Professional Documents
Culture Documents
Biochemistry Lab Guide OBE
Biochemistry Lab Guide OBE
EXPERIMENT NO. 1
PHYSICAL CHEMISTRY
Learning Outcomes
At the end of the experiment the student must be able to:
I. Discussion:
A. Titration
Titration is a process of ascertaining the quality of a constituent present in a compound by
using a standard solution.
A. Direct Method – this is a method in which the standard acid solution is added to an
alkali solution until the end is reacted.
II. PROCEDURE:
a. Measure 10 ml of sodium hydroxide of unknown strength and place in a 250 ml
Erlenmeyer flask.
b. Add 2 drops of phenolphthalein
c. Mix by shaking
d. Titrate with 0.1N HCl until the solution becomes colorless.
e. Take note of the volume of the standard acid used and determine the normality of the
solution using the equation:
N1 V 1 = N 2 V 2 or Na V a = N b V b
Write your results and observations on a space below:
1
B. DETERMINATION OF pH
The pH of a solution is compound logarithm of the reciprocal of the hydrogen ion concentration
expressed as:
pH = Log 1/H+
Pure water is slightly ionized and at 25°c contains 0.00000001 or 10 -7 per liter. The pH of water
is therefore:
pH = Log 1/10-7
pH = -Log 10-7
pH = 7
If the hydrogen ion concentration (H +) of a solution is greater than in pure water, the pH is a
smaller number than 7, such solution is acidic. Conversely, if the (H +) is less than in pure water
the solution is basic. The range of pH values fall between 0 – 14
pH is detected and measured by using a pH meter, but the most common detectors are the color
changes of acid base indicators. An acid base indicator is a weak organic acid. The ionized and
unionized forms of the indicators have different colors. The colors of indicators solution is
therefore a measure of pH.
pH Determination
Procedure:
1. Prepare 1 ml in four clean test tubes of the following samples below and label:
2. Determine the approximate pH of the sample by adding 1 drop in each test tube using
the following indicators:
2
Observed the color product in each test tube. Tabulate your results by recording the color of each
Indicator.
SAMPLES INDICATORS
Phenolphthalein Congo Red Phenol Red Methyl Orange
1. 0.1N HCl
2. 0.1N NaOH
3. Fresh Milk
4. Urine
5. Orange
Juice
6. Soft drink
Determine the accurate pH of the samples using the pH meter by following these steps.
Operatory Procedure:
1. Set the temperature control knob at about 25°C.
2. Dip the electrode of the pH meter into the standard buffer solution.
3. Turn the knob to position “pH” and check if the pointer gives the correct reading of pH of the
buffer solution.
4. If the reading is not correct, turn the standardization knob until the accurate reading is
obtained.
5. Remove the buffer solution and rinse the electrode with distilled water. Wipe the electrode with
tissue paper.
6. Dip the electrode into the first solution and turn the knob to the position “pH”. Get the pH
reading on the scale of the instrument and record it.
7. Turn back the knob to off position and removed the electrode from the test solution.
Samples pH meter
1. 0.1N HCl
2. 0.1N NaOH
3. Fresh Milk
4. Urine
5.Orange Juice
6. Soft drink
Activity Sheet Number 1
3
Name:_______________________________________________________Section__________________ Date _______________
Instruction: Based on the results of your performed experiments, discuss among your group mates
all of your observations and answer the following questions:
2. How does the pH of your assigned samples as obtained from the pH values (indicators)
compared with the pH obtained from the pH meter?
3. What indicator/s is/are used when titrating a weak acid against a strong base? A weak base
against the strong acid?
4
C. BUFFERS
A buffer is one that resists a change in pH when a small amount of acid or base is added. A buffer
solution contains a weak Bronsted acid and its conjugate base.
If a small amount of strong acid is added to the buffer solution, some of the anions from the buffers
salt will combine with the added protons to form more dissociated acid.
Similarly, if the small amount of the base is added to the buffer solution, some of the buffer acid
reacts with the pH- to form more buffer salt.
PROCEDURE:
3. To the first set add 0.1ml of 0.1N NaOH to each sample. Determine the pH using the pH meter.
Record your result.
4. To the second set add 0.1ml of 0.1N HCl to each sample. Determine the pH using the pH meter.
Record your results.
5
Activity Sheet Number 2
Name:_______________________________________________________Section__________________ Date _______________
Instruction: Based on the results of your performed experiments, discuss among your group mates
all of your observations and answer the following questions:
2. How would you correlate the buffer systems involved in whole blood and plasma?
3. What are the two factors that determine the effectiveness or capacity of buffer solutions?
D. COLLOIDS
6
The word colloids include any dispersion of very small particles that are larger than simple
molecules. Colloidal size lies between those of true solution which are homogenous and coarse
mixtures which separate on standing. This includes particles with dimensions in teh range of 10 oA
to 10,000 oA or .001 micron to 1 micron.
A natural colloidal substance which composes the protoplasm of the living cells is called biocolloids.
Biocolloids include nearly all of the energy foods such as starches, proteins, fats and biocatalysts
such as enzymes, hormones and vitamins.
7
Precipitation with Electrolytes:
2. Add saturated solution of ammonium sulfate drop by drop counting the number of drops until a
permanent precipitate is formed.
REVERSABILITY
1. Decant the supernatant liquid from each of the two test colloids prepared in letter B procedure.
2. Add an excess of water and note whether the two colloids are reversible.
8
Activity Sheet Number 3
9
E. DIALYSIS
The rate of dialysis depends on many factors: the area of the dialyzer, the size of the pores,
the temperature, the electric charges and the relative concentration of solution on the two
sides of the membrane.
Procedures:
1. Pour some colloid on solution (5% in equal parts of ether and alcohol) into a clean and dry
hard test tube taking care not to form air bubbles.
2. Wet completely the inner side of the test tube and pour the excess of colloidion back to the
bottle.
3. Allow to drain and rotate the tube slowly in horizontal position until the ether has partly
separated.
6. Loosen the film around the tip of the tube and allow cold water to run between membranes.
If necessary, loosen the membrane from the side of the tube with a smooth glass stirring
rod.
7. Rinse the colloidion bag thus prepared with distilled water several times.
8. Take 3ml of distilled water and treat it with 2 drops of silver nitrate t.s., If negative use
about 2/3 of a beaker with distilled water
9. By means of stirring, suspend the dialyzing bag containing equal volumes of 1% starch and
1% sodium chloride solutions.
11. Test the dialyzing by using silver nitrate t.s. and iodine solution.
10
Activity Sheet Number 4
Name:_______________________________________________________Section__________________ Date _______________
Instruction: Based on the results of your performed experiments, discuss among your groupmates
all of your observations and answer the following questions:
1. Which of the substances inside the bag diffuses out of the colloidion mixtures? Explain why?
3. What are the factors that affect the rate of dialysis? Explain why?
11
F. DIFFUSION THROUG GELS
All solutes tend to diffuse through solutions until the composition is homogenous
throughout. Small molecules and ions move with sufficient velocity to distribute themselves
throughout the solvent rapidly. On the other hand, macromolecules move slowly because of
their latge molecule size.
Procedure:
1. Put 2ml of 10% copper sulfate solution in a test tube. Incline the test tube and carefully add 3ml
of distilled water. Take note the time for the entire body of fluid to have the same color.
