HACETTEPE UNIVERSITY

FACULTY OF MEDICINE

PHASE I
BIOCHEMISTRY
LABORATORY GUIDE
2023-2024

Name :

Number :
Contents 1

General Laboratory Instructions 2

General Laboratory Procedures 3

Experiment I- Acid-Base Titration 9

Experiment II- Spectrophotometry 11

Experiment III- Enzymes (Urease) 14

Experiment IV - The Oxidative Enzymes and The Electron Transport Chain 19

Experiment V - Paper Chromatography – Transamination 21

1
 GENERAL LABORATORY INSTRUCTIONS

1. Always read the experiments carefully and thoroughly before coming to the
lab.

2. Be in the lab on time. Wear your lab coat to protect yourself. Remember at
all times that the laboratory is a place for serious work.

3. Do only the experiments in the lab manual that are approved by your teacher.

4. Read the label on a reagent bottle twice before removing any of its contents
to be sure that you have the right bottle

5. Do not touch and never taste chemicals. If an acid or other corrosive chemical
is spilled, wash it off immediately with water.

6. Report any accident or injury, however minor, to your teacher.

7. Never return unused chemicals to the stock bottles, and never mix different
chemicals.

8. Keep your apparatus and desktop clean. Avoid spillage, but if you do spill
something, clean it up immediately. Return all material to its proper place
after use.

9. Bring permanent marker and millimetric graph paper to the laboratory.

2
 GENERAL LABORATORY PROCEDURES

1. CLEANING THE GLASSWARE

All the equipments you use should be clean to obtain accurate results. The provided glassware
is clean. However, if you are going to reuse a glassware, you must wash it first with tap water
and then with distilled water.

Always use distilled water for your experiments, never use tap water.

When mixing the tube content, you must cover the tube opening with a piece of parafilm
and turn the tube upside down. Avoid shaking the tube randomly or turning the tube upside
down by closing the tube opening with your thumb.

2. MEASURING THE VOLUME

Much of the glassware used in the laboratory is marked with a graduated scale for volume
readings. Erlenmeyer flasks and beakers have approximate graduations for rough estimates.
Most of the volume measurements will be made with a graduated cylinder, but you have to
use a pipet or buret for more precise work (figure 5). The least volume that you can measure
with a 50 ml graduated cylinder is 0.5 ml, while 0.05 ml can easily be read on a 50 ml buret.
Thus, the buret is more precise.

Before using glassware to measure a volume, you have to check the graduations on it.

You should prefer the one with the most proper graduation to the volume you want to
use.

For example, you should not use a 25 ml graduated cylinder for 5 ml or a 10 ml pipet for 1
ml. The correct choices are 5 or 10 ml pipets for 5 ml and 1 or 2 ml pipets for 1 ml.

3
Figure 1. Various labware

4
 Figure 2. The appropriate method of reading the level of a pipet

The quality of your data will depend on how accurate you measure the volumes.

After choosing the proper glassware, fill it with the liquid you need. Read the markings at the
level of the bottom of the meniscus, the lens-shaped surface of the liquid. As on Fig.2, your
eye must be at the same height as the bottom of the meniscus.

5
 A pipet is used to measure small amounts of solution very accurately. Use a clean pipet for
every solution. Do not dip a used pipet into another liquid. If you have to reuse the pipet, fill
it first with tap water and then with distilled water.

Figure 3. i) The pipet bulb ii) The pipet pump iii) Automatic pipet

A pipet bulb or pipet pump is used to draw solution into the pipet (Figure 3). Always use
either of them and never pipet by mouth. How to use a pipet bulb:

1. Push glass pipet into opening at bottom

2. Pinch valve "A" while squeezing main bulb

3. Then, pinch valve "B" to draw liquid up

4. Then, pinch valve "C" to dispense liquid

You will use both of pipet bulb and pump in the lab. You will also be given automatic pipets
in certain experiments.
3. FILTERING

Filtering is a method of separating solid particles from a liquid. The method consists of pouring

6
the solid-liquid mixture into a filter paper cone supported by a funnel. The preparation of the
filter paper is shown on Figure 4. Always wet the filter paper by distilled water before
starting to filter your mixture.

