Experiment No.

BLOOD SAMPLE HANDLING

Blood is by far the most frequent body fluid used for analytical purposes. Three general procedures for
obtaining blood are:
• Skin puncture
• Venous puncture
• Arterial puncture

Blood oxygenated by the lungs is pumped from the heart to all organs and tissues for their metabolic
needs. Arterial blood is uniform in composition throughout the body but the composition of venous blood
varies depending on the metabolic activity of the organ or tissue being perfused. Site of collection can affect
the venous composition.
VENOUS PUNCTURE:
The relative ease of obtaining venous blood sample makes this a primary source of specimen for clinical
laboratory analysis. Whole blood without anticoagulant yields Serum, with anticoagulant Plasma.
• Heparin in the form of lithium salt is an effective anticoagulant in small quantities without significant
effect on many determinations and is the ideal universal anticoagulant for blood.
• For glucose measurements fluoride maybe added to heparin. Fluoride inhibits glycolysis of the
blood cells that may otherwise destroy glucose at the rate of about 5% per hour.
CENTRIFUGATION:
• Handle the sample with gloves.
• Put the sample in centrifuge tube and balance with distilled water.
• Program the machine for five minutes at 3500rpm and press the start button.
• Centrifuge the given blood sample for five minutes.

PREPARATIONFOR VENOUS PUNCTURE

1. Verify the precipitation slip or outpatient clinic requisition form.
2. Identify the patient.
3. If a fasting patient is required (usually 08 hour fast), confirm that fasting order has been followed
properly.
4. Address the patient and inform the patient what is to be done. Reassure the patient to avoid as
much tension as possible
5. Position the patient properly depending on whether the patient is sitting or prone, for easy
comfortable access to the antecubital fossa
6. Assemble the equipment and supplies including collecting tubes, tourniquet, preparations for
cleansing the area, syringes if necessary, sterile blood collection needle and holder used to secure
the needle and evacuated collection tube.

TECHNIQUE FOR VENOUS PUNCTURE:

• Ask the patient to make a fist to make the veins more palpable
• Select a suitable vein for puncture. Veins of the antecubital fossa in particular the median cubital
and cephalic veins are preferred over wrist, ankle and hand veins.
• Cleanse the venepuncture site with 70% Isopropyl alcohol solution. Begin at the puncture site and
cleanse in a circular motion. Allow the area to dry. Do not touch the swabbed area with any unsterile
object.
• Apply a tourniquet several inches above the puncture site. Never leave the tourniquet in place
longer than one minute. Anchor the vein firmly both above and below the puncture site. Use either
the thumb and middle finger or thumb and index finger.
• Perform the venepuncture.
• Enter the skin with the needle at approximately 15 degrees angle to the arm with the level of the
needle up. Follow the geography of the vein with the needle.
• Insert the needle smoothly and fairly fast to minimize patient discomfort. Do not bury the needle.
• If using a syringe, pull back on the barrel with a slow even tension as blood flows into the syringe.
• Do not pull back too quickly to avoid haemolysing the blood or collapsing the vein.
1
 • Release the tourniquet when the blood begins to flow.
• After all the blood has been drawn, have the patient relax his or her fist.
• Do not allow the patient to pump the hand.
• Place a clean sterile cotton ball lightly over the skin. Withdraw the needle and apply pressure to the
site
• Dispose off contaminated material such as needle, syringes, cotton etc.
• Initial the labels and record the time the specimens were drawn.
• Dispose the syringe properly with a syringe cutter.

HANDLING OF HIGH RISK SAMPLES

1. Identify that the sample is high risk (HIV, HCV, HBV etc)
2. Wear gloves
3. Avoid any needle pricks from the syringe containing the high risk specimen.
4. Transfer the sample from the syringe to a collection tube and label it as high risk sample.
5. Dispose the needle proper with a needle cutter.
6. Dispose off the waste in disinfected container/plastic bags with the red color coding on it.

Categories, color coding and types of containers recommended for segregation of

biomedical waste by regulations of the ministry of environment and forest, Government of
Pakistan.
Color Types of Waste category Treatment as per
coding container schedule
Yellow Plastic bag Cat-1 human anatomical Incineration/deep burial
waste
Cat-2 animal waste
Cat-3 microbiological waste
Cat-6 soiled waste
Red Disinfected Cat-3 microbiological waste Autoclaving/microwaving/
container/plastic Cat-6 soiled waste chemical treatment
bags Cat-7 solid waste
Blue/White/ Plastic Cat-4 waste sharps Autoclaving/microwaving/
translucent bag/puncture proof Cat-7 plastic disposable tubing chemical treatment and
Containers etc distractions/shredding
Black Box Cat-5 discarded medicines Disposal in secured
Cat-9 incineration ash landfill
Cat-10 (solid) chemical waste

2
 Experiment No.2

SAFETY IN LABORATORIES

Importance:
Laboratories can be dangerous places to work and all users need to be aware of the potential hazards and steps
to be taken in cases of emergency.
Personal protection:
1. Goggles or safety spectacles: Goggles should always be worn when carrying out any procedures where
there is a risk of splashes from reagents into eyes.
2. Gloves:Heavy-duty gloves must be worn when handling corrosive substances such as strong acids or alkalis.
Skin contact with other chemicals should also be avoided to avoid the risk of absorption through the skin.
3. Laboratory coats: Lab coats are meant to protect the worker from chemical splashes and infectious
material. Cotton is better material for a lab coat than nylon as it has a greater absorptive capacity and is
generally more resistant to chemical splashes.
4. Face Masks: They need to be worn when there is a risk of dust from chemicals or an aerosol of micro-
organisms
Dangers to avoid:
Poisoning often arises from the accidental transfer of a compound to the mouth. The risk can be greatly
reduced by always keeping three simple rules in the laboratory.
1. NO smoking
2. NO eating and drinking
3. NO mouth pipetting

3
Chemical Hazards:
• All chemicals should be considered as potentially dangerous and handled accordingly. Contact with skin and
clothing should be avoided and even if a chemical is thought to be harmless it should not be tasted or smelt.
• A Corrosive Substances is one that destroys living tissue e.g., strong acids or alkalis and inherent dangers of
strong acids or alkalis coming in contact with the skin are only too obvious.
• An irritant Substance can cause local inflammation but not destruction of the tissue and the dangers in this
case are more subtle and not always appreciated. Occasional contact with skin has no detectable effect but
repeated exposure can suddenly give rise to irritation as in case of organic solvents.
• Compounds are graded as toxic or highly toxic depending on the dose required to kill 50 percent of a
population of animals (LD50). They are dangerous not only if swallowed but also if skin absorption or
inhalation occurs.

Biological Hazards:
• Micro-organisms: Infection can occur from a cut or by ingestion, but this can be prevented by taking the
precautions already discussed. Less obvious is the risk of infection by inhaling the particulars from an aerosol
and this is probably the biggest danger when handling bacteria and viruses.
• Aerosols are formed whenever a fluid surface is broken, as e.g., with careless pipetting. Aerosols containing
micro-organisms are highly dangerous if they have a diameter of 1—5 m as this size readily penetrates the
lungs.

Animal and body Fluids:

Infection can also occur when working with tissues, body fluids and whole animals. Same precautions should be used
when handling these materials as with the direct manipulation of micro-organisms.
Animals
There are a large number of diseases that affect animals and can be transmitted to man, so even apparently healthy
animals need to be handled with care.
Animals can also cause allergies in some individuals but there is little that one can do about this except to avoid direct
exposure as far as possible.
Body Fluids:
Blood and plasma, are frequently used in biochemical experiments, also need to be treated with caution as they are a
potential source of viral infection. A much greater risk to the laboratory worker is a serum from hepatitis patients. All
human fluids should therefore be treated with the greatest care and strict precautions taken.
Spillage and Waste Disposal
Spillages
Reagent bottles should be carried firmly and well supported. The working area needs to be kept as clear as possible by
keeping the bottles and flasks away from the edge of the bench and placing them on a shelf when not required. Don’t
leave pipettes in flasks or bottles which are then very susceptible to a knock.

4
Waste Disposal
Small quantities of many materials may be washed down the sink with plenty of water but some reagents must not be
disposed of in this way. Chemicals such as acids or alkalis need to be neutralized first before washing down the sink.

5
 SPECIAL INSTRUCTIONS

• All students must be in white coats before entering the laboratory.

• All students must switch-off their mobile during practical classes.

• The water taps should be closed after finishing your work.

• The taps for gas should be turned off after you finish your experiment.

• The switches for electricity should be turned off in a state of emergency.

• Each student is supposed to write down the experiment in practical notebook with experiment No. and
date.

• The practical notebook should be checked and signed by the teacher concerned on the same day or
maximum within one week’s time.

• Every student must wash glassware, i.e., test tubes etc., after finishing their experiment.

• The working place should be cleaned during and after finishing the experiment.

• While working in a laboratory, it is important to become familiar with the layout of the room and the
location of the safety equipment.

EXPERIMENT No. 3

WATER

A. Types of water in nature:

1. Rain water: It is the purest water when freshly coming from uppermost layers, before it gets in contact
with the lower atmospheric contaminants.
2. Surface water: This represents water collections passing down from tops of the mountains to hills and
valleys with solutes and various vegetables, animals and bacterial contaminants. This includes ocean, sea,
lake water and river.
3. Subsoil water: Water that has passed through layers of earth and collect down at various depths to form
deeply running channels and reservoirs of various kinds and characters. This includes wells, spring water,
mineral water and natural fountains.
B. Soft and hard water
In accordance with the property of lather formation with soap, water has been divided into:
6
a) Soft: Easily forms lather
b) Hard: Does not form lather readily with soap. This property is attributed to the presence of dissolved
salts mainly of calcium and magnesium ions causing water hardness because of the formation of
insoluble Ca2+ and Mg2+ salts of the fatty acids in soaps thus preventing lather formation.
Hard water is divided into two distinct types:
(a) Temporary hardness: is due to the presence of Ca2+ and Mg2+ bicarbonate, this can be treated by
simple boiling when Ca2+ and Mg2+ soluble bicarbonate will be converted to insoluble carbonate
with loss of CO2, water then becomes soft.

(b) Permanent hardness: is due to the presence of soluble chlorides and sulfates of Ca2+ and Mg2+
hence the hardness could not be removed by boiling, water has to undergo other forms of treatment to
remove hardness such as lime treatment, or ion exchange resin treatment.

Tap water: Ordinary tap water contains a variety of impurities including particulate matter (sand, silt,
etc.); dissolved organic, inorganic and gases; and micro-organisms (bacteria, viruses, protozoa, algae). The
natural degradation of micro-organisms leads to the presence of byproducts called pyrogens.
Tap water should never be used for the preparation of any reagent solutions. For most of the laboratory
procedures, it is recommended that some type of purified water should be used.

Water purification techniques:

There are different water purification technologies; for most procedures carried out in a biochemistry
teaching laboratory, water is purified by distillation.
Water that is purified only by ion exchange will be low in metal ion concentration, but may contain
organics that are washed from the ion-exchange resin. These contaminants increase the ultraviolet
absorbance properties of water. If sensitive ultraviolet absorbance measurements are to be made, distilled
water is better than de-ionized.
Water Distillation (Laboratory Grade Water)
This has been traditionally used for most general laboratory work including qualitative analysis, non-
critical media & reagents, glass washing and rinsing. The use of all glass distilling apparatus made from
carefully tested neutral-glass, will produce more reliable quality especially for “Reagent Grade” water.

7
Preliminary Examination of Water “Laboratory Grade”
Laboratory Grade (L.G.) water is that water which is used in the laboratory for preparing various
solutions, used either for analytical purposes or various reagents. Such type of water should fulfill the
following requirements as regards physical and chemical limits.
Physical Examination:
1. Aspect: Absolutely Clear
2. Color: Colorless
3. Odor: Odorless
4. Taste: Tasteless
5. pH: Within neutrality ± 0.2
Chemical Examination:
1. Barium chloride test: (for carbonates, phosphates and sulfates)
Take 2 ml of 10% barium chloride solution and add 2 ml of water in a test tube, shake it, if no turbidity;
test is negative (-ve) and it is L. G. water.
2. Silver nitrate test: (for chlorides)
Take 2 ml of 2% silver nitrate solution, shake it with 2 ml of water, if no turbidity; test is negative (-ve)
and it is L. G. water.

