIZED WATER AND CAREFULLY BLOT THE PROBE
DRY USING A KIM WIPE. pH meters should be calibrated before each use (before
each series of samples, not between each sample itself) or when measuring a large
range of pH. The standard buffer solution is taken in a beaker. The temperature of
buffer solution is noted and electrodes are dipped in the solution. The selector
switch is now turned to proper range 0-7 or 7-14. The meter will show the pH of
buffer. Set the pointer to the exact value of pH of buffer. Bring the selector switch
again to zero, clean the electrodes with water. Collect sample water in a glass or
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Biochemistry Laboratory Manual PHL 224
plastic container. Collect enough so the probe tip can be submerged in sample;
either rinse the probe with deionized water (and blot dry) or with sample before
inserting the probe into the collection vessel. Submerge the probe into the sample
and wait until the pH reading on the meter stabilizes. Many meters have automatic
temperature correction (ATC), which calculates the pH taking into account
temperature, if your meter does not have this feature, you may need to adjust a
knob on the meter to correct the pH for temperature. Record the measurement
when the pH reading is stable.
2. Measurement of pH by pH paper strips: Using Litmus paper is simple. First
of all, it is necessary to immerse an end of it in the liquid you wish to examine and to
remove it immediately. The pH of the liquid is determined by comparing the color
of the paper to the scale of colors which is printed on its packet
Precautions:
1. The pH meter must be calibrated by checking against a standard buffer of know pH.
2. Keep the electrodes immersed in water when not in use.
Results: The pH of tap water & distilled water by pH meter was found to be ______ &
____ respectively and by pH paper strip was found to be ____ & ____ respectively.
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Biochemistry Laboratory Manual PHL 224
Experiment No. 2 (B)
Objective: To prepare standard buffer solution (phosphate, citrate and carbonate
buffer) and measurement of pH.
Principle: Buffer is the solutions that resist changes in pH when small amounts of acid
or base are added. There are two types of buffer.
a. Weak acid and the salt of the same weak acid, (for example a solution containing
ethanoic acid and sodium ethanoate). This gives a buffer solution with a pH less
than 7
b. Weak base and the salt of the same weak base (for example ammonia and
ammonium chloride solution). This gives a buffer with a pH greater than 7
The first (acidic) buffer works in the following way.
If an acid is added it combines its free hydrogen ions with the ions from the salt of the
weak acid making molecular weak acid that cannot affect the pH.
If a base is added the OH- ions from the base react with the H+ ions that are present
from the weak acid dissociation. Having been removed from the solution this stimulates
the weak acid to produce more H+ ions (Le Chatelier's Principle) and the original pH is
re-established.
Materials required:
1.
2.
3.
4.
5.
6.
7.
8.
9.
Potassium dihydrogen phosphate (KH2PO4)
Disodium hydrogen phosphate (Na2HPO4)
Sodium carbonate (Na2CO3)
Sodium Bicarbonate (NaHCO3)
Citric acid
Sodium citrate
Distilled water
pH meter
Glassware
Experimental Procedure:
1. Preparation for Phosphate Buffer:
Solution I: 1.36 g KH2PO4 was dissolved in sufficient water to produce 100 ml.
Solution II: 3.58 g of Na2HPO4 was dissolved in sufficient water to produce 100 ml.
Then, 96.4 ml of solution I was mixed with 3.6 ml of solution II.
2. Preparation for Carbonate Buffer:
0.84 g of NaHCO3 and 1.06 g of Na2CO3 was dissolved in sufficient water to produce
50 ml.
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3. Preparation for Citrate Buffer:
Solution I: 0.21 g of citric acid was dissolved in sufficient water to produce 10 ml.
Solution II: 0.29 g of sodium citrate was dissolved in sufficient water to produce 10
ml.
Then, 2.8 ml of solution I and 2.2 ml of solution II was mixed in sufficient water to
produce 100 ml.
4. Measurement of pH: The pH meter is put on and allowed to warm and calibrate
the probe using two standard solutions (pH 4, 7, and 10 buffers are recommended,
dependant on the range). Calibration procedures vary by instrument, so following
the manufacturer's instructions is highly recommended. BE SURE TO RINSE THE
PROBE THOROUGHLY BETWEEN BUFFERS USING DEIONIZED WATER AND
CAREFULLY BLOT THE PROBE DRY USING A KIM WIPE. pH meters should be
calibrated before each use (before each series of samples, not between each sample
itself) or when measuring a large range of pH. The standard buffer solution is taken
in a beaker. The temperature of buffer solution is noted and electrodes are dipped in
the solution. The selector switch is now turned to proper range 0-7 or 7-14. The
meter will show the pH of buffer. Set the pointer to the exact value of pH of buffer.
Bring the selector switch again to zero, clean the electrodes with water. Collect
sample water in a glass or plastic container. Collect enough so the probe tip can be
submerged in sample; either rinse the probe with deionized water (and blot dry) or
with sample before inserting the probe into the collection vessel. Submerge the
probe into the sample and wait until the pH reading on the meter stabilizes. Many
meters have automatic temperature correction (ATC), which calculates the pH
taking into account temperature, if your meter does not have this feature, you may
need to adjust a knob on the meter to correct the pH for temperature. Record the
measurement when the pH reading is stable.
Precautions:
3. The pH meter must be calibrated by checking against a standard buffer of know pH.
4. Keep the electrodes immersed in water when not in use.
Results: The pH of phosphate, citrate and carbonate buffer was found to be ______,
____ & ____ respectively.
Dr. Gamal Gabr
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Biochemistry Laboratory Manual PHL 224
Experiment No. 3
Objective: To detect the presence of amino acids qualitatively by Ninhydrin test,
Xanthoproteic test, Hopkins-Cole Test, Millon’s test, Pauly’s test, Ehrlich test,
Nitroprusside test, lead acetate test and Sakaguchi test.
Principle: Amino acids of the general formula RCH(NH2)COOH are amphoteric,
behaving as aliphatic amines in some reactions and as carboxylic acids in others.
Ninhydrin test: Amino acids react with ninhydrin (triketohydrindene hydrate)
at pH=4. The reduction product obtained from ninhydrin then reacts with NH 3 and
excess ninhydrin to yield a blue colored substance.
Xanthoproteic Test: Amino acids containing an aromatic nucleus form yellow
nitro derivatives on heating with concentrated HNO3. The salts of these derivatives are
orange in color. Apply this test to tyrosine, tryptophan and glutamic acid.
Hopkins-Cole Test: The indole group of tryptophan will react with glyoxylic
acid in the presence of concentrated H2SO4 to give a purple color. Glacial acetic acid,
which has been exposed to the light, always contains glyoxylic acid CHOCOOH as an
impurity. Apply this test to tyrosine, glycine and tryptophan.