Figure 4. Preparation of the filter paper

7
 4. THE EXPERIMENT REPORT

The reports must be prepared individually and will be collected at the end of the experiment.
All reports should be written in English.

Never describe the experimental procedure in your report since it is written on the lab
manuals. Graphics must be plotted on millimetric graph papers. Pencil is must be used for
the preparations of the graphs.
HER DENEYİN SONUNA RAPOR FORMU KOYULABİLİR, MİLİMETRİK KAĞIT VS. DE)

Name: Date

Number:

Phase/ Group:

Laboratory/Desk number:

Name of the experiment:

Purpose: (one or two sentence)

Findings:

Measurements, tables, graphs, calculations

Conclusion (one or two sentence)

8
 EXPERIMENT I ACID-BASE TITRATION

PURPOSE: The purpose of this experiment is to determine the concentration of an unknown

acid solution by using a volumetric method.

PRINCIPLES: Volumetric analysis involves the determination of the concentration of a

substance by reacting it with a measured volume of a known standard solution. This method is
commonly called titration.

An acid-base titration is the determination of the concentration of an acid or base by exactly

neutralizing the acid/base with an acid or base of known concentration (standard solution). This
allows for quantitative analysis of the concentration of an unknown acid or base solution. It
makes use of the neutralization reaction that occurs between acids and bases and the knowledge
of how acids and bases will react if their formulas are known. Neutralization is achieved at the
end point.

The endpoint can be detected by using an indicator, a chemical compound that exhibits a change
in color as a result of concentration changes occurring near the equivalence point.

Acid-base indicators are generally complex organic compounds of high molecular weight. Each
acid-base indicator has a certain pH interval over which it exhibits a color change.

In this experiment methyl red will be used as an indicator. This indicator is red in acidic medium
(pH lower than 4) and yellow in basic medium (pH higher than 6).

PROCEDURE:

1. Preparation of the buret:

1. Fill the buret with tap water. If there is any leakage consult your instructor.

2. Wash the buret first with tap water, then with distilled water, and finally rinse it with the
solution to be filled into it. Then fill the buret with the solution.

2. Determination of the concentration of unknown base:

1. Take two Erlenmeyer flasks. Pipet 10 ml of standard acid solution (0.1 N HCl), add 25
ml of distilled water and 2-3 drops of methyl red indicator into each of them. Your acid
solution is diluted 35/10 times.

2. Take another Erlenmeyer flask. Pipet 10 ml of unknown base, NaOH solution, in it. Add
90 ml of distilled water and mix thoroughly. Now your base solution is diluted 10 times.
Fill your buret with this solution (do not waste the remainder of the base since it will be
used to titrate the unknown acid).

3. Titrate the acids in the two Erlenmeyer flasks with the diluted NaOH solution in the buret
until an “onion skin” color develops. When this color is obtained, stop the titration and

9
note the amount of NaOH (ml) used from the buret. Take the average of the volumes of NaOH
used for each titration, and calculate the concentration of the base solution in the buret using the
following equation.

Vacid x Nacid = Vbase x Nbase

V: Volume, N: Normality

After you found the concentration of the unknown base, (NaOH) solution, you will use it as
standard base for determining the concentration of unknown acid.

3. Determination of the concentration of unknown acid

1. Take two Erlenmeyer flasks. Pipet 10 ml of unknown acid, 25 ml of water, 2-3 drops of
methyl red into each; and titrate them with standard NaOH solution in the buret as
described before.

2. Calculate the concentration of the unknown acid by using above formula.

10
 EXPERIMENT II
SPECTROPHOTOMETRY

PURPOSE: You will investigate the rules governing the absorption of light, and application
of these rules to quantitative chemical analysis in this experiment.