EXPERIMENT NO. 4

“SOLUTIONS” WORKSHEET FOR 1ST YEAR (MBBS & BDS)

1.PERCENT SOLUTIONS

2.MOLARITY NUMERICALS

3.DILUTION NUMERICALS

8
4.MOLALITY NUMERICALS

5.NORMALITY NUMERICALS

6.CONVERSION OF MEASURING UNITS

9
 PERCENT SOLUTIONS

1. Prepare 0.9% w/v% NaCl solution.

2. Prepare 0.5% w/v% AgNO3 solution.

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MOLARITY NUMERICALS

1. Suppose you had 2.00 moles of solute dissolved into 1.00 L of solution. What's the molarity?
2. What is the molarity when 0.75 mol is dissolved in 2.50 L of solution?
3. Suppose you had 58.44 grams of NaCl and you dissolved it in exactly 2.00 L of solution. What would be the
molarity of the solution? (NaCl, mol. wt = 58.44g/mol)
4. Calculate the molarity of 25.0 grams of KBr dissolved in 750.0 mL. (KBr, mol. wt = 119g/mol).
5. 80.0 grams of glucose (C6H12O6, mol. wt = 180. g/mol) is dissolved in enough water to make 1.00 L of solution.
What is its molarity?
6. Calculate the molarity when 75.0 grams of MgCl2 is dissolved in 500.0 mL of solution. (MgCl2, mol. wt =95.2
g/mol).
7. 100.0 grams of sucrose (C12H22O11, mol. wt. = 342.3 g/mol) is dissolved in 1.50 L of solution. What is the
molarity?
8. 49.8 grams of KI is dissolved in enough water to make 1.00 L of solution. What is the molarity? (KI, mol. wt
=166 g/mol).
9. When 2.00 grams of KMnO4 (mol. wt = 158.0 g/mol) is dissolved into 100.0 mL of solution, what molarity
results?
10. How many grams of KMnO4 are needed to make 500.0 mL of a 0.200 M solution? (KMnO4, mol. wt =
158g/mol).
11. 10.0 g of acetic acid (CH3COOH) is dissolved in 500.0 mL of solution. What molarity results? (CH3COOH, mol.
wt =60 g/mol).
12. How many mL of solution will result when 15.0 g of H2SO4 is dissolved to make a 0.200 M solution? (H2SO4,
mol. wt = 98g/mol).
13. Sea water contains roughly 28.0 g of NaCl per liter. What is the molarity of sodium chloride in sea water? (NaCl,
mol. wt = 58.44g/mol).
14. What is the molarity of 245.0 g of H2SO4 dissolved in 1.00 L of solution? (H2SO4, mol. wt = 98g/mol).
15. What is the molarity of 5.30 g of Na2CO3 dissolved in 400.0 mL solution? (Na2CO3, mol. wt =106 g/mol).
16. Determine the number of moles of solute to prepare these solutions:
a. 2.35 liters of a 2.00 M Cu(NO3)2 solution.
b. 16.00 mL of a 0.415-molar Pb(NO3)2 solution.
17. Determine the grams of solute to prepare these solutions:
a. 0.289 liters of a 0.00300 M Cu(NO3)2 solution. (Cu(NO3)2, mol. wt = 187.56g/mol).
b. 16.00 milliliters of a 5.90-molar Pb(NO3)2 solution. (Pb(NO3)2, mol. wt =331 g/mol).
c. 508 mL of a 2.75-molar NaF solution. (NaF, mol. wt =42 g/mol).
18. Determine the final volume of these solutions:
a. 4.67 moles of Li2SO3 dissolved to make a 3.89 M solution.
b. 4.907 moles of Al2O3 to make a 0.500 M solution.
c. 0.783 grams of Na2CO3 to make a 0.348 M solution. (Na2CO3, mol. wt = 106g/mol).
19. How much CuSO4.5H2O will be required to prepare 1000ml of 0.5M solution? (CuSO4.5H2O, mol. wt = 250).
20. How much NaCl will be required to prepare 200ml of 0.2M solution? (NaCl, mol. wt = 58.5 g/mol).
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21. How much CaCl2 is required to prepare 100ml of 0.1M solution? (CaCl2, mol. wt = 111g).

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DILUTION NUMERICALS

1. 53.4 mL of a 1.50 M solution of NaCl is on hand, but you need some 0.800 M solution. How many mL of 0.800
M can you make?
2. 100.0 mL of 2.500 M KBr solution is on hand. You need 0.5500 M. What is the final volume of solution which
results?
3. A stock solution of 1.00 M NaCl is available. How many milliliters are needed to make 100.0 mL of 0.750 M
4. 2.00 L of 0.800 M NaNO3 must be prepared from a solution known to be 1.50 M in concentration. How many mL
are required?

MOLALITY NUMERICALS

1. Suppose you had 2.00 moles of solute dissolved into 1.00 L of water. What's the molality?
2. What is the molality when 0.75 mol is dissolved in 2.50 kg of solvent?
3. Suppose you had 58.44 grams of NaCl and you dissolved it in exactly 2.00 kg of pure water (the solvent). What
would be the molality of the solution?
4. 100.0 grams of sucrose (C12H22O11, mol. wt. = 342.3 g/mol) is dissolved in 1.50 L of water. What is the molality?
5. 49.8 grams of KI is dissolved in 1.00 kg of solvent. What is the molality? (KI, mol. wt = 166g/mol)

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NORMALITY NUMERICALS

1. What is the normality of the following?

a. 0.1381 M NaOH

b. 0.0521 M H3PO4

2. What is the molarity of the following?

a. 0.3181 N HCl
b. 0.1115 N Sr(OH)2

3. What is the equivalent weight of the following?

a. C6H5COOH (mw = 122.12 g/mol)
b. Na2CO3 (mw – 105.99 g/mol)

CONVERSION OF MEASURING UNITS

1. Convert serum calcium level 10mg/dL to mmol/L. (Ca, atomic mass = 40g).
2. Convert 120mg/dL of glucose into mmol/L (glucose, mol. wt = 180g).
3. Convert serum magnesium 2.2mmol/L into mg/dL and mEq/L. (magnesium, atomic. mass = 24.3g).

14
 EXP 2. SOLUTIONS
“A solution is a mixture where all particles exist as individual molecules or ions.”
Homogeneous means that the mixture is the same all the way through. You could take two same-sized samples: one
from the bottom and one from the top and they would be identical. Homogeneous mixtures do not settle out if left to
sit undisturbed, whereas a heterogeneous mixture would. Blood is a good example of a heterogeneous mixture.
An example of a solution is salt water. All solutions are not liquid systems. Metal alloys such as steel or brass are
solid solutions. Air is an example of a gaseous solution.
SOLVENT
Substance that holds another substance in solution is called a solvent, it has the same physical state as that of the
resultant solution. Examples: water, alcohol.
SOLUTE
A solute is a substance which dissolves in the solvent and looses its physical state to form a solution. Examples:
sugars, salts.
IMPORTANT TERMS
COLLOID
A colloid is a type of mechanical mixture where one substance is dispersed evenly throughout another. Because of this
dispersal, some colloids have the appearance of solutions. Colloids are intermediate between homogeneous and
heterogeneous mixture
GEL A gel (from the lat. gelatus—frozen, immobile) is an apparently solid, jelly-like material formed from a
colloidal solution. By weight, gels are mostly liquid, yet they behave like solids due to the addition of a gelling agent
SUSPENSIONS
Suspensions are most likely heterogeneous. The particles in suspensions are very large and can many times be seen
by the naked eye. They usually settle out after a period of time because of their insolubility. Dust particles in the air
would be an example. Suspensions are necessary for the dispensing of certain types of medicines such as milk of
magnesia, which must be shaken well just before swallowing it.
STANDARD SOLUTION
It the solution in which exact concentration of the solute is known. For example: standard solutions are used in
diagnostic kits in the lab, to estimate the concentration of unknown solution.
CONCENTRATION OF A SOLUTION
The concentration of a solution may be defined as the amount of solute present in the given quantity of the solution.
PERCENT SOLUTIONS
W/W % is the weight of solute dissolved in 100 grams of solution (not the solvent). A 20% of w/w solution contains
20 grams of solute in 80 grams of solvent.
W/V % is the weight of the solute dissolved in 100ml of solution (not the solvent). A 20% w/v solution contains 20
grams of solute dissolved in a final volume of 100ml solution.
V/V % is the volume of solute dissolved in 100ml of solution (not the solvent). A 20% v/v solution contains 20 ml of
solute dissolved in the solvent so that the final volume of solution is 100ml.
MOLAR SOLUTION:

15
Number of moles of the solute dissolved per liter of the solution.
A 1M molar solution contains 1 gram mole of the solute dissolved in solvent that the final volume of the solution is 1
liter. A millimolar (mM) solution contains 1/1000 of a mole of solute dissolved in solvent so that the final volume of
the solution is 1 liter. For example 1 M NaOH solution contains 40g of NaOH per 1000 ml of the solution, while 0.1M
NaOH solution contains 4g of NaOH per 1000ml of the solution.
The volumes are not necessarily additive. The only volume of importance is the final solution's volume. You add
enough water to get to that volume without caring how much the actual volume is.
EXAMPLES
• Suppose we had 1.00 mole of sucrose (it's about 342.3 grams) and proceeded to mix it into some water. It
would dissolve and make sugar water. We keep adding water, dissolving and stirring until all the solid was
gone. We then made sure that when everything was well-mixed, there was exactly 1.00 liter of solution.
What would be the molarity of this solution?
The answer is 1.00 mol/L. Notice that both the units of mol and L remain. Neither cancels. Replace the M
with mol/L when you do calculations. The M is just shorthand for mol/L.
• A molar solution of sodium chloride is made by placing 1 mole of a solute into a 1-liter volumetric flask.
(Taking data from the example above we will use 58 grams of sodium chloride). Water is then added to the
volumetric flask up to the one liter line. The result is a one molar solution of sodium chloride.
MOLAL SOLUTION
It is the number of moles of solute dissolved in 1000g or 1 kg of the solvent.
A 1m molal solution contains 1 gram mole of solute dissolved in 1000g of solvent.
EXAMPLES:
• Suppose we had 01 mole of sucrose (it's about 342.3 grams) and proceeded to mix it into exactly 1.00 liter
water. It would dissolve and make sugar water. We keep adding water, dissolving and stirring until all the
solid was gone. We then made sure everything was well-mixed.
What would be the molality of this solution? Notice that one liter of water weighs 1000 grams (density of
water = 1.00 g / mL and 1000 mL of water in a liter). 1000 g is 1.00 kg, so:
The answer is 1.00 mol/kg. Notice that both the units of mol and kg remain. Neither cancels.
replace the m with mol/kg when you do calculations. The m is just shorthand for mol/kg.
Note that the solvent must be weighed unless it is water. One liter of water has a specific gravity of 1.0 and
weighs one kilogram; so one can measure out one liter of water and add the solute to it. Most other solvents
have a specific gravity greater than or less than one. Therefore, one liter of anything other than water is not
likely to occupy a liter of space.
• To make a one molal aqueous (water) solution of sodium chloride (NaCl) , measure out one kilogram of water
and add one mole of the solute, NaCl to it. The atomic weight of sodium is 23 and the atomic weight of
chlorine is 35. Therefore the formula weight for NaCl is 58, and 58 grams of NaCl dissolved in 1kg water
would result in a 1 molal solution of NaCl.
NORMAL SOLUTION
It is the number of gram equivalents of solute dissolved per liter of the solution.
A 1N normal solution contains 1 gram equivalent of solute dissolved in the solvent so that the final volume of the
solution is 1 liter. A mEq is 1/1000 of a gram equivant.
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"Normality" refers to the activity of a reagent: how many moles of active species are there per liter? For acids and
bases, normality refers to how many moles of H+ or OH- there are per liter. Thus, for hydrochloric acid (HCl) and
sodium hydroxide (NaOH) the normality is equal to the molarity. But for diprotic substances like sulfuric acid
(H2SO4) or barium hydroxide Ba(OH)2, the normality is twice the molarity. For a triprotic substance, normality
would be three times the molarity and so forth.

EXAMPLES
• What is the normality of 2.4M H2CO3?
Normality is equal to the number of equivalent weights of solute per Liter of solution.
Molarity is equal to the number of moles of solute per Liter of solution, and to determine the number of
moles, you take the weight of the solutes in solution divided by the weight of one of those solute molecule
(the molecular weight).
How does one relate equivalent weights to moles? The equivalent weight of a compound is equal to its
molecular weight divided by its valence. The valence in this context means the same thing as the number of
substitutable groups (H+s or OH-s). Carbonic acid is a diprotic acid that dissociates to release 2 ionizable H+
s. So along those lines, a 1 M solution of H2CO3 is equivalent to a 2N solution of H2CO3. If 1 molar H2CO3
= 2N H2CO3 , a 2.4 M H2CO3 with 2 ionizable groups x 2.4, or 4.8N.
• What is the normality of the following?
a. 0.1381 M NaOH
b. 0.0521 M H3PO4
The normality of 0.1381 M NaOH = 0.1381/1L * 1 = 0.1381 N, i.e. the normality is same as molarity in this
case.
The valency of H3PO4 is 3, so in this case:
The normality of 0.0521 M H3PO4 = 0.0521/1L * 3 = 0.1563N

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CALCULATIONS
1. To calculate the molarity of “x” grams of a substance in “y” ml of solvent.
Molarity = moles/L
1 mole = 1 MW
Moles of the substance = x/MW
Molarity = x/MW/y in liters
2. How many grams of a substance are required to make “a” ml of “b” M, solution?
Molarity = moles/L
Molarity * L = moles = grams/MW
Grams of substance required = M * L * MW
3. MOLARITY AND WATER MOLECULES
Several substances exist in solid state in the form of crystals. Some of them hold water molecules as integral
part of their crystal lattice. Water of crystallization is water that occurs in crystals but is not covalently bonded
to a host molecule or ion. When we prepare their molar solution, we should also include the molecular weight
of water molecules.
• For example:
How much CuSO4.5H2O will be required to prepare 1000ml of 0.5M solution? (MW of the compound is
250).
Answer is:
1 mole of CuSO4.5H2O = 250g
1M solution of CuSO4.5H2O = 250g/L
0.5 M CuSO4.5H2O = 250/2 =125g/L
So 125g of CuSO4.5H2O will be required to prepare 0.5M solution.

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DILUTION OF SOLUTIONS
To dilute a solution means to add more solvent without the addition of more solute. Of course, the resulting solution is
thoroughly mixed so as to ensure that all parts of the solution are identical. The fact that the solute amount stays
constant allows us to develop calculation techniques. First, we write:
moles before dilution = moles after dilution
From the definition of molarity, we know that the moles of solute equals the molarity times the volume.So we can
substitute MV (molarity times volume) into the above equation, like this:
M1V1 = M2V2
The "sub one" refers to the situation before dilution and the "sub two" refers to after dilution. This equation is the
dilution equation.
EXAMPLE:
• 53.4 mL of a 1.50 M solution of NaCl is on hand, but you need some 0.800 M solution. How many
mL of 0.800 M can you make?
Using the dilution equation, we write:
(1.50 mol/L) (53.4 mL) = (0.800 mol/L) (x)
Solving the equation gives an answer of 100. mL.