12
Name:_______________________________________________________ Date Performed:_______________
EXPERIMENT NO. 2
ELEMENTS FOUND IN PHYSIOLOGICAL COMPOUNDS
Learning outcomes
At the end of the experiment the student must be able to:
Procedure:
13
Activity Sheet Number 5
Name:_______________________________________________________Section__________________ Date _______________
Instruction: Based on the results of your performed experiments, discuss among your groupmates
all of your observations and answer the following questions:
14
Test for Nitrogen
Procedure A
1. Burn hair, feather or horn and note the odor produced.
2. Describe the odor produced and account for it.
Procedure B
1. Place about 0.5 gm of egg albumin in a porcelain dish.
2. Place about 1 gm of soda lime. Mix and grind the mixture.
3. Place the mixture in dry test tube and heat gently over a Bunsen burner lame. Note the odor
of the gas evolved.
4. Test the gas evolved by:
a. Exposing previously moistened red litmus paper over the fumes. Take note of the
changes in color.
b. Observed the odor of the fumes.
c. Exposing a glass rod previously dipped in concentrated hydrochloric acid.
Procedure:
15
Test for Phosphorous
Procedure:
1. Place in a porcelain crucible about 0.5 gm of casein
2. Add equal amount each of potassium hydroxide and potassium nitrate.
3. Heat the mixture strongly. Cool, dissolve in water and filter.
4. Place 2 ml of the filtrate in a test tube.
5. Acidify by adding 5-10 drops of concentrated nitric acid.
6. Add about 15 drops of ammonium molybdate.
7. Note the change produced.
OSMOSIS
Osmosis is the passage or movement of the solvent molecules from more dilute solution to
the concentrated when these are separated by a semi-permeable membrane. In the
following procedure, the stroma of the erythrocytes acts as a semi-permeable membrane.
1. Prepare 3 clean and dry test tube labeled 1, 2 and 3 each containing 5ml each
of the following solutions 0.3, 0.9 and 5% NaCl.
2. To each test tube, add 2 drops of blood
3. Allow the test tube to stand for 3-5 minutes.
4. Examine each suspension under the microscope
16
Activity Sheet Number 6
Name:_______________________________________________________Section__________________ Date _______________
Instruction: Based on the results of your performed experiments, discuss among your groupmates
all of your observations and answer the following questions:
1. What are the factors that play the dominant role in this experiment?
3. What is an NSS?
4. What is the effect of the following solution on the red blood cells; Draw the cells found in each
tube.
a. Hypotonic;
b. Hypertonic;
c. Isotonic salt?
17
ADSORPTION
Adsorption occurs when a layer of ions, molecules or aggregates of molecules are condensed on the
surface of the solid. This arises from the valence forces or other attractive forces of the atoms or
molecules in the outermost layer of the solid. The amount of adsorption depends upon the content
of the surface and the surface adsorbed.
Procedure:
4. Filter and note the color of the filter and set it aside. Then allow the residue left in the filter
paper to dry.
5. Pour 10ml of the 95% ethyl alcohol over the residue in the filter paper and collect the filtrate in
another clean test tube.
18
SURFACE TENSION
The surface of the liquid possesses special properties which are due to the unbalanced forces of
molecular attraction at the surface. The molecules at the surface are pulled inward by then other
molecules of the liquid and the liquid tends to adjust itself to give the minimum surface area.
When the two partially miscible liquids are placed in contact, each dissolve to a certain extent in the
other causing marked changes in surface tension. The addition of the third component may lower
the surface tension considerably. Soaps, detergents and salts of bile acids are specially considered.
Procedure:
1. Prepared 2 test tubes labeled no. 1 and no. 2. To each test tube, place 1ml distilled water and
1ml CHCl3. Add 1ml of soap solution to test tube no. 2 Shake both test tube then let stand for a
few minutes (Note the time it takes for the drop to coalesce in each test tube)
2. Put 5ml of bile solution in a dry evaporating dish, sprinkle a p[inch of sulfur powder on the
surface of the solution. Observe. Repeat the test tube using distilled water.
19
Name:_______________________________________________________Section__________________ Date _______________
Instruction: Based on the results of your performed experiments, discuss among your groupmates
all of your observations and answer the following questions:
b. Absorbent
c. Elution
d. Adsorbate
e. Eluate
DONNAN’S EQUILIBRIUM
20
The presence of a non-diffusable ion in one side of a semi-permeable membrane wills at
equilibrium, produce an unequal distribution of the diffusible ion’s in both sides of the
membrane.
Procedure:
Reagents:
Albumin solution
Dilute one fresh egg white 5 times with distilled water
Hydrochloric Acid 0.1N
Add 0.83ml of concentrated HCl to 50ml distilled water and dilute to 100ml
Sodium Acetate 0.1N
Dissolve 0.82 g NaC2H3O2 in distilled water to make 100ml solution
Sodium Hydroxide 0.1N
Dissolve 0.10 g NaOH in distilled water to make 100ml solution
Buffer Phosphates (sorenses)
a. M/15 Potassium Dihydrogen phosphates dissolve 9.00 grams of KH 2PO4 in distilled
water to make 1 liter of solution.
b. M/15 Disodium hydrogen phosphates dissolve 11.88 grams of Na 2HPO42H2O in distilled
water to make 1 liter solution
Bile Solution
Dilute bile from fresh milk fish with sufficient distilled water
Name:_______________________________________________________ Date Performed:_______________
21
EXPERIMENT NO. 3
CHEMISTRY OF CARBOHYDRATES
CARBOHYDRATES
Learning outcomes
At the end of the experiment the student must be able to:
1. Discuss the properties of carbohydrates
2. Differentiate the types of carbohydrates based on their physical and chemical
characteristics
3. Perform the tests in classifying the different types of carbohydrates
Carbohydrates are organic compounds containing carbon, hydrogen and oxygen in which
the ration of hydrogen to oxygen is 2:1 the same molecule as a molecule of water. They are
polyhydric alcohols containing potential aldehyde or ketone groups. The reaction described
here are some of the chemical reactions attributed either to the carbonyl group or the
carbinol group.
- Physical Properties -
Take a small amount of the following sugars and explain its forms, color, odor and shapes.
Solubility
22
Prepare seven (7) clean and dry test tubes labeled as to content. Observe and record the
results obtained.
General Principle: Sugars upon addition of mineral acids such as HCl or H 2SO4 undergo
dehydration to form furfural or furfural derivatives which in turn condenses to form characteristic
colors.
Procedure:
1. To 5 ml of the test solution in a test tube, add 5 drops of Molisch reagent.
2. Mix thoroughly by inversion
3. Incline the tube and pour about 5 ml concentrated acid down the sides of the test tube forming
a layer of acid beneath the sugar solution.
4. A reddish-violet ring at the junction is a positive reaction.
B. Seliwanoff’s Test for Resorcinol-HCl test – this is a test for the ketone sugars.
23
Procedure:
1. To 2.5 ml of Seliwanoff’s reagent in a test tube add 5 drops of the unknown sugar solution and
boil.
2. The appearance of a red color is a positive reaction.
3. Note the time of appearance of the red color.
4. Do the test also with sucrose, glucose and maltose.
To confirm whether the red color is due to precipitate formed with fructose, add 1 ml of methyl
alcohol. If the precipitate dissolves, then the result is definitely due to fructose.
Write your results and observations below:
24
Name:_______________________________________________________Section__________________ Date _______________
Instruction: Based on the results of your performed experiments, discuss among your groupmates
all of your observations and answer the following questions:
2. Why do all sugars give positive Molisch Test? Does the test confirm the presence of sugars?
Explain.