Millon’s Test: Phenols only give this reaction; tyrosine is the only common
phenolic amino acid. Millon’s reagent is concentrated HNO3, in which mercury has
been dissolved. A yellow precipitate of HgO in a test is NOT a positive reaction but
usually indicates that the solution is too alkaline. Apply this test to tyrosine,
phenylalanine, glycine and β-naphtol.
Pauly Test: The imidazole ring of histidine, in the presence of sodium nitrite,
reacts with sulfanilic acid forming a yellow product. This is a diazo coupling reaction.
Ehrlich Test: Aromatic amines and many organic compounds (indole and urea)
give a colored complex with this test. Apply this test to urea, glycine and tryptophan.
Nitroprusside Test: Thiol groups give a red color with sodium nitroprusside in
the presence of excess ammonia. Apply this test to cystine, cysteine and methionine.
Lead Acetate test: Sulfur containing amino acids, such as cysteine and
methionine, are degraded in strongly alkaline media to release sulfide ion (S2-) in the
form of H2S (hydrogen sulfide). The sulfide ions can react with lead (II) acetate to form
a brownish-black precipitate.
Sakaguchi Test: The only amino acid, which contains a guanidine group, is
arginine. Arginine gives a red color with α-naphthol, in the presence of an oxidizing
agent like Bromine solution. Apply this test to arginine.
Materials required:
Glassware: Beaker, water bath, test tubes, graduated pipettes.
Chemicals: 0.2% ninhydrin solution (triketohydrindene hydrate), NH3, conc HNO3,
NaOH (20%, 40%), glyoxylic acid, glacial acetic acid, conc H2SO4, Millon’s reagent, 5%
Dr. Gamal Gabr
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Biochemistry Laboratory Manual PHL 224
sodium nitrite, 1% sulfanilic acid, sodium nitroprusside, lead (II) acetate, α-naphthol,
Bromine solution
Experimental Procedure:
S. No. Test
Observation
Ninhydrin test: To 1ml solution blue color formed
1.
2.
3.
4.
5.
6.
7.
add 5 drops of 0.2% ninhydrin
solution in acetone. Boil over a
water bath for 2 min. Allow to
cool
Xanthoproteic test: To 2ml of
solution in a boiling test tube,
add an equal volume of conc.
HNO3. Heat over a flame for 2
min and observe the color. Now
COOL THOROUGHLY under
the tap and CAUTIOSLY run in
sufficient 40% NaOH to make
the solution strongly alkaline.
Hopkins-Cole Test: To a few ml
of glacial acetic acid containing
glyoxylic acid, add 1-2 drops of
the amino acid solution. Pour 12ml concentrated H2SO4 down
the side of the sloping test tube
to form a layer underneath the
acetic acid.
Millon’s test: To 2ml of amino
acid solution in a test tube, add
1-2 drops of Millon’s reagent.
Warm the tube in a boiling
water bath for 10min.
Pauly’s test: Into clean test tube,
dispense 1mL of 1% sulphanilic
acid and 2 drops of 5% sodium
nitrite. Mix for 1 min. Add
about 0.5mL of amino acid
solution.
Ehrlich test: To 0.5ml of the
amino acid solution, add 2ml
Ehrlich reagent
Nitroprusside test: Add 2ml of
the amino acid solution into test
Dr. Gamal Gabr
Yellow color formed
Inference
Amino acid present
Amino acid present
purple color at the Amino acid present
interface
A brick red color is a Amino acid present
positive reaction.
Yellow
formed
product Amino acid present
A colored complex Amino acid present
formed
Red color formed
Amino acid present
18
Biochemistry Laboratory Manual PHL 224
8.
9.
tubes. Add 0.5ml fresh sodium
nitroprusside
solution
and
shake thoroughly. Add 0.5ml
ammonium hydroxide
Lead acetate test: Everything brownish-black
needed to carry out this test will precipitate
be in the hood and should not
remove anything from the hood.
A toxic, stinky gas will be made
(in small, but immensely smelly
quantities) and you don’t want
to smell it. Dispense about
0.5mL of the amino acid
solution only into a clean test
tube (found in the hood, where
you will leave it when you are
done). Add 0.5mL of 20% NaOH
and insert the test tube in a
boiling water bath for 1 min.
Add 2 drops of lead (II) acetate
solution.
Sakaguchi test: 1ml NaOH and Red color formed
3ml of the arginine solution is
mixed and 2 drops of αnaphthol
is
added.
Mix
thoroughly and add 4-5 drops
Bromine solution
Amino acid present
Amino acid present
Results: The amino acids were found to be present in given sample on the basis of
above chemical tests.
Dr. Gamal Gabr
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Biochemistry Laboratory Manual PHL 224
Experiment No. 4
Objective: To carry out qualitative tests to detect lipids in the given sample using
Sudan III reagent.
Principle: Sudan red is a fat-soluble dye that stains lipids red.
Materials required:
Glassware: test tubes, graduated pipettes.
Chemicals: Sudan III reagent, distilled water
Experimental Procedure:
1.
To a test tube, add equal parts of test liquid and water to fill about half full.
2.
Add 3 drops of Sudan III stain to test tube. Shake gently to mix.
3.
A red-stained oil layer will separate out and float on the water surface if fat is
present.
Results: The lipids were found to be present in the given sample on the basis of above
qualitative test.
Dr. Gamal Gabr
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Biochemistry Laboratory Manual PHL 224
Experiment No. 5
Objective: Estimation of glucose content by kit method and glucometer in blood &
urine.
Principle: the oxidation of glucose is catalysed by glucose oxidase (GOD). The
resultant hydrogen peroxide (H2O2) is oxidatively coupled with 4-aminophenazone and
phenol in the presence of peroxidase (POD) to yield a red quinonemine dye, the
concentration of which at 546 nm is proportional to the concentration of glucose.
Mutarotse
α-D-glucose
β-D-glucose
GOD
β-D-glucose + H2O + O2
D-gluconic acid + H2O2
POD
H2O2 + 4-aminophenazone + phenol
quinonemine + 4 H2O
Materials required:
Reagent composition: (Kit supplied by Crescent Diagnostics, Jeddah)
1. Phosphate Buffer (pH 7.5)
0.1 mol/l
4-aminoantipyrine
0.25 mmol/l
Phenol
0.75 mmol/l
Glucose oxidase
15 KU/l
Peroxidase
1.5 KU/l
Mutarotase
2 KU/l
2. Glucose standard
100 mg/dl or 5.5 mmol/l
Experimental Procedure:
1. Glucose estimation by Kit Method
Pipette into cuvettes
Micro
Macro
Blank
standard
sample
Blank
standard
sample
Test sample
----0.01
----0.025
Standard
--0.01
----0.025
--Distilled water
0.01
----0.025
----Reagent
1.0
1.0
1.0
2.5
2.5
2.5
Mix and incubate for 10 min at 20-25C or 5 min at 37C
Measure the absorbance of the sample (As) and standard (Astd) against the reagent blank.