CONVERSIONS
CONVERSION OF mg/dl TO mmol/l:
• Convert dl to ml and then L
• Determine the molar quantity by:
• Given mass/molecular mass = moles
• So mmol/L = mg /100ml * 10 / molecular mass

CONVERSION OF mg/dl INTO mEq/L:

• mEq/L = mg/dL * 10 / equivalent weight

CONVERSION OF mmol/L INTO mEq/L:

• mEq / L = mmol/L divided by valency
• mmol/ = mEq/L * valency

19
EXPERIMENT # 1
How much CaCl2 is required to prepare 100ml of 0.1 M solution?(CaCl2, mol. wt = 111g)

TO CALCULATE MASS OF THE CHEMICAL REQUIRED

Molarity = moles/L
Moles required = molarity * volume in L
Moles required = grams of substance required/MW
Therefore:
Grams of substance required/MW = molarity * volume in L
Grams of substance required = molarity * volume in L * MW
Grams of CaCl2 required = molarity * volume in L * MW
= 0.1 * 0.1 * 111
= 1.11g
STEPS
1. To make 100ml of 0.1 M solution, first calculate the weight of chemical needed. (1.11g of CaCl2 in this
case).
2. Weigh out the quantity of the chemical, using a balance.
3. Transfer the weighed chemical to a clean, dry volumetric flask.
4. Slowly add distilled water to the flask.
5. Keep adding the water until you reach the 100ml mark on the neck of the flask.
6. Place the stopper on the flask and gently swirl the flask until all the chemical is dissolved.
If you don’t have a volumetric flask, you can use a graduated cylinder instead.
Just add the chemical to the graduated cylinder and then add distilled water until you reach the 100ml mark.

20
EXPERIMENT # 2
Preparation of 150ml of 0.09M HCl solution from available HCl solution (Specific gravity = 1.2; purity = 37%)
Some substances are available in liquid form e.g., HCl, H2SO4 etc.
To calculate the mass of the substance in a specific volume of the available liquid, we have to consider:
The specific gravity of that liquid
Purity of that liquid
Preparation of 150ml of 0.09M HCl solution from available HCl solution (Specific gravity = 1.2; purity = 37%)
CALCULATIONS:
Purity of 37% means that each 100ml of available HCl contains 37ml of pure HCl and rest is impurities.
Specific gravity of available HCl = 1.2 means that each ml of HCl contains 1.2g HCl.
So each gram of available HCl solution contains 37% pure HCl
The concentration of pure HCl in the available solution = 37 / 100 * 1.2 = 0.444g / ml
1ml of available HCl solution contains HCl = 0.444g
1000ml -------------------------------------- = 0.444 * 1000 = 444g
Molecular weight of HCl = 36.5
Number of moles of HCl in 444g = 444 / 36.5 = 12.16 moles
So molarity of available HCl solution = 12.16moles/L = 12.16 M
Dilution equation: M1V1 = M2V2
M1 = 12.16 moles/L M2 = 0.09 moles/L
V1 = unknown V2 = 150ml
V1 = M2V2/M1
= 0.09 x 150 / 12.16 = 1.12ml
Therefore 1.12ml of the available HCl is required to prepare 150ml of 0.09M HCl.

21
 Experiment No.5

CARBOHYDRATES

Carbohydrates are aldehyde or ketone derivatives of polyhydric alcohols. These are widely distributed in
animals and plants where they fulfill both structural and metabolic growth. They are also called
saccharrides.
Classification
On basis of structure, Carbohydrates are classified as:
• Monosaccharides.
• Disaccharides.
• Oligosaccharides.
• Polysaccharides.
• Derived carbohydrates.
Monosaccharides: These are simple sugars which cannot be further hydrolyzed into simpler compounds.
These may be divided into trioses, tetroses, pentoses and hexoses depending on the number of carbon
atoms and as aldoses or ketoses depending on whether aldehyde or ketone groups are present.
Example Aldoses Ketoses
Trioses Glyceraldehydes dihydroxyacetone
Tetroses Erythrose Erythrulose
Pentoses Ribose Ribulose
Hexoses Glucose Fructose

Disaccharides: These yield two molecules of monosaccharide units which may be same or different when
hydrolyzed.
Maltose → Glucose + Glucose
Lactose → Glucose + Galactose
Sucrose → Glucose + Fructose
Oligosaccharides: These yield 2 –10 monosaccharide units on hydrolysis e.g. maltotriose yields three
molecules of glucose.
Polysaccharides: These yield more than ten molecules of monosaccharide residues on hydrolysis.
These are further subdivided into:
• Homopolysaccharides
• Heteropolysaccharides
Homopolysaccharides: These have same monosaccharide units
e.g. starch, glycogen, dextrin, etc.
Heteropolysaccharides: These have different units of monosaccharides e.g. mucopolysaccharides,
mucilages and hemicellulose.
Derived carbohydrates: These are derived from carbohydrates by various chemical reactions.
1. Sugar acids are formed by oxidation of sugars:

22
 a. Oxidation of aldehyde group of sugars by mild oxidizing agents like bromine water, oxidizes –
1
CHO → -COOH & form sugar acids; e.g., glucose → gluconic acid is formed.
b. Oxidation of C # 1 & 6 of sugars by strong oxidizing agents like HNO3, oxidizes –CHO & -
CH2OH → both to –COOH, e.g.,
glucose → glucaric acid is formed.
c. When last carbon atom of glucose is oxidize by an enzyme but
–CHO group is protected from oxidation, -6CH2OH → –COOH, e.g., glucose → glucoronic
acid is formed.

2. Reduction product e.g. glycerol and ribitol are derived from glyceraldehydes and ribose respectively.
3. Amino sugars have -NH2 group at carbon No. 2 e.g. glucosamine and galactosamine derived from
glucose and galactose respectively.
4. Deoxy-sugars have less number of oxygen atoms than other sugars e.g. deoxyribose present in DNA.

Properties of Carbohydrates:
Physical Properties:
• These are soluble in water.
• Mono and disaccharides are soluble in cold water whereas polysaccharides in hot water.
• These are mostly white in colour and sweet in taste.
Chemical Properties:
These are due to the presence of hydroxyl group and carbonyl group.
• Properties due to hydroxyl group:
- Glycoside formation.
- Ester formation.
- Dehydration.
• Properties due to carbonyl group:
- Reducing properties.
- Oxidation properties.
- Osazone formation.
- Effect of alkalies

23
 Experiment No.6

To study the dehydration property of carbohydrates by performing Molisch’s test.

Reagents:
1. Conc. H2SO4
2. 5 % Alcoholic α –naphthol (indicator)
3. Carbohydrate solution
Principle:
When carbohydrates are treated with non-oxidizing agent like H2SO4, dehydration of carbohydrates take
place and a colorless hydroxy methyl furfural compound is formed which is very reactive and condensed
with an indicator to give violet colored ring. Pentoses give furfural compounds.
H –C═O
|
H – C – OH HC4 3
CH
|
HO – C – H +Con H2SO4
| -3 H2O O
H – C – OH HOH2C6 C5 2
C 1
C H
| O
H – C – OH Hydroxymethyl furfural compound
|
CH2OH
D-Glucose

Procedure:
Take 3ml of carbohydrate solution in a test tube. Add 3-4 drops of 5% alcoholic alpha naphthol and mix it
thoroughly. Take 3 ml of concentrated H2SO4 in another test tube. Pour slowly and gently the acid by
incling the test tube into the test tube containing solution. For a few seconds roll this test tube in your
palms.
A violet colored ring is formed at the junction of two solutions. The same procedure will be applied with
the rest of the solutions. If green colour appears, it will be due to the presence of impurities in sulfuric acid
solutions.
Discussion:
When carbohydrates like glucose, galactose, fructose, maltose, lactose, sucrose, starch and dextrin are
treated with non-oxidizing agents like H2SO4, dehydration of carbohydrates takes place and a colorless
hydroxy- methyl furfural compound is formed which condenses with an indicator (alcoholic α–naphthol) to
give violet coloured ring at the junction of two solutions.

24
Conclusion:
It is a general test for all the carbohydrates; it is used to identify carbohydrates in an unknown solution.
Clinical Importance of Molisch’s Test:
Molisch’s test forms the basis of clinical test to diagnose impaired absorption of carbohydrates from upper
small intestine. The test is carried out by determining the total xylose excreted in five hours followed by an
oral dose of 5 grams of the xylose. Xylose is selected in this test because it is not metabolized to any extent
in the body and secondly there is no mechanism for its absorption in the kidney. During this period normal
persons excrete 23% - 50% of the dose where as those with steatorrhea due to malabsorption excrete less
than 20%.

25
Perform Molisch’s test with the following sugar solutions:
A - Glucose E - Lactose
B - Galactose F - Sucrose
C - Fructose G - Starch
D - Maltose H - Dextrin

VIVA QUESTIONS

1. Definition of carbohydrates.
2. Classification of carbohydrates.
3. Define aldoses classify them according to number of carbon atoms.
4. Define ketoses classify them according to number of carbon atoms.
5. Properties of carbohydrates due to hydroxyl group?
6. Properties of carbohydrates due to carbonyl group?
7. What is the principle of Molisch’s test? Name the indicator.
8. Molisch’s test is based on which property of carbohydrates?

26
 Experiment No.7

CARBOHYDRATES

Glucose: Glucose is widely distributed in nature as such or in combination with other sugars. In fruits it
is present in combination with fructose. Commercially it is prepared by hydrolysis of starch, which is a
polymer of -D-glucose.
Sources: Fruit juices, cane sugar, maltose and lactose.
Properties:
• It is a dextrose sugar (because of dextrorotation).
• It is monosaccharide.
• It is an aldohexose as it has 6 carbon atoms.
• It is a reducing sugar.
• It forms osazone with phenylhydrazine hydrochloride.
• It is the sugar present in blood.
• It is utilized by the cells.
Straight chain formula of glucose:

Clinical Significance: Normally it is not present in the urine but sometimes it appears in the urine of
poorly controlled patients of Diabetes Mellitus. Presence of glucose in urine is called glycosuria & it
results from hyperglycemia.
Fructose: It is also called fruit sugar. It is the sweetest of all sugars.
Sources: Fruit juices, honey, hydrolysis of cane sugar, and inulin which is a polymer of D-fructose.
Properties:
• It is called levulose because it s levorotatory.
• Fructose is a ketohexose, as it has 6 carbon atoms.
• It is a reducing sugar and forms osazone with phenylhydrazine hydrochloride.
Straight chain formula of D- Fructose
CH2OH

27
 C O

HO C H

H C OH

H C OH

CH2OH
D-fructose
Clinical Significance
1. Hereditary fructose intolerance: It is an autosomal recessive inborn error of fructose metabolism. The
enzyme deficient is aldolase-B enzyme; hence fructose-1-PO4 cannot be metabolized. Accumulation of
fructose-1-PO4 inhibits glycogen phosphorylase. This leads to accumulation of glycogen in the liver and is
associated with hypoglycemia.
2. Fructosuria: This is a benign metabolic defect due to deficiency of fructokinase enzyme. There is no
abnormality other than the excretion of fructose in urine.
Galactose:
Sources: Hydrolysis of lactose (milk sugar), legumes.
Properties:
• It is an aldosehexose sugar.
• It is a reducing sugar and forms osazones with phenylhydrazine hydrochloride.
• Galactose and mannose are epimers of glucose.
• It forms mucic acid crystals on oxidation with HNO3.
• It is synthesized in mammary gland for the synthesis of lactose in milk in lactating mothers.
• It is a constituent of galactolipids and glycoproteins.

Straight chain formula of D- Galactose

H C O

H C OH

HO C H

HO C H

H C OH

CH2OH

28
 D-galactose

Clinical Significance:
Hereditary Galactosemia: It is a serious inborn error of metabolism resulting from the deficiency of
enzyme galactose-1- phosphate uridyl transferase. When this enzyme is deficient / absent, gal-1-PO4 will
accumulate in liver, RBCs, spleen, kidney, heart and cerebral cortex. It will inhibit galactokinase as well
as glycogen phosphrylase enzymes, resulting in hypoglycemia. Free galactose accumulates, leading to
galactosemia. It is partly excreted in urine, causing galactosuria and part of it is reduced to galacitol or
dulcitol. The accumulation of dulcitol in lens results in cataract.
Tests based on Dehydration property of carbohydrates
• Molisch’s Test
• Rapid Furfural test.
• Seliwanoff’s test
Rapid Furfural Test: (Specific for keto-sugars, i.e., fructose and sucrose)
Reagent: 5 % alcoholic α-naphthol (indicator)
Principle: When carbohydrates are treated with conc. HCl, dehydration of keto-sugars (fructose and
sucrose) takes place and a colorless hydroxy methyl furfural compound is formed, which condenses with
an indicator to give deep purple color.
Procedure: Take 1 ml diluted sugar solution (1:10) + 4 drops of alcoholic α-naphthol (indicator); + 5 ml
conc. HCl and boil. Boiling can be done on naked flame for 30 seconds or in a boiling water-bath for two
minutes.
Precautions: Avoid spurting of contents of test tube, while heating, by removing the test tube for a few
seconds while boiling.