3. Is glucose normally positive with this test? Account for the result obtained with glucose.
25
Procedure:
1. Place about 50 mg of Galactose in a clean and dry test tube.
2. Add 1 ml of distilled water and 1 ml of concentrated nitric acid.
3. Heat on a boiling water bath for 1 ½ hours.
4. Allow it to stand overnight.
5. Observe the crystalline precipitate formed and draw the crystals seen under the
microscope.
Procedure:
1. To 1 ml of 9% sugar solution, add 0.5 ml of phenylhydrazine mixture.
2. Shake well and heat on a boiling water for ½ hour.
3. Allow to cool slowly and examine the crystals under the microscope.
4. Perform the test with the following solutions: glucose, maltose, fructose and lactose.
26
Principle Involved:
Sugars having a free or a potentially free aldehyde or ketone group are capable of reducing certain
oxidizing agents such as Cu++, Hg+, Bi+, Ag+ are oxidized to the corresponding sugar acids. The
metallic ions are reduced to a lower valence state.
The reagents used are solution of metallic salts either in acid or alkaline medium, certain organic
compounds containing alcoholic groups such as Rochelle salt and sodium citrate or glycerol maybe
added.
A. Trommer’s Test
Procedure:
1. To 1 ml of 5% glucose solution add 0.5 ml of NaOH and mix.
2. Add drop by drop dilute solution of CuSO 4 until there is a slight permanent precipitate of
Cu(OH)2.
Write your results and observations below:
27
B. Fehling’s Test
Procedure:
1. Mix 1 ml of Fehling’s A and B in a test tube.
2. Add 5 ml of water and boil. If a precipitate forms, the solution must not be used.
3. To the warm Fehling’s solution, add the unknown solution drop by drop and heat the
mixture after each addition.
4. A positive reaction test is indicated by the production of a yellow to brick red
precipitate.
5. Do the test with at least 3 sugars and starch.
28
Activity Sheet Number 9
Name:_______________________________________________________Section__________________ Date _______________
Instruction: Based on the results of your performed experiments, discuss among your groupmates
all of your observations and answer the following questions:
1. Why is it important that the Fehling’s solution for use as reagent be freshly mixed?
3. Give the chemical composition of the two Fehling’s reagent and the role played by each.
C. Benedict’s Test
29
Procedure:
1. Place about 2.5 ml of Benedict’s reagent in the test tube.
2. Boil. The solution must remain clear. If precipitate is formed, the reagent must be
changed.
3. Add 1 drop of the sugar solution and boil over direct flame for 2 minutes or place in a
water bath for 5 minutes.
4. Reduction is manifested by the formation of colored precipitate like the one in Fehling’s
test.
5. Perform the test using the following sugar solution.
2. Fructose
3. Sucrose
4. Lactose
5. Starch
6. Galactose
30
Instruction: Based on the results of your performed experiments, discuss among your groupmates
all of your observations and answer the following questions:
3. Name the other sugar that showed a positive result and explain why?
D. Barfoed’s Test
31
Procedure:
1. To about 3 ml of the Barfoed’s test reagent add 0.5 ml of the sugar solution in a test
tube.
2. Place on a warm water bath for 5 minutes.
3. Remove from the water and cool.
4. Observe at once and after 5 minutes of cooling.
5. Reduction is indication of a formation of colored precipitates similar to the one seen in
Fehling’s and Benedict’s test.
6. Perform this test using the sugars used in the Benedict’s test.
2. Fructose
3. Sucrose
4. Lactose
5. Starch
6. Galactose
32
Instruction: Based on the results of your performed experiments, discuss among your groupmates
all of your observations and answer the following questions:
1. How does the condition of Barfoed’s test different from the Benedict’s test?
4. Can the Barfoed’s test be used in place of the Benedict’s test for the detection of sugar in the
urine?
5. What precipitate results when reducing sugars are heated with Fehling’s, Benedict’s and
Barfoed’s reagents.
E. Nylander’s Test
33
Procedure:
1. Place 1 ml of sugar solution in a clean test tube. Use at least 5 sugar solutions.
2. Add 0.5 ml of the Nylander’s solution.
3. Heat for 5 minutes in boiling water bath.
4. Note the change in color while boiling and after cooling for a few minutes.
34
Instruction: Based on the results of your performed experiments, discuss among your groupmates
all of your observations and answer the following questions:
1. Give the importance of the reagents and the role of each component.
E. Picric Acid
35
Procedure:
1. Place 5 ml of the sugar solution to be tested in a test tube.
2. Add 2 ml of aqueous solution of picric acid.
3. Add 2 ml of 10% sodium hydroxide.
4. Heat in a boiling water bath.
5. Reduction is indicated by the formation of a mahogany red color.
D. Inversion of Sucrose
Procedure:
1. Take about 25 ml of sucrose solution in a beaker or flask; add about 1 ml of
concentrated sulfuric acid.
2. Boil for 15 minutes.
3. Cool and neutralized with 15% NaOH using litmus paper as indicator.
4. Take a portion and perform the phenylhydrazine test.
5. In another portion, perform the Benedict’s test.
36
Instruction: Based on the results of your performed experiments, discuss among your groupmates
all of your observations and answer the following questions:
Fermentation Test
37
Fermentation is the process of converting hexone sugars to alcohol and carbon dioxide
through the action of a mixture of enzymes called zymase which is present in yeast. Hexoses
are readily fermented while pentoses are not fermented by yeast. For dissacharides to be
fermented they must be hydrolyzed to their monossacharide component by the enzyme
found in yeast. The chemical step in the fermentation of lactose by yeast juice is very similar
to the aerobic breakdown in muscle extracts.
Procedure:
1. Mix 10 ml of sugar solution with an equal volume of yeast solution in a beaker and
transfer the mixture to fermentation tube or saccharometer.
2. See to it that the long arm is filled with the solution and no bubbles are present.
3. Set aside in warm place in about 12 hours. If the sugar is fermentable, carbon dioxide
gas will be collected in the upper portion of the tube.
4. At the end of the incubation period, add 1.2 ml of 10% NaOH solution in the long arm by
means of a pipette.
5. Fill the bulb portion with water, place the thumb tightly over the opening of the
apparatus and invert the saccharometer.
7. To 5.5 ml of the filtrate, add several drops (enough to give a yellow color of the whole
mixture) of a strong solution of I and II.
8. Warm gently. Note the iodoform odor and examine the iodoform crystals under the
microscope. What does a positive test here indicate?
38
Instruction: Based on the results of your performed experiments, discuss among your groupmates
all of your observations and answer the following questions:
POLYSACCHARIDES
39
A. Starch
Preparation of Starch:
Prepare a raw potato, comminute and grind. Mix with a little of water. Strain through gauze
to remove coarse particles. Wash by repeated decantation. Drain the starch and dry in the
air. This starch maybe used for experiment.
Procedure:
1. To 5 ml of dilute starch paste solution, add a drop of iodine solution. Observe the color
2. Heat the mixture and observe what happens.
3. Cool again and observe the changes.
Hydrolysis of Starch
40
Procedure:
1. Place 20 ml of starch paste solution in a small beaker. Add 5 ml concentrated HCl and
boil the mixture. At interval of 1 minute transfer a drop of a mixture in a watch glass
over a white paper and test with iodine solution. Continue the test until it gives a
negative color with iodine. Then test it with Benedict’s reagent. Outline the hydrolysis of
starch.