Dr. Gamal Gabr
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Biochemistry Laboratory Manual PHL 224
Calculation:
As
Glucose (mg/dl) =
X concentration of standard
Astd
To convert mg/dl to mmol/l divide by 18
Results: The glucose levels in blood & urine were found to be ____ & ____
by kit method and ____ & ____ by glucometer respectively.
Dr. Gamal Gabr
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Biochemistry Laboratory Manual PHL 224
Experiment No. 6
Objective: Enzymatic hydrolysis of starch by α & β amylase.
Principle: The reducing groups released from starch are measured by the reduction of
3,5-dinitrosalicylic acid. One unit releases from soluble starch one micromole of
reducing groups (calculated as maltose) per minute at 25°C and pH 6.9 under the
specified conditions.
The rate at which maltose is released from starch is measured by its ability to
reduce 3,5-dinitrosalicylic acid. One unit releases one micromole of β-maltose per min
at 25°C and pH 4.8 under the specified conditions.
Materials required:
Reagents
α -amylase
 0.02 M Sodium phosphate buffer, pH 6.9 with 0.006 M sodium chloride
 2 N Sodium hydroxide
 Dinitrosalicylic acid color reagent. Prepare by dissolving 1.0 gm of 3,5dinitrosalicylic acid in 50 ml of reagent grade water. Add slowly 30.0 gms sodium
potassium tartrate tetrahydrate. Add 20 ml of 2 N NaOH. Dilute to a final volume of
100 ml with reagent grade water. Protect from carbon dioxide and store no longer
than 2 weeks.
 1% Starch. Prepare by dissolving 1.0 gm soluble starch, (Merck) in 100 ml 0.02 M
sodium phosphate buffer, pH 6.9 with 0.006 M sodium chloride. Bring to a gentle
boil to dissolve. Cool and bring volume to 100 ml, with water, if necessary. Incubate
at 25°C for 4-5 minutes prior to assay.
 Maltose Stock Solution. Prepare by dissolving 180 mg maltose (MW 360.3) in 100 ml
reagent grade water in a volumetric flask.
β amylase
 0.016 M Sodium acetate, pH 4.8
 2 N Sodium hydroxide
 Dinitrosalicylic acid color reagent: same as above
 1% Starch: Prepare by dissolving 1.0 gram of soluble starch (Merck) in 100 ml of
0.016 M sodium acetate buffer pH 4.8. Bring to a gentle boil to dissolve. Cool and, if
necessary, dilute to 100 ml with reagent grade water. Incubate at 25°C for 4-5
minutes prior to assay.
 Maltose stock solution, 5 micromoles/ml. Prepare by dissolving 180 mg maltose
(MW 360.3) in 100 ml reagent grade water. Incubate at 25°C for 4-5 minutes prior to
assay.
Dr. Gamal Gabr
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Biochemistry Laboratory Manual PHL 224
Enzyme
Dilute to a concentration of 1-10 micrograms/ml. A minimum of three different
concentrations in this range should be run.
Mg/ml=A280 X 0.38
Experimental Procedure:
α -amylase
1. Adjust spectrophotometer to 540 nm and 25°C.
2. Using the maltose stock solution prepare a maltose standard curve as follows: In
numbered tubes, prepare 10 maltose dilutions ranging from 0.3 to 5 micromoles per
ml. Include two blank tubes with reagent grade water only. Into a series of
corresponding numbered tubes pipette 1 ml of each dilution of maltose. Add 1 ml of
dinitrosalicylic acid color reagent. Incubate in boiling water bath for 5 minutes and
cool to room temperature. Add 10 ml distilled water to each tube and mix well. Read
A540 versus micromoles maltose.
3. Enzyme assay: Pipette 0.5 ml of respective enzyme dilutions into a series of
numbered test tubes. Include a blank with 0.5 ml reagent grade water. Incubate
tubes at 25°C for 3-4 minutes to achieve temperature equilibration. At timed
intervals, add 0.5 ml starch solution (at 25°C). Incubate exactly 3 minutes and at
timed intervals add 1 ml dinitrosalicylic acid color reagent to each tube. Incubate all
tubes in a boiling water bath for 5 minutes. Cool to room temperature and add 10 ml
reagent grade water. Mix well and read A540 versus blank. Determine micromoles
maltose released from standard curve.
β amylase
1. Prepare a maltose standard curve as above
2. Enzyme assay: same as above
Calculation:
Results:
Dr. Gamal Gabr
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Biochemistry Laboratory Manual PHL 224
Experiment No. 7
Objective: Effects of temperature & pH on the activity of enzymes (catalase from
fresh potato).
Principle:
Enzymes are biological catalysts that carry out the thousands of chemical reactions that
occur in living cells. They are generally large proteins made up of several hundred
amino acids and often contain a nonproteinaceous group called the prosthetic group
that is important in the actual catalysis.
Each enzyme is specific for a certain reaction because its amino acid sequence is
unique and causes it to have a unique three-dimensional structure (tertiary or
quaternary structure). The business end of the enzyme molecule, the active site, also
has a specific shape so that only one or a few of the thousands of compounds in the cell
can interact with it. If there is a prosthetic group on the enzyme, it will form part of the
active site. Any substance that blocks or changes the shape of the active site will
interfere with the activity and efficient of the enzyme. If these changes are large
enough, the enzyme can no longer act at all and is said to be denatured. There are
several factors that are especially important in determining the enzyme’s shape and
these are closely regulated both in the living organism and in laboratory experiments to
give the optimum or most efficient enzyme activity: salt concentration and temperature.
In this exercise, you will study the enzyme catalase, which accelerates the
breakdown of hydrogen peroxide, a common end product of oxidative metabolism, into
water and oxygen according to the summary reaction:
Catalase
2H2O2
2H2O + O2
This catalase-mediated reaction is extremely important in the cell because it prevents
the accumulation of hydrogen peroxide, a strong oxidizing agent that tends to disrupt
the delicate balance of cell chemistry.