Observation:
• Purple color appears within 30 seconds after boiling on flame or after two minutes in a boiling
water-bath ⎯⎯ fructose is present.
• No purple color within 30 seconds after it starts boiling on naked flame or two minutes in boiling
water-bath ⎯⎯ glucose is present.
Conclusion: This test is specific for keto-sugars, i.e., fructose and sucrose.

29
 Seliwanoff’s test:
Specific test for keto-sugars (fructose and sucrose)
Seliwanoff’s reagent: Conc. HCl, and resorcinol (indicator)
Principle: When carbohydrates are treated with conc. HCl acid, dehydration of keto-sugars (fructose and
sucrose) takes place and a colorless hydroxy methyl furfural compound is formed which condenses with
resorcinol indicator to give cherry red color.
Procedure: Take 5 ml of Seliwanoff’s reagent, add 0.5 ml of sugar solution and boil. Boiling can be done
on naked flame for 30 seconds or in a boiling water-bath for two minutes.
Precaution: On direct flame, avoid spurting of contents of test tube, by removing the test tube for a few
seconds intermittently while boiling.
Observation:
• Cherry red color appears within 30 seconds after it starts boiling on naked flame or within 2
minutes in boiling water-bath ⎯⎯ fructose is present.
• No red color seen within 30 seconds after it starts boiling on naked flame or two minutes in boiling
water-bath ⎯⎯ glucose is present.
Conclusion: It is a specific test for keto-sugars i.e. fructose and sucrose. Keto-sugars are more reactive so
they give a positive test within 30 seconds of boiling.

Viva questions:
1. What is the principle of Seliwanoff’s test?
2. Name the tests based on dehydration properties of carbohydrates.
3. What are the chemical properties of glucose.
4. What are the chemical properties of fructose.
5. Write down the straight chain formula of glucose and fructose.
6. Define epimers?
7. Define enantiomers?

30
 Experiment No.8

BENEDICTS QUALITATIVE TEST

REDUCING SUGARS:
Reducing sugars are those which have free functional aldehyde or ketone group. They can readily reduce oxidizing
ions such as silver mercury copper bismuth in alkaline medium. They include:
Monosaccharides:
Glucose
Galactose
Fructose
Disaccharides
Lactose
Maltose
Non-reducing sugars include:
Sucrosez(it has to be hydrolysed first)
Starch and dextrin (they have no functional aldehyde or ketone group)
TESTS BASED UPON REDUCING PROPERTY:
1. Benedicts qualitative and quantitative test - It contains copper ions in weak alkaline medium
2. Barfoeds test - It contains copper ions in acidic medium
3. Fehlings test - It contains copper ions in a strong alkaline medium
4. Nylanders test - It contains bismuth ions in alkaline medium
5. Silver mirror test
6. Osazone test

EXPERIMENT: To study the reducing property of carbohydrates by Benedicts qualitative test

Benedicts solution is commonly employed for the qualitative determination of reducing sugars especially glucose in
urine of diabetic patients. Alkaline solution of CuSO4 provides cupric ions to be reduced.
Composition of Benedict’s qualitative reagent
1. Copper sulphate: It provides copper ions to be reduced
2. Sodium carbonate: it provides weak alkaline medium
3. Sodium citrate: This is called Rochelle salt of the reagent. It prevents precipitation of cupric carbonate by forming
soluble slightly dissociable complexes which dissociates sufficiently to provide supply of readily available cuprous
ions for oxidation.
4. Distilled water
Principle:
When carbohydrates are treated with benedicts reagent, the reducing sugars reduce copper sulphate into cupric oxide
and then into cuprous oxide in hot alkaline medium. Red color precipitates of cuprous oxide are formed .Amount of
precipitates depends upon the concentration of reducing sugar in a solution.
Chemical reaction:

31
CuSO4 +Na2CO3 → Na2 SO4 + CuCO3
CuCO3 → CuO+ CO2
2CuO+R-CHO →R-COOH+Cu2O
Procedure:
Take 5ml of benedicts qualitative reagent in a clean and dry test tube. Now add 8 drops of original carbohydrate
solution. Mix it and then boil it in a water bath for 2 to 3 minutes. Now allow it to cool at rooms temperature .Red
precipitates appear showing positive test for reducing sugar. The color of solution varies from bluish green, yellow
orange and red depending on concentration of sugar present in the solution.

CONCLUSION:
This test is specific test to detect presence of reducing sugars as well as to estimate the approximate concentration of
the sugar present in a given solution or in urine.
Characteristics of Benedicts Qualitative Reagent:
1. Simple test and easy to perform.
2. Very sensitive to detect sugars even in minute quantity.
3. Specific only for reducing sugars.
4. It is a stable reagent.

VIVA QUESTIONS:
Q1.Define reducing sugars.
Q2.Define reducing property.
Q3.Name the reducing sugars.
Q4. Why is sucrose a non-reducing sugar?
Q5.Name the non-reducing sugars.
Q6.What is the significance of Benedicts test in Diabetes Mellitus?
Q7.What are the components of Benedicts qualitative reagent?
Q8.What is the significance of sodium citrate in benedicts qualitative reagent
Q9.What is the color of precipitates of cuprous oxide?

32
 Experiment No.9

EXPERIMENT: TO FIND THE APPROXIMATE CONCENTRATION OF GLUCOSE IN A URINE

SAMPLE BY BENEDICT’S TEST

The reducing property, reducing sugars, and chemical composition of the Benedict’s Qualitative reagent and
importance of its components has been discussed earlier.
Chemical reaction
CUS04 + Na2CO3 Na2SO4 + CuCO3
CuCO3 CuO +CO2
2CuO + R-CHO R-COOH + Cu2O
(Red ppts)
Procedure:
Take 5 ml of Benedict's qualitative reagent (BQR) in six clean dry test tubes and add 8 drops or 0.5 ml of carbohydrate
solution in each. Mix and boil them in a boiling water bath for 2 - 3 min. Now allow them to cool at room temperature.
Red color precipitates, appear showing a positive test, and solutions in different colors. The color of solution may vary
from bluish green, yellow, brown, orange, brick red, depending upon the concentration of reducing sugar in a solution.
Conclusion:
This is a specific test for reducing sugars so it is applied to detect reducing sugars in a given solution, as well as to
estimate the approximate concentration of sugar in the solution. The test is negative for sucrose due to the absence of
free aldehyde or keto group.

Clinical Significance:
• This test is used to estimate the approximate concentration of glucose in the urine of patients with Diabetes
mellitus.
• Diabetes Mellitus is the commonest cause of Glycosuria.
• Glycosuria is also seen in the patients with a low renal threshold for glucose. ( renal threshold value for
glucose is 180 mg% )
• Glycosuria may occur in pregnancy. (Gestational Diabetes) It is a feature of congenital and acquired disorders
of proximal renal tubular function
CLINI TEST:
This Test is convenient standardized test method for the detection of glucose in urine. The tablet which is used in this
test contains anhydrous cupric sulfate, sodium hydroxide, citric acid and sodium bicarbonate. 5 drops of urine is
mixed well with 10 drops of water in a test tube. One tablet is added and the mixture is allowed to stand undisturbed.
Change in color is observed, and matched with the chart provided by the manufacturer for the interpretation of results.
CLINISTIX TEST:
Glucose is the commonest and most important reducing sugar (abnormally) present in urine. It can be detected by
Clinistix reagent strips. This is an enzyme test. The strip is impregnated with glucose oxidase, peroxidase and
Orthotoluidin. Glucose in the presence of glucose oxidase is converted to gluconic acid and hydrogen peroxide

33
(H2O2).H2O2 in the presence of peroxidase converts orthotoluidin to its blue color oxidized form. The formation of
blue color takes place in less than one minute, after it is dipped in fleshly provided urine.

Approximate concentration of Glucose in urine

S.No Color of solution gm% of glucose in solution Clinical results

1. Unchanged blue Nil --
2. Bluish green with red ppt Traces +
3. Green with red ppt 0.5 +
4. Yellow with red ppts 1.0 ++
5. Orange with red ppts 2.0 +++
6. Brick red with red ppts 2.5 ++++

VIVA QUESTIONS
1. What is the composition of Benedict's qualitative reagent?
2. Define Glycosuria?
3. What are the pathological conditions other than Diabetes mellitus which can show positive Benedict’s
test?
4. What is the normal fasting & random blood glucose level?
5. What is the normal urine glucose level?

34
 Experiment No.10

EXPERIMENT: TO STUDY THE REDUCING PROPERTY OF CARBOHYDRATES BY BARFOED’S

TEST

Reagent : Barfoed’s reagent has two components;

1 Cu-acetate: To provide “ Cu++ ” ions to be reduced by monosaccharides.
2 CH3COOH: Provides acidic medium.

Principle
When Carbohydrates are treated with Barfoed’s reagent, “Cu++” are reduced to Cu2O in acidic medium by
monosaccharides by boiling it only for 30 sec, red ppts appear. Color of the solution remains blue. If boiling is
prolonged more than 30 sec; disaccharides also show positive results because they are hydrolyzed into
monosaccharide units in acidic medium. Since acidic medium is unfavorable for reduction, only strong reducing
carbohydrates i-e, monosaccharides react very fast and give a positive result only in 30 seconds and cool it for 15
minutes or cool it under tap water, if red ppts of Cu2O do not appear, put the test tube again in the boiling water bath
for 15 minutes. Note the appearance of ppts.

Chemical reaction
COOCH3

Cu +2H2O Cu(OH)2+2CH3COOH
COOCH3 (cupric hydroxide)

Cu(OH)2 CuO+ H2O

O (Cupric oxide)

2CuO +R - C H Cu2O + RCOOH

(Cuprous oxide) (Sugar acid)
Red PPts

Procedure
35
Take 5 ml of Barfoed’s solution in a test tube, add 0.5 ml of sugar solution, mix & boil it for 30 seconds, allow the
tube to cool for about 15 minutes and examine. Reduction is indicated by the formation of red precipitate of cuprous
oxide, with monosaccharide sugar solutions. If the ppt does not appear after boiling for 30 sec and cooling for 15
minutes, this means sugar solutions is of disaccharide.

Significance
This test is basically meant for the detection of monosaccharide in acidic medium but this can also be used for
differentiating between monosaccharide and disaccharides by controlling time of heating. It can not be used for the
detection of glucose in urine, because of the presence of chlorides in urine, as chloride ions interfere with this test.
This test differs from Benedict’s test in that the reduction of cupric ions is carried out in mildly acidic medium.
Conclusion
This test is specific for monosaccharide if boiled for 30 seconds. It differentiates monosaccharide from disaccharides
in an unknown solution.

Viva questions
1. What is the composition of Barfoed’s reagent?
2. Describe principle of Barfoed’s test?
3. What is the color of precipitate and solution at the end?
4. What is the significance of Barfoed’s test?
5. Can this test be used for the detection of glucose in urine sample If not, why?
6. What is the difference between Barfoed’s test and Benedict’s test?
7. Why monosaccharides give positive results earlier than disaccharides?
8. What are the factors on which Barfoed’s test depends?
9. Can this test be used to distinguish between Galactose and Lactose?

36
 Experiment No.11

TO STUDY THE PROPERTY OF OSAZONE FORMATION OF MONOSACCHARIDES

Osazones which are actually phenylosazones are yellowish, crystalline compounds and are produced as a result of
heating sugar solutions with phenylhydrazine. These crystals have characteristic structural arrangement, melting point
and specific time of formation Osazones are valuable in identification of reducing sugars in an unknown solution.
Reagents
1. Phenylhydrazine hydrochloride.
2. Anhydrous sodium acetate.
3. Glacial acetic acid.

Principle
When reducing sugar are treated with phenylhydrazine hydrochloride, greenish yellow crystalline derivatives called
osazones are formed. These crystals are formed only by those sugars which contain a potential aldehyde or ketogroup
Each of the reducing sugars give rise to osazone which is typical for that sugar and in a specific time.

Chemical reaction
This reaction occurs best at pH 5 and to bring this pH near 5 Sodium acetate & glacial acetic acid are added. In this
reaction only C No.1 & C No.2 of glucose & glactose are involved. Other sugars react in the same way to form
osazone. Osazone of fructose, glucose & mannose form identical osazones termed as glucosazone because these three
sugars differ from each other at C.No1&2 and after reaction with phenylhydrazine at C.No1&2 this difference is no
more present and rest of the structure is also same. Sucrose however lacks potential aldehyde or ketone group, and
does not form osazone. It can form osazones only after hydrolysis.

Glucose + 3(C6H5NHNH2) → glucosazone+ C6H5NH2+NH3+2H2O

(Phenylhydrazine) (aniline)
Procedure
Take 3 clean & dry test tubes Mark them ½ inch from the bottom. Add a powdered mixture of phenylhydrazine HCl &
anhydrous sodium acetate upto the mark in each test tube. Now add 5ml of carbohydrate solution and 10 drops of
glacial acetic acid in each test tube. Mix them thoroughly and place all the test tubes in boiling water bath. Greenish
yellow crystals are formed with in specific time for each sugar. Note the time of formation of crystals. Osazones of
monosaccharides are formed in hot solution. Then put a drop of each solution formed on a glass slide. Cover it with
coverslip & examine each slide under microscope.
Note the arrangement of crystals formed.

Osazone Colour Time Shape Arrangement of crystals

37
Glucosazone Greenish 5 minutes Thin needles Broom or tree like
yellow

galactosazone Greenish 20 minutes Thick needles Mostly scattered

yellow

Fructosazone Greenish 2 minutes Thin needles Broom or tree like

yellow

Conclusion

This is a specific test for reducing sugars which contain potential aldehyde or ketone group e.g glucose, galactose,
fructose, maltose & lactose.