41
Instruction: Based on the results of your performed experiments, discuss among your groupmates
all of your observations and answer the following questions:
1. In the iodine test, what difference was observed using plant and animal starch? Explain the
difference.
2. Account for the disappearance of the color upon heating the iodo-starch complex.
B. Cellulose
42
Solubility of Cellulose
Procedure:
1. Take a few shreds of cotton and test the solubility in water, dilute and concentrated
acids and alkaline.
2. What can you conclude with regard to the solubility of cellulose?
Formation of Amyloid
Formation of Amyloid
Procedure:
1. Add by little 2.5 ml of sulfuric acid to 1.5 ml of distilled water, cooling the solution under
the water after each addition.
2. Place about 0.5 of filter paper
3. Stir for 5 to 10 minutes until it is dissolved. Pour 0.5 ml of the solution to 2.5 ml of water
and note the production of flocculated precipitate, which is the Amyloid.
4. Add a drop of iodine to a small portion of the precipitate.
5. Pour the remaining portion in 7.5 ml of water and boil for 5 to 15 minutes.
6. Cool and neutralized with NaOH.
7. Test with Benedict’s and other reducing agents.
Glycogen
43
Procedure:
1. Cut the fresh crystals into small pieces.
2. Mix with sand and grind in mortar.
3. Place in a beaker and add 25 ml of water.
4. Heat for 20 minutes, adding water from time to time to maintain the volume.
5. Acidify with 5% acetic acid.
6. Boil for 1 minute, cool and filter.
7. Add four volumes of 95% alcohol.
8. Allow the precipitate to settle, pour off the supernatant fluid and filter.
9. Dissolve the precipitate in hot water to make a glycogen solution.
10. Test a small portion of the solution of glycogen with benedict’s reagent and iodine t.s.
Acidify the remaining solutions with HCl and boil for 10 minutes. Cool and neutralize
solutions with HCl and boil for 10 minutes. Cool and neutralize with 10% NaOH and test
with Benedict’s reagent.
Compare the results of your reduction test with that was obtained previous to hydrolysis.
C. Hemicellulose
Gum Arabic
Physical Appearance
Examine the sample as to form, color, odor, and taste.
A. Cold water
B. Hot water
C. Alcohol
44
1. To 20 ml of gum Arabic solution, add 5 ml of concentrated HCl solution. Heat for 5
minutes, cool and neutralize with NaOH solution.
2. Test with Benedict’s reagent.
Galactam
Agar
45
A. Cold water
B. Hot water
C. Alcohol
Pectin
Preparation of Pectin:
1. Take the inner white ring of orange and chop into fine pieces.
2. Place in a beaker and cover with distilled water.
3. Allow to stand until the next laboratory period.
4. Drain through cheese cloth and squeeze out of the liquid.
5. Boil the pulp with water for 2 hours maintaining the volume by adding more water.
6. After 6 hours reduce the volume to just sufficient amount to cover the pulp.
7. Filter through cheese cloth.
8. Cool the filtrate and add twice its volume of 95% alcohol.
9. Filter and dry the precipitated pectin.
46
Instruction: Based on the results of your performed experiments, discuss among your groupmates
all of your observations and answer the following questions:
1. What substance is responsible for the starch iodine complex color formation?
2. Give the color reaction produced when starch is subjected to Benedicts test and Iodine tests.
I. Discussion:
47
Chromatography of Sugars
A mixture of sugar in the solution can be separated by the use of paper chromatography if they
posses different partition coefficients between the mobile phase and the water saturated cellulose.
The solvent is stationary usually water bound to cellulose. The other usually organic, moves over
the filter paper by capillary actions. When the partition is in favor of the aqueous phase, the solute
tends to remain near the point of application. On the other hand, if the partition favors the organic
phase, the solute will move with solvent phase.
II. Procedure:
1. Cut a piece of whatman no. 1 filter paper 1.5 x 8.0 cm.
2. Draw a light pencil line across the width of the paper 2.5 cm from the end of the strip.
3. With a pointed end of applicator stick, place a small drop of a solution containing a mixture of
glucose.
4. Allow the spot to dry in air.
5. When dry, put the other end of the strip in a razor slit in a rubber stopper.
6. Insert the stopper tightly into a 25 x 200 mm test tube containing 5 ml of developing solvent (a
mixture of n-butanol, glacial acetic acid and water 4:1:5 parts by volume)
7. The end of the strips should be placed into the solvent 1-1.5 but the solvent level must be below
the carbohydrates spot in the starting line. The paper should not touch the sides of the test
tubes.
8. The solvent will rise by capillary action. When it reaches within a few centimeters, remove the
strip, mark the solvent boundary with a pencil and allow the strip to dry in air.
9. When the strip is dry, spray with CuSO4 – Na2CO3 – Sodium Citrate spray reagent.
10. Place the moist strip in an oven at 105°c for 5-10 minutes. The carbohydrates will appear as
yellow spots which will turn blue when exposed to light.
11. Draw a pencil around each spot and place a dot at the point where the color is most dense.
12. Calculate the R.f. value for each carbohydrate by measuring the distance. The carbohydrate
moved from the starting line and the distance the solvent boundary moved from the starting
line.
The R.f. value in this solvent has two, found to be Glucose 0.18, fructose 0.23, ribose 0.31
48
EXPERIMENT NO. 4
CHEMISTRY OF LIPIDS
The brain has a high content of lipids. It contains complex lipids such as the phosphatides,
cerebrosides, spinosides. This contain fatty acids component of the C-16 to C-24 carbon chains,
chiefly in the form strearic, linoleic, oleic and arachidoic acids. The steroid, alcohol, cholesterol is
abundantly accuring in the brain.
Precautions:
Keep flammable solvents and extracts away from open flames.
Procedure:
Isolation of Triglycerides
1. Take the first 2/3 portion (from A) and evaporate the extract over hot water bath (without
flame) or over hot plate to a syrupy consistency.
2. Add 30 ml of alcoholic KOH, stir and transfer the mixture into an Erlenmeyer flask
3. Place a few glass beads to prevent bumping during heating. Cover the mouth of the flask with
funnel and put the flask over low heat for 30 minutes with occasional shaking.
4. Add distilled water if necessary to replace the lost volume. Continue heating until saponification
is complete. Add now the saponified mixture and 30 ml distilled water. Remove any insoluble
portion that may separate out.
5. Transfer the saponified solution into an evaporating dish. Heat again until the alcohol is driven
off.
6. Evaporate to a thicker consistency. Finally, add 50 ml of water and divide the solution into:
a. 20 ml portion
b. 30 ml portion
49
(Use the 20 ml portion)
Salting-out
Take the 5 ml of the solution and add a little NaCl. The salt will dissolve; add some more until no
more dissolves. Note the precipitate formed. The soap has been salted out. The precipitate should
dissolve in water.
Insoluble Soaps
To 2.5 ml portion of the soap solution in two tubes, add to the point of saturation, 0.5N CaCl 2 to the
first and 0.5M MgSO4 to the second tube. Test the solubilities of the precipitate in water.
Solubility Test: Test the solubility of the fatty acids in water chloroform.
Translucent Tests
Place a small amount of the precipitate in 5 ml of CHCl 3. Add Hubi’s iodine reagent drop by drop and
shake between additions. The iodine reagent will decolorize if unsaturated fatty acids are present.
