Hayat Private University For Science & Technology

Faculty of Science
Department of Pathological Analysis

Biochemistry
Laboratory Manual

by
Kutaiba Ibrahim Alzand, Ph.D
Suher Muneer Alsaaty, Msc
 Safety & Laboratory Information

1. Read the experiment before coming to the laboratory session.

2. No unauthorized experiments are allowed.

3. To do an experiment, have at least one other person in the laboratory with you.

4. Keep your desk and apparatus neat and clean. Do not discard any chemicals down the
sink unless instructed to do so. At the end of the laboratory period, clean and put away
all apparatus, and wash and dry the desktop. This will be graded.
You will need to provide dishwashing soap and a towel for your lab drawer.

5. Never leave spilled chemicals or water on a desktop or floor. Clean up immediately.

6. Wash your hands thoroughly when exiting and entering the lab. Biochemical
experiments are very sensitive to contamination & the chemicals can harm you without
any symptoms being apparent.

7. Record your observations and results directly in your notebook immediately after you
obtain them. Do not put them on odd pieces of paper.
8. The chemical reagents necessary for the experiments will be made available in
labeled bottles placed in the hoods or in the appropriate location.

A. Read the label carefully before removing the material.

B. Never return unused chemicals to the stock bottles. This may lead to contamination
of the entire supply of reagent.
C. Be careful not the mix stoppers or caps.
D. Return reagent bottles to proper place.
E. If a solution should be kept cold make sure and place the container in the
refrigerator.

9. Leave any food, beverages outside the lab. Never drink water or eat ice in or from
the laboratory. Never taste a chemical. Never mouth pipette.

10. Know the properties (i.e., flammability, toxicity, and reactivity) of the chemicals
used. All hazardous chemicals must be handled in the fume hood.

11. Do not use cracked or broken glassware.

12. Consult the teaching assistant before using any unfamiliar equipment.

13. Do not remove any chemicals or equipment from lab without specific permission
from the instructor.

14. Learn proper exit routes from the building for a fire or other emergency. Know
where fire extinguishers, first-aid kits, showers and eye wash stations are located in
the laboratory and/or building and be familiar with the guidelines for their use.

15. Report any injury, no matter how slight, to your instructor.

16. If you have any health conditions, which may impact your presence or work in the
lab, inform the instructor.

17. Never pour water into conc. acid. Pour the acid slowly into the water with
stirring. (In a fume hood).
 Writing a Formal Lab Report

1. Introduction
1.1. Purpose or objective of the experiment
1.2. Background and theory
2. Materials and Methods
3. Experimental Procedure
4. Results
5. Discussion
6. Conclusion
7. References
A. Units, Amounts and Concentrations

Units and abbreviations

The common units, and their respective abbreviations, used in biomedical sciences are
based on the International System of Measurements (SI units): grams (g), metres (m) etc.
Often it is necessary to deal with large quantities e.g. 1500 g or, more commonly, very
small quantities, e.g. 0.0000015 g. In such cases, such numbers are either expressed by
use of powers of 10 (positive or negative) or by use of the appropriate prefix.
Thus: 1500 g = 1.5 x 103 g = 1.5 kg (kilograms)

and 0.0000015 g = 1.5 x 10-6 g = 1.5 µg (micrograms)

Laboratory scientists most commonly use the prefix notation, particularly those that
differ by a factor of 1000 in magnitude, so you should try to become familiar with them.
Use of the prefixes can greatly simplify calculations, especially mental ones.

Note also that biomedical scientists normally express volumes and concentrations in
terms of litres rather than in cubic measurements:
 
e.g. 1 litre, rather than 1 dm3
1 millilitre (1 ml) rather than 1 cm3
A concentration of 1 milligram per litre is also still commonly expressed in the form 1
mg/l rather than 1 mg.l-1 or 1 mg.dm-3.