Viva questions
1. Enlist the crystal arrangements of the oszaones of 3 monosaccharides.
2. Why do glucose & fructose give same osazones?
3. Why do glucose & fructose form osazone crystal earlier then maltose and lactose?
4. What is the principle of osazone test?
5. Name the reagents used in osazone test?

38
 Experiment No.12

EXPERIMENT: TO STUDY THE OROPERTY OF OSAZONE FORMATION OF DISASACCHARIDES

Osazones which are actually phenylosazones are yellowish, crystalline compounds and are produced as a result of
heating sugar solutions with phenylhydrazine. These crystals have characteristic structural arrangement and specific
time of formation Osazones are valuable in identification of reducing sugars in an unknown solution.
Reagents:
1. Phenylhydrazine hydrochloride. 2. 2. Anhydrous sodium
acetate.
3. Glacial acetic acid.
Principle:
When reducing sugars are heated with phenylhydrazine hydrochloride at a constant pH of 4.5 and a constant
temperature of 100 C, greenish yellow crystalline derivatives called osazones are formed. This reaction occurs best at
pH 5 and to bring this pH Sodium acetate & glacial acetic acid (buffer pair) are added.
These crystals are formed only by those sugars which contain a potential aldehyde or ketogroup. Sucrose lacks
potential aldehyde or ketone group, and thus does not form osazone. It can form osazones only after hydrolysis.
Each of the reducing sugars give rise to osazone which is typical for that sugar and in a specific time. The
disaccharides take longer than monosaccharides to form osazones because they are weaker reducing sugars. The
osazones formed by disaccharides are soluble in hot water and thus the solution needs to be cooled to observe the
crystals formed.

Chemical reaction:

Lactose+3(C6H5NHNH2)→ lactosazone+ C6H5NH2+NH3+2H2O

(Phenylhydrazine) (aniline)

Maltose +3(C6H5NHNH2)→Maltosazone+ C6H5NH2+NH3+2H2O

(aniline)
Procedure:
Take 2 clean dry test tubes. Mark them ½ inch from the bottom. Add a powdered mixture of phenylhydrazine HCl &
anhydrous sodium acetate (oszaone mixture= two parts anhydrous sodium acetate + one part phenylhydrazine) upto
the mark. Now add 5ml of carbohydrate solution and 10 drops of glacial acetic acid in each test tube. Mix them
thoroughly and place all the test tubes in boiling water bath. Heat the solutions for 35 and 45 minutes respectively and
then let the solutions cool down & observe for the formation of greenish yellow crystals. Then observe these crystals
under the microscope after putting a drop of each solution on the glass slide. Note the arrangement of crystals formed.

Osazone Colour Time Shape Arrangement of crystals

39
 Lactosazone Greenish 35 minutes Thin needles Hedge hog or fluffy ball
yellow

Maltosazone Greenish 45 minutes Thick needles Rosette shaped or sunflower

yellow shaped

Precautions
1. The solutions should be cooled down to room temperature.
2. Waterbath should be boiling before the test tubes are put in it and should be kept boiling throughout the procedure.
Conclusion:
This is a specific test for reducing sugars which contain potential aldehyde or ketone group

Viva questions:
1. Enlist the crystal arrangements of the two disaccharides
2. Why do maltose and lactose form osazones later than fructose and glucose?
3. Why do we need to cool down the solutions to observe the crystals?

40
 Experiment No.13

EXPERIMENT: TO STUDY THE GENERAL PROPERTIES OF POLYSACCHARIDES i.e STARCH AND

DESTRIN

Polysaccharides:
They are the condensation products of more than ten monosaccharide units.
Types:
1. Homopolysaccharides: On hydrolysis they yield only one type of monosaccharide
units.These include starch, glycogen, cellulose, dextrin.
2. Hetropolysaccharides: On hydrolysis they yield different types of monosaccharides
and other substances.
General Properties.
1. Either insoluble in cold water or when soluble they form colloidal solution.
2. They are fermented by yeast.
3. They can be hydrolysed by acid or enzymes.
4. Usually have no reducing property. However some low molecular weight dextrins have reducing property
due to potentially free carbonyl group.
1. STARCH:
Compositions: Homopolymer of glucose.
Sources: It is the most important dietary source of carbohydrate in cereals, potatoes, legumes, and other vegetables in
the form of starch grains.
Each starch grain consists of two parts.
1. Amylose: It forms inner portion of grain, it’s soluble in cold water and gives deep blue colour with Iodine.
Consists of about 300 units of glucose arranged in an unbranched chain coiled like a left handed helix. There is a turn
after every 6 glucose units. Its mol.wt. is about 50000 and the glucose units are joined by α1-4 glycosidic linkage.
2. Amylopectin: It forms outer portion of starch grain, is insoluble in cold water but in hot water it just swells up
without dissolving. It consists of many branched chains of glucose, each having about 25 glucose units joined byα1-4
glycosidic linkage and α1-6 glycosidic linkage. Its molecular weight is 20 times that of amylase. It gives violet
colour with Iodine
2.DEXTRINS :
Dextrins are formed as an intermediate products during hydrolysis of starch by acid or enzyme. Not
fermentable by yeast and readily soluble in cold water. Various dextrins give different colored solutions with iodine
depending on their molecular complexity. The high molecular weight dextrins give violet color. Intermediate dextrins
give red color and low molecular weight give no color at all. Dextrins have a slight reducing property.
Physical properties
Colour: Starch: Milky white
Dextrin: White
Solubility: Starch: soluble in hot water
Dextrin: soluble in cold water
41
Chemical properties:
Molisch’s test: Starch: Positive
Dextrin: Positive
Benedict’s test: Starch: Negative
Dextrin: Positive
Iodine test: 2 ml of iodine solution and few drops of original solution
Starch: deep blue
Dextrin: red colour
a) Effect of heat on I2 test:
Perform I2 test then heat it colour disappear then cool it colour reappears. The mechanism by which iodine
gives colour with starch & dextrin is known as “adsorption’’ which is a physical phenomena. When we heat the
solution colour disappear because kinetic energy of molecules breaks the I2-film. On cooling this film is again formed
& colour reappears.
b) Effect of alkali & acid on I2 test
Perform iodine test then add 1—2 drop of NaOH colour disappear, then add 1—2 drop of HCl colour reappears.
Explanation: when NaOH is added it converts free iodine into Na iodide & Na Iodate. So no free iodine is available to
form film and colour disappears.
On adding HCl these iodine salts are broken down. Iodine is again free to form film so colour reappears.
I 3 I2 + 6 NaOH → 5 NaI + NaIO3 + 3H2O
II 5 NaI + NaIO3 + 6HCl → 3I2 + 6 NaCl+ 3H2O
4.Half saturation with (NH4)2So4 test:
Procedure: 5 ml of original solution + 5ml saturated solution of ammonium sulphate mix thoroughly, wait for 5—7
minutes then filter it & perform iodine test on the filtrate.
Explanation: Starch is precipitated by half saturation therefore it gives negative iodine test on filtrate which shows that
half saturation with ammonium sulphate test is positive.
Dextrin is not precipitated by half saturation with ammonium sulphate due to low molecular size so it gives colour
with iodine so half saturation with ammonium sulphate is negative.
5.Full saturation with ammonium sulphate test:
Procedure
5 ml original solution + dissolve solid ammonium sulphate in it till no more is dissolved. Waite for 5—7 minutes,
filter it and perform iodine test on the filtrate.
Explanation: Same as above.
Starch Dextrin

Half saturation test with (NH4)2 So4 Positive Negative

(I2 test on Filtrate-ve) (I2 - test on filtration+ve)

Full saturation test with (NH4)2 So4 Positive Positive

(I2 - test on Filtration-ve) (I2 - test on filtration-ve)
42
 Experiment No.14

HYDROLYSIS OF SUCROSE BY ACID

PRINCIPLE
Sucrose is a disaccharide composed of glucose and fructose.It is a nonreducing sugar but after hydrolysis by a strong
acid into its component sugars it acquires reducing properties hence a positive benedicts test and osazone formation
occurs.

REAGENTS
1. Conc HCl
2. Solid Na2CO3

PROCEDURE:
1. Take 10ml of sucrose solution.

2. Add five drops of con. HCl

3. Boil for one minute in a water bath

4. Neutralize with adding solid Na2CO3 till effervescence stops.

5. Now perform the following tests on the resulting solution.

a) Benedict’s test
b) Osazone test

CONCLUSION:
Sucrose after hydrolysis acquires reducing properties as glucose and fructose both are reducing sugars.

43
 SCHEME FOR UNKNOWN CARBOHYDRATES

MOLISCH TEST

+VE -ve
+ve
Carbohydrates Non-Carbohydrates
carbohydrates

IODINE TEST

-ve (Mono and disaccharides) +ve ( polysaccharides)

BENEDICTS TEST Starch (blue) Dextrin (red)

Test Starch Dextrin

+ve Colour Milky white White
Reducing sugars Solubility Hot H₂O Cold H₂O
B.Q. Test -ve non – +ve reducing
reducing
Half Positive Negative
Saturation
BARFOED’S TEST
Full Positive Positive
Saturation

+ve -ve -ve

monosaccarides disaccharides (Non-reducing sugars-sucrose)

SUCROSE HYDROLYSIS BY
HCL
SALIWANOFF TEST OSAZONE TEST

BENEDICTS TEST (+VE)

+ve (keto sugars- -ve (aldo sugars- glucose, OSAZONE TEST (+VE)
fructose) galactose) SALIWANOFF TEST (+VE)

OSAZONE TEST

44
 Name: ---------------------------
Roll No/ Batch: ------------------

Date: -------------------

Experiment: To Study The Hydrolysis of Starch by Conc. HCl

Starch is a polymer of glucose. It is readily hydrolyzed by acid and enzyme. The acid used is HCl and the
enzyme is amylase which is of two types i.e; α amylase present in humans and animals, β amylase present in plants. In
humans α amylase is present in salivary and pancreatic secretions.
Principle:
Heating of starch in the presence of concentrated HCl causes its hydrolysis into glucose. Because of free
aldehyde group in glucose it is a strongly reducing monosaccharide hence benedict’s, barfoed’s, and osazone tests
become positive.
Reagents:
• 1% starch paste
• Concentrated HCl
• Iodine Solution
• Benedict’s Quantitative Reagent
Preparation of Starch Paste:
1 gm of starch powder plus 10 ml of cold water. Stir it to get a uniform suspension. Pour this in 75 ml of boiled water
and keep on stirring. Boil to make a uniform mixture. Cool it and add cold water to make the volume 100 ml. This is
1% Starch Paste.
Procedure:
Fill 2/3 of a water bath with water and boil it. Make 2 sets of test tubes, each having 6 test tubes. Add 2 ml of
I2 in each test tube of set # 1. Add 5 ml. of Benedict’s Qualitative Reagent in each test tube of set # 2. Now take 20 ml
of 1% starch paste in a beaker, add 15 drops of concentrated HCl. Mix it well. Now fill 2/3 of a test tube with this
mixture and place test tube in the boiling water bath. Just after placing the test tube in the boiling water bath take out
starch solution with a dropper from the bottom of the test tube and add 2-3 drops in the 1st test tube of set # 1 and 8
drops in 1st test tube of set # 2. The Iodine solution will give blue color. Boil the 1st test tube of set # 2 for 3 minutes.
After an interval of 3-4 minutes again, take out some starch solution from the bottom of the test tube, add 4-5
drops in 2nd test tube of set number 1. If violet color appears then add 8 drops in 2nd test tube of set number 2 and boil
for 3 minutes. Now repeat this at an interval of 3-4 minutes till Iodine solution gives no color and B.Q.T. shows large
amount of red ppts of Cu2O.
Precautions:
1) Take the sample from the bottom of the test tube.
2) Add starch paste to B.Q.T after getting proper colour with I2.

45
 Time in min. Substrate Product I2 Test B.Q.R

00 Starch + HCl Soluble Starch Deep Blue -

Soluble starch + Amylo dextrin + +

3-4 Violet
HCl traces of glucose -

Amylodextrin + Erythrodextrin I +
4-8 Red +
HCl glucose +
Erythrodextrin I + ErythrodextrinII +
8-12 Reddish brown ++
HCl glucose + +
ErythrodextrinII + Achrodextrin +
12-16 Bornwish yellow +++
HCl glucose + + +

Achrodextrin + Resitant Dextrin No color with Full of brick red

16-20
HCl +-glucose+ +++ iodine ppts.

Achromic Point:
This is the point at which I2 gives no colour with starch because the starch is converted to glucose.

Chromic Period:
It is the total time period to reach the achromic Point. It is noted from placing the test tube in boiling water
bath till achromic Point.

Difference Between Starch Hydrolysis by Acid and Enzymes:

Hydrolysis by Acid By Enzymes

• At 1000C • AT 370C
• With Conc. HCl • With amylases.
• End products: Resistant dextrin and • End Products: alpha-limit dextrin,
glucose maltose, isomaltose, maltotriose
• Break α1-4; α 1-6 glycoside bond • Breaks α 1-4 glycosidic linkages
• No specific site of action • Acts on non-reducing end.

