Name: __________________________ Date: ___________

Course and Year: _________________ Rating: _________

Experiment No. 2
pH and Buffers
Discussion:
pH: The pH of a solution is the common logarithm of the reciprocal of the Hydrogen ion
concentration expressed as pH = -log [H]. Pure water is slightly ionized and at 25°C contains 1𝑥10−7 = 7.
If the concentration of a solution is greater than that in pure water, the pH is a smaller number than 7. Such
a solution is acidic. Conversely, if the [OH] 1s less than in pure water, the solution is basic.
pH is detected and measured by using a pH meter, but the most common detectors are the color
changes of acid-base indicators. The color of the indicator solution is therefore a measure of the pH.
Buffer: A buffer is one that resists a change in pH when a small amount of acid or base is added.
A bufter solution contains a weak Bronsted acid, HA and its conjugated base. An example is the mixture
of acetic acid and sodium acetate.
There are two factors that determine the effectiveness or capacity of a buffer solution. The first is
the molar concentration of the buffer components. The buffer capacity is directly proportional to the
concentration of the buffer compounds. The second factor is the ratio of the conjugated base to the
concentration of the weak acid. The most effective buffer is one equal concentration of basic and acidic
components to react with added acid and alkali, respectively.
Buffers are significant in Biochemistry. Many biological reactions of interest occur in the pH range
of 6 to 8. Specific enzyme reactions that might be used for analyses may occur in the range of pH 4 to 10
or even greater. The proper sclection of buffers for the study of biological reactions or for use in clinical
analyses can be critical in determining whether or not they influence the reaction.
Biological buffers includes the carbonates, phosphates, and proteins. They fulfill the very vital
function of maintaining the pH even though large amount of acids and bases are constantly introduced by
digestion of food, absorption, muscular activity, and respiration.
The pH of a buffer may be calculated using the Henderson-Hasselbalch equation.
Objectives: To know which is the effective buffer and the pH of different samples.
Materials and Apparatus:
pH meter, pH paper, test tubes, graduated cylinder, defibrinated blood, fresh milk, freshly voided
urine, distilled water, 0.1M sodium acetate solution, phosphate buffer pH 7.0, albumin solution, 0.1N
NaOH, 0.2 N HCL.
Procedure:
A. pH Determination
Prepare the following samples: (a) defibrinated blood (b) fresh milk (c) freshly voided urine.
Determine the approximate pH of the sample usiny pH paper by following these steps:
a. Dip a piece of the paper in the ssample for about 10 seconds. Remove the pH paper and place on
a watch glass.
b. Match the color produced in the pH paper with the chart to determine the pH of the sample.
Determine the accurate pH o the samples using pH meter Record results in table 1.
I. Results

Sample Color Approximate pH Accurate pH (from pH meter)

Defibrinated blood Red/deep red - 7.4 pH

Fresh milk Creamy white 7.0 pH 8.2 pH

Freshly voided urine Light yellow 7.0 pH 7.2 pH

B. Buffers
Prepare duplicate samples of the following: 10mL of freshly boiled and cooled distilled water,
10mL 0.1M sodium acetate solution, 10mL phosphate buffer pH 7.0, 10mL albumin solution. Determine
the original pH of each sample.
To the first set, add 0.1mL of 0.1 N NaOH to each sample. Determine the pH using the pH meter.
Record your results.
To the second set, add 0.1mL of 0.1 N HCl to each sample. Determine the pH using a pH meter.
Record your results.
3. Table of Results

Sample Original pH pH with added 0.1N NaoH pH with added 0.1N HCl

Sodium acetate 10.5 pH 11.3 pH 3.2 pH

Albumin solution 8.6 pH 9.2 pH 3.0 pH

Phosphate buffer 8.3 pH 8.2 pH 3.3 pH

Freshly boiles & cooled 8.7 pH 10.6 pH 3.0 pH

distilled water
4. Which sample exhibited buffer action?
The most effective buffer is phosphate buffer. There was only minimal changes in original pH
and the one added with 0.1 N of NaOH.
5. What is the biochemical relevance of pH?
Biochemical processes and pH is relevant to one another and it is important because it involves
our body’s processes and it is dependent on the pH, cells and orgnanims.
6. What is meant by buffer action?
Buffer action contain substances that present major changes in solution pH when small amount of
acids or bases are added to it.
7. What are the buffer systems in whole blood and plasma? Describe how it works?
Carbonic acid, a weak acid and bicarbonate, and a base maintain the blood’s pH. The bicarbonate
neutralizes excess acid in the blood while neutralizing excess base is the work of carbonic acid. (1 pt)
8. What are the limitations of the use of phosphate buffers?
The limitations of phosphate buffers are: the phosphate buffers do not eliminate H+ ions, the supply
of buffer molecules is limited and phosphate buffer only provide temporary solutions to acid-base
imbalances. (2 pts)

Exercises:
1. Calculate the ph of the following solutions
a. 0.0105 M HCl b. 0.023 M NaOH

2. Describe the preparation of a buffer composed of H2PO4 and HPO4 with a pH of 7.09, K1 =
6.2𝑥10−8
3. The normal pH of blood is 7.4 the principal buffer system in the plasma is the bicarbonate buffer.
To maintain the normal pH of the blood, what should be the ratio of the components? pKa = 6.1.
4. A buffer solution is 0.20M in acetic acid and sodium acetate. Calculate the change in the pH upon
adding 1.0mL of 0.10M HCl to 10mL of this solution.
 Experiment No. 1
BIOCHEMICAL PROCESSES
Discussion:
A. Diffusion. The tendency of the solute to spread thoroughly the solution until the composition is
homogeneous is called diffusion. Small molecules and ions move with sufficient velocity to
distribute themselves throughout the solvent rapidly. The rate at which substance diffuses across a
uniform cross-sectional area depends not only on the molecular size and shape but also on the
concentration gradient of the substance. In the absence of any other influencing factor, particles of
matter move spontancously from a region of high concentration toward one of lower concentration.
B. Osmosis: Osmosis is the passage of a solvent through a semi-permeable membrane. Such
membrane is permeable only to the solvent, not to the solute. In Osmosis, the solvent will follow
from a region of lower concentration to a region of high solute concentration. To prevent osmosis
to occur, sufficient pressure must be applied to the more concentrated solution. This pressure is
called osmoticpressure.
C. Dialysis: Dialysis is common biochemical method of separation and purification by selective
passage of ions and small molecules through a semi-permeable membrane that will not allow
colloids to pass through. Some of the membrane used in dialysis is: Viking sausage casings,
collodion and cellophane. These membranes contain small pores which allow the ions, but not the
large colloidal particles to diffuse. The kidneys are made up of a fine network of dialyses tubing’s.
They excrete liquids, salt and small waste molecules but at the same time prevent the loss of protein
from the body fluids.
The rate of dialyses depends on many factors: the area of the dialyzer: the size of the pores, the
temperature, the electric charges, and the relative concentration of the solution on the two sides of the
membrane.
D. Surface Tension: Surface tension is the tendency of liquid surface to contract. The surface
molecules feel an unbalanced attraction and are pulled inward. This inward pull causes the
molecules at the surface to come closer together. The surface are tends to become smaller giving
rise to the membrane effect.
Objectives: __________________________________________________________________________
__________________________________________________________________________
Materials and Apparatus:
10% CuSO4 solution, 10% Prussian blue solution, 0.9% NaCl, milk, Fehlings A & Fhelings B,
trichloroacetic acid, test tubes, cellophane, microscope and Bunsen burner.
Procedure:
A. Diffusion
Put a 2 mL of 10% CuSO4 solution in a test tube. Incline the test tube and carefully add 3 mL of
distilled water. Note the time it takes for the entire body off fluid to have the same color.
1. Observation: The rate of diffusion or for the fluid to have the same color is slower since it has a
lighter color.
Repeat the test using 2 mL of 10% Methylene blue solution instead of 10% CuSO4
2. Compare the results: The rate of diffusion of methylene and distilled water is faster compared to
Tube 1 because it has darker color.
B. Osmosis
Prepare 4 test tubes containing 5 drops of defibrinated blood. Label test tube no. 4 as “control”.
To test tubes no. 1, add 1 mL 0.9% NaCl solution, to test tube no. 2, add 1 mL of 0.1M NaCl solution and
to test tube no. 3 add 1 ml of 1.0M NaCl.
Examine the results of cach test tube under a microscope. Record you observation.
3. Observations: The shape of the RBC is determined by the concentration of NaCl (sodium chloride).