Catalase is found in animal and plant tissues and is especially abundant in plant
storage organs such as potato tuber, corns and the fleshy parts of fruits. Catalase has
been isolated from potato tubers. The activity and efficiency of enzymes are influenced
by various factors, including temperature and pH conditions. Temperatures above
60º C/140ºF damage (denature) the intricate structure of enzymes, causing reactions to
cease. Each enzyme operates best within a specific pH range, and is denatured by
excessive acidity or alkalinity.
The higher the temperature of water, potato and H2O2, the rate at
which the Enzyme will work will be faster therefore producing more
oxygen. The reaction will be the same without the catalase (potato). Therefore
in both experiments the Enzyme will work more rapidly and produce more
oxygen. The optimum pH condition for Catalase is pH 7.52.
Dr. Gamal Gabr
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Biochemistry Laboratory Manual PHL 224
Materials required:
Fresh potato, 1% H2O2, Distilled water, Crushed ice
Homogenizer, Thermometer, Measuring cylinder, Beaker, Water bath etc.
Experimental Procedure:
Extraction of Catalase:
1. Peel a fresh potato tuber and cut the tissue into small cubes. Weigh out 50 gm of
tissue.
2. Place the tissue, 50 mL of cold water and a small amount of crushed ice into a
prechilled blender.
3. Homogenize for 30 seconds at high speed.
From this point on, the enzyme preparation must be carried out in an ice bath!
4. Filter the potato extract through cheesecloth and pour the filtrate into a 100 mL
graduated cylinder. Add cold distilled water to bring up the final volume to 100 ml.
This extract will be arbitrarily labeled 100 units of enzyme per mL (100 units/mL).
Effect of temperature
Using 40 mL of a 1% H2O2 solution as the substrate and 5 mL aliquots of the 100units/ml-enzyme solution, measure the enzyme activity. Run the reactions in the
different temperature water baths. The catalase and substrate (H2O2) should be brought
to the testing temperature before they are used. Allow 10 minutes for equilibration.
Record the exact temperature and your data in a table in your notebook. Also test the
activity of the enzyme that has been boiled. DO NOT boil the H2O2. These assays
should be run in duplicate.
Effect of pH
Obtain five 50-mL beakers and label them as follows:
1. pH 4
2. pH 6
3. pH 7
4. pH 8
5. pH 10
Into each beaker, pour 10 mL of enzyme preparation and 30 mL of buffer solution at the
appropriate pH. Using 40 mL of a 1% H2O2 as the substrate, measure the enzyme
activity in the usual manner.
Results:
Dr. Gamal Gabr
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Biochemistry Laboratory Manual PHL 224
Experiment No. 8
Objective: Estimation of Serum Glutamate Oxaloacetate Transaminase (SGOT/AST)
& Serum Glutamate Pyruvate Transaminase (SGPT/ALT) activity by kit method in
blood.
Principle:
For AST
AST catalyzes the following reaction:
AST
L-Aspartate + 2-Oxoglutarate
Oxalacetate + L-Glutamate
In the present method a diazonium salt is used which selectively reacts with the
oxalacetate to produce a color complex that is measured photometrically.
For ALT
In this procedure ALT catalyzes L-alanine and a-ketoglutarate to form pyruvate and
glutamate. The pyruvate is then reacted with 2 4-dinitrophenylhydrazine (2 4-DNPHine) to form 2 4-DNPH-one. The addition of sodium hydroxide dissolves this complex
allows 2 4-DNPH-one to be measured at 505 nm.
ALT
L-Alanine + a-ketoglutarate
Pyruvate + Glutamate
H+
Pyruvate + 2 4 - DNPH-ine
Pyruvate + 2 4-DNPH-one
Materials required:
Reagent composition:
For AST
1. AST Substrate: 33 mM Aspartic acid 5 mM ketoglutaric acid phosphate buffer pH
7.4.
2. AST Color Reagent: 0.25% w/v Diazonium salt preserved with formalin.
3. AST Standard: A lyophilized serum with AST (SGOT) value provided in each lot
Reconstitute with distilled water let stand until dissolved and swirl to mix. Stable for
5 days at 2 - 8°C after reconstitution. Aliquot into small portions and keep frozen.
For ALT
1. ALT Substrate: 0.2 M L-alanine 2.0 mM a-ketoglutarate 100 mM phosphate buffer at
pH 7.4 + 0.05 0.2% v/v preservatives.
2. ALT Color Reagent: l.0mM 2 4-dinitrophenylhydrazine in 1N Hydrochloric Acid
preservative.
3. ALT Color Developer: 0.5N sodium hydroxide.
4. ALT Standard: Solution of sodium pyruvate in 100 mM phosphate buffer at pH 7.4 .
Dr. Gamal Gabr
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Biochemistry Laboratory Manual PHL 224
Experimental Procedure:
For AST
1. Place 0.5 ml of AST substrate into test tubes labeled "Blank" "Standard" and "Test".
Warm vials in 37°C heating bath for at least 4 min.
2. At timed intervals add 0.1 ml (100µl) of samples into their respective tubes gently
mix and return to 37°C heating bath for exactly 10 min. Use distilled water for
sample blank.
3. After 10 min and in the same timed sequence add 0.5 ml of AST Color Reagent. Mix
gently and immediately return to 37°C heating bath for another 10 min.
4. After 10 min add 2.0 ml of 0.1 N Hydrochloric acid and mix by inversion.
5. Set the wavelength of the spectrophotometer at 530 nm and zero the instrument
with the Blank. Read and record the absorbance of all tubes.
NOTE: The final color developed in the reaction must be read within
60 minutes.
For ALT
1. Label test tubes "Blank" "Standard" "Test".
2. Transfer 0.5 ml of ALT substrate to each tube and place in a 37°C heating bath for 35 min.
3. At timed intervals (about 15-30 seconds) add 0.1 ml (100µl) of sample to the
correspondingly labeled tube. Mix and immediately return to 37°C healing bath for
exactly 30 min.
4. After exactly 30 min add 0.5 ml of ALT Color Reagent to each tube maintaining the
timed interval sequence. Mix and return to 37°C heating bath for exactly 10 min.
5. After exactly 10 min add 2.0 ml of ALT Color Developer (maintaining the same
timed intervals). Mix and return to 37°C heating bath for 5 min.
6. Zero the spectrophotometer with the reagent "blank" at 505 nm. Read and record
absorbance of all tubes.
Calculation:
Abs. of sample
AST (IU/L) =
X Concentration of standard (IU/L)
Abs. of standard
Abs. of sample
ALT (IU/L) =
X Concentration of standard (IU/L)
Abs. of standard
Results: The SGOT & SGPT activity in blood sample was found to be ____
& ____ respectively.