This is due to the adsorption of the iodine by the double bonds of the fatty acids. A central test
should be done by shaking CHCl3 with the iodine reagent but to which no fatty acids has been added.
Write your results and observations below:
50
Solubility Test: Add a few drops of the solution into 3ml portion of water and chloroform.
Acrolein Test: Place about 0.5 gm of powdered KHSO 4 in clean, dry hard test tube. Add about 10
drops of the glycerol solution. Heat the solution, cautiously at the first then more strongly. Note the
odor produced.
Benedict’s Test: Test your solution with litmus paper for any activity if acidic, neutralize with a
few drops of dilute sodium carbonate solution. Add 5 drops of the glycerol solution into a test tube
containing 5 ml of Benedict’s reagent. Boil the mixture for 2 to 3 minutes. Observe the results,
compare with the results obtained from the tests for sugars
51
A. Directly from the acetone-ether filtrate from the isolation of lecithin. The filtrate, however, contains
saponifiable lipids.
Procedure:
Evaporate the ether-acetone filtrate over a hot water bath or hot plate until syrupy. The residue
should not carbonize. Cool and dissolve the residue in 8 ml of CHCl 3. Filter and get a clean solution. The
chloroform solution is ready for the color reaction.
Procedure:
Evaporate the ether-acetone filtrate from the isolated lecithin (b) to a syrupy consistency. Cool and
add alcoholic KOH and follow the procedure. When the saponification is complete, add 30 ml of ether. The
ether extracts contain cholesterol, the aqueous portion contain soaps and glycerol.
Separate the ether layer, transfer into an evaporating dish and heat over a hot plate or water bath
until dry. Dissolve the residue in 8 ml CHCl3. Filter if necessary.
The CHCl3 extracts of cholesterol from the isolation of cholesterol may now be used for the color
reactions.
Note: use dry test tubes, the CHCl3 solution must be free from water.
Salkowski Test
Place the 4 ml of the extracts in a test tube and add equal volume of concentrated sulfuric acid. Note
the colors produced in the CHCl3 layer and I acid layer.
Liebermann-Burchard Test
To the remaining 4 ml of the extract, add about 10 drops of acetic acid anhydride and 2 drops of
concentrated sulfuric acid. Note the final color produced.
This is a very sensitive test for cholesterol and is utilized for the quantitative termination of
cholesterol.
52
Activity Sheet Number 16
Name:_______________________________________________________Section__________________ Date _______________
Instruction: Based on the results of your performed experiments, discuss among your groupmates
all of your observations and answer the following questions:
1. Using the general formula for triglycerides, write the equation involved in saponification.
4. Compare the properties of glycerol and fatty acids as performed in the laboratory.
6. Why will glycerol gives positive Benedict’s test after being heated?
7. Write the chemical composition of lecithin. Relate the composition with the test performed.
53
10. Why is alcoholic medium used in the alkaline hydrolysis of lipids?
54
EXPERIMENT NO. 5
NUCLEIC ACIDS
Learning outcomes
At the end of the experiment the student must be able to:
1. Perform qualitative test for RNA
2. Investigate the products of hydrolysis of Nucleic acid.
I. Discussion:
Nucleic Acid
Nucleic acids are polyanionic molecules of high molecular weight. These polymers are composed of
a sequence of polynucleotide.
There are two types of nucleic acids, the ribonucleic acid (RNA) and deoxyribonucleic acid (DNA)
which on hydrolysis yields the sugar ribose and deoxyribose respectively. Nucleic acids are found in the
chromatin granules of the nucleus (DNA) and cytoplasm (RNA). They are responsible for the storage and
transmission of genetic information.
Nucleic acids are mostly conjugated with proteins such as histines to form nucleoproteins which on
hydrolysis yields protein and nucleic acids, DNA can be isolated from thymus or muscle tissue, RNA from
yeast
The main difference between DNA and RNA lies in their sugar components and in one of their
pyrimidine bases, DNA contains d-deoxyribose and thymine while RNA has d-ribose and uracil instead of
thymine.
Various physio-chemical studies on DNA show that aqueous solution of DNA has very large
molecular weight and very rigid structure.
Procedure:
55
A. Test for the presence of Purines
Add 3 ml of 10% NH4OH to 2 ml of the solution. Mix 2-3 drops of
AgNO3 solution.
Place the test tubes on a boiling water bath until a color develops.
Result and Observation:
56
Physical Property of DNA
1. Determination of Viscosity (at room temperature)
Use the DNA solution prepared above. Draw the solution into a 1 ml pipette. Record the time in
seconds required to drain the pipette. Perform three (3) trials.
Note: Hold the end of the pipette into the solution during the determination. Compare the time requires for
distilled water to fall the same distance. Perform three (3) trials also
57
Instruction: Based on the results of your performed experiments, discuss among your groupmates
all of your observations and answer the following questions:
58
EXPERIMENT NO. 6
PROTEINS
Learning outcomes
At the end of the experiment the student must be able to::
1. To be able to test for the presence of different elements in protein sample.
2. To perform the color reaction test for proteins to determine other elements
present in the sample.
I. Discussion
Proteins
Proteins are nitrogen-containing compounds composed of amino acids joined covalently by peptide
bonds. Each amino acid has a basic group and acidic group plus a characteristic radical. The chemical
properties of these amino acids are due to these groups and the chemical properties of proteins in turn are
dependent upon these amino acids. Upon hydrolysis with acids, alkali or enzymes finally to alpha amino
acids.
II. Procedure
2. Mix 0.2 gm of casein with 1 gm of soda lime and heat strongly. Note the odor of the vapor. Take a
confirmatory test for the gas evolved.
59
Instruction: Based on the results of your performed experiments, discuss among your groupmates
all of your observations and answer the following questions:
60
2. Add slowly 0.1M HCl from a buret with constant mixing until the pH of the solution is 4.6 (use pH
meter or an appropriate indicator). The acid should be added at a rate of about 5 ml per minute while
mixing the solution in order to avoid momentary acidity.
3. Allow the suspension to stand until the precipitated casein settles. Filter the precipitate.
4. Decant and discard the supernatant.
5. Wash the precipitate with water until the washing is no longer acid to litmus paper.
6. Dry the precipitate by pressing between filter papers.
7. Weigh the dried precipitate, using the density of milk as 1.03 g/ml, calculate the % by weight of casein
isolated.
61
Instruction: Based on the results of your performed experiments, discuss among your groupmates
all of your observations and answer the following questions:
62
2. Soak in water for 1 day.
3. Grind the beans finely with water and blend with more water using the waring blender.
4. Extract the milk through cheesecloth and discard the insoluble residue.
5. Add 1N HAC until precipitate is complete. Allow the mixture to stand for sometime.
6. Strain through cheesecloth and squeeze the water out as much as possible.
7. Weigh the dry precipitate.
63
Instruction: Based on the results of your performed experiments, discuss among your groupmates
all of your observations and answer the following questions:
64
These tests are based on the production of colored end products as a result of the reaction between
chemical reagents with one or more radicals of the reaction between chemical reagents with one or more
radicals or groups present in the complex protein molecule. The test yield varying degrees or intensities of
colors depending on the nature and the amount of groups contained in the particular protein examined. It
should be emphasized that these color tests are not specific for proteins because other substance may
respond to these tests as long as they have the radicals or group similar to proteins.
1. Million’s Test
To 5 ml of protein in a test tube, add 3-4 drops of Million’s reagent. Mix and bring the mixture
gradually to boiling point over a low flame.