Test tube No. Results

1. 1 (0.9%) Normal (isotonic) – biconcave disk

2. 2 (0.1M) Swell (hypotonic)

3. 3 (1.0M) Shrink (hypertonic)

4. 4 (control) Normal (isotonic)

C. Dialysis
Soak a piece of cellophane (12x12 inches) in water for a few minutes to soften it. Fold the soaked
cellophane like a filter paper to fit into a glass funnel and pour 50 mL of milk in it.
Collect all the edges of the cellophane and tie with a string to secure them.
(Note: Leave an air space above the milk)
Suspend the bag from an air stand into a 500 mL beaker so that it will almost touch the bottom.
Add distilled water to the beaker until it is in level with the surface of milk.
Allow the milk to dialyze for 1 hour with constant stirring of the water outside. Test the dialysate
for protein and sugars.
a. Test for Proteins
Put 1 mL of the dialysate in a test tube and add 1 ml of 25% trichloroacetic acid solution.
4. Results: The solution is clear. (Negative because the protein was not able to pass through because it
is large.)
To check the sensitivity of the test, dilute 1 drop of milk to 10 mL of distilled water. Put 1 mL of diluted
milk in a test tube and add 1 mL of 25% trichloroacetic acid solution.
5. Compare the results with a: The solution is cloudy compared to the other one. (Positive because the
result was cloudy. There were particles of milk that passed through).
 b. Test for Sugars
Mix 1 mL Fehlings A and 1 ml Fchlings B in a test tube and heat in a boiling water bath. Add 1 mL of the
dialysate and continue to boil for 1 minute.
6. Results: There was a reddish precipitation observed. (The result is positive because of the presence of
glucose precipitate and fracture.
D. Surface Tension
Prepare two test tubes labelled no. 1 and no. 2. To test tube, place 1 mL, distilled water and 1 mL,
chloroform. Add 1 ml of soap solution to test tube no. 2. Shake both test tubes, then let stand for a few
minutes. Note the time it takes for the drops to coalesce in each test tube.
7. Results: In test tube 1, the H2O and CH3Cl3 were not mixed and both of them are colorless. In test tube
2, the soap solution subsided at the bottom while CH3Cl3 and H2O were mixed. Coagulation can be seen
in test tube 2.
Put 5 mL dilute bile solution in a dry evaporating dish. Sprinkle a pinch of sulphur powder on thesurface
of the solution.
8. Observation: Some of the sulphur powder subsided to the bottom of the solution with the bile.
Repeat the test using distilled water instead of bile solution.

9. Compare the results: The sulphur powder settled at the surface of the water.

10. What is the effect of hypertonic, hypotonic, and isotonic salt solution on the red blood cell.

Solution Effect on the RBC

Hypertonic The RBC will shrink.
Hypotonic The RBC will swell and later on, burst.
Isotonic The RBC will be normal in size.

11. How does the kidney maintain the body internal environment?
The kidney maintains the body’s internal environment by maintaining the volume of body fluids,
maintaining the balance of salt ions in body fluids and excreting wastes.
12. What is the role of bile salts during digestion?
Bile salts play a vital role in mixing liquids together to form emulsion (emulsifying) fats to help in
absorption.

13. What is surfactant?

It is also known as surface active agent. Surfactant is a substance that lowers the surface tension
between liquids. It may also act as detergent, wetting agents, etc.
14. Explain how soap lowers the surface tension in water.
The soap separates the water molecules from each other. The polar end of the soap is attracted to the
polar water molecule. When a number of soap is attached to a water molecule, it forms a unit. This
unit have a water force of attraction since only the non-polar sides are exposed. Thus, the surface
tension is lowered.
 Chromatographic Separation and Identification of Compounds
Discussion:
One method of separating compounds is by paper chromatography. It involves the distribution of
the solute between a polar liquid phase, which is strongly absorbed on the cellulose fibers of the paper, and
the electing solvent. The mobile phase moves up the paper, and the compound is carried to a particular
position on the paper. The location of the compound depends on several factors namely: the pH, the
temperature, the concentration of the solvent and the time of chromatography.
This experiment concerns the separation of mixtures of amino acids obtained from the hydrolysis
of proteins and peptides. Identification will be made using the Rf values of the known amino acids. Since
amino acids are colorless, their location on the developed chromograph will be made visible by staining
each spot with ninhydrin, a substance that reacts with amino acids to form colored products.
Objectives: To know the different distance travelled by amino acids and solvent, their Rf value of a specific
spots (amino acids) and to know the unknown amino acids. Additionally, to know the importance of Rf
value in the analysis of amino acids.
Materials and Apparatus: Graduated cylinder, beaker, Whatman filter paper No. 1(16.50 cm long and
8.0 cm wide), mixture of Butanol, Acetic acid and distilled water (4:1:5 by volume), 0.5% glycine, 0.5%
lysine, 0.5% aspartic acid, MSGc(pure sodium), 0.2% ninhydrin solution, aluminium foil.

Procedure:
A. Preparation of Developing Chamber
Pipette 8 mL of the solvent and introduce it into a dry 250 ml beaker. Avoid splashing the liquid
on the sides of the beaker. Cover with a piece of aluminium foil and let it stand for 10 min for
atmosphere inside to become saturated with solvent vapor.
B. Preparation of the Paper Chromatogram
1. With minimal handling (finger print can obscure the result) cut a piece of Whatman filter paper
no.1 (16.50 cm long and 8.0 cm wide). With a pencil, draw a line 6 m from the lengthwise edge
of the paper and 1 cm from each crosswise edge, for handling.
2. Mark hghtly with a pencil 5 equidistant spots along the lengthwise line of the filter paper
3. Gently and quickly touch the first mark with a point of a fine capillary tube (0.5 mm diameter)
containing 0.5% glycine. Apply approximately 20 micrograms of the sample on the mark,
allowing the spot to dry before cach application. The wet area should not be more than 2 mm
in diameter.
4. Repeat step no. 3 on the other marks using different amino acid for each mark.
5. Set aside the paper for a few minutes to allow the spots to dry.
C. Development of the Spot
1. Once all the spots are dry, roll the paper into the cylinder and carefully staple the ends
together. The edges of the paper should not touch.
 2. Put the cylindrical paper upright into the beaker with the spotted edge at the bottom. The solvent
should wet the lower of the paper without reaching the spots. Put the aluminum foil cover in
place and let stand for 30-45 minutes or until solvent’s front is 1 cm away from the upper edge.
3. Remove the paper from the beaker and open up. Lay the paper flat on a dry towel to dry. Quickly
mark the position of the solvent front with the pencil.
4. Spray the paper very lightly and evenly with 0.2% ninhydrin solution and drag it in the oven at
90°C for 3 or 5 minutes. Ninhydrin will react with amino acid upon heating to produce a
characteristic purple, red-brown or yellow color.
5. Once you have the developed and dried chromatogram, draw a circle around each spot that
appears above the starting line and note the color of each. Make a dot in what you think is the
center of each spot. Some spots elongate, showing a definite head and an extended tail, the dot
should be in the center of the head.
6. Measure the distance in mm from the starting linc to the dot in the center of each separate
component, and also measure the distance from the line to the solvent front. Calculate the Rf
values for every component spot that appears on the chromatogram. Show all calculations.
D. Calculation of the Rf (rate of flow) value
1. Calculate the R-value of cach amino acid and using the values obtained, identify the amino
acid in the unknown solution given to you.
𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑎𝑚𝑖𝑛𝑜 𝑎𝑐𝑖𝑑 𝑖𝑛 𝑚𝑚
Rf =
𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 𝑖𝑛 𝑚𝑚

Spots Distance travelled by Distance travelled by Rf value

amino acid solvent

Lysine 6.3cm – 63mm 6.5cm – 65mm 0.969

Glycine 6.25cm – 62.5mm 6.6cm – 66mm 0.946

Aspartic Acid 6.5cm – 65mm 6.8cm – 68mm 0.955

Glutamic Acid 6.3cm – 63 mm 6.7cm – 67mm 0.940

Unknown (Lysine) 6.5cm – 65mm 6.7cm – 67mm 0.970

*Lysine *1 – 63/65
– Polar enough to interact with water but is basic *2 – 62.5/66
– Proton acceptor *3 – 65/68
– Hydrophobic *4 – 63/67
– Nitrogen makes the Lysine a proton acceptor *5 – 65/67
2. Identify the unknown through the Rf value obtained. The unknown is Lysine.