Dr. Gamal Gabr
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Biochemistry Laboratory Manual PHL 224
Experiment No. 9
Objective: Estimation of LDH activity by kit method in blood & urine.
Principle: Lactate dehydroenase (LDH) catalyses the conversion of Lactate to
Pyruvate accompanied by the simultaneous reduction of NAD to NADH. LDH activity
in the serum is directly proportional to the increase in absorbance due to reduction of
NAD.
LDH
Lactate + NAD
Pyruvate + NADH
Materials required: (Diagnostic kit supplied by Reckon diagnostics Ltd.)
1. Micropipettes
2. Cuvette
Preparation of the working reagent:
1 LDH (Coenzyme) x14
2 LDH (Buffered substrate) x1
Reconstitute one vial of the 1LDH with 1.1 ml of the 2 LDH. Mix them gently to dissolve
the contents. Use within five minutes.
Reaction Parameters:
Type of reaction
Flow cell temperature
Wavelength
Interval time
Delay time
Reagent volume
Factor
Sample volume
Zero setting with
Path length
:
:
:
:
:
:
:
:
:
:
Kinetics/ increasing OD
37C
340 nm
30 seconds
60 seconds
1.0 ml
3376
50 l (0.05 ml)
Distilled water
1.0 cm
Experimental Procedure:
Pipette into test tube
Working reagent (ml)
Sample (ml)
Test
1.0
0.05
Mix and read the first absorbance of the test exactly at one minute and thereafter at 30,
60, and 90 seconds at 340nm. Determine the mean change in absorbance per minute.
Dr. Gamal Gabr
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Biochemistry Laboratory Manual PHL 224
Calculation:
LDH activity = Δ A/minute x F
Where F
TV
SV
6.22
= 1/6.22 X TV/ SV X 1000 = 3376
= Total volume (1.05 ml)
= Sample volume (0.05 ml)
= Milimolar extinction coefficient of NADH at 340 nm
Results: The LDH activity in blood & urine was found to be ____ & ____ respectively.
Dr. Gamal Gabr
30
Biochemistry Laboratory Manual PHL 224
Experiment No. 10
Objective: Estimation of alkaline phosphatase activity by p-nitrophenol method in
blood & urine.
Principle: Phosphatases are enzymes which catalyse the splitting of a phosphate from
mono-phosphoric esters. Alkaline phosphatase (ALP), a mixture of isoenzymes from
liver, bone, intestine and placenta, has maximum enzyme activity at about pH 10.5.
Serum ALP measurements are of particular interest in the investigation of hepatobillary
and bone diseases.
Paranitrophenyl phosphate, which is colourless, is hydrolysed by alkaline
phosphatase at pH 10.5 & 370C to form free paranitrophenol, which is coloured yellow.
The addition of NaOH stops the enzyme activity and the final colour shows maximum
absorbance at 410 nm.
Materials required:







2-amino 2- methyl 1-propanol (AMP) buffer pH 10.5: Add 116 ml of AMP to 600 ml
of distilled water. Mix and adjust the pH to 10.5 with 6 M HCI and then make up to
1 litre with distilled water. Stable for 6 months at 2-80C.
Magnesium chloride (1.5 mmol/l): Dissolve 300 mg of magnesium chloride
hexahydrate in distilled water and make up to 1 litre. Stable for 6 months at room
temperature (25-350 C)
Substrate: Dissolve 83.5 mg of disodium paranitrophenyl phosphate in 1.0ml
magnesium chloride solution. Stable for 24 hours at 2-80C. This solution should be
colourless; do not use it if the OD at 410nm > 0.800.
Sodium hydroxide 0.25 M: Dissolve 10 g of NaOH in about 800 ml of distilled water
and then make up to 1 litre with distilled water. Store in a polythene bottle at room
temperature (25-35°C). Stable for 6 months.
Stock paranitrophenol (PNP) 10.8 mmol/l: Weigh out 150 mg of PNP and dissolve
in about 80ml of NaOH (0.25M) and then make up to 100 ml with the same NaOH
solution. Store in a brown glass bottle at room temperature (25-35C). Stable for 3
months.
Working PNP 54 m mol/l: Pipette 0.5 ml of the PNP stock solution into a 100ml
volumetric flask and make up to the mark with NaOH solution (0.25 M). Prepare
fresh before use.
Equipment, glassware and other accessories
Experimental Procedure:
The protocol of the procedure is described below.
Dr. Gamal Gabr
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Biochemistry Laboratory Manual PHL 224
Preparation of standards (SI -S6)
Pipette the following into appropriately labeled test tubes & Mix well
S1
S2
S3
S4
S5
Working PNP solution (ml)
0.5
1.0
2.0
3.0
4.0
NaOH solution (ml)
4.5
4.0
3.0
2.0
1.0
Activity (U/L)
40
80
160
240
320
S6
5.0
400
Set the spectrophotometer to zero absorbance at 410 nm against 0.25M NaOH and
measure the absorbance of the above standards.
Enzyme measurement in test
Pipette the following into another set of appropriately labeled 18 x 150 mm tubes.
Blank
Test
AMP buffer (ml)
1.4
1.4
Mix and Incubate at 370C for 5 minutes
Test Sample (ml)
0.05
Substrate (ml)
0.1
0.1
Mix and Incubate at 370C for 15 minutes
NaOH (ml)
4.0
4.0
(Note: NaOH should be added to each tube in sequence maintaining timed intervals)
Mix and cool the tubes to room temperature (25-35C). Measure the absorbance of test
at 410nm, setting the spectrophotometer to zero with the blank.
Calculation:
The working PNP concentration is 54 m mol/L. Standard S1 contains 0.5ml PNP
54
Concentration of PNP in SI = ---------------- x 0.5 = 0.027m mol.
1000
ALP activity in U/L = Liberation of 1 m mol of PNP per minute at 37C incubation per
litre serum.
In the assay protocol, 0.05 ml serum is mixed with reagent and incubated for 15 minutes
and the total volume is made up to 5.55ml. But the total volume in the case of each
standard (SI to S6) is 5.0 ml.
Test absorbance 0.027
5.55
1000
PNP (umol/L) or ALP activity (U/L) = -------------------- x -------- x --------x----------Std absorbance 15
5.0
0.05
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Biochemistry Laboratory Manual PHL 224
Test absorbance
= ----------------------- x 40
Std absorbance
i.e, ALP activity equivalent for S 1 = 40 U/L. Similarly the ALP activities represented by
other standards are: S2 = 80, S3 = 160, S4 = 240, S5 = 320 and S6 = 400 U/L.