65
Instruction: Based on the results of your performed experiments, discuss among your groupmates
all of your observations and answer the following questions:
2. Xanthoproteic Reaction
66
To 3 ml of protein solution in a test tube, add 1 ml of concentrated HNO 3. Mix and warm the
mixture, observe. Cool the solution and add an excess of concentrated NaOH solution.
67
Name:_______________________________________________________Section__________________ Date _______________
Instruction: Based on the results of your performed experiments, discuss among your groupmates
all of your observations and answer the following questions:
68
Mix 2 ml of protein solution and 2 ml of Hopkin’s Cole reagent. Incline the tube and allow 3 ml of
concentrated sulfuric acid to flow down the sides of the tube to form a layer of acid beneath the protein
mixture.
69
Name:_______________________________________________________Section__________________ Date _______________
Instruction: Based on the results of your performed experiments, discuss among your groupmates
all of your observations and answer the following questions:
4. Biuret Test
70
To 3 ml of protein solution in a test tube, add an equal volume of 10% NaOH solution, mix
thoroughly and add 0.5% Copper sulfate drop by drop until a purplish violet color is observed.
Repeat the test on UREA. Fill the round bottom of the test tube with urea. Heat until it melts. What
fumes are given off/ what do you call the white residue? Perform the test on the residue. What color is
produced?
71
Activity Sheet Number 22
Name:_______________________________________________________Section__________________ Date _______________
Instruction: Based on the results of your performed experiments, discuss among your groupmates
all of your observations and answer the following questions:
2. If the protein is completely hydrolyzed, will the hydrolysate yield a positive result? Why?
72
Add 2 drops of 1% solution of alpha Napthol in alcohol to 3 ml of protein solution in a test tube. Mix
and run 2-3 ml of concentrated sulfuric acid down the sides of the test tube containing the mixture.
6. Sakaguchi test
To 3 ml of protein solution, add 1 ml of 10% NaOH solution in a test tube. Add about 15 drops of
alkaline hypobromite. A color develops which fades immediately. However it can be prevented by addition
of 40% urea solution.
8. Ninhydrine Test
1. To 2 ml of protein solution, add 0.5 ml of a 0.1% Ninhydrine solution.
2. Heat to boiling for about 3 minutes and allow cooling.
73
Name:_______________________________________________________Section__________________ Date _______________
Instruction: Based on the results of your performed experiments, discuss among your groupmates
all of your observations and answer the following questions:
74
1. To 1 ml of glacial acetic acid, add 2 drops of protein solution.
2. Mix well and in an inclined position pour concentrated sulfuric acid down the sides of the test tube
until a colored layer forms at the junction.
75
EXPERIMENT NO. 7
Learning outcomes
At the end of the experiment the student must be able to:
1. To be able to conduct different methods of testing to precipitate protein in identifying the
presence of the different substances present in the sample.
2. Identify the different protein precipitate reaction as to color and solubility.
I. Discussion
II. Procedure.
Fill 6 test tubes with 2 ml of dilute egg albumin solution, add the following reagents drop by drop.
b. Lead acetate
c. Silver nitrate
d. Copper sulfate
e. Ferric chloride
f. Barium chloride
76
Name:_______________________________________________________Section__________________ Date _______________
Instruction: Based on the results of your performed experiments, discuss among your groupmates
all of your observations and answer the following questions:
1. Why is the egg albumin in a good antidote for lead and mercuric poisoning?
Salting-out Process
77
Add solid ammonium sulfate to the pint of saturation to 10 ml of saturation to 10 ml of egg albumin
solution, keeping the temperature below 40°c. Filter and test the precipitate with Million’s reagent and the
filtrate with Biuret test.
Precipitated by Alcohols
Place in each of the three test tubes, 5 ml of 95% alcohol. To the first add 1 drop of dilute HCl. To
the second, add 2 drops of 10% NaOH. Leave the third neutral. Add to each tube drops of egg albumin
solution.
78
Name:_______________________________________________________Section__________________ Date _______________
Instruction: Based on the results of your performed experiments, discuss among your groupmates
all of your observations and answer the following questions:
1. Why alcoholic solution used to disinfect areas of the skin before surgery?
79
Place 12 ml of dilute egg albumin solution in each of the 6 test tubes. Add the following reagents
drop by drop at first until an excess is added. Observe the changes.
Heller’s Test
Place the 5 ml of concentrated nitric acid in a test tube. Incline the tube and allow dilute egg
albumin in solution to flow slowly on the sides of the tube. At the point of contact, what do you
observed?
Robert’s Test
Place 5 ml of Robert’s reagent in a test tube. Allow the albumin solution to flow slowly on the sides
of the tubes. What is observed at the junction of the two liquid?
80
Name:_______________________________________________________Section__________________ Date _______________
Instruction: Based on the results of your performed experiments, discuss among your groupmates
all of your observations and answer the following questions:
1. What is common between the two tests? Give the principle involved in each tests.
81
Preparation of Alkali Metaproteins
1. Place the white of an egg in an evaporating dish. Add 10% NaOH solution drop by drop with
constant stirring. The mass gradually thickens and finally assumes the consistency of a jelly. Do not
add an excess of the alkali or the jelly will dissolves.
2. Cut into small pieces and wash with running water until free from adherent alkali.
3. Add a small amount of water and dissolve with the aid of gently heat.
4. Cool and divide into two parts.
5. To the first part, neutralize with dilute HCl. Note the odor of the gas liberated and the precipitate
formed.
6. Filter off the precipitate and test as follows:
Solubility Result
Precipitate in water
Precipitate in HCl
Precipitate in NaCl
Precipitate in NaOH
Solubility Test
Use coagulated egg white prepared by boiling the egg for 10-15 minutes with the following solvents
Solvents Observation
1. Water
2. NaCl Solution
3. Dilute HCl
4. Dilute NaOH
82
EXPERIMENT NO. 8
MILK EXAMINATION
Learning outcomes
At the end of the experiment the student must be able to:
1. To test the properties of milk using different qualitative analysis.
2. Discuss the significance of the reaction produced in different procedures
I. Procedure
1. Reaction of Milk: Test a sample of milk with red and blue litmus papers, congo red and
phenolphthalein.
Results and observations
2. Place a drop of fresh whole milk on a side and cover with cover slip. Examine under the microscope.
Illustrate below:
3. Determination of Specific Gravity: Determine the specific gravity of both fresh whole milk and
skimmed milk. Which has higher specific gravity? Why?
Results and observations:
4. Coagulation Test: Place 2 ml of fresh milk in a test tube, acidify with dilute acetic acid and heat to
boiling. Observe.
Results and observations:
5. Film Formation: Place 10 ml of the fresh milk in a small evaporating dish and boil. Note the
formation of a film on the surface. Remove the first film and heat again. Try to obtain a film.
Results and observations:
Question:
What do you think is the film made of?
83
To about 5 ml, ad 6-8 drops of dilute HNO 3 to form a precipitate. Filter and test the filtrate for the
presence of the following salts:
A. Guiac Test
To 3 ml of water and a drop of milk and enough alcoholic solution of Guiac until turbidity is
produced. Mix thoroughly and note any change in color. If no color appears within 5 minutes add
hydrogen peroxide until color is produced.
B. Benzidine Test
To 2 ml of dilute milk, add 2 drops of saturated solution of Benzidine in glacial acetic acid and 1
drop of hydrogen peroxide.