3. Of what importance are the Rf values in the analysis of amino acid?

The importance of Rf value is that, it serves as a basis in identifying several amino acids and its
corresponding value.
4. What part of the amino acid of protein reacts with the ninhydrin solution?
α – amino acid reacts with ninhydrin to give a colored product. *Free carboxylic grow and amino
groups on proteina and peptides also reacts.
5. Give other developing solvents that can be used in paper chromatography.
Other solvents that can be used are acetone and small alcohols because both have a polar and non-
polar groups (ampiphatic).
 ENZYMES
Discussion:
An enzyme is defined as a protein synthesized in the ving cells, which catalyzes thermodynamically
possible reactions so that the rate of the reactions is compatible with biochemical process needed tort
the maintenance of the cell.
Some enzymes owe their specific reactivity only to their specific protein structure. Others however
are conjugated proteins that require the presence of a unique non protein unit to become active enzymes.
The protein portion of a conjugated enzyme is known as apoenzyme and it’s non- portion is termed a
cofactor. There are two types of cofactors if the cofactor is an organic unit; it is commonly called a
coenzyme. If the cofactor is a metal ion, it is called a metal-ionactivator. Some enzyme require both types
of cofactor some of the meal-ion activators are Na+, K+, Mg2+, Co2+ and Z2+. Many trace metal ions
found in the body are important in enzyme reactions. Some vitamin or their derivatives are coenzymes.
Since vitamins and metal ions are essential for proper cnzyme function, and the reason why they are
essential components of the diet.
The substrate is the primary species acted on by an enzyme. Some enzymes are named after the
substrate on which they act by simply adding —ase to the root of the name of the substrate.
Objectives: To be able to identify the enzyme’s specificity based on their protein structure.
Materials:
10% NaOH, % CuSO4, 3% H2O2, 0.5% Benzidine, phosphate buffer, pH 6.7, 0.9% NaCl, iodine
solution, starch solution, test tubes, beaker.
Procedure:
I. Preparation of Catalase
Peel a potato and grate into 100mL of distilled water. Let stand for 10 minutes, stir occasionally,
Strain through a cheese cloth and finally filter the extract through filter paper. Use the extract in the
following tests.
A. Biuret Test *Test to detect the presence of peptide bonds
*Blue-violet complex
*CuSO4

To a 2 mL of the extract, add 2 mL of 10% NaOH. Mix thoroughly and add 2 to 3 drops of 1% CuSO4
solution.
1. Observations: The solution turned *purple because of the presence of *peptide bonds. The test used
was the Biuret test which indicatrd that there is a peptide bond present.

2. What is the biuret test for?

It is a general test for proteins. It gives color reaction due to the presence of peptide linkage.

B. Test for Catalese Activity

To 5mL of the extract, mix 1mL of 3% H2O2.

3. Observations: The color is tinge of orange-brown and is lighter and clear in color compare to
Benzidine. *Presence of bubbles.

Test for the combustibility of the gas evolved by holding a glowing splinter over the mouth of the test tube.
4. Observations: The flame glowed longer because the catalayse acted as a catalyzing agent producing,
water and oxygen. Oxygen is a factor of combustion.
Add 1 mL of 0.5% Benzidine solution to the solution in the test tube.
5. What color is developed? Darker compared to H2O2 and is cloudy. A ppt was also formed after a
while.

II. Preparation of a dilute solution of Salivary Amylase

Rinse your mouth several times with water. Then collect Iml of saliva. Prepare a 1:8 solution of the
saliva by adding 0.25 saliva to 19.75 mL of distilled water. Use the prepared solution in the following test.
III. Test for Specificity of Enzyme Action
A. Prepare 2 test tubes, each containing 2 mL of 0.02M phosphate buffer, pH 6.7 and 1 mL of 0.9%
NaCl solution
To test tube number 1, add 1 mL cooked starch and 1 mL of the solution of salivary amylase. To
test tube no. 2 and 0.1% glycogen solution and 1 mL of the salivary amylase solution. Let the test tubes
stand for 15 minutes at room temperature.
B. Stir the mixture in test tube no.1 and place 1 drop in an evaporating dish or spot plate. Add 1 drops
of iodine solution.
6. What is the color developed?

Repeat this test at 5 minutes interval for 1 hour.

C. Perform the test done in b on the solution in test tube no. 2

Result
A. Test tube no. 1 B. Test tube no. 2

Time interval Color of I2 Color of I2

5 mins Light yellow Light yellow

10 mins Dark yellow Light yellow

15 mins Dark yellowish brown Dark orange

20 mins Orange Light orange

25 mins Brownish orange Light orange

 30 mins Yellow orange Light orange

35 mins light yellow Tinge of yellow

40 mins Brownish yellow Brownish orange

45 mins Brownish orange Dark orange

50 mins orange Light orange

55 mins Brownish yellow Dark orange

60 mins Dark orange Dark orange

*Darker – glycogen

*Lighter – starch (iodine test)

– Turns into glucose

7. Define the following terms:

a. Enzymes – a biomolecule that catalyzes a specific chemical reaction.
b. Substrate – a molecule that is acted upon, and chemically changed by an anzyme.
c. Cofactor – an inorganic ion or a coenzyme required for an enzyme activity.
8. What type of enzyme is catalase? Catalase acts as the catalyzing enzyme in the decomposition of
hydrogen peroxide into water and oxygen.
9. What is the specificity of salivary enzyme? Salivary amylase is specific for degredation of starch or
amylase into glucose.
10. What mechanism is involved in the hydrolysis of starch by salivary analyse? Absorption of the free
enzyme onto the surface of substrate, reaction of the absorbed enzyme with the substrate, and
liberation of the product. *Breakdown of starcg into simple sugars.
 Factors Affecting Enzymes Activity
Discussion:
The increase in temperature in an enzyme activity will increase the rate of enzyme reaction due to
an increase of collision of activated molecules. For a 10C rise in temperature, the rate of an enzyme action
is doubled. But as the temperature is increased beyond 30°C, the reaction rate decreased due to the
denaturation of the protein portion of the enzyme.
Enzyme action is usually demonstrated by the increased formation of the products and decreased
concentration of the substrate. In the digestion of starch by salivary amylase. There is a decrease in the
substrate concentration as indicated by the change in color of the starch iodine reaction.
Objectives: To determine the effects of certain factors on enzyme action and to understand the changes in
temperature and pH.
Materials: Test tubes, 1% cooked starch solution, iodine solution
A. Temperature
Procedure
1. Prepare 3 test tubes, label them accordingly
2. For test tube number 1, place 5 mL of 1% cooked starch solution + 1 mL saliva. Place in a
water bath at 10 °C
3. For test tube number 2, place 5 mL of 1% cooked starch solution + 1 mL saliva. Place in a
water bath at 30°C
4. For test tube number 3, place 5 ml of 1% cooked starch solution + 1 mL saliva and place in
water bath at 40°C.
At 5 minutes interval, take a drop of the reaction mixture from the test tube number 1 (Stir the
contents of the test tube before taking a drop), and test with iodine solution. Take note of the color of the
mixture. Perform the todine test for a period of 1 hour. Do the same with the other two test tubes.

10C 30C 40C

Time Color with Iodine Time Color with Iodine Time Color with Iodine
5 mins Light brown 5 mins No change in color 5 mins No color change
10 mins Brown 5 mins Gray 5 mins Cloudy white
15 mins Violet 5 mins Dark gray 5 mins Shade of gray
Yellow green w/ black
20 mins Blue-violet 5 mins Darker gray 5 mins
pigment
Black green w/ black
25 mins Blue-violet 5 mins Grayish black 5 mins
pigment
30 mins Brown-black 5 mins Black 5 mins Brown-black
35 mins Brown-black 5 mins Black 5 mins Brown-black
40 mins Brown-black 5 mins Black 5 mins Darker brown
45 mins Brown-black 5 mins Black 5 mins Dark brown
 50 mins Brown-black 5 mins Brownish black 5 mins Black
55 mins Brown-black 5 mins Brownish black 5 mins Black
60 mins Dark purple 5 mins Dark 5 mins Black

11. Show the different stafes of starch hydrolysis through the action of salivary analyse
STARCH SOLUBLE STACH AMYLODEX TRIN ERYTHRODEXTRIN

GLUCOSE MALTOSE ACHRODEXTRIN

B. Effect of pH
Procedure
Label 3 test tubes and mix the substances indicated below
Acid or Base or
Test tubes 1% starch solution 0.1M NaCl saliva
water added