46
 Experiment No.15

TO STUDY THE PROPERTIES OF LIPIDS

LIPIDS:
They are a heterogenous group of substances which are very poorly soluble in water. These are soluble in organic
solvents.
Significance:
They are important as energy storing compounds. One gram of fats provides 9 kilocalories of energy. They form an
integral part of cell membrane. They act as carriers for fat soluble vitamins (vitamin A,D,E and K). Subcutaneous fate
gives shape and contour to the body and provide insulation. Lipid hormones perform various biological activities.
Classification:
• Simple lipids

• Compound lipids

• Derived lipids

PHYSICAL PROPERTIES:
1.SOLUBILITY:
They are soluble in organic non-polar solvents like
alcohol, ether, chloroform, acetone etc. They are not soluble in polar solvents like water.
TESTS:
1. Take 2 ml of distilled water. Add a drop of oil. Heat the mixture. Shake well and allow to stand for a while. Oil
remains insoluble and separates from water quickly. This shows that temperature of the solvent has no effect on
solubility of lipids and that the specific gravity of lipids is less than water as oil tends to float on the surface of water.
2. Take 2 ml of ether in a test tube. Add four drops of oil and shake well. Oil gets dissolved.
Now pour this solution on a filter paper. Ether will evaporate rapidly leaving behind a greasy spot on the filter paper.
2.LITMUS PAPER TEST:
Dip the litmus paper strip in the given oil sample. Since lipids are generally neutral in pH so they do not change the
color of litmus paper.
3.RANCIDITY:
When fats are exposed to air, light and high temperature, they undergo partial hydrolysis and oxidation which leads to
loss of oxidation properties and bad odour.
TYPES OF RANCIDITY
1. Oxidative
2. Hydrolytic
TEST:
Smell the fresh oil and then rancid oil. Odour is changed in case of rancid oil.
CHEMICAL PROPERTIES
Emulsification
Fats can be emulsified i-e, broken up into droplets either mechanically by agitation or spontaneously.

47
Water molecules due to their high surface tension have a tendency to come together and form a separate layer. That is
why water and oil emulsion is unstable.
In the presence of substances that lower the surface tension of water e.g, sodium carbonate, soap, bile salts etc, the
tendency of water molecule to coalesce is decreased, and the emulsion becomes stable.

Tests: Take two test tubes.

1. In the first one put 5ml of distilled water. Add 3 drops of oil. Shake the tube. An unstable emulsion is formed.
Oil globules reappear after some time.
2. Put a drop of oil in another test tube. Add 4 drops of oleic acid and 6 drops of 5% Na2CO3.Shake well. Stable
emulsion is formed, which separates after a few minutes.
Clinical application: Bile salts lower the surface tension and thus they help in absorption of fats from intestines.
Saponification.
When fats are boiled with strong alkali, (i-e, NaOH), they are readily hydrolyzed into glycerol and fatty acids. These
fatty acids form salts with alkali metals i-e soaps.
Saponification number: It is the number of milligrams of potassium hydroxide required to neutralize fatty acids
observed from 1 gm of fats.
Tests: Take 3 watch glasses.
1. In the first watch glass, put 5 drops of 0.5% Na2CO3.Add 9 drops of oil. No Saponification occurs and drop
remains clear.
2. In the second watch glass, put 6 drops of 0.5% Na2CO3.Add 2 drops of CaCl2.Add 6 drops of oil and 3 drops
of oleic acid. Emulsion is formed and oil spreads slowly.
3. Put 6 drops of 0.5% Na2CO3.Add 2 drops of CaCl2, 6 drops of oil, 7 drops of oleic acid. Mixture becomes
white and remains undissolved. This shows that soap is formed.
Note: CaCl2 is used to form insoluble soap of calcium.
Iodine absorption test.
This test is used to identify the presence of unsaturated fatty acids. Double bond of unsaturated fatty acids
undergoes halogenations. Degree of halogenation is used as the index of the degree of unsaturation of fatty acids.
The number of grams of iodine absorbed by 100gms of a fat is known as its iodine number.
Fats containing increased number of unsaturation in fatty acids have increased iodine number.
In olive iodine number is 79—88.Cotton seed oil has 103—111.
The saturated fatty acids do not absorb iodine.
Test: Take 1ml of oil in a test tube. Add 5ml of 14%Na2CO3.Add iodine solution dropwise, shaking after each
addition. The color of iodine disappears being absorbed by oil.
Count the number of drops needed to produce the end point i-e yellow color shows iodine number.
TESTS FOR CHOLESTEROL
Cholesterol is a steroid alcohol present in various tissues of the body. It is essential for the production of vit-D1,
steroid hormones, bile acids, and is an important constituent of the cell membrane.
Normal range is 150-250mg/dl. Serum cholesterol levels are increased in pancreatic diseases e,g diabetes mellitus
and liver disorders. This leads to atherosclerosis.
48
 Levels are decreased in:
• Hyperthyroidism
• Severe liver damage.
• Malnutrition.
• Chronic anemia.

Dietary sources: Egg yolk, liver, kidney, brain, milk. butter.

Leiberman-Buchard test.
Dissolve 0.5ml of cholesterol in 2ml of chloroform in dry test tube. Add 10 drops of acetic anhydride and 2 drops
of conc.H2SO4.Shake well. Deep blue color appears due to oxidation of cholesterol by conc. H2SO4.

Salkowski’s test.
Take 2ml of chloroform in a test tube. Add 0.5ml of cholesterol. Shake well. Add 2ml of conc.H2SO4.Different
layers will be observed. Upper layer will be red, then yellow, then ultra fluorescent green color is observed.

VIVA QUESTIONS
1. Is the specific gravity of lipids equal to water?
2. Why lipids are soluble in non polar solvents?
3. What is the pH of lipids?
4. Why water and oil emulsion is stable in the presence of sodium carbonate?
5. What is the role of bile salts in human body?
6. How is soap formed?
7. What is iodine number?
8. What is the clinical si8gnificance of cholesterol?

49
 Experiment No.16

TO STUDY THE PROPERTIES OF DENATURATION AND HEAT COAGULATION OF PROTEINS

PROTEINS:
Proteins are nitrogenous compounds made up of a variable number of amino acids joined together by peptide linkage.
The amino acids possess an amino group –NH 2 and a carboxyl group –COOH. The acid amide bond formed between
two amino acids is called the peptide bond. Peptides containing less than 100 amino acids is called polypeptide and
polypeptides containing more than 100 amino acids (high molecular weight polypeptide) are called protein.
CLASSIFICATION OF PROTEINS:
1. SIMPLE PROTEINS: These are proteins made up of amino acids only. They can be subdivided on the basis of
their solubility and heat coagulation into albumins, globulins, prolamin, protamine, histones and albuminoids.
2. CONJUGATED PROTEINS: These are proteins made up of amino acids and a non protein prosthetic group based
on which they are subdivided into glyco, muco, nucleo, lipo, chromo and metallo proteins.
3. DERIVED PROTEINS: Primary derived proteins are obtained from denaturation of the native protein whereas
secondary derived protein are obtained from hydrolysis of the native protein (primary proteoses, secondary proteoses
and peptones)
INTRODUCTION
DENATURATION:
When a protein is heated its physical, chemical and biological properties are changed due to breaking up of certain
bonds. The proteins become insoluble. This process is known as Denaturation. This can be brought about by many
physical and chemical agents,
X-rays, salts of heavy metals, ultraviolet rays, vigorous shaking and stirring. This process can be reversed by certain
manipulations. It can be detected by Hellers Ring Test.
COAGULATION:
When the coagulable proteins are heated at their isoelectric pH a series of changes occur involving dissociation of
protein subunits (disruption of the quaternary structure) uncoiling of polypeptide chains (disruption of the secondary
and tertiary structure) and matting together of the uncoiled polypeptide chains. This process is called
COAGULATION. And this is an irreversible process. It can be detected by Heat Coagulation test.
PRINCIPLE:
When a certain protein is heated in an acidic medium it becomes denatured. This is a reversible process as the primary
structure is retained. When the coagulable proteins are heated at their isoelectric pH, their coagulation occurs and they
form an insoluble mass. This process of coagulation is irreversible.
A.HELLERS RING TEST
REAGENTS:
1. Conc. Nitric acid
PROCEDURE:
1. Take 3 ml of original solution in a test tube.
2. Add 3ml of conc. nitric acid slowly along the wall of the inclined test tube.
INTERPRETATION:

50
If a white ring is formed at the junction of the two solutions, it indicates denaturation of proteins brought about by the
acid.
B.HEAT COAGULATION TEST
PROCEDURE:
1. Fill 2/3rd of the test tube with the given solution.
2. Hold the test tube near it bottom; incline it slightly and heat the upper portion of the fluid (Do not boil.)
3. Add a few drops of dilute acetic acid after a white precipitate appears and then reheat the solution.
INTERPRETATION
1. If heat coagulable proteins (albumin and globulins) are present, a dense coagulum will be formed in the upper part
of the solution. It can be compared with the lower part of the solution which serves as the control.
2. In case of non-coagulable proteins (gelatin, proteoses, peptones and polypeptides) no coagulum will be formed.
3. If turbidity is due to phosphates it disappears on addition of acetic acid whereas if it is due to proteins, it persists.
PRACTICAL APPLICATIONS OF HEAT COAGULATION TEST:
1. This is the most widely used test to detect the presence of proteins in the urine. The proteins which commonly
appear in the urine are albumin and globulins and since both are heat coagulable their presence in the urine can be
conclusively established by the heat coagulation test.
2. This test is also used to differentiate precipitates in urine due to excessive excretion of phosphates in some
conditions.

51
 Experiment No.17

PRECIPITATION REACTIONS OF PROTEINS

Proteins are large biomolecules which can be identified on the basis of size shape and charge. Most of the proteins
when dissolved in water form colloidal solutions. The stability of a colloidal solution depends on the electrical charges
on the surface of the dissolved particles which prevent their coagulation and precipitation. Hence proteins can be
precipitated by neutralization of the electrical charge which they carry and also by dehydration.
1. PRECIPITATION BY ACIDS OR ALKALOIDAL REAGENT:
In acidic medium proteins become positively charged. In such a medium the negatively charged anions of alkaloid
reagent bind with positively charged protein cations resulting in their precipitation.
TEST : OBSERVATION INFERENCE
3 ml O.S+ 1ml of 3 % TCA White ppt Proteins present
Clinical importance: Its used for estimation of CSF protein
2.PRECIPITATION BY SALTS OF HEAVY METALS:
In alkaline medium the proteins become negatively charged anions .Positively charged heavy metal cations bind with
the negatively proteins resulting in their precipitation. Salts of heavy metals include mercuric chloride, silver
nitrate,CuSO4,lead acetate and zinc sulphate.
TEST OBSERVATION INFERENCE
3ml O.S+2 drops Na2CO3+ White ppt Proteins can be precipitated by metals.
1 drop lead acetate

3. PRECIPITATION BY ORGANIC SOLVENTS:

When organic solvents like alcohol or acetone are added to the protein solution in water, the water molecules are
available for keeping the proteins in solution are decreased.
TEST OBSERVATION INFERENCE
1 ml O.S +absolute alcohol White ppt Proteins are precipitated
dropwise

CLINICAL IMPORATNACE: Fixing of tissues for histological examination utilizes this test.
4. PRECIPITATION BY STRONG MINERAL ACIDS:
Strong acids precipitate proteins by denaturation and coagulation.
TEST OBSERVATION INFERENCE
Hellers test: 3ml White ring at junction of two Concentrated. acid causes denaturation

52
conc.HNO3+3 ml O.S layers
alongside of test tube

5.PRECIPITATION BY CONCENTRATED SALT SOTUIONS:

High concentration of neutral salts like ammonium sulphate remove the water of hydration from the protein molecules
thus reducing their solubility and hence get precipitated. Examples: Ammonium sulphate, sodium sulphate and sodium
chloride. Albumin is not precipitated by half saturation with ammonium sulphate and globulin is precipitated by half
saturation. Both are precipitated by full saturation.

A. TEST :PRECIPITATION BY HALF SATURARTION WITH AMMONIUM SULPHATE:

Take 5ml of protein solution and add 5ml of saturated solution of ammonium sulphate. Mix it well and wait for 5 to 7
minutes. Filter it and add solid NaOH till effervescence stops. Perform biurette test on the filterate. It will be positive
for albumin and negative for globulin.
B. TEST:PRECIPITATION BY FULL SATURATION WITH AMMONIUM SULPHATE:
Take 5ml of protein solution and dissolve solid ammonium sulphate till no more can be dissolved. Wait for 5 to 7
minutes. Filter it and add solid NaOH till effervescence stops. Perform biurette test on the filterate. It is negative for
both albumin and globulin.
Half saturation Biurette test Full saturation Biurette test
Albumin: Negative Positive Positive Negative
Globulin Positive Negative Positive Negative

53
 Experiment No.18

COLOUR REACTIONS OF PROTEINS

Proteins are polypeptides of amino acids joined by peptide bonds. Because of the presence of amino acid residues
proteins react with different chemical reagents to form a characteristic colored complex. These tests are known as
colour reactions of proteins. Most of the color reactions are due to the presence of specific amino acid in the proteins.
Biuret test:
It is a generalized test for all proteins and their derivatives due to the presence of two or more peptide bonds in a
protein molecule. Peptide linkage is formed by condensation of carboxylic group of one amino acid and amino group
of another amino acid
O H

C—N--
Peptide linkage
The name of the test comes from the compound biuret potassium cupric hydroxide formed as the final product of the
reaction.
All substances which contain two carbonyl – CO—NH2 groups joined together either directly or through a single
atom of nitrogen or carbon give this test positive e.g oxamide.

Chemical reaction:
2NaOH + CuSO4 Cu (OH)2 + Na2SO4

Cu(OH)2 + A compound having peptide bond Violet or colored complex.