PRINCIPLES: When a beam of light passes through matter, a portion is absorbed. If this
absorbed portion is in visible region, the matter exhibits a color. Visible region extends from
380 nm to 780 nm.

The concentration of a colored solution will be determined by measuring the amount of light
absorbed at a certain wavelength and by comparing it with the absorbances of standard
solutions. The concentration of a substance is directly proportional to the amount of light
absorbed (Lambert-Beer’s law).

Absorbance of a solution is measured by an instrument called “spectrophotometer”. The

components of the spectrophotometer are shown in Figure 1.

Figure 5. Spectrophotometer

Slit
“wavelength selector”

Light source Monochromator Sample Detector monitor

“lamp” “prism or grating” “cuvette” “photocell” “meter”

Figure 6. Components of a spectrophotometer.

In order to effectively use a spectrophotometer we must first adjust the machine to zero
absorbance using the blank.

The blank is a solution which contains everything except the compound of interest.
Preparation of blank tube is given in Table 1.

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PROCEDURE:
The concentration of a methylene blue solution will be determined by using a standard
solution. A standard solution is a solution containing a precisely known concentration of
an element or a substance i.e., a known weight of solute is dissolved to make a specific
volume.
The wavelength at which the standard solution gives maximum absorption will be determined
by the spectrophotometer. Then various concentrations of the standard solution will be
prepared according to Table 1, the absorbances will be measured for each concentration and
the standard calibration curve will be constructed.

1. Operation of the spectrophotometer:

1. Turn on the POWER.

2. Set the wavelength to the required wave-length
3. Open the lid of the sample compartment,
4. Insert a blank in the cell holder, and press Auto-zero button.
5. Insert unknown sample in the cell holder.
6. Read out the absorbance from the screen.

2. Determination of the absorption spectrum of the standard solution:

1. To obtain the absorbances of the standard solution at different wavelengths, change the
wavelength at intervals of 25 nm (in the range of 500-750 nm), and record the
corresponding absorbances.
2. Remember to set the instrument to zero absorbance using the blank solution, at each
wavelength.
3. Plot the results on a graph paper, marking the wavelengths on the x axis and the
absorbances on the y axis. Draw a continuous curve through the points. The peak of the
curve is the wavelength at which your solution absorbs light maximally.
4. Use this wavelength in the next step.
3. Determination of the concentration of the unknown solution:
Dilute the standard solution properly to obtain three more standards as described below.

Table 1. The volume of each solution (ml) that you should add into each tube.
Tube 1 Tube 2 Tube 3 Tube 4 Tube 5 Tube 6 Tube 7
Solutions, ml Blank Std.1 Std.2 Std.3 Std.4 Sol.1 Sol.2
Standard solution, (4 mg/L) - 2 4 6 8 - -
Distilled water 8 6 4 2 - - -
Solution 1 - - - - - 8 -
Solution 2 - - - - - - 8
Dilution None 1/4 1/2 3/4 None None None
Concentration of methylene blue 0 1 2 3 4
(mg/L)
? ?
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Using distilled water as blank, measure the absorbances of your standard and unknown
solutions in the wavelength you found in the first step.

4. Drawing a standard calibration curve and finding the concentration of solution 1

Determining the concentration of an unknown solution is done by the use of a calibration curve.

A calibration curve may be prepared by measuring the absorbances of a series of standard

solutions; and by plotting absorbance versus concentration.

Calibration curve is a straight line since there is a linear relationship between absorbance and
concentration.

Now, if you measure the absorbance of the unknown solution, you can obtain the concentration
of it from the calibration curve.

Figure 7. Standard calibration curve

Plot the results on graph paper, marking the absorbances on the ordinate and concentration on the
abscissa.

Draw the calibration curve as described above, plotting the concentrations of your standard
solutions calculated from the dilution factors versus obtained absorbances. Find the concentration
of the unknown sample using this curve.

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 EXPERIMENT III- ENZYMES (Urease)

PURPOSE:
The purpose of this experiment is to observe some kinetic properties of an enzymatic reaction involving
urea as the substrate and urease as the enzyme.