1 10mL 1mL 1mL of 0.05M HCl 2mL

2 10mL 1mL 1mL distilled water 2mL

3 10mL 1mL 1mL of 0.05M NaOH 2mL

Mix well by shaking each test tube, and place in a water bath maintained at 37C. Recording the time
at 3 minutes intervals, test for the presence of starch using 0.001 N iodine solution. Record the time need
for the blue color of starch with iodine to fail to appear. This means that starch has been completely
hydrolyzed to glucose.
1. Results
Color of saliva extract with Iodine
Time (min)
Test tube 1 Test tube 2 Test tube 3

White surrounded by black

3 Violet No color change
ring

Light yellow surrounded by

6 Deep blue No color change
black ring

Light yellow surrounded by

9 Deep blue No color change
dark ring

Light yellow surrounded by

12 Deep blue No color change
black ring

Yellow surrounded by black

15 Deep blue No color change
ring

18 Dark blue Dark yellow No color change

2. In what test tube is there evidence of unhydrolyzed starch after 18 min? Test tube 1 & 3
The statch was not really hydrolyzed because the color was still blue.
3. Observation: The longer the time observed, the darker the color becomes.
4. What is the optimum pH of salivary amylase? The optimum pH ranges from 6 to 7 but is most active
at pH 6.8
 CARBOHYDRATE
Discussion:
A carbohydrate is a polyhydroxy aldehyde, a polyhydroxy belong on a compound that yields these
open end hydrolysis. They are principal constituents of plants, composing of 60% to 80% of their weight.
Starch and cellulose are both polymers of glucose, which is produced in photosynthesis. There are three
classes of carbohydrate based on the products formed when they are hydroprolysed.
1. Monosaccharide are the simplest carbohydrate which cannot be broken down into simpler
carbohydrates.
2. Disaccharides yields two monosaccharides upon hydrolysis.
3. Polysaccharides yield more than two monosaccharides upon hydrolysis.
Only a few classes of carbohydrate are of special biological importance. These include the
hemolysis and glucose, also known as deodrose or grape sugar, fructose or levolose, galactose.
Glucose is a normal constituent of blood and tissue fluids of the human body. D-galactose, also
called as brain sugar is found as cerevrosides in the brain and as ganglioside in the nerve tissue. Ketoses
like ribose and deoxyribose are constituent of nucleic acid also present in some enzymes.
Common reactions carbohydrate includes the following:
1. Molisch Test – is a general test for carbohydrate and monosaccharide will give positive result rapidly.
2. Reduction Test – all sugars that can form a free aldehyde group in solution will read the with the
Fehling’s reagent or Benedict’s to form a brick red brown, green or ocassionally yellow precipitate.
This sugars are called reducing sugars.
3. Barfoed’s Test – this test is used to distinguish between reducing monosaccharide and reducing
disaccharides. Only reducing monosaccharide will form precipitate of Cu2O within 5 minutes.
Materials:
Molisch reagent, Benedict’s reagent, Fehling’s reagent, Barfoed’s reagent and test tubes
Procedure:
1. Molisch Test
Label six test tubes within number 1 to 6. Place 1 mL of each of the following solutions into the
five of the test tubes. Add distilled water to the sixth test tubes to seve as the control.
a. 1% xylose solution
b. 1% glucose solution
c. 1% fructose solution
d. 1% sucrose solution
e. 1% starch solution
f. Distilled water (the control)

To each test tube, add 3 drops of Molisch reagent. Shake each test tube to ensure mixing. With
the test tube held at 45 angles, carefully and slowly run 10 drops of concentrated H2S04 solution down
the wall of the test tube so that two layers form. Note the color at the point where he two layers meet.
1. Results: Table 2: Benedict ’s Test and Fehling’s Test

Observation
Sugar
Benedict’s test Fehling’s test

Xylose (+) Brick red precipitate (+) brick red ppt

Glucose (+) Brick red ppt (+) brownish red ppt

Fructose (+) Brick red ppt (+) red orange ppt

Suctose (-) No ppt; blue solution (-) no brick red ppt

Starch (-) No ppt; blue solution (-) no brick red ppt

Water (-) No ppt; blue solution (-) no brick red ppt

Lactose (-) - (+)

2. Which carbohydrate gave positive results? Xylose, glucose, and fructose gave of positive results in
both of the tests
3. Did any carbohydrate(s) require an extended time to give positive results? Xylose needed more time
(Fehling’s test) to see the positive results. *Starch

II. Reduction test:

A. Benedict Test

Place 1mL of each of the following into seven test tubes:

1. 1% xylose solution 5. 1% lactose solution

2. 1% glucose solution 6. 1% starch solution
3. 1% fructose 7. Distilled water (the control)
4. 1% sucrose solution

Add 5 mL of the Benedict’s reagent to each solution. Shake the solution and place all 7 test tubes
in a boiling water bath at the same time. Remove the test tubes after 5 minutes and allow them to cool. After
15 minutes, observe any changes that occurred in each test tube.
B. Fehling Test
Dilute 2 ml, Fehling’s solution (a mixture of 1mL Fehling’s A and 1 mL Fehling’s B) with 8 mL
water. Put 1 mL of the diluted solution in the seven test tubes. Take 1 of the test tubes, while heating on a
water bath; add 1% glucose solution drop by drop. Note the result.
Repeat the test on the other remaining test tubes, using different 1% sugar solution of xylose,
fructose, sucrose, lactose, starch and the t control, water. Record your observation in the table provided.
Table 1: Molisch Test
4. Results

Sugar Observation

Xylose Monosaccharide (+) purple ring was observed

Glucose Monosaccharide (+) purple ring was observed

Fructose Monosaccharide (+) purple ring was observed

Sucrose (glycosidic) Disaccharide (+) purple ring was observed

Lactose (lactic acid) Disaccharide (+) purple ring was observed

Starch (glycosidic) Polysaccharide (+) purple ring was observed

Water - (-) no purple ring was observed

5. Which carbohydrate(s) are reducing sugars? Xyclose, glucose, and fructose are the reducing sugars.
*Lactose
6. Which are non-reducing disaccharides? Non-reducing monosaccharide? Sucrose is the non-reducing
sugar because it is made up of fructose and lactose which is not free from aldehyde and ketone groups.
III. Barfoeds Test
Prepare six test tubes; label them from 1 to 6. Place 1mL each of the following 1% sugars in the test tubes,
xylose, fructose, lactose, maltose, sucrose, glucose, and starch. To each solution, add 5 mL of the Barfoeds
reagent. Shake each sample. Place the test tubes in a boiling water bath at the same time, remove them after
5 mins and let cool after 15 mins. Make your observation and record them in the table provided.
7. Table 3: Barfoed’s Test

Sugars Observation

Xylose (+) brick red ppt

Glucose (+) brick red ppt

Fructose (+) brick red ppt

Lactose (-) no brick red ppt observed

Maltose (-) no brick red ppt observed

Sucrose (-) no brick red ppt observed

Starch (-) no brick red ppt observed

8. Which carbohydrate is a reducing monosaccharide? Xylose, glucose and fructose are the reducing
monosaccharide because they showed brick red ppt. (3 pts)
9. What can be learned about the sugar by performing both Benedicts and Barfoed test?
Not all sugars have their reducing property, it will depend on their components. Also, not all sugars
are capable of transferring hydrogen (electrons) to other compounds. (?)
IV. Picric Acid Test
Label seven test tubes from 1 to 7. Place 1 mL of each of the following sugars: xylose, fructose,
galactose, maltose, sucrose, lactose, and starch solution. Add 1 mL of saturated picric acid solutions to each
test tube and 2 drops of NaHCO3 solution. Mix thoroughly. Boil each test tube for 1 minute and observe
the changes in color.
10. Results: Table 4: Picric Acid Test

Sugar Observation

Xylose No mahogany red color change occurred

Fructose After an hour, the solution changed into a mahogany red color

Galactose After 45 mins, solution changed to mahogany red

Maltose No mahogany red color change occurred

Sucrose After an hour, solution changed into mahogany red color

Lactose No mahogany red color change occurred

Starch After an hour, solution changed into dark yellow color

11. What is the principle describes by the color changes of some sugars?
For the presence of carbohydrates, the picric acid test is a very sensitive test. It is also based on the
dehydration of carbohydrate by sulfuric acid to produce an aldehyde.
12. Which of the tests performed is commonly used in clinical laboratory for analysis of urine sugars?
The Fehling’s and Benedicts are commonly used for urine analysis. They are used as reagents and
there is also a present of CuSO4 and it reduced by glucose to form colored precipitate (cuprous oxide)
that is determinant for glucose concentration level in the urine. (0.5)
 Specific Reaction of Carbohydrate
Discussion:
Carbohydrates are commonly isomers, but certain reactions due to particular arrangements of
atoms in the molecule distinguish them from each other.
Seliwanoff Test
This test will distinguish between ketohexoses and aldohexoses. Ketoses will give a cherry red
color within a few minutes (about 2 mins) while aldoses require a larger time. Disaccharides and
polysachharides will eventually hydrolyze to hexoses which in time will also form red colored solutions.
Iodine Test
This test will detect the presence of polysaccharides. Polysaccharides will combine with iodine to form a
blue, red, or purple color as a sign that the test is positive. Iodine is adsorbed onto the surface of the
polysaccharide, forming a colored complex.