84
Name:_______________________________________________ Date Performed:_______________
EXPERIMENT NO. 9
ENZYMES
Learning outcomes
At the end of the experiment the student must be able to:
1. Determine different properties of enzymes
2. Determine the biological activities of enzyme on varied conditions
I. Discussion
Enzymes
Enzymes are proteins which catalyze biological reactions. The activity of the enzymes is
altered to a large extent by changes in the condition of the reaction medium. The change maybe a
deviation from the optimum condition or the addition of a substance that can influence activity. In
either case, the result would be an enhancement or a decrease in the activity of the enzymes.
The activity of the enzymes is affected by several factors:
a. Substance concentration
b. Enzyme concentration
c. Temperature
d. pH of the reaction
e. Presence of activators and inhibitors
Enzyme action is usually demonstrated by the increased formation of the products and the
decrease in concentration of the substrate. In the digestion of starch by salivary amylase there is a
decrease in the substrate concentrate as determined by change in the starch-iodine reaction.
II. Procedure:
Observation:
When the solution gives no color reaction with iodine, test with Benedict’s test. Explain changes
observed.
85
To 5 ml of water, add half a volume of saliva and boil the mixture for 2-3 minutes. Add to the
mixture a small amount of starch and shake thoroughly the final mixture.
Repeat Iodine test at intervals of 3 minutes. Repeat the same using the Benedict’s test.
Results:
3. Determination of Oxides
Put slices (10) from Chico, guava and potato. Put them on a watch glass and examine at
intervals. Note the changes.
4. Effect of pH
Prepare 2 test tubes containing equal amounts of egg white. Place the test tubes in a hot
water bath. When the egg white is coagulated, cool and mark the height of the following solidified
egg white. Then add the following reagents to each test tube.
Test Tube 1 = 5 ml of 2% pepsin + 10 drops of 0.4% HCl
Test Tube 2 = 5 ml of 2% pepsin + 10 drops of 0.4% Na2CO3
Keep the test tube for 1 hour in a beaker of water controlled at 40°c. Determine the extent
of digestion in each test tube by noting the amount of protein dissolved or disintegrated. Perform
Biuret test on 1 ml of the supernatant liquid from each test tube. Compare the colors obtained.
86
Name:_______________________________________________________Section__________________ Date _______________
Instruction: Based on the results of your performed experiments, discuss among your groupmates
all of your observations and answer the following questions:
5. Preparation of Catalase
87
Peel a potato and grate into 100 ml of distilled water. Let it stand for 10 minutes. Stir
occasionally, strain through cheesecloth and finally filter and extract through filter paper. Use the
extract in the following tests:
A. Biuret Test = to 10 ml of the extract add 2 ml of 10% NaOH solution. Mix thoroughly and
add 2-3 drops of 1% Copper sulfate. Observe the appearance of a violet color.
Results and observations:
B. Test for Catalase Activity = Prepare 3 test tubes labeled 1, 2 and 3 respectively. Place 5 ml
of potato extract into tube no. 1, 5 ml of boiled extract in a test tube no. 2 and 5 ml of water
in test tube no. 3. Now add to each test tube 5 drops of an 8% solution of water and add 1 ml
of Benzidine solution. Note the formation of a blue to green solution.
88
Name:_______________________________________________________Section__________________ Date _______________
Instruction: Based on the results of your performed experiments, discuss among your groupmates
all of your observations and answer the following questions:
89
EXPERIMENT NO. 10
SALIVARY DIGESTION
Learning outcomes
At the end of the experiment the student must be able to:
1. Discuss the role of salivary enzymes in digestion
2. Determine the properties of salivary enzyme using test experiments
Procedure:
1. Microscopic examination
Stain unfiltered saliva with methylene blue and examine a drop under the microscope. Illustrate
below:
2. Reaction
Test the reaction of saliva on the following:
3. Specific gravity
The urinometer cylinder is partially filled with saliva and urinometer is introduced. Make own
reading.
Readings:
What are the different organic and inorganic solids present in saliva?
For Chloride: acidify with nitric acid, heat and then add silver nitrate solution
90
For Phosphates: acidify with nitric acid, heat and then add ammonium molybdate solution
For Sulfates: acidify with HCl, than add NaCl solution and warm
For Calcium: acidify with acetic acid, then add ammonium oxalate solution
a. Precipitation of mucin
Add 1-2 drops of dilute acid to a small amount of saliva in a test tube. What precipitate is
formed? Write your observation:
b. Biuret test
Place a small amount of saliva in a test tube and render it alkaline with a 3 drops of
KOH. To the mixture, add a few drops of dilute copper sulfate solution. Write your result:
91
c. Million’s test
To a small amount of saliva in a test tube, add a few drops of Million’s reagent. Note the color of
the precipitate formed. Gradually heat the mixture and note the changes in color of the
precipitate.
8. Influence of dilution
Set on your test tube rack, 6 test tubes each containing 9 ml of water. To test tube 1, add 1
ml of saliva then mix thoroughly. Transfer 1ml of solution from test tube 1 to test tube 2. Mix the
content of test tube2 and transfer 1 ml of this to test tube 3 and so on and so forth until you have
dilutions of decreasing strength. To each tube, add a small and equal amount starch paste, shake
well and allow them to remaining in the water bath at 40°c for 20 minutes. Test the mixture with
iodine and Fehling’s solution. Tabulate your result.
9. Influence of temperature
Prepare 4 test tubes and place 5ml of starch paste in each test tube. Immerse the first tube
in cold running water. Keep the second tube at room temperature.
Place the third tube in water bath set at 40°c. Add to the contents of these three tubes, a
small amount of saliva (2ml) and to the content of the 4 th tube add a small amount of boiled saliva.
Shake to mix the contents of the four tubes. Note in which test tube there is more rapid
digestion in terms of unit of time (in terms of minutes)
92
Name:_______________________________________________ Date Performed:_______________
EXPERIMENT NO. 11
URINE
Learning outcomes
At the end of the experiment the student must be able to:
1. Describe the physical and chemical properties of urine
2. Perform the physical, chemical and microscopic examination of urine
3. Relate the findings of urine tests to actual clinical conditions
Discussion:
Urine
The composition of urine carries from time to time within 24-hour period and is dependent
on several factors such as the physiological state of the individual and the nature of diet. Therefore,
quantitative estimation of the constituents should be done on a 24 hour sample rather than on
samples collected at random preferably before breakfast is satisfactory although it is more accurate
to do a quantitative and qualitative determination on a 24-hour urine sample. Samples voided at
random should be examined within 1-2 hour after voiding
Procedure
Physical Examination
Color -
Volume/unit of time -
Odor -
Transparency -
Specific gravity -
93
Specific Determination of Gravity
Procedure:
1. Fill the cylinder nearly full with urine sample
2. Place the urinometer to float
3. Determine the reading at an eye level
4. Take care that the urinometer does not touch the cylinder
5. Make 3 readings and get the average
Results:
Chemical Tests
Benedict’s Test
a. Place 5 ml. Of Benedict’s reagent in a test tube.
b. Add o.5 ml. (or 10 drops) of urine.
c. Mix thoroughly by shaking.
d. Place in a pan of boiling water for 5 minutes.
e. Remove from boiling water boiling and read results immediately.
INTERPRETATION:
94
B. TEST FOR BLOOD
Guaiac’s Test
NOTE: Albumin is present if the cloudiness in the boiled portion persists after
addition of acetic acid.