PRINCIPLE:
Urease is an enzyme that converts urea to ammonia (1). The ammonia causes the pH of the
medium to be more basic, by forming ammonium (2). The amount of ammonia formed from urea
decomposition can be measured by titrating it with HCl.

The following properties of this reaction will be investigated:

1) The disappearance of substrate urea with respect to time,

2) Dependence of this disappearance to
a) initial substrate concentration, and
b) enzyme concentration.

In order to follow the consumption of the substrate, urea, the product of the reaction, ammonia
will be determined at certain intervals. The amount of ammonia produced will be calculated by
titration with HCl. Since two moles of ammonia is produced for each mole of urea used, the
amount of urea consumed will be calculated.

The enzymatic reactions will proceed in the tubes prepared according to Table I. The reactions
will start upon addition of urease into the tubes. At the 15th and 30th minutes, some aliquots of
the reaction mixtures will be transferred into Erlenmeyer flasks which contain mercuric chloride
(HgCl2) and methyl red. HgCl2 inhibits the urease enzyme, therefore the reaction will be
terminated. Ammonia in these aliquots will be determined by titration with HCl. Methyl red is
the indicator of the titration.
The reaction will continue in the tubes, it will only be terminated in the aliquots transferred
into the Erlenmeyer flasks.

1 mole of urea is hydrolyzed by urease to form 2 moles of ammonia and 1 mole of

OH- ions.

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 PROCEDURE:

1. Prepare 5 Erlenmeyer flasks marked as blank, 1, 2, 3, 4. Add 2 drops of HgCl2 and 2 drops
of methyl red in each. You will use these flasks during the titration of 15 th minute reaction
mixtures.

2. Prepare the blank tube according to Table 1. As soon as you add urease, take 5 ml of this reaction
mixture into the Erlenmeyer flask marked ‘blank’. You will later titrate this with
0.02 N HCl until you obtain the color of the onion skin.

3. Prepare the reaction mixtures for 5 tubes according to Table 1.

• The effect of substrate concentration on the reaction rate will be observed in the tubes
1, 2, 3 and 4 (Substrate concentration is maximum in Tube 4).

• Timing will start as soon as the urease is added. For convenience add urease within 2
minutes intervals into each tube, and mix.

Table 2. Preparation of reaction tubes

Solutions, ml Blank* Tube 1 Tube 2 Tube 3 Tube 4

Urea, 0.25 M 12 0.5 1 4 12

Tris Buffer 0.05 M, pH 7.2 2 13.5 13 10 2

Urease, 4 mg/ml 1 1 1 1 1

* A blank titration is necessary to find out the ammonia produced by the enzymatic reaction as
soon as the enzyme is added into the incubation mixture.

4. At the 15th minute, take 5 ml of the mixture from tube 1 to add into Erlenmeyer 1. Within 2 minutes
intervals do the same for the following tubes, 2, 3, 4.

5. Save the rest of the mixtures in the tubes and continue keeping time for 30 th minute analyses.
Prepare four Erlenmeyer flasks as described in step 1 (omitting the Blank) for 30th minute analyses.

6. At the 30th minute, take 5 ml of the mixture from tube 1 into Erlenmeyer 1 of 30th min analyses.
Within 2 minutes intervals do the same for the following tubes, 2, 3, 4.

7. Titrate the mixture in these flasks with HCl. Record HCl volume used for each sample (15 and 30
min) in addition to blank (0 min) in Table 2

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 Table 3. Volume of HCl (VHCl) used in the titrations
Sample Sample Sample Sample
Blank
1 2 3 4
VHCl 0 15
min min
(ml)
30
min

8. After completing the procedures described, calculate the amount of urea (micromole) used at 15 th
and 30th minutes in the total reaction mixture for each tube (Refer to the Calculations section).
Fill in Table 3.

Table 4. Amount of urea used at the 15th and 30th minutes.