Objectives: To determine the identity of an unknown carbohydrate by carrying out a series of chemical
reaction.

Materials:
Test tubes, Seliwannog’s reagent, iodine solution.

Procedure:

A. Seliwanoff’s Test
Prepare 5 test tubes. Label them accordingly. Place in each test tube 10 drops of the following 1%
sugar solution: glucose, fructose, sucrose, maltose, xylose. To each test tube add 3 mL of the Seliwanoff
reagent and shake. Place all the test tubes into the boiling water bath at the same time. Observe the changes
in each test tube during the first 15 mins. Record the color and time of its formation in each solution tested.

Sugar Color Time

Glucose (-) Tinge of orange 10 mins

Fructose (+) Deep cherry red 2 mins and 27 sec

Sucrose (+) Deep cherry red 2 mins and 30 sec

Maltose (-) Tinge of orange 10 mins

Xylose (-) Tinge of green 10 mins

B. Iodine Test
Prepare 5 test tubes, label them accordingly. Place into the test tubes 10 drops of the following 1%
sugar solution: xylose, sucrose, starch, glycogen and glucose. To each test tube add 1 drop of Iodine
solution. Note the color formed.
Results
Sugar Color Time

Glucose Light yellow 3 mins and 48 sec

Starch Purple color 1 min and 56 sec

Sucrose Light yellow 1 min and 59 sec

Glycogen Light yellow 5 mins and 36 sec

Xylose Light yellow 3 mins and 27 sec

3. Which carbohydrate(s) gave a positive iodine test? The starch gave a positive result because of the
presence of amylopeatin and the change of color.
4. What is responsible for the intense blue color formed in the iodine test? Amylose. When starch is
mixed with iodine in water, intensely blue color is formed. And it is also the amylose that gets stuck
in the coil of betaamylose molecules.
C. Muric Acid Test

Prepare 4 test tubes. Label them from 1 to 4. Place in test tube number 1, a pinch of galactose,
number 2, a pinch of glucose, number 3, a pinch of lactose, and number 4, 1 mL of water and 1 mL conc.
Nitric acid, HNO3. Heat the test tubes in water bath for one hour and then allow them to cool at room
temperature. Induce crystal formation by scratching the inner side of the test tube with a clean stirring rod.
If no cystals appear, let stand until the next laboratory period. Examine the crystals under a low power
microscope and draw or sketch as seen.
Drawing

Confirm the solubility of the crystals by adding 2 mL water to the test tube where crystals have formed.
Note carefully which test tube shows water insoluble crystals.
6. Observations:
________________________________________________________________________________
________________________________________________________________________________
________________________________________________________________________________
D. Reaction with phenyldydrazine
Prepare 6 test tubes. Label each test tube from number 1 to 6. Into each of the test tubes, place 1
mL of 1% solution of numner 1, glucose, number 2, fructose, number 3, galactose, number 4, sucrose,
number 5, maltose, and number 6, the unknown solution.
Add a pinch of phenyldydrazine to each test tube. Mix thoroughly and stopper the test tubes with
cotton. Place them in boiling water bath or half and hour. Allow to cool slowly at room temperature.
Observe each test tubes for the formation of crystals.
While boiling the water with the test tubes, fructose, glucose, and unknown (glucose) formed crystals
while boiling. Crystals were formed in galactose and maltose during the cooling down process.

Examine the cyrstals formed under a low-power microscope.

9. Describe and draw the crystals.

10. Which sugar form characteristic osazone crystals? Glucose, fructose and the unknown formed osazone
crystals. (2 pts)

11. How do the crystals differ? They differ in size and shapes.

12. Identify the unknown sugar. Glucose was the unknown sugar.
 PROTEINS
Discussion:
Proteins are complex nitrogenous organic compound which when hydrolyzed yield alpha amino
acids. They are main constituents of living cells and serve many functions as catalyst to control the rate of
biochemical reacttons, as regulators for various body functions, to transport oxygen within the body, and
as defence mechanisms in the body. Amino acids are organic compounds that contain both an amino (-NH2)
and a carboxyl (COOH-) group. The general formula is RCH(NH2)COOH.
Proteins are coagulated by a variety of agents like salt of heavy metals, alcohol, mineral acids and
heat. These compounds also give characteristic color reactions.
Objectives: To know and understand different kinds of test in identifying the presence of protein and to
know its importance.
Materials and Apparatus:
Beaker, test tubes, Bunsen burner, distilled water, egg, 10% NaOH, dil. CuSO, solutions, Millions
reagent, conc. HNO2, NH4OH solution, 1% HgCl2 solutions.
Procedure:
A. Burning Test for Proteins Place a small amount of egg on an evaporating dish and apply heat gently. 1.
Note the odor. “The Swmeil_ W strong / pungent:
B. Preparation of Egg Albumin Sample Separate the white from the yolk of an egg. Place 150 ml of
distilled water in a 250 ml
beaker. Add the egg white to the water while stirring the mixture. Observe the appearance of the
mixture. wee
2. Observation We mixture was ou Small bubbles on the Surface. a _

3. What do you call the mixture formed between egg albumig and water? __ Clbia, 4 substance that
contss's ts. of“ particles disperser through out. _Qnother Supstance wihich ave to small for
resolution - incapable of

Rr assing Wo ugh A SSmipemeable mb rane . Filter the mixture and use the filtrate forthe following
proccdures.
C. Color Reactions and Proteins. . ium hydroxide
1. Biuret Test reagent: NadH , CuSO4 Detects {protein peptiae bond vioret
repens

4 wdense colo 25 A cage ? raise pH te reach

Place 3 ml of egg albumin tn a tes : Wuecteeodes , na test tube. Add 3 ml of 10% NaOH, then add a very
dilute
solution of © uSO, drop by drop, shaking the mixture after each addition
4 What do you observe Mtpr aoding CaSO4, tne michare shoud _o lightt purple,
Win TREANS , tree is ce of peptiole bond.
‘ _ presence of peptiole ‘ If Milon’s Test Atha cd. Ayrasine(pnenolic aid) A” ink rea (EOGemy
MNtrosrius S yin’ ate (yellow) /” oa SAiam nitrate & heahns Cpink~ reat Place a ml of egg algun in a lest
tube. ‘Add 5-drops of Millon's reagent. Heat in
boiling water bath for 1G minutes, then allow to cool in running water.
5 Record your observations. After heating in a toniling water for_10 minules,
Tertni"G WAS ovserved wetn tne mixture.
Then add 4 drops of freshly prepared 0.1% NaNOvand warm gently. 6 Observations: After 9Ading
NaN02- nisin cao ie tne toe Hinge Of orange in a markler of Seconois - tyrosine Askects. ominn reagent:
NHOS & Naot NA 0}
{{{ Xanthoproteic Test tryptopnan ) acids emtaining phenylalanine aromorhic nuclease ae ial
Place 3ml of egg albumin in test tube. Add 3 drops of conc. HNO; er}
7. What do you observe? After adding NHOs , there were YAS nothing observa: Apply heat to the
mixture. .

8. Note the change in color. Abie heorting tine mixture it became Alo Cool and make solution slightly
basic by adding NH, OH solution.

9. Note the changes observed. Afver the Hh crop, she. mi xture developed a foul dor. On the SOth Are
p the mixtwe became tvtally orange not D. Precipitation Reactions. “olicones she presence f
arowarh'c Amino sci, specifically, Colenaturation of fenic bord tyrosine - :

I. By Heat Place 3ml of egg albumin in test tube and apply heat for a few minutes.
10. What do you observe? ‘The. wixtut is Govoly whi ond formed. ould les «
a
11. With Strong Mineral Acid feagent: nitric aciol — tt a
Place 2 ml of egg albumin solution in a test tube. Hold the tube in an inclined position. “> / Add 5 ml
conc. HNO3 slowly along the side of the test tube, (DO NOT SHAKE). Observe the 7% color at the
junction of the two layers. .
11. Observations: yellow hetero genous mixture Was observed »
III. With Salt of Heavy Meta) wie MmEAounc Ce once Hg ele
Place 2 ml of cgg albumin in a test tube. Add 1% HgCh drop by drop with shaking . Count the number of
drops to produce a reaction. Oy
~“
12. How many dropkot HgClare needed to produce reaction? A precipi hate was observed _ at trol drop of
HgCla And we aaded excess drop to observe
Hf the precipitate increaces % decreases:
a a TEIN Ot
26
Add an excess of the rcag
cnt. Note the _ decreased t Note whether the amount of precipwate 1s increased or ecre .
13. Wnte you observations. The gmount Tr — aftr adding enuss naer ; FF pre api rae pan ation
1hcveasreol
1V. With Alkaloid Reagents.
Tame head i ite ye -
Place 2ml of egg albumin solution in a test tube Add 10% tannic acid solution drop by
drop shaking after cach addition. Count the number of drops needed to produce a precipitate Then add an
excess of the reagent.
c usually B regs
14. Observations. a
ae At the +hira drop, a brown precipitate was Q teas of reagent ana ‘tre precipitate
V. Test for Sulfur. The lead acetate test. presence of sulfide group Oy
Place Iml each of egg albumin solution and a pinch of gelatin in separately labcled test tubes. Add 5 drops
of 10% NaOH and 3 drops of 5% lead acetate solution into cach of the test tubes. Shake and heat in
boiling water bath. Describe the color of the precipitate formed
oO
15. Results . 7
The ¢gq albumin Sdluten tumed golden yellow to black precipitate while heatag ye +e { Sh sol ‘ th a : - <
,
The 49 alowmin was she first to react: 1, 2. VI. Hopkin’s Cole Test tes? for alheayeuiie add or tyrptephan
Place Iml each of egg albumin and 5%gelatin solution in separately labeled test tubes. Add 5 drops of
Hopkin’s Cole reagent. Incline the test tube and carefully allow Iml of
concentrated H2SO-to slide down the side of the inclined test tube. DO NOT SHAKE. Take not of the
color at the junction of the two liquids.
a 16. Observations 2a
With the Int of egg albumin, Olight purple ring was observa while the 5% elahh solution hat no reactim
and remasnca calories - 17. Why do all proteins give a positive Biuret test? Because all proteins have pep
tole
—_bonols_anat Biuret rest _cledects the pretence of — peptide. bone. 18. Why does HNO stain the skin
with a yellow color? ic on the
iNn_are
e : On ‘ « _ws A ‘s 19. Why is egg albumin used as an n antidote for lead or mercury poisoning Present
1A our SKin.
E in has sulfur containing proteuns which react with heavy metaly heloing to keep tnem from reacting
with similar probeins in the bo cy: "yoo d ‘ 7
~
20. What is denaturation? Denaturation is the alteration gf a protem shape thro
—Some_form_of eternal” chess ti_such aoitry itil a lenge beable Yo — CQ, out fh cellular function.
21. What is the principle of the biuret test? It ts a general test for proteine _tt giver.
-falor reaction duc te -prescace of pephicle linkage-in—polypspiatie tr prnsein« — The name of the tst
Came from he compounal Biuret, which 1s the Simplest Compeurol shal gives & typical peste reaction.—

Nance: a Course and Year:

—<——____ Date:
—__—.1- Rating:
Experiment No.
LIPIDS
Discussion:
This group of biomolecules constitute physiological and chemical substances classifi which are insoluble
in water but are soluble in tetrachloride, alcohol and benzene. They are c animal cells. In the human boy,
they are foun the nervous tissues.
a large heterogenous group of unrclated
ed together because they are fat-like, substances organic solvents such as chloroform, ether, carbon.
ssential constituents of practically all plants and
d mostly in the cell membranes, in the brain and in
Fatty acids are constituents of lipids; those found in nature have even numbers of carbon atoms in the
chain. The double bonds in unsaturated fatty acids are readily attacked by halogens to give addition
products. This is the basis of iodine number determination which is a test used to detect the degree of
unsaturation of fats and fatty acids.
Fats are hydrolyzed by dilute acids completely to fatty acids and glycerol: by alkalies to soap and
glycerol; by enzyme lipase into a mixture of fatty acids, glycerol and glycerides. The glycerol released
can be detected by dehydration. The product formed is acrolcin or propenal which has a pungent irritating
odor.
Fats develop rancid odor and taste when exposed to air at room temperature. The double bonds of
unsaturated fatty acids combine with oxygen to form peroxides, volatile aldehydes, ketones and acids,
responsible for the rancid odor. The change maybe catalysed by bacteria.
Objectives: - To describe the characteristics of Weide , discuss the princes ple th each of “the +tesk
employed end dis Hinguish differen prope. ties of” Lipide” through the diffrent Jest performed.
aterials: Distilled water, ethanol, ether, chloroform, 5%HCI, coconut oil, glycerol, KHSO4,
phenolphthalein, methyl orange, pH paper
Procedure: I. Solubility Test
A. Pipet | ml of the following solvents in separate vials or test tubes, distilled water, ethy1 alcohol, ether,
chloroform, 5% HCL and 5% NaOH.
B. Using a pipet or a dropper, add 1-2 drops of oil into each test tubes and shake thoroughly. Record the
time required for the oil to dissolve.
resu ly: non-polar , x 7 they do ot direc lye. tn polar CO! CnT | / te Lyy made od lona ? ; , Ww / Mt é é Ht
Go f Ph) Ls 4 L ‘

~~ Solvent a | Time ‘Observations

Disulled war [mine | insoube Epler
re $7 bea Slightly soluble = = non-polar, polar | —— | 16 see —| Solute = non-polar se Yager a tm
ragNaoH | $0 a a polar (Hy jo f- oer insoluble = polar
C. On different spots of a piece of bond paper, place 3 drops of cach of these mixtures — oil and ethanol;
oi] and ether. Allow the solvents to evaporate and compare the solubility of the oi! in the
two solvents.
2. Observations:
__ Ether and chloroform are solube which means Hey art non-polar and ethanol
18 - sist Soluble Which means_it is nan-po
ern like 7,
concep is “ike I. ae Test
Prepare 2 test tubes. To test tube #1, place two drops glycerol and a pinch of KHSO,. and
Ilpe
to test tube #2, place 2 drops oil and a pinch of KHSO,
Heat each test tube over a low flame. Note the odor produced.
3. Observations:
The oder was pus
_the simplest
makes
Arwlein
a pungent ,
rr
Loch unsaturated aldthyde - Arco fern in the buming fat breaking clon fo @rrol cua.
4. What is the odor due to? -
wee
oe pungent - suffocating o dor.
5. Why is the acrolein test a general test for fats?
Because arcolein js present in fats that hove. un dev g one. g tof fi peat
fats contain glycerol cthen' ficated by carbonic ad ges.
II. Test for Rancidity Pa Pty cect
4
rah on
«
le js a
cause of the | arwlein rs the result of glycere/
clear a
a. Prepare 6 test tubes. Label the test tubes accordingly.
b. To test tubes labelled 1,2,3 place 5 drops of rancid coconut oil.
4a
6. Observations
Almost. all reachone resulted te puisledive_t tons, which _mé€a art rance
bss die -
lso
4T7|
lar and polar. Jn the »_ solubility, the Test for
that y's
or yetlow a th dlorles«. oud we
ne thot ‘o/ ol
29
c. To the reaction of fresh coconut o1! with phenolphthalein, methyl orange, and pH paper. Do the
same reactions with the rancid coconut oil
‘—
7. Test Sample
Saute. Color Reactions | Test tube #1 7 P —phicnol py ne' WY yellow ish—whi'te oudy w/ eoa ileted arts
Test tube #2 +MO rancid oil cei) aa) Wack red-orange — acid Test tube ‘s elt _| #5at pH indicator —
moderately addic Test tube EJ ; no cotor chanae, bubble present
Test tube #5 +MO pail Ire red orange — basic
Test tube #6 +pH #@ ok pl indicator — weakly acidic
8. What type of rancidity occurs in vegetable shortenings and how can it be prevented? Oxidative ro
neidity 7 ceurs because the oxygen molecules srferacted with _ Ne_moltculte o at causes
changes/damage fo the_substonce. We coun it by light - proof packaging 1 oxygen - free octmes
phere, and by addin of
IV. The Grease Spot test anh’ox! dl ants.
Indicator
a. Place a small drop of vegetable oil on a piece of filter paper. Holder the paper up to the light.
b. Describe your observations: is tr u : ts te ‘ght. Paper is macle of fibers and +here _areIittle pockets
of air between the fibers: Vihen dhe gil ceineein axmbacet with the Paper, -iny droplets of orl fille
the little gaps. AS a vesuld, the Might hoe V. Test foy-Onsaturation to pass from the air, t te qreage.
dsepret of sortrvot L
Place 5 drops of olive oil, corn oil, coconut oil, and oleic acid in 4 separate test tubes. Add bromine in
CCla shaking it until a reddish brown color persists. Record the number of drops of bromine used to
produce a reaction.
9. Table of Results
77 Sample tested Numberof drops Bromine used
yr vy AMAT AK f
hive, “oil adn 800 Lorn ok aot 31S a Palm oil Peers ns ASF 186

— Silence ~ N
“ONISS EY RR: pone: preg nn prey Alueniiniss VI OAKS ONG TO OZ INEgY ‘dnoss sDADOIO OY
AO “OTRHUOTE aNRy pe Ue IO UNAOIDAY IGA RUE mE
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oINyxTU B ssonpoid sursO1d JO SISA[OIPAL OEE SACS ree GE Alne BE ree (AM AL “UIBAXO
“UOSOIPAY UOGLES JO PUNOMIIOD GORISGNS [O(AOTOTy LMA Vb Ch A
‘OPLIQIATSLE OYL JO QOUEPIAD SF PITT SUEUIOL Olle Vee KA war mime ey bf ep Mims
ou0}90k ay daypy laded prog 40 waded sayy fo avard @ Oye (6A Be ALL hr ON OCF
quojaoe UY “jsa1 SJods ISEOIG IY SI SOPLIGDA [BLY fO orIGsaId ry) ACAES CA I I AVP} pur
plow oPAKOGLeD (rego BHO] SOY PTE (OMIA LA fOr SHIGE VE VEN HT! Ave gE
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on+oHe+ (HOD) < Tt (ued wey) xe 4 O~ ) yy N OH Suronpas Jo souasaid Vyy JO VOUAPIA’ [ANSTIA
P SHAS Yop ESEE EE ve A snl ye JO UONRULIOY BIJ, “MOTIIA OF BBUIEID aq, (HP 1 POP HME
Corned oe ne Aqysry @ “aprxo (Jf) taddoo on pronpor sr nor (yy ryddery os Aiello een Lene SPAYOPIE
S_IRBNS VY VPIXO MRO MONNYOS Wapato ope cerepe de ners cee @ Ul ‘UO! (JJ) Loddon SER
{HOD HOTINIOS IST OT DOT En) CRIA AIRVRIVSE phy sree ree 9 0} pasn st 3S} JOIpauag oy edns
Breronprye er vy oy ve ve Merde vy 4 ited uleys-uddo Ue UT “WHAIKD [[RHIS TOAD OF
HONINTOS Oe) or SoA pes Spee fates -
SPOON OL IR IpPA Ode » fret cog cog per tern yy v4 Med
Vel anny ser eowely 4
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OF 31
The linkage formed when two amino acid combine 15 called a peptide link or an amide functional
group. O H || —C—N— Peptide link Biuret, a compound after which the protein test 1s named, contains
an amide functional group O H O H,N—C — N—C—NH, Buiret Objectives:
To_know 4nemn is presence of fat proteanc, and carhohyorates in Our ood ~nd mils SAampON
AitCerenk fects tnat wnill le performed -
Materials: Test tube, Molisch reagent, Benedict reagent, iodine solution, 2% CuSOz solution. 6M
NaQH, conc. HNOs, 0.1 ninhydrin solution, acetone, mortar and pestle. Procedure:
A. Solid Foods / Grind about 2 grams of the solid in a mortar, then add acetone to cover the solid
to a depth of about 2 cm. Continue to grind the food for about 2 minutes. Filter through cheese cloth and
perform the grease spot test using the acetone filtrate.
deterd
1. Observation: . A ronslucent, eerk Was olsesved inthe wase spot test of Our faa wamole ,
witich 1 Ane DiSCaAtts- Wherefore , the loiscurt contains Sat -
Spread the solid retained by the cheese cloth ona piece of filter paper and allow acetone to evaporate. Add
samples of this solid to 1ml of distilled water in cach of six test tubes and perform the three carbohydrates
test (the Molisch, Benedict and lodine tests). and the three
protein tests (xanthoprotic, biuret, and ninhydrin tests).
2. Give the description of the food sample.
The food sample that was used was Marie Gold Biscuit which § round ancl its _
inaredients are wheat flour, Sugar, palm oil, mnvert sugar, lactose, Ammoniah: Car bonak, goolium by
carbonan + salt, mano & diglyceride of fatty Quads k sodium StHoy! p-lactaak 3. Carbohydrate test be
dacten (emul sifper)calci um carbonate -
iL Test Result/Observations Molisch Test @) A purple ring was observed at the junction
{ Ke
| Benedict Test) The calor change Prom uct green whieh mnaicates traces of vaducing MOAN: lodine
Test (4) | After adding 1 drop of lndine solution the color changra ty 1o'et
—7

4 Result of Fat test Moe bentdcaict ond loading tect tuMmed out posshve which WO cates 4ne prestn &
of Sar: : |
5 Protein [cst Test _ Xanthoprotic [cst Biuret Lest Ninhydrin Test
_Result/Observations /
A Wellw color was omewd si‘ as‘stst;!;!OC~™
_A__purple Color Was opeenved — —s—“—sSSSSSSCSCS purple Color Wos observed.
| eo
6 Conclusions: Ail fo of SONA food containin _protesns _gawe Color. veactons Aue to ore or more
(adicals °F groups Present in the complek protein yal ecules -
B. Testing Milk
1. Place 1ml of milk in cach of three clean test tubes. Perform the three carbohydrates tests (the
Molisch, Benedict and lodine Test).

2. Mix | ml of milk with 2 ml acetone. If necessary filter the solution through two layers of cheese
cloth. Place a few drops of the filtered solution in the center of a piece of clean bond paper.
Compare the results with that of the grease test obtained in A.

3. Using | ml samples of milk, carry out the three protein tests ( the xanthoprotic, Biuret and ninhydrin
tests). Record all observations.
Testing Milk 1. Source and Description of the milk sample
Thesampie of our milk was sterilized mille. Wren Steri Zaton ocours, ait mi lero organism
present and bacterial sports are killed so that st Can he ctored for a long pericod at
ambient temp - two of the Components of the milk sample were Satured rat ancl whole rot wri ch 2. Test
for detecting Carbohydrates: mean +nat lipids are present:
Test Result/Observations | Molisch 7 Test G) | A pwole precdpitote was observed: | Benedict Test (+)
When Paced in “Ne Odiling wWOter bath the iolur Soin Aumned oF se aon | Iodine Test (-) _[+ne milk
sot'n pecame yellow iMdicating @ negative vesuit
3. Grease Spot Test
Observations:
An Anis ket, Me result chould yidd a postive result betause milk has fats which _Inecans they Owe Mon
volatile However in our exdenmrent, our regu, war Regahve pEecausy the filkeved sola Hwapor RA AMI
CALLy-
Conclusions:
Mi nnd of tla naturally antains fat Wich Means they owe non n_solattle_n_ nature tinat aes a wandatens
qiéloreron alnuing Rant 4 pass Threw (t=
33
4 Tests for detecting, Protein
| Test . es -Result/Observations: - | Xanthoprotic best _ A Yellow) color Was olnserved a _ Paiuret Pest |
A purple color was oloceaverd - a aa [ Ninhydrin Test__1&_ourple__eotor was observed - TT
5 Summarize what you have learned about the milk sample
~
“The talk Sample uni cin WS Clem zed mile Wad two positive results vo the est foe _carbubyscat
“ence for jO ANE DECAUSE Wame Woy is or aetecn NQ puiysaccnars dés soe aie athy sx cw Wm 4s
NOL Ove semt in tne mik- it ws also posts men the GQtase Spot
“jyeat OC oN of “We. “ests for grioteins: Therefore tine Cheri zed onic hows gre presence of Car
looltyalc ates, ips, andl proves:

Name Date Course and Ycar ating

Experiment No. Analysis of Urine
Discussion:
[he kidney is the major excretory organ of the body, and the fliud separated from plasma by its activity is
called urine. The kidney may be regarded as a filter through which the wastes, products of metabolism
pass, removing it from the blood. The removal of the waste prodcts of metabolism 1s essential for the
maintenance of the normal composition of blood.
A normal adult excretes about 600 to 2000 ml of urine a day. The volume varies with the volume of water
intake, the nature of the food in the diet, and the temperature of the environment.
The normal constituents of urine are water, nitrogenousorganiccompounds, urea, and unc acid, creatinine,
hippuric acid. purines and amino acids. : non- nitrogenousorganicsubstances. glucose, glucoronic acid,
acetone bodies, oxalate organic acids and organic sulphur compounds, Inorganicsalts of sodium,
potassium, ammonium tons, and traces of iron, copper, zinc, chlorides, phosphate, sulfates.
The presence of the following constituents in a sample of urine — glucose, proteins, blood, ketone bodies,
bile, pus and calculi, usually implies a pathological condition.
The Physical Characteristics of Urine.
1. Color ~ the color of normal urine is “amber yellow” which is duc to presence of the pigment
urochrome, a compoumd of urobilinogen. On standing, urine usually darkens due to the oxidation of
urobilinogen.

2. Odor — fteshly voided urine has an aromatic odor, but on staning develops an ammoniacal odor duc
to the hydrolysis of urea to ammonia.

3. Transparency - normal urine is clear and transparent, but on standing, clouds appear due to
precipitation of some components.

4. pH ~ normal urinc has a pH ranging from 4.8 to 7.5

5. Specific gravity — the specific gravity of urine ranges from 1.02 to 1.030. It is influenced by the
amount of solid and fluid intake, time of day and pathological condition.

Objectives: __b be abie to drfferentiaty oa normal _unne from Q urine with proolems or —Lomplicahons
ty Know the Physical chacacterishcs of urine and we be able ®
Some tr ™m™/ ad: ferent ty a G- esd. Materials and Apparatus: pe«-fo ff pes of
10% HCL, NaNO; crystals, phenol red, dil acetic acid, 1% urease solution. conc. Hel, uric acid, 5%
NasCO3;, 0.05% CuSQ,, 10% NaOH, sat. picric acid, Benedict's reagent, 5%
(NH4).SO4, 5% Na nitroprusside, conc. NH,OH, conc. HNO, beakers, medicine droppers, set of test
tubes.
=5
Procedure:
| Physica! Characteristics Examine the urine specimen as to color, odor, transparency and pH
Properties Observations Lignt yclow Oromab e Transparency syaw(ceark transparent) pH ‘ia II. Tests for
Normal Urine Constituents:
UREA e Place 10 drops of urine in a test tube, add 5 drops of 10% HCI, then add a few crystals of
Sodium nitrite. Note the evolution of N> gas. 1. Observations:
Jnere were Dulobles olpserwo this 16 because Of the N2 gasin the TiNe -
To 5 ml of fresh urine, add a few drops of phenol red solution. If the solution is pink, add a few drops of
dilute acetic acid until! it turns yellow. Then add 2 ml of fresh 1% urease solution and note the time
required to produce a red color.
2. Observations:
AURIC ACID e Measure 5.0 ml if the urine sample and place it ina SO m! beaker. Add | ml of
concentrated HCl, stir well and set aside until the next laboratory period. Examine the characteristic
pigmented crystals that have formed on the sides of the beaker 3. Observations:
e Dissolve a few of the uric acid crystals in 2 ml of 5% Na2CO; solution. Test its reducing property by
adding 0.05% CuSO4. Boil in a water bath for 15 minutes. 4. Observations:
CREATININE _ . © Place 5 ml of urine ina test tube, add 1 ml of saturated picric acid and | ml of 10%
Na OH. Notc the color formed. 5. Observations:
Mier _odding the picric_acid d ty unne the color changed fnm light yeliow te yellow. ber adding tne
NaOH ,-the yellow calor changed te veo -
e Acidify the solution and take note of the color change. 6. Observations:
Mer OGGing HCA_ana picnic add ty urine , the color changed from ved _ —tr_rea orange -
Neel Akal Z 1g ' mais 36
Hl Tests for Patholopical Constituents GLUCOSE ~ Performed Benedict's test to normal urine and three
samples of urine for people with diactes Of varying dcyrees of severity. Heat Sml of Benedict's reagent to
boiling, add & drops of the urine sample and continue heaing, for 2-3 min. Observe and record the result
Perform the test on the other urine sample
Te fast test tube had a nequh ve resut, lbccause afier heahna fur 3 ming
Interpretation of Resuts: HS color CtaAued the Same. The Inol
Blue solution Negative tes} 4+uwoe 4umed Pattive because the Greenish-blue or greenish Trace color
Unanged +t? greenish yellow acter Greentsh-ycllow + heahvig for B ming. ANOKANZE precipita
Ycllow ++ Was Also owserveA ct we wottem Orange ++-++
Brick red det
ACETONE BODIES: Rothera’s Nitroprusside Test
• Place 5 ml urine in a test tube and add 5 ml of 5% (NHa4)2SOs solution. Shake until the urine Is
saturated. Add 2-3 drops of 5% sodium nitroprusside and !-2 ml] of conc. NH40OH. Mix
thoroughly and let it stand. Note the formation of a purple tinge that
gradually deepens 7. Observations:
_Atter adding @ mi of conc. NH4OH the uring soluhon turned pale
+o brown , winich means the, result is negative- (normal urine)
e Repeat the test on the other urine sample 8. Results: . Uinen the Urine Solution (Aoloetec) was added
with 2 ml of conc. N40, a purple Ange calor wac olos tive twat ina cae o post The result:
ALBUMIN: Heller’s Ring Test____
e Place 1.5 ml of conc. HNO3 in a test tube. Pour Iml of the urine down the side of the tube such that it
forms a separate layer. The presence of protein is shown by a fluffy zone (sometimes a white precipitate)
at the urine-acid interface.
9. Observations: wht No fiuffy zone or “precipitate was observed, Wich means that aloumin wat
nok present in Unine-
e Repeat the test on the other urine sample 10. Observations: . The some result appeared. No fuffy zone or
wnik predpitak WO ohservedl wiih means What ollpwnin was hot vesemt in urine -
Kerosi
I Table of Results: ee ' Urine Sample | Observation/Results cer .
| Glucose {
Norma! Urine
fee l ; r L negative blue solution 2 ee ua Sa cea | — positi ve Oreensn lution -— Acetone
Normal Urine
hegative, pole brown was ‘the cater of sohchon
2. 9 ti - _ : Positive the Color changed info & tinge of purple Albumin Norma! Urine I ; 5 negative, no
fluffy zone/white preopi tate thserved 2 negabre, no fluffy zone/ white e preci pl Cate. 3 _J

3. Why must a 24 hour period sample of urine be used for examination if a detailed composition

is to be determined? Qver the 24 hr pero , the camposita on and concentration of tanne Changes , that's
why
th is imporiant 40 haw 94 br arme Sample Sor a detarea A examination necause_it “reveals Now wists +o
ARAN ing Physintogic Needs over along perioc .
13. What is Glucosuria? Albuminuria?
_Glucosuria is an Abnormal presence of glucose in the urine. tng from the # of carb opydraxk or from a
meee Sease, SUCh
rageshion of large amoun ll While Albumnunor is a presence Uf protcin_in_unine.chiefy
as daheses mellitus.
‘Qlbumn but alo globuba, ucvally indwative.of Akease, but tomehmer retalang frm a kmpeorary er
Fansient dye funen vn -
14. What are ketone bodies? How are they formed?
Are two products of tipict Dumvatk metabolicm, deta - ~hydroxy bu bume acd and ed “atette acid, etm
‘hich ich_aochphe tay st spon tancoudly Ketone booctice aur faened from acchyl- CoA in the liver & ave
on zed hy the muycc s.. Excessive _produch
read -b they excretion m uring, as wm iabekt mestrfus . Alc? ealied ace ibn bodes °
t sy What are the conditions leading to ketosis? _h condtion winch the concentration of ketone bodies
inthe blvd, Hssue and
_unite is anomaly Wiah. Ketos ocours when tne Ipady dors not Nave suffiaent —aecese +n glucose »
a