Construct a calibration graph by plotting the equivalent activity of ALP of the standards
against their corresponding absorbance values. The measurable range with this graph is
from 10 to 400 U/L.
Plot the absorbance values of test on the calibration graph and read off the
concentrations.
Once linearity is proved, it will be enough if a single standard is set up every time that
sample are analysed. Use standard S6 in the assay and calculate the results using the
formula:
Test Absorbance
----------------------- x 400 U/L
Std Absorbance
Results: The alkaline phosphatase activity in blood & urine was found to be ____ &
____ respectively.
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Biochemistry Laboratory Manual PHL 224
Experiment No. 11
Objective: Estimation of total cholesterol, HDL-Cholesterol & Triglycerides content
by kit method in blood.
Principle: Cholesterol reacts with hot solution of Ferric Perchlorate, Ethyl Acetate and
Sulphuric acid (Cholesterol reagent) and gives a purple colored complex which is
measured at 560 nm.
For Total Cholesterol
Materials required:
Kit contents:
Reagent 1: Cholesterol reagent
Reagent 2: Working Cholesterol standard (200 mg %)
Reagent 3: Precipitating reagent
All reagents were ready for use.
Experimental Procedure:
Pipette into 3 test tubes labeled blank (B), standard (S) and test (T) as shown below
in Table:
Reagent
2 ml Procedure
3 ml Procedure
B
S
T
B
S
T
Cholesterol reagent
2 ml
2 ml
2 ml
2 ml
2 ml
2 ml
Working Cholesterol
10 µl
10 µl
standard (200 mg %)
Specimen / serum
10 µl
10 µl
Distilled water
1 ml
1 ml
1 ml
Mix well and keep the tubes immediately in the boiling water bath exactly for 90
seconds. Cool them immediately to room temperature under running tap water.
Measure the OD of S and T against B on a colorimeter with a yellow green filter or
on a spectrophotometer at 560 nm.
Calculation:
Absorbance of Test
Total Cholesterol (mg/dl) =
Dr. Gamal Gabr
X 200
Absorbance of Standard
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Biochemistry Laboratory Manual PHL 224
For HDL-Cholesterol
Principle: High Density Lipoproteins (HDL) was obtained in the supernatant after
centrifugation. The cholesterol in the HDL fraction was also estimated by this
method.
Materials required:
Reagents: Reagents supplied in kit were used directly.
Kit contents:
Reagent 1: Cholesterol reagent
Reagent 2: Working Cholesterol standard (200 mg %)
Reagent 3: Precipitating reagent
Experimental Procedure:
Step 1: HDL-cholesterol separation
Pipette into a centrifuge tube:
Serum
0.2 ml
Precipitating reagent
0.2 ml
Mixed well and allowed to stand at room temperature for 10 minutes. Centrifuge at
2000 rpm for 15 minutes to get a clear supernatant. If the supernatant is not clear (high
TG level) dilute the sample 1:1 with normal saline and multiply the result with 2.
Step 2: HDL-cholesterol estimation
Pipette into 3 test tubes labeled blank (B), standard (S) and test (T) as shown below
in Table:
Reagent
2 ml Procedure
3 ml Procedure
B
S
T
B
S
T
Cholesterol reagent
2 ml
2 ml
2 ml
2 ml
2 ml
2 ml
Working Cholesterol
10 µl
10 µl
standard (200 mg %)
Specimen from step 1
80 µl
80 µl
Distilled water
1 ml
1 ml
1 ml
Mix well and keep the tubes immediately in the boiling water bath exactly for 90
seconds. Cool them immediately to room temperature under running tap water.
Measure the OD of S and T against B on a colorimeter with a yellow green filter or on a
spectrophotometer at 560 nm.
Calculation:
Absorbance of Test
HDL-Cholesterol (mg/dl) =
X 50
Absorbance of Standard
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Biochemistry Laboratory Manual PHL 224
For Triglycerides
Principle: Lipase hydrolyses triglycerides sequentially to Di- and mono-glycerides
and finally to glycerol and free fatty acids. Glycerol kinase (GK) using ATP as PO 4
source converts liberated glycerol to glycerol-3-phoshate. Glycerol-3-phoshate oxidase
(GPO) oxidizes glycerol-3-phoshate formed to dihydroxy acetone phosphate and
hydrogen peroxide is formed. Peroxidase (POD) uses the hydrogen peroxide formed,
to oxidize 4-aminoantipyrine and TOOS (N-ethl-N-sulphohydroxy propyl-m
Toluidine) to a purple colored complex. The absorbance of the colored complex is
measured at 546 nm (530-570 nm or with yellow filter) which is proportional to the
triglycerides concentration.
Lipase
Triglyceride + H2O
Glycerol + fatty acids
GK
Glycerol + ATP
Glycerol-3-phoshate + ADP
GPO
Glycerol-3-phoshate + O2
dihydroxy acetone phosphate + H2O2
POD
H2O2 + 4-aminoantipyrine + TOOS
Quinoneimine + H2O
Materials required:
Kit content:
1 – Triglycerides (Enzyme, Chromogen)
2 – Triglyceride (Buffer)
* Triglyceride standard (conc. 200 mg/dl)
Preparation of working reagent: Dissolve the content of one vial labeled (1) containing
Triglycerides with 1.1 ml of second vial labeled (2) containing Triglyceride (Buffer).
Mix gently to dissolve.
Reaction Parameters
Type of reaction
: End point
Wavelength
: 546 nm (530-570 nm)
Flowcell temperature : 30C /37C
Incubation
: 15 minutes at 37oC
Standard concentration : 200 mg/dl
Sample volume
: 10 l (0.01 ml)
Reagent volume
: 1.0 ml
Zero setting with
: Reagent Black
Light Path
: 1.0 cm
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Biochemistry Laboratory Manual PHL 224
Experimental Procedure:
Pipette into 3 test tubes labeled blank (B), standard (S) and test (T) as shown below
in Table:
Reagent
1 ml Procedure
0.5 ml Procedure
B
S
T
B
S
T
Triglycerides reagent (1)
1 ml
1 ml
1 ml
0.5 ml 0.5 ml 0.5 ml
Triglycerides standard (2)
10 µl
5 µl
Specimen (serum)
10 µl
5 µl
Mix well and incubate at 37 oC for 15 minutes and read the absorbance of S and T
against B at 546 nm or with yellow filter (530-570 nm). The final color was brownish
purple and stable at room temperature.
Calculation:
Absorbance of Test
Triglycerides (mg/dl) =
X 200
Absorbance of Standard
Results: The total cholesterol, HDL-Cholesterol & Triglycerides levels in blood were
found to be _____, _____ & _____ respectively.