Time Tube 1 Tube 2 Tube 3 Tube 4

15th min

30th min

9. For each tube draw the graph of urea consumption versus time. From these graphs find the urea
consumption in 1 hour for each tube (as micromole / hour) and fill in Table 4. The value will be
the rate (V1, V2, V3, V4) of each tube.

Table 5. Reaction rate in each tube

Tube Tube 2 Tube 3 Tube 4

1 V2 V3 V4
V1
Urea consumption
(µmol/h)

10. Calculate the initial urea concentrations (S1, S2, S3, S4) in the tubes 1, 2, 3, 4. Fill in Table 5.

Table 6. Initial substrate concentrations

S1 S2 S3 S4

Urea (mM)

By using the data, draw the graphs of V versus [S] and 1/V versus 1 /[S].

Find the Km and Vm of this reaction through these graphs.

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CALCULATIONS

1. Amount of urea used for the total reaction mixture:

You titrated an aliquot of 5 ml from the reaction mixture with HCl. Total reaction mixture is 15.

1 ml of HCl (0.02 N) titrates 20 µmol of ammonia (known data). Therefore, each 1 ml of HCl used
corresponds to 10 µmol of urea.

VHCl used for titration of NH3 produced by urease =VHCl used for the titration of the sample − VHCl used for the titration of the Blank

Urea consumed (micromoles, µmol) = VHCl used for titration of NH3 produced by urease x 10 x  15 / 5

Urea Concentration ( C ) in each tube:

According to following equation, you can calculate the urea concentration in each tube.

V1.C1 =V2.C2

To calculate the urea concentration in tube 1:

Urea stock solution is 0.25 M. You transfer 0.5 ml from this solution into tube 1. Then you add
buffer and enzyme to tube 1. Its final volume will be 15 ml.

You get 0.5 ml of 0.25 M urea

0.25 x 0.5 = C2 x15

C2 = 0.25 x 0.5 = 0.0083 M = 8.3 mM

15

17
EXPERIMENT IV -THE OXIDATIVE ENZYMES AND THE
ELECTRON TRANSPORT CHAIN

PURPOSE:
The oxidation of succinate in the electron transport chain (ETC) will be studied, using methylene
blue instead of molecular O2 as the last electron acceptor.

PRINCIPLE:
The oxidation of many metabolites in the living organism occurs by the removal and transfer of
hydrogen atoms to molecular oxygen and the formation of water. Reaction of Oxygen and
hydrogen occur through a set of electron acceptors known as the electron transport chain. In the
meantime, free energy is conserved as chemical energy. This occurs within the mitochondria.

Figure 8. Electron Transport Chain

Many metabolites transfer their electrons to the ETC by NAD-dependent enzymes; however,
succinate uses an uncommon pathway and passes its electrons to the ETC through FAD- linked
succinate dehydrogenase.
In this experiment, succinate oxidation will be studied. Methylene blue will be used as the last
electron acceptor instead of molecular oxygen. Methylene blue is an ionic dye that is blue in
its oxidized form and bleaches on reduction (leuco-methylene blue). Methylene blue takes over
electrons from the components in the ETC before cytochromes and bleaches to leucomethylene
state.
Succinate dehydrogenase activity will be followed by fading of the blue color under anaerobic
conditions. As methylene blue is an autoxidant dye, that is, on contact with oxygen, it is directly
oxidized and becomes blue. Therefore, the experiment must be carried out under anaerobic
conditions. Mineral oil is added to stop oxygen reaching into the tubes.
In addition, the effects of cyanide and malonate on the oxidation of succinate will be studied.

PROCEDURE:
For mitochondria isolation, the bovine heart is chopped, minced, and washed first with tap water
and then with phosphate buffer (0.1 M, pH = 7.4). During this procedure all soluble coenzymes
such as NAD and NADP will be washed out of the tissue.