The commonly used prefixes are:

The prefixes centi (10-2) and deci (10-1) are only commonly used in specific cases e.g. cm
Concentrations

The determination of the concentration of a substance in a biological fluid is central to

many areas of medical and dental practice (e.g. electrolytes in serum or glucose in
urine). It is important, therefore, that concentrations are expressed in clear unambiguous
terms. There are several ways of doing this. The simplest is to express the concentration
as the weight or mass of the substance per unit volume:

e.g. 10 g/l or 20 mg/ml or 2 µg/ml

Another way is to express the concentration of a solution or mixture in terms of per cent
(%). This is a somewhat outdated method but you may still come across it, particularly
if you read the older medical literature, so you should know what it means. Note that
there are different types of % concentration:

% (v/v) (volume by volume)

% (w/v) (weight by volume)
% (w/w) (weight by weight)
1. A 1% (v/v) concentration is obtained by diluting 1 volume of a substance into 100
volumes (total) of solution, e.g. 1 ml ethanol diluted with water to a final volume of
100 ml gives a 1% (v/v) ethanol solution.

2. A 1% (w/v) concentration is obtained by dissolving 1 g of substance in a final

volume of 100 ml solution, e.g. 1 g glucose dissolved in water to a final volume of
100 ml solution gives a 1% (w/v) glucose solution.

3. A 1% (w/w) concentration is obtained by mixing 1 g of substance with something

else in a total weight of 100 g, e.g. 1% (w/w) salt in sand.

Another old method of expressing concentration that you may still see (just look at the
side of a tube of toothpaste) is "parts per million (ppm)". One ppm is one part of
anything in one million parts of total material, e.g. 1 g of compound X in a million g
total, or 1 litre of Y in a million litres total.
Moles and molarity

By far the most important unit defining an amount of a biological substance is the mole,
with the corresponding concentration being the molarity. As far as possible, the
concentrations of specific compounds in serum, urine etc, are now expressed as
molarities in clinical laboratories.

One mole of a substance is the molecular weight of that substance expressed in grams.
Thus, the molecular weight of glucose is 180, so:

1 mole of glucose = 180 g

1 mmol glucose = 180 mg
1 µmol glucose = 180 µg
100 µmol glucose = 18,000 µg = 18 mg

Note the abbreviations:

1 mmol = 1 millimole;
2 mmol = 2 millimoles;
5 µmol = 5 micromoles.
Concentrations in molarities are given by expressing the number of moles of the
substance present in a defined volume of solution:

A 1 molar (1 M) solution contains 1 mole per litre (1 mol/l)

A 1 millimolar (1 mM) solution contains 1 millimole per litre (1 mmol/l)

Note: mol and moles mean the same thing (an amount) and moles/litre (long winded
but correct) is a concentration and can be expressed as mol/l, or mol.l-1, or (best and
simplest of all) — M. Ensure you know the distinction between concentrations and
amounts.

So, if you dissolve 0.5 mol of a compound in one litre of solvent the concentration of
the compound is 0.5 mol/l, or 0.5 M. 100 ml of the solution contains 0.05 mol.
Prefix notation

The value of the prefix notation can now be seen as it allows rapid mental calculations to
be performed (after much practice!). The following concentrations are all the same:

0.5 M, 0.5 mol/l, 0.5 mmol/ml, 0.5 µmol/µl, 0.5 pmol/pl

You see that by scaling both the units (amount and volume) in the concentration up or
down by a factor of 1000, the value of the concentration remains the same. If you only
scale one of the terms (amount or volume), then you can express the same concentration in
yet more ways:

0.5 mol/l = 0.5 mmol/ml = 500 µmol/ml = 500,000 pmol/µl

Now, let’s say you wanted to determine the amount of cholesterol in the blood of a newborn
infant. You know it will be around 5 mM. The detection limit of the method you are going
to use is about 20 nmol and you can only take 20 µl of blood. Will this be enough?

5 mM cholesterol contains 5 mmol/l, or 5 µmol/ml, or 5 nmol/µl.

It is easy to see now that 20µl contains 100 nmol of cholesterol - ENOUGH.
Examples

1. How many µmol are dissolved in 2 l of a 20 mM solution?

20 mM = 20 mmol/l, so 2 l contain 40 mmol. 40 mmol = 40,000 µmol.

2. The molecular weight of NaCl is 58. How many mg are in 50 µmol of NaCl?

1 mol of NaCl is 58 g, so 1 µmol is 58 µg, so 50 µmol is 2,900 µg = 2.9 mg.

3. What is the molarity of a 1% (w/v) solution of glucose? (molecular weight = 180)

1% (w/v) contains 1 g in 100 ml and, therefore, 10 g in