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Biochemistry Laboratory Manual PHL 224
Experiment No. 12
Objective: To estimate protein in the given sample by Folin--Lowry’s method.
Principle: Lowry’s method of protein estimation is the most widely used and
accepted method for accurate determination of protein concentration. The method is a
combination of biuret reaction and folin-ciocalteau reaction. In the first step of the
reaction protein binds to copper in alkaline medium and produces Cu++ In the second
step, Cu++ catalyses oxidation of aromatic amino acid by reducing
phosphomolybdotungstate to heteropolymolybdanum blue. This reaction produces
strong blue color, which predominantly depends upon tyrosine and tryptophan content
of protein and to a lesser extent cysteine and other residues in protein.
Materials required:
Preparation of the reagents:
1. Alkaline sodium carbonate solution (2 % Na2CO3 in 0.1 N NaOH): 100 ml of 0.1 N
NaOH solution was prepared by dissolving 400 mg of NaOH in distilled water and
the volume was made upto 100 ml. Then 2 g of Na2CO3 was dissolved in 100 ml of
0.1 N NaOH solution.
2. Copper sulfate sodium tartarate solution (0.5 % CuSO4 in 1 % Na-K tartarate):
Sodium potassium tartarate solution: 0.5 % copper sulfate solution was mixed with 1
% Na-K tartarate.
3. Alkaline solution: Prepared on the day of use by mixing 50 ml of reagent 1 and 1ml
of reagent 2.
4. Folin ciocalteau phenol reagent: The commercial reagent was diluted with
2 volumes of distilled water on the day of use.
5. Standard protein: Bovine serum albumin (0.5 mg/ml)- 5 mg of bovine serum
albumin was dissolved in 10 ml of distilled water to get a solution of 2.0 mg/ml
of protein.
Experimental Procedure:
5 ml of alkaline solution was added to 1 ml of protein sample and allowed to stand for
10 minutes. 0.5 ml of diluted folin reagent was added and the tube was shaken to mix
the solution. After 30 min check the absorbance of unknown sample against blank at
750 nm and determine the concentration of the unknown sample using the standard
curve.
Preparation of Calibration standard curve of protein:
5 ml of bovine serum albumin solution (0.5 mg/ml) was prepared and different
volumes were taken in 6 test tubes. To all tubes distilled water was added to make up
the volume in each tube to 1 ml. The protein concentration in the above six tubes was
estimated in the same way as for the sample. A graph was plotted between
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Biochemistry Laboratory Manual PHL 224
concentration of protein and optical density. The calibration standard plot was used to
calculate the concentration of protein in each ml suspension of the sample.
Results: The total protein levels in given sample was found to be _____.
Dr. Gamal Gabr
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Biochemistry Laboratory Manual PHL 224
Experiment No. 13
Objective: To estimate the Urea in blood.
Principle: Urea reacts directly with diacetyl monoxime under strong acidic conditions
to give a yellow condensation product. The reaction is intensified by the presence of
ferric ions and thiosemicarbazide. The intense red color formed is measured at 540nm/
yellow green filter.
Materials required:

Stock acid reagent: Dissolve 1.0g of ferric chloride hexahydrate in 30 ml of distilled
water. Add 20 ml orthophosphoric acid and mix. Store in a brown bottle at room
temperature (25-350C).Stable for 6 months.

Mixed acid reagent: Add slowly 100 ml of Conc. H2S04 to 400 ml distilled water
taken in a 1-litre flat-bottom conical flask kept in an ice cold water bath. Mix well and
add 0.3ml of stock acid reagent. Mix and store in a brown bottle at room temperature
(25-350C). Stable for 6 months.

Stock color reagent – A: Dissolve 2g diacetyl monoxime in distilled water and make
the volume up to 100 ml in a volumetric flask. Store in a brown bottle at room
temperature (25-350C). Stable for 6 months.

Stock color reagent – B: Dissolve 0.5 g thiosemicarbazide in distilled water and make
up to 100 ml in a volumetric flask. Store in a brown bottle at room temperature (25350C). Stable for 6 months.

Mixed color reagent: Mix 35 ml of stock color reagent A with 35 ml of stock color
reagent B and make up to 500 ml with distilled water. Store in a brown bottle at room
temperature (25-350C). Stable for 6 months.

Stock urea standard: Weigh 1.0g of analytical-grade urea and dissolve in 100ml of
benzoic acid (1g/dl). Use a 100ml of volumetric flask for preparing this. Store at
room temperature (25-350C). Stable for 6 months.

Working standard 50mg/dl: Dilute 5.0ml of stock urea standard to 100 ml with
benzoic acid. Store at room temperature (25-350C). Stable for 6 months.

Equipment, glassware and other accessories:
Experimental Procedure:
The protocol of the procedure is described below.
Dilution of Standards (S1-S3) & Test
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Biochemistry Laboratory Manual PHL 224
Pipette the following into appropriately labeled test tubes & Mix Well
Distilled Water (ml)
50 mg/dl Urea (ml)
Test sample (ml)
S1
1.9
0.1
-
S2
1.8
0.2
-
S3
1.7
0.3
-
Test
1.9
0.1
Color Development
The color reagent is prepared fresh at the time of analysis by mixing distilled water,
mixed acid reagent and mixed color reagent in the ratio 1:1:1.
Pipette the following into another set of appropriately labeled test tubes.
Color reagent (ml)
Respective diluted standard ml)
Diluted test (ml)
Blank S1
3.1
3.0
0.1
-
S2
3.0
0.1
-
S3
3.0
0.1
-
Test
3.0
0.1
Mix all tubes well. Keep them in a boiling water bath for 15 minutes. Remove from
water bath and cool the tubes for 5 minutes. Set the spectrophotometer to zero with
blank at 540nm and measure the absorbance of the other tubes.
Calculation:
Concentration of standards:
S1 = 50 mg/dl
S2 = 100 mg/dl
S3 = 150 mg/dl
Plot the absorbance values of standards against their respective concentrations. The
measurable range with this graph is from 10 to 150 mg/dl. A calibration graph should
be constructed whenever a new set of reagents is prepared. Plot absorbance values of
test on the calibration graph and read off the concentrations.
Once linearity is proved, it will be enough if S3 is set up every time that sample are
analyzed and the results calculated using the formula:
Absorbance of test
Urea in test sample = ----------------------------- x 150 mg/dl
Absorbance of Standard
Results: The urea level in blood was found to be _____.
Dr. Gamal Gabr
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Biochemistry Laboratory Manual PHL 224
Experiment No. 14
Objective: To test the given urine sample for the presence of ketone bodies - Rothera's
test.