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Therefore, the enzymes that need these coenzymes for their activity will not be working (!). The
washed tissue is homogenized and then the homogenate is centrifuged to remove debris and large
cellular organelles such as the nucleus. The supernatant contains the enzymes, flavoproteins and
other substances necessary for the oxidation of succinate.
1. The tubes are labeled.
2. The solutions except the mitochondria homogenate are pipetted into each tube as shown in
Table 1.
3. The tubes are covered with parafilm and the contents mixed thoroughly.
4. Then mitochondria homogenate is added.
5. The tubes are covered with parafilm and the contents mixed, again, thoroughly.
6. 1-2 ml mineral oil is pipetted into each tube to cut off the contact with air.
7. The time is recorded and the time required for the color of each tube to resemble the
color of the control tube (Tube 5) is determined.
8. Tube 5 contains 0.2 ml methylene blue instead of 1 ml and corresponds to the color
when methylene blue is reduced by 80%.
9. For a better comparison, 2 ml inactive mitochondria homogenate is added to Tube 5.
10. Inactive homogenate is obtained by boiling the active sample.
11. After the bleaching times are determined, the auto-oxidation of the dye in Tube 1 and
Tube 3 will be observed by pipetting out the mineral oil layer and shaking the tubes
vigorously.

Table 7. Preparation of the tubes

Solutions, ml Tube 1 Tube 2 Tube 3 Tube 4 Tube 5 Tube 6 (Control)

Succinate, 0.1 M 1 1 1 1 - -

KCN, 0.05 M - - - 1 - -

Malonate, 0.1 M - 1 0.1 - - -

Glutamate, 0.1 M - - - - 1 -

H2O 3 2 2.9 2 3 4.8

Methylene blue, 1/5000 1 1 1 1 1 0.2

Mitochondria homogenate 2 2 2 2 2 2 (inactive homogenate)

DISCUSSION: What do you expect to see in each tube? Compare your results with what you
expect theoretically.

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 EXPERIMENT V - PAPER CHROMATOGRAPHY –
TRANSAMINATION

PURPOSE: In this experiment you will observe the transamination reaction catalyzed by
glutamate-pyruvate transaminase (alanine amino transferase, alanine transaminase, ALT). The
reaction is shown below:

COOH COOH COOH COOH

ALT
C H N2 H + C 0 C 0 + C H N2 H

CH 3 ( CH2)2 C H3 (CH2)2

COOH COOH
L-alanine -ketoglutarate Pyruvate L-glutamic acid

ALT is found in large quantities in liver therefore, you will use liver tissue extract as the source
of the enzyme.

PRINCIPLE:
Chromatography refers to processes that permit the resolution of a mixture as a consequence of
differences in rates at which the individual components of that mixture migrate through a
stationary medium under the influence of a mobile phase. Therefore, it is one of the most effective
techniques to separate, isolate and identify chemical compounds.
There are many kinds of chromatography: adsorption, partition, affinity, ion-exchange, and
molecular sieve; and many specialized techniques for using them: column, paper, thin layer, and
gas chromatography.
Compounds that are soluble in both water and organic solvents are readily separated by partition
methods. One of the most common types of partition chromatography is paper chromatography.

THEORY OF PARTITION CHROMATOGRAPHY:

When a compound is shaken with two immiscible solvents (solvents that do not mix easily, like
water and oil) it will distribute itself unevenly between the two phases. At equilibrium, the ratio
of the concentration of the compound (x) in the two solvents is constant and is known as the
partition coefficient ().
Concentration of x in solvent 1
partition coefficient () = ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯
Concentration of x in solvent 2