95
D. Test for Indican
Obermeyer’s test
1. Place 2 ml of urine in a test tube
2. Add equal amount of Obermeyer’s reagent
3. Add I ml of chloroform
4. Shake by inverting the tube
Gmelin Test
96
Activity Sheet Number 29
Name:_______________________________________________________Section__________________ Date _______________
Instruction: Based on the results of your performed experiments, discuss among your groupmates
all of your observations and answer the following questions:
1. What are the substances that are responsible for the normal color of urine?
a. Polyuria
b. Anuria
c. Nocturia
d. Oliguria
e. Isestinuria
f. Hypotenuria
g. Hyperstenuria
97
Name:_______________________________________________ Date Performed:_______________
EXPERIMENT NO. 12
STOOL ANALYSIS
Learning outcomes
At the end of the experiment the student must be able to:
1. Describe the physical and chemical properties of stool
2. Perform the physical, chemical and microscopic examination of stool
3. Relate the findings of stool exams to actual clinical conditions
Procedure:
Macroscopic Appearance
Color –
Consistency –
Odor –
Microscopic Examination
1. Place a small amount of feces in a clean slide
2. Add a drop of water and emulsify
3. Break the mass into finer particles by means of a glass rod
4. Examine under LPO and HPO
Reaction
1. Mix thoroughly a small amount of feces with distilled water
2. Test the reaction with red and blue litmus paper, congo red and phenolphthalein.
98
Chemical Examination of Stool
A. Chloride
Acidify with nitric acid and then add silver nitrate solution. Note the precipitate formed
B. Phosphates
Acidify with nitric acid, heat and then add few drops of ammonium molybdate solution
C. Sulfates
Acidify with HCL and add barium chloride solution and then heat. Note the precipitate
formed
D. Calcium
Acidify with acetic acid and then a few drops of ammonium oxalate. Note the production of
a precipitate.
A. Benzidine reaction
1. Make a thin suspension of feces in 2 ml of water
2. Shake with 2 ml of ether to remove the fat. Then discard this ethereal extract
3. Add 1-2 drops of acetic acid to the residue and extract again with 2 ml of ether
4. Evaporate the ether extract in hot water bath
5. Dissolve the residue with 2 drops of water
6. Add a drop of a saturated solution of benzene in glacial acetic acid and drop of hydrogen
peroxide
Results:
B. Ortho-toluidine test
1. Place1 ml of fecal suspension in a test tube
2. Add 1 ml of ortho-toluidine in a glacial acetic acid
3. Then add 1 ml of hydrogen peroxide and observe
Results:
99
C. Guiac test
1. Place 1 ml of fecal suspension on a watch glass
2. Add a solution of Guiac in glacial acetic acid drop by drop
3. Gradually add hydrogen peroxide or old turpentine. Observe
Results:
A. Gmellin’s Test
1. Using an evaporating dish or mortar, mix 7 ml of 10% barium chloride with a piece of
stool about a size of marble
2. After an emulsion is made, filter the mixture
3. When the funnel is completely drained, add a few drops of concentrated nitric acid to the
filter paper
4. Note the play of colors produced
Results:
Results:
100
C. Schlesinger test (urobilin)
1. Shake a sample of feces with ether to remove fat.
2. Extract the residue with 5% acid alcohol
3. Neutralize the yellow fluid that results with ammonia or sodium hydroxide solution
4. Mix with equal quantity of 1% zinc acetate in alcohol
5. Remove the precipitate, if any by filtration
6. Examine the clear filtrate against a dark background for the green fluorescence that
indicates the presence of urobilin
Results:
Result:
Draw all microscopic elements that you may also see from fres stool specimen under the
microscope:
101
Activity Sheet Number 30
Name:_______________________________________________________Section__________________ Date _______________
Instruction: Based on the results of your performed experiments, discuss among your groupmates
all of your observations and answer the following questions:
1. What is the significance of testing the presence of occult blood in the stool?
4. What are the substances that contribute to the normal color of the stool?
5. Enumerate the substances which when ingested may alter the color of the feces.
102
Name:_______________________________________________ Date Performed:_______________
EXPERIMENT NO. 13
BLOOD
Learning outcomes
At the end of the experiment the student must be able to:
1. Discuss the physical characteristics of blood.
2. Differentiate the solid, liquid and gaseous components of blood.
3. Perform blood tests that aids in diagnosis of disease.
Discussion:
BLOOD
Blood is the circulation tissue of the body. It consists of solid elements (RBC, WBC, Platelets)
suspended in a liquid medium, the plasma. Because of its numerous functions, its composition is
complex. It has access to all cells of the body, thus it contains countless substances in dispressed or
dissolve state, nutrients, enzymes, hormones, vitamins, metabolites, electrolytes, etc. as well as
intermediary and waste products of cell metabolism
Cellular Elements
Stain prepared blood smear with Wright’s stain and examine the cellular elements of the blood
under the microscope.
RBC:
WBC:
Neutrophils:
Eosinophils:
Basophils:
103
Plasma Preparation
1. Make a venipuncture and withdraw 5ml of blood
2. Transfer the blood to a test tube containing the specified and quantity of anticoagulant
3. Mix the blood and the anticoagulant by completely inverting the test tube several times. Do
not shake
4. Centrifuge the blood at full speed for about 10 minutes
5. Collect the clear fluid and transfer it into a clean test tube
Drawing:
Serum preparation
1. Make a venipuncture and withdraw about 5 ml of blood
2. Transfer the blood to a serological tube
3. Place the tube containing the blood in a beaker or test tube rack
4. Let it stand for at least 30 minutes. This allows the blood to clot
5. Centrifuge at full speed for 10 minutes
6. Remove the test tube from the centrifuge
7. With a quick deliberate action, pour the serum into another test tube
Drawing:
A. Capillary Method
Sterilize the finger with alcohol and puncture as for blood counts. Fill a capillary tube with
the blood and note the time. After 2-3 minutes, carefully break off the end if the tube a small
piece at a time, as half minute interval and ends are slowly separated
B. Drop Method
Sterilize the finger with alcohol, dry and puncture as in blood count. Place several drops on
a clean slide and note the time
Draw a needle through another of the drops at half minute intervals and note the time when
threads of fibrin clining to the needle. Note the time
104
Test for the Extraction of Blood
A. Guiac test
To a solution of blood in attest tube, add solution of Guiac in glacial acetic acid (1.50) drop
until the fluid becomes turbid
Result:
B. Benzidine reaction
Place 3 ml of a dilute solution of blood in a test tube. Then add 3 ml of saturated solution of
benzidine in glacial acetic acid
105
TABLE OF CONTENTS
Preface
Experiment 5 – Proteins 47
Experiment 8 – Enzymes 60
106
PREFACE
Biochemistry Laboratory Manual for Medical Technology Students has primarily been
prepared to provide students with basic concepts about the principle and skills involved in
procedures, and also to provide them with enough review materials in preparation for their future
professional subjects
It is with this ides in mind that the laboratory manual has been prepared to provide a brief
and concise presentation on the fundamental knowledge and principles of clinical and laboratory
procedures. Efforts have been exerted to present the topics in the simplest and clearest possible
way, so that the students may not find much difficulty in grasping and absorbing the ideas that the
lesson would like to impart
The author does not claim originality of the ideas, particularly of the procedures presented
in the manual. These have merely been taken from various authorities in the field, compiled and
reconstructed to provide the students with a simplified knowledge of the subject matter
AUTHOR
107