Principle: The three main ketone bodies are acetone , acetoacetic acid (diacetic acid)
and beta-hydroxybutyric acid. Testing for ketone bodies should be done on fresh urine
or the specimen kept at 4C. Acetone and acetoacetic acid react with sodium
nitropruside in the presence of alkali to produce a purple color.
Materials required:


Rothera's Reagent (Dry mixture): Pulve210rize 7.5g sodium nitropruside with 200g
ammonium sulphate. Store in a clean amber bottle at 250-35C. Stable for 6 months.
Ammonia concentrated, specific gravity 0.91
Experimental Procedure:
To about 5ml of urine taken in an 18 x 150mm glass tube, add about one teaspoon of the
mixture, mix well, then add 0.5 to 1.0 ml of concentrated ammonia down to the side of
the tube so that it layers on top of the urine. Observe for any colour change within 30-60
seconds.
If acetone and diacetic acid are present, then a purple (permanganate calomel red)
colour will form at the junction of the two layers within 30-60 seconds. The result can be
graded from trace to 3+ based on the intensity of the color formed, as detailed below.
No change in colour - Negative
Pinkish ring - +
Red ring - ++
Deep purple ring - +++
Results: The ketone bodies were found to be present in given urine sample on the
basis of above chemical tests.
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Biochemistry Laboratory Manual PHL 224
Carbohydrate Report
Unknown ( )
Student name …………………..
Arabic Date……………………..
No.
1.
Test
Student No. ………………
English Date……………..
Observation Inference
Solubility:
Compound + water
Molisch test:
Procedure: Mix 2ml of sugar sample with 5
2.
drops of Molisch's Reagent in a test tube. Add
gently through the side by tilting the tube,
about 2 ml of concentrated H2SO4 so as to form
a bottom layer.
Benedict’s test:
Procedure: Take 5 ml of Benedict's qualitative
3.
reagent, add 8 drops of sugar solution. Boil over
a flame for 2 minutes or place in boiling water
bath for 3 minutes. Allow to cool.
Fehling’s test:
Procedure: 2 ml of Fehling's solution A + 2ml of
Fehling's solution B + 2 ml of Sugar solution Boil.
Barfoed’s test
5.
Procedure: Sugar solution + Barfoed’s reagent
(Cu Acetate/Acetic acid), heat for 3 min. on
boiling W.B
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Biochemistry Laboratory Manual PHL 224
Seliwanoff's Test
Procedure:Sugar solution+ few crystals of
6.
Resorcinol + Equal volume of conc.HCl and
warm on W.B.
Bial's test:
7.
Procedure: 2 ml of sample solution is placed in a
test tube and 2 ml of Bial's reagent is added,
solution is heated gently in hot W.B
Osazone test
Procedure:
1- Add 10 drops of glacial acetic acid to 5 ml of
sugar solution in test tube.
2- Then add a knife point of phenyl hydrazine
hydrochloride and double the amount of
8.
sodium acetate crystals.
3- Mix and warm a little to see that the solids
are dissolved.
4- Filter the solution, and keep the filtrate in a
boiling W.B for 20 min.
5- Cool slowly in water bath not under the tap
to have better crystals.
6- Examine the crystal under microscope.
Hydrolysis of sucrose (Inversion test)
9.
Procedure: add 2 drops of HCl and 1 drop of
Dr. Gamal Gabr
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Biochemistry Laboratory Manual PHL 224
thymol blue to 5 ml of sucrose solution.
The development of pink color indicates that the
solution is acidic.
Divide it in to two equal parts
Boil one portion for about one min., cool under
tape water.
Neutralize both portions by adding 2% sodium
carbonate drop by drop.
Iodine test:
Procedure: iodine is added to an peeled potato
Iodine/Potassium iodide test:
10.
Procedure: Two ml of a sample solution is placed
in a test tube. Two drops of iodine / potassium
iodide solution and one ml of water are added.
Results:.
Dr. Gamal Gabr
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Biochemistry Laboratory Manual PHL 224
Amino Acids Report
Unknown ( )
Student name …………………..
Arabic Date……………………..
No.
1.
2.
3.
4.
5.
6.
7.
Test
Student No. ………………
English Date……………..
Observation
Inference
Ninhydrin test: To 1ml solution add 5
drops of 0.2% ninhydrin solution in
acetone. Boil over a water bath for 2 min.
Allow to cool
Xanthoproteic test: To 2ml of solution in
a boiling test tube, add an equal volume
of conc. HNO3. Heat over a flame for 2
min and observe the color. Now COOL
THOROUGHLY under the tap and
CAUTIOSLY run in sufficient 40% NaOH
to make the solution strongly alkaline.
Hopkins-Cole Test: To a few ml of
glacial acetic acid containing glyoxylic
acid, add 1-2 drops of the amino acid
solution. Pour 1-2ml concentrated H2SO4
down the side of the sloping test tube to
form a layer underneath the acetic acid.
Millon’s test: To 2ml of amino acid
solution in a test tube, add 1-2 drops of
Millon’s reagent. Warm the tube in a
boiling water bath for 10min.
Pauly’s test: Into clean test tube, dispense
1mL of 1% sulphanilic acid and 2 drops
of 5% sodium nitrite. Mix for 1 min. Add
about 0.5mL of amino acid solution.
Ehrlich test: To 0.5ml of the amino acid
solution, add 2ml Ehrlich reagent
Nitroprusside test: Add 2ml of the amino
acid solution into test tubes. Add 0.5ml
fresh sodium nitroprusside solution and
shake thoroughly. Add 0.5ml ammonium
hydroxide
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Biochemistry Laboratory Manual PHL 224
8.
9.
Lead acetate test: Everything needed to
carry out this test will be in the hood and
should not remove anything from the
hood. A toxic, stinky gas will be made (in
small, but immensely smelly quantities)
and you don’t want to smell it. Dispense
about 0.5mL of the amino acid solution
only into a clean test tube (found in the
hood, where you will leave it when you
are done). Add 0.5mL of 20% NaOH and
insert the test tube in a boiling water bath
for 1 min. Add 2 drops of lead (II) acetate
solution.
Sakaguchi test: 1ml NaOH and 3ml of
the arginine solution is mixed and 2
drops of α-naphthol is added. Mix
thoroughly and add 4-5 drops Bromine
solution
Results:.
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Biochemistry Laboratory Manual PHL 224
Attendance Sheet
Group No. (
Week
)
Student Name:
Date
Exp.
Marks
1st
22d
3rd
4th
5th
6th
7th
8th
9th
10th
11th
12th
13th
14th
Total
marks
Dr. Gamal Gabr
48