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In partition chromatography one solvent, usually water is held on the stationary supporting phase
that is in the form of a column or film of inert material. The other phase consists of a mobile,
water saturated, organic liquid that flows past the stationary phase. The components of a mixture
are separated if their partition coefficients between the solvents are sufficiently different.
PAPER CHROMATOGRAPHY:
Cellulose in the form of paper sheets makes an ideal support medium where water is adsorbed
between the cellulose fibers and forms the stationary hydrophilic phase. Partitioning occurs
between the bound water and the mobile phase.
The mixture is spotted onto the paper, dried, and the chromatography progresses by allowing the
solvent to flow along the sheet. The solvent front is marked and, after drying the paper, the
positions of the compounds present in the mixture are visualized through a suitable staining
reaction (or as in the case of keto acids, colored complexes of the constituents are formed during
the application).
The ratio of the distance which the sample travelled to the distance which the solvent travelled
(solvent front) is known as the Rf value (Fig. D).
Rf is constant for a particular compound, solvent system, and paper under carefully controlled
conditions of solute concentration, temperature, and pH.
PROCEDURE:
1. Prepare the incubation mixtures as shown in Table 1 by pipetting the enzyme extract last.

Table 8. Preparation of the reaction tubes

Solution, ml Tube 1 Tube 2 Tube 3 Tube 4
0.2 M -ketoglutarate - 0.1 - 0.1
0.2 M L-alanine - - 0.1 0.1
0.2 M pyruvate 0.1 0.1 - -
0.2 M L-glutamate 0.1 - 0.1 -
0.2 M sodium arsenite 0.2 0.2 0.2 0.2
Liver extract containing enzyme 0.3 0.3 0.3 0.3

• Incubate the tubes at 370C for 45 minutes. (During this period, prepare your
chromatography papers, and apply the standards as described below).
• At the end of the incubation period pipet 2 ml of ethanol into each tube and mix by
shaking. Filter them into four clean test tubes.
• Apply the clear filtrates to the amino acids (AA) and keto acids (KA) papers.

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2. Chromatography:

• Get two chromatography papers. Mark one as AA and the other as KA at the right top corner.
Please hold the papers from sides. Never touch them with your fingers. Use a pencil for
marking the application spots as shown in Figure 1 (A).

• Apply the filtrates of each tube to the papers by using capillary tubes, Fig 1 (B). For each
application use a clean capillary tube. Do not contaminate the capillary tubes with other
solutions. Sample taken 5-6 mm high in capillary tube is adequate for application. The duration
of the application should be less than a second and the diameter of the spots should not exceed
2-3 mm.

• For AA paper, apply the samples once and dry the spots. To the 5th and 6th points, apply
glutamic acid and alanine standard solutions and dry the spots.

• For KA paper, apply the samples for 5-6 times. Dry the spots after each application. Then
apply 2,4-dinitrophenyl hydrazine (DNPH) onto each spot and dry again. Repeat this
procedure 5-6 times. To the 5th and 6th points on KA paper apply -Ketoglutarate and pyruvate
standard solution and dry the spots.

• After the papers are dried, insert two pins across each paper for supporting them in the tanks,
Figure 1 (C). The pins should be 2-3 cm distant from each other.

• Place AA paper into the AA tank. Mobile phase in this tank is propyl alcohol - H2O mixture,
(3: 1 by volume).

• Place KA paper into KA tank which contains n-butanol saturated with %3 ammonia as a
mobile phase.

• Place the papers into the tanks carefully. Sides of the papers should not touch the walls of
glass tanks.

• Pipet 4 ml of solvent to the bottom of AA and KA tanks without touching your pipets to the
papers you placed. Then you close the covers of the tanks and wait for the development of
chromatograms.

• When the solvent travels to the front of the paper, take the papers out the tanks. Mark the
solvent fronts with pencil while they are wet.

• Dip the AA paper into ninhydrin solution and dry it completely.

• Dry the KA paper.

• Measure the distance travelled by the solvent front (b) and the distance travelled by each spot
(a) (distance between origin and the center of the spot). Calculate the Rf values for each spot,
Figure 1 (D).

• Comparing the Rf values of the standards with the Rf values of the spots for each tube,
determine the amino acids and keto acids in these four incubation mixtures.

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 45 mm

75 mm

4 mm

12 mm

5  7  7  7  7 7  5
(mm)
(A) (B)

Solvent
Chromatography front
paper

b a
Pin Application
point

Solvent (D)

(C ) a
Rf =
b
a : The distance which the sample travelled
b : The distance which the solvent travelled

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