Reagents:
• Protein solution.
• 10 % NaOH solution.
• 1 % CuSO4 solution.
Procedure:
Take 2 test tubes labeled as A and B.
A: Put 6 ml of 10 % NaOH in test tube A. Add 2 drops of CuSO4 soln. Also add 3 ml of proteins soln. Mix
thoroughly. Proteins give violet color. Pentoses, peptones and egg albumin give rose color.
B: In test tube B add 6 ml of 10 % NaOH in A test tube add 2 drops of CuSO4 soln. add 3 ml of distilled water. Mix
thoroughly. Blue color showing proteins not present.
Precautions:
1. Avoid excess of CuSO4 solution other wise blue color of copper will obscure the violet or rose tinge.

54
2. If original solution contains much of ammonium SO4 it must be treated with some amounts of strong NaOH before
performing Biuret test.
Xanthoproteic test:
In xanthoproteic reaction proteins react with conc. Nitric acid to form metaproteins. This gives white precipitates on
boiling. The aromatic amino acids containing benzene ring, with conc. nitric acid undergo nitration and give yellow
color. In the alkaline medium this yellow nitrobenzene compound is freely ionized to form orange colored complex.
This test is specific for aromatic amino acids excluding phenylalanine. Tyrosine and tryptophan give positive color but
nitration of phenylalanine does not occur under these conditions.
Reagents:
• Conc HNO3
• 40% NaOH solution

Procedure:
Take 3 ml of the given soln. in a test tube. Add 1 ml of Conc. Nitric acid. Mix .White ppts are formed. Heat the
solution for 1 minute. Cool under tap water. Some of the ppts dissolve. The soln. becomes yellow. Add about 2 ml of
40 % NaOH soln. until orange color is developed. Orange colored ppts indicate aromatic amino acids.
Modified Millon’s test:
This test is positive for the amino acids containing a hydroxyphenyl group. Mercuric sulphate in the presence of
sulphuric acid reacts with hydroxyphenyl group of tyrosine and forms nitrated mercury phenolate ion which gives red
colored complex with NaNO2 soln.
Reagents:
• HgSO4 reagent (10% mercuric sulphate in 10 % H2SO4)
• NaNO2 solution
Procedure:
Take 3 ml of given soln. in a test tube. Add 2 ml of HgSO4 reagent. Mix and boil in boiling water bath for 2—3
minutes white ppts are formed which turn yellow on cooling under tap water for 2—3 minutes. Add 2 drops of NaNO2
solution and warm gently for 1 minute red ppts are formed indicating the presence of tyrosine
Ninhydrin test
Principle: Ninhydrin is a powerful oxidizing agent and causes oxidative decarboxylation of alpha amino acids
producing CO2 and ammonia. This is one of the most sensitive test for amino acids having at least one free carboxyl
and one free amino group. It is given positive by all the free aminoacids, proteins, peptones as well as ammonia and
other primary amines.
Proline and hydroxyproline do not respond as they are imino acids. Their amino group is a secondary amine.

Reaction:
alpha amino acid + ninhydrine → aldehyde + hydindantin + CO2 +NH3
Hydindantin + Ninhydrine + ammonia → Violet blue colored complex + 3H2O
Proline & Hydroxyproline give yellow color.
Reagents:

55
 • Ninhydin soln.
Procedure:
Take 1 ml of given protein soln. add 2 drops of ninhydrin soln. mix and boil for 1—2 minutes allow to cool . Violet
blue color develops.
Lead acetate Test:
Reagents:
• 40 % NaOH soln.
• Lead acetate soln.
Principle:
Organic sulphur present in aminoacids such as cysteine or cystine when boild with strong alkali is converted into
sulphide. When lead acetate soln is added lead sulphide is precipitated which is black in color. Methionine dose not
respond as the sulphur group in this amino acids is not released when treated with NaOH.
Procedure:
Take 2 ml of given soln in a test tube add 2 ml of 40% NaOH solution. Boil for 2 minutes. Cool and add 0.5 ml of lead
acetate soln and worm slightly. Cool it brown or black ppt of lead sulphide are formed indicating sulphur containing
amino acids .

56
 Experiment No.19

TO STUDY THE PROPERTIES OF THE PROTEIN CASEIN

INTRODUCTION
Casein is a conjugated protein which contains phosphate as prostheitc group. It is the chief component of milk
proteins and constitutes about 80% of the milk proteins( remaining proteins are lactalbumin and lactglobulin).Casein
is a phosphoprotein(the phosphoric acid is released during digestive hydrolysis and it confers acidic properties to
casein) It is held in solution as a calcium salt in milk.
Casein is soluble at neutral or physiologic pH (7.4) Its isoelectric pH 4.7 at which it has minimum solubility. Most
proteins can be precipitated by heating them at their isoelectric pH. However casein is peculiar that it is precipitated at
its isoelectric pH even at room temperature.
Tests for casein:
1.Heat Coagulation Test
This test is negative for casein as heat is not sufficient to break the bond between casein and phosphate group.
2. Half saturation test with ammonium sulfate
As casein is fully precipitated due to high molecular weight so it does not pass into the filtrate. Hence this test is
positive for casein (but negative for albumin)
3.Full saturation with ammonium sulphate
This test is also positive for casein as well as for albumin so it cant help to differentiate between the two proteins..
4. Color Reactions:
Biuret test +ve
Lead acetate test +ve
Xanthoproteic test +ve
Ninhydrin test +ve
Modified Millons test +ve
5. Neumann’s test
This test is specific for casein and detects the presence of
phosphate group in casein.
Procedure
1.Place 2 ml of casein solution in a test tube.
2. Add one drop of conc. nitiric acid and about 12 drops of strong sulphuric acid.
3. Heat on low flame with constant agitation. Fumes of nitric acid will evolve. Heat until all the nitric acid has
evolved.
4. If the fluid becomes brown or black discard the solution & start again.
5. Allow the test tube to cool down to room temperature.
6. Add 5ml of distilled water and a drop of methyl red.
7. Add strong ammonia drop by drop until the color of the solution turns yellow.
8. Now add 2ml of ammonium molybdenate solution and boil it-yellow precipitate of ammonium-phosphomolybdate
are formed which indicate the presence of phosphorous in casein.

57
Precautions
Failure to perform this test could be due to taking supernatant solution only or taking inadequate casein solution or not
heating sufficiently and strongly.

58
 Experiment No.20

SCHEME FOR UNKNOWN PROTEINS

BIURET TEST

-VE +VE
Non-proteins Proteins

HEAT COAGULATION TEST

-VE +VE

CASEIN ALBUMIN

1.HALF SATURATION TEST +VE -VE

2.FULL SATURTION TEST +VE +VE

3.COLOUR REACTIONS:

• Xanthoproteic test +VE +VE

• Modified Millons test +VE +VE

• Lead Acetate test +VE +VE

• Ninhydrin test +VE +VE

4.NEUMANN’S TEST +VE -VE

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 Experiment No.21

PHYSICAL PROPERTIES AND INORGANIC CONSTITUENTS OF URINE

Urine is an excretory product of the body formed by the kidneys. Majority of the waste products are excreted
through urine.

Composition:

The composition of urine varies and chiefly depends upon diet and weather conditions. It consists of organic and
inorganic constituents.
For analysis, urine must be collected early morning and generally a mid stream sample is recommended. The
urine analysis should be done as soon as possible to avoid bacterial growth.
Physical characteristics:

1) Volume:
The average daily volume of urine passed by a normal adult is 800-1200 ml. It greatly depends upon the
amount of fluid intake.
< 600 Oliguria
< lOO Anuria
>1500Polyuria
2) Color:
The color of a freshly passed sample of urine is amber yellow. It is mainly due to urochrome. Urine when passed is
clear but if allowed to stand for sometime it becomes turbid. The turbidity is due to ppts of phosphates and uric
acid.
3) Odour:
The odour of normal sample of urine is faintly aromatic due to the presence of certain volatile acids
4) Specific gravity
Adults with normal diet and fluid intake will produce urine of specific Gravity 1.015-1.025.It is measured by
Urinometer.
Subject to great variations
If there is excessive water intake it may be as low as 1.003 or in case of excessive perspiration as high as 1.040.
To Measure Specific Gravity:
Take 100ml of urine in a burette. Immerse a urinometer in it. Note the reading and room temperature. Calculate the
temperature difference between room temperature and the temperature at which urinometer is calibrated. For every 3
degree rise in room temperature one unit is added to the right digit of the observed sp. gravity urinometer reading.
This is the calculated specific gravity. 5)pH
The PH of Urine is the ability of kidneys to maintain hydrogen ion conc., in plasma and ECF. Normally fresh urine
is acidic with pH range of 4.8-7.0. Urine on standing becomes alkaline due to bacterial action on Urea and
formation of ammonia.
Inorganic constitution of Urine:

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 1) Ammonia:

It is derived from glutamine and other ammo acids in kidney. Normal adults excrete 0.3-1.2g (0.7g average) of
ammonia nitrogen or amino acid nitrogen.

Experiment

3 ml of urine + 1 ml 5% NaOH added on to phenophthalein soaked filter paper; heat it over fumes

Procedure Observation Inference

3ml Urine+lml 5% NaOH+ Pink color on filter Ammonia present
Heat paper and the color liberated by heat. Ammonia
disappears quickly being alkaline gives pink
color with phenophthalein

2) Inorganic Sulphate:

It is the major form of sulphur present in Urine (80%).Sulphur excretion varies directly with protein metabolism. The
main source of sulphur is sulphur containing amino acids such as cysteine and methionine.

Procedure Observation __________ Inference

3.0 ml Urine +1.0 ml conc. White ppt. Inorganic Sulphates are
HCl +1 ml 10% BaCl2 precipitated as BaSO4.HCl is
added to prevent
precipitation of phosphates.

3) Chlorides:

Chloride is excreted mainly in the form of Sodium Chloride. Normally 5-25mg of NaCl is excreted per day.

Procedure _________________ Observation ____________ Inference ______________

3.0 ml Urine+1.0 ml Conc. White ppt. Chlorides are precipitated as
HNO3+1.0 ml AgNO3 AgCl. Conc. HNO3 is used to prevent the precipitation
of phosphates

4) Calcium and Phosphates:

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 Daily excretion of Calcium in normal adult is around 300mg per day depending upon the diet. Normal excretion
of phosphates in urine during 24 hrs varies from 400mg to 2.0g. (Average 1.0g)

Experiment:
Take 1.0 ml of urine in test tube. Add 3.0 ml of strong ammonia and boil. Filter it by using filter paper. Discard
the filtrate. Add 5 ml of acetic acid (1:5 dilutions) to the ppt. on the filter paper. Collect solution in a test tube and
divide into 2 equal parts.
Procedure Observation Inference

a) For Calcium first part+2.0 White turbidity Calcium Oxalate found.

ml of 2% Potassium Oxalate

b) For Phosphates Second Canary Yellow ppt. Phosphate in urine reacts with
part+1.0 ml cone, canary Ammonium Molybdate to form
yellow HNO3+ 2 ml of 4% Ammoniumphospho Molybdate
Ammonium Molybdate

Questions:

1. What is the average daily output of urine?

2. What is the specific gravity of urine?
3. What is the pH of normal urine?
4. What are the normal inorganic constituents of urine?
5. Ammonia is used as a nitrogen source in which metabolic pathway?

62
 Experiment No.22

TO STUDY THE ORGANIC CONSTITUENTS OF URINE

1.Urea

It is the principle end product of protein metabolism. 80-90 percent of total nitrogen of human
urine is present as urea. The daily excretion is approximately 30g/day. This amount is directly
related with intake of protein. Uremia; Increased amount of urea in blood.

SPECIFIC UREASE TEST

PROCEDURE OBSERVATION INFERENCE

Solution turns pink due to Urea present is hydrolysed
alkalinity of the product by the enzyme urease to
3ml of urine + 4 drops of phenolphthalein+Urease
form ammonium carbonate
powder mix and keep it at room temperature for 10
min

2.Uric Acid

It is the end product of purine bases (adenine and guanine).The daily excretion is around 0.5mg/day.It
mainly depends on dietary intake.

Hyperuricemia: Increased uric acid level in blood

Gout: Deposition of uric acid crystals in joints of the body.

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 3.CREATININE
It is an anhydride of creatine. Normal daily excretion is1 to 2 grams. The amount depends on muscle mass diet and
tissue metabolism.
Increased in: renal failure, pneumonia, leukemia and fever.
Decreased in: Starvation, anemia and muscular atrophy.

PROCEDURE OBSERVATION INFERENCE

PROCEDURE OBSERVATION INFERENCE

a)Benedicts uric acid test: Blue color seen Uric acid reduces
3ml urine+few drops of Benedicts uric acid Phopsphoric acid to blue
reagent + few drops NaOH and mix. Phosphotungstic acid.
b)Schiffs test
Soak filter paper with AgNO₃ solution and Uric acid reduces silver
few drops of urine on filter paper +few drops Brown black stain of metallic nitrate to metallic silver in
of Na₂CO₃ silver on filter paper alkaline medium.

Jaffe’s test (picric acid test)

10ml of saturated picric acid solution + 5 ml 5%
NaOH.Mix and divide into two parts Orange red color Creatinine picrate is formed.
1) In the first test tube add 5 ml urine Acts as control
2) In the second test tube add 5 ml water No color

Sodium niroprusside test:

5 ml urine +few drops of sodium nitroprusside + few drops Red color soon turns Creatinine is present
of 10% NaOH. yellow

4.PIGMENTS
a)Urochrome: This is the main pigment which gives yellow color to the urine. It is the product of endogenous
metabolism and is unaffected by diet. Its amount is constant in urine.
b)Urobilinogen and urobilin:

64
It is formed by the breakdown of Haemoglobin. Its amount increases in fever, liver diseases, malaria, pneumonia and
anemia. Its excretion is 104 gm/day
c)Uroerythrin:
It is present in small amounts in normal urine and is red in acidic in medium. Its derived from melanin.

65
 Experiment No.23

TO STUDY THE ABNORMAL CONSTITUENTS OF URINE

Examination of urine for changes in physicochemical characteristics is an important test for suspected kidney
damage as well as systemic diseases.
Usually the urine analysis is carried out with properly preserved 24 hour urine specimen. The preservative used is
10 ml conc. HCl.
A 24 hour urine sample is usually collected by discarding the first morning sample and collecting the urine up to
and including the first sample of next morning.
PHYSICAL CHARECTERISTIS AND PATHOLOGICAL CONDITIONS:
1. COLOR:
Normally fresh excreted urine is amber yellow in color. Variation in color of urine in various pathological
diseases is due to presence of certain abnormal metabolites excreted in urine.

COLOR CAUSE
Orange Concentrated urine
Dark yellow / yellow brown Bile pigment
Colorless Dilute urine
Reddish brown color Due to the presence of hemoglobin
Milky Fat
Black brown on standing Melanin
Orange Urobilinogen
Cloudy Presence of pus cells or bacteria / calcium and Magnesium phosphate
Red Porphyria

Certain drugs can also cause change in urine color.

COLOR DRUGS
Dark yellow Vitamin B complex
Bright orange red Rifampicin
Red brown Methyl dopa
Red purple Phenolphthalein

2. VOLUME:
Volume of urine excreted in 24 hours is influenced by fluid intake, temperature and sweating. Normal output is
800-1200ml/day.

POLYURIA OLIGURIA ANURIA

66
Diabetes mellitus Diarrhea , vomiting Acute tubular necrosis
Diabetes insipidus Fever Bilateral stones
Chronic glomerulonephritis Shock Blood transfusion reactions
Alcohol consumption Acute nephritis
Drugs like diuretics Cardiac failure

3.ODOUR:

Normal urine Aromatic odour

Fruity or sweet smell Diabetic ketoacidosis
Foul smell Bacterial infections

AMINO ACID DISORDERS ODOUR

Maple syrup urine disease Maple syrup
Methionine malabsorption Cabbage-like
Phenylketonuria Mousy
Tyrosinaemia Rancid

4. SPECIFIC GRAVITY:
It gives an indication of urinary total solute concentration. Normal specific gravity of urine is 1.010-1.030.

LOW SPECIFIC GRAVITY HIGH SPECIFIC GRAVITY

Diabetes insipidus Fever, vomiting , sweating
High fluid intake Nephrosis
Glomerulonephritis Diabetes mellitus
Pyelonephrits CCF

3.pH:
It is the ability of the kidney to maintain normal hydrogen ion concentration. Normal is 4.8-8.0.

More acidic (ph less than 4.8) Alkaline (ph more than 8.0)
Fever Metabolic alkalosis
Diabetes mellitus
Acidosis

ABNORMAL ORGANIC CONSTITUENTS OF URINE:

67
 1. PROTIENS:
Normal urine does not contain any proteins. Presence of proteins in urine is called protienuria.
Physiological protienuria:
Pregnancy, after severe exercise, prolonged exposure to cold.
Pathological Protienuria:
Pre-renal causes:
Leukemia, hypertension, multiple myeloma
Renal causes:
Glomerulonephritis, pyelonephritis, nephrotic syndrome
Post-renal causes:
False albuminuria, urinary tract infections (UTI)
TESTs
PROCEDURE OBSERVATION INFERENCE
a)Sulphosalicylic acid test: White ppt. Proteins present
3ml urine + 20% sulphosalicylic acid
(drop wise)
Heller’s Test: White ring at the junction of 2 Proteins in urine are
3ml of conc. HNO₃ + 3ml urine layered layers denatured
carefully over the acid
c)Heat Coagulation Test: Turbidity in upper part due to Proteins confirmed.
Take 10ml urine in the test tube. Heat coagulation of proteins
only upper portion to boil. Add 1%
acetic acid along the side of tube.

BENCE JONES PROTEINS:

These proteins are of diagnostic importance in cases of multiple myeloma and myelogenic osteosarcoma. It
appears to be present in small amounts in bone marrow and in certain WBCs.
Detection of Bence Jones Proteins:
Heat the suspected urine sample very gently, carefully noting the temperature. At a temperature as low as 40
degrees celsius a turbidity maybe observed and as temperature is raised to about 60 degrees a flocculent
precipitate forms and clings to the side of test tube.
2.GLUCOSE:
Glycosuria: presence of glucose in urine.
Physiological Causes:
Pregnancy, after severe exercise
Pathological Causes:
Diabetes mellitus
Severe infections
Nephrosclerosis

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 TESTS:
BENEDICT’S QUALITATIVE TESTS:
PROCEDURE OBSERVATION INFERENCE
5ml Benedict’s qualitative reagent +8 Green or yellowish green or Glucose is present
drops urine and boil for 2 min orange red ppt.

FOR OTHER CARBOHYDRATES:

Salivnoff’s test:
PROCEDURE OBSERVATION INFERENCE
5ml Salivnoff’s reagent + 5ml urine No cherry red color The type of CHO is an aldo
and boil for 30 sec. sugar

Tollen’s test:
PROCEDURE OBSERVATION INFERENCE
2ml urine + 2ml conc. HCl and add a Reddish brown color It indicates the presence of
pinch of floroglucinol. Mix and boil for galactose and lactose.
30 sec.

3.BLOOD:
Hematuria: presence of blood in urine. In haematuria, besides hameoglobin, unruptured red blood cells are also
present which can be detected microscopically.
Causes:
Lesions of kidney, Renal, ureteric, vesical stones and infections
Hemoglobinuria: Presence of pigment alone in urine
Causes
Jaundice, mismatched blood transfusion, yellow fever, malaria, typhoid

TEST:
BENZIDINE TEST:
PROCEDURE OBSERVATION INFERENCE
3ml saturated solution of Benzidine in Immediate blue or green color Blood is present
glacial acetic acid + 2ml urine + 3%
H₂O₂

69
 Experiment No.24

TO STUDY ABNORMAL ORGANIC CONSTITUENTS OF URINE

1) Ketone bodies:
In impaired carbohydrate metabolism excess of fat is catabolised to produce ketone bodies which gets accumulated in
blood and excreted in urine. The presence of ketone bodies in urine is called ketonuria. The ketone bodies include
acetone, acetoacetate and beta hydroxy butyrate.
Causes:
• Diabetes mellitus
• High fat diet
• Starvation
• Fever
• Glycogen storage diseases
• Pregnancy and lactation
Tests:

Procedure Observation Inference

Rothera’s Test Purple colored ring at the Test is positive. Shows the
10ml of urine+add solid junction of 2 layers presence acetic acid and
(NH4)2SO4 till no more is acetone
dissolved .Now add 2-3
drops freshly prepared 5%
Na Nitroprusside soln. Add
3 ml of Conc. Ammonia.
Wait for 5-10 min

Gerhardt’s Test: Wine red color Ferric Acetoacetate formed.

5 ml Urine+FeCl3 drop wise
few drops

2. Bile Salts and Bile pigments:

Bile is formed in liver. It is a golden yellow, viscous, alkaline fluid. Urine containing bilirubin has a typical brown
color.

Cause of bile salts and bile pigments in urine:

Obstructive Jaundice
Haemolytic Jaundice
Infective Hepatitis
Tests:

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 Bile salts:
These are salts of glycocholic or taurocholic acid
Types:
Primary bile salts: Taurocholic acid Glycocholic acid
Secondary bile salts: Chenodeoxycholic acid, Glycodeoxycholic acid

Procedure Observation Inference

Hay’s Test Sulphur powder sinks to Lowering of surface tension

1)5 ml Urine+Sulphur powder bottom due to presence of Bile salts
2) 5ml water+Sulphur powder
Sulphur powder remains on the No bile salts present to lower
surface of water the surface tension

For Bile Pigments:

Bilirubin and Biliverdin
Tests:

Procedure Observation Inference

Fouchet’s Test:
5 ml Urine+ 5 ml of 10%
BaCl2 filter and to the ppt
on filter paper, add few
drops of Fouchet’s reagent.
Color changes from yellow to
light green
Bile pigments are present. Bile
pigments are absorbed on
BaSO4 ppt and are oxidised to
colored products by
trichloroacetic acid and FeCl3
of Fouchet’s reagent

Gmelin’s Test:
5ml Conc. HNO3+5 ml Urine
from the side of test tube
Different colored rings are
formed at the junction of 2
layers.
Bile pigments are present. Bile
pigments in urine are oxidized
to various colored products e.g
Biliverdin(Blue)
Bilitusin(Red)

71
CMH MEDICAL COLLEGE LAHORE

DATE LAB NO. ____

PATIENTS NAME AGE/SEX _______
CLINICAL FEATURES_____________________________________

MEDICAL OFFICER

SPECIMEN: URINE

PHYSICAL EXAMINTAION

COLOR pH
APPEARANCE SPECIFIC GRAVITY

CHEMICAL EXAMINATION

SUGAR ALBUMIN
BLOOD CREATININE
URIC ACID ACTONE
BILEPIGMENT UROBILINOGEN

MICROSCOPIC EXAMINATION

PUS CELLS RBCs

EPITHELIAL CELLS GRANULAR CASTS
CRYSTALS BACTERIA

CLINICAL BIOCHEMIST
Experiment No. 25

SPECTROPHOTOMETERY

It is a technique which uses spectrophotometer to find out concentration of solute in solution in relation to the
absorption of light.

SPECTROPHOTOMETER
A spectrophotometer is a photometer (a device for measuring light intensity) that can measure intensity as a function
of the color, or more specifically, the wavelength of light. It is quantitative study of electromagnetic spectra.
Spectrophotometry deals with visible light, near-ultraviolet, and near-infrared.

72
MONOCHROMATIC LIGHT
Light of a particular wavelength.

COMPONENTS:

• Light source.
• Spectral isolation
• Fibre optics
• Cuvette
• Photodetector
• Meter or recorder

Light source:
Types of light sources used in spectrophotometer includes
1. INCANDESCENT LAMP: light source for measuring invisible portion of spectrum is tungsten light bulb.
U-V region of the spectrum is the low pressure mercury vapor lamp.
2. LASER: (Light amplification by stimulated emission of radiation). It is a device that controls the way that
energized atoms release photons.

Spectral isolation:
A system for radiant energy of a desired wavelength and excluding that of other wavelength is called a
monochromator.
Various ways include
• Filters colored glass
• Prisms
• Diffraction grating
• Holographic grafting

Fibre optics
Also known as light pipes, are bundles of thin, transparent fibres of glass, quartz or plastic that are enclosed in
material of a lower refractive index and they transmit light throughout their length by internal reflection.
Advantage is better directional control beam of light with in geometrical confines of an instrument.

Cuvette;
It is a small vessel used to hold a liquid sample to be analyzed.
They may be round, square or rectangular.
They are constructed from glass, silica, {quartz} or plastic.
In visible portion of light ordinary borosilicate glass cuvettes are used.
In U-V region quartz cells are required.
Plastic can be used in both.
Avoid keeping alkaline solutions for long because it dissolves glass.

Photo detectors:
Devices that convert light into an electric signal that is proportional to
number of photons striking the phototube-Types include:
• Photomultiplier tube
• Photodiode
• Barrier layer cell

73
DIAGRAMATIC REPRESENTATION OF A SPECTROPHOTOMETER

source of light monochromator cuvette photodetector

WORKING OF A SPECTROPHOTOMETER:

Beam of monochromatic light enters the solution, some of which is absorbed and remainder passes through and strikes
detector and is converted to an electrical signal.

BEER-LAMBERT’S LAW:
It states that the concentration of a substance is directly proportional to the amount of light absorbed or inversely
proportional to logarithm of transmitted light. Mathematically Beer’s law is

ABSORBANCE (A) = abc

A=Absorbance
a=proportionality constant/absorbtivity
b=light path
c=concentration expressed in mol /l

Since absorbtivity (a) remains constant for each test, and light path (b) remains constant for the particular cuvette size.
Absorbance becomes directly proportional to concentration.
To calculate the concentration of an unknown sample it is compared with a calibrating solution.
Rearranging equation;

A=abc to a=A/bc

As, a and b are constant so

A∞c

Au = As
Cu Cs

Where, Au = Absorbance of unknown

Cu = Concentration of unknown
As = Absorbance of standard
Cs = Concentration of standard

Solving for the concentration of unknown,

Cu = Au × Cs OR cu = Au × Cs
As As

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CONCENTRATION OF UNKNOWN SAMPLE = AU × Cs
As

Where, factor (K) = Cs

As

Hence, Cu = Au×K

75
METHODS FOR SPECTROPHOTOMETRY:
1. End point method
a. With standard
b. With factor
Used for estimation of cholesterol, glucose, calcium, phosphate, bilirubin, urea and uric acid.
2. Kinetic method
Used for measurement of enzymes: e.g. ALT, AST, CK, LDH and Amylase.
3. Two point kinetic method
a. Urea
b. Creatinine

IMPORTANT TERMS

1. BLANK
It is used to set zero on spectrophotometer.
a. Sample blank (sample without reagent)
Unwanted substances in the sample (e.g. lipids, hemolyzed RBC etc), may alter the reading. We
subtract the absorbance of sample blank from the absorbance of sample to get the actual absorbance of
sample.
b. Reagent blank (reagent without sample)
2. SAMPLE concentration is unknown
3. STANDARD concentration is known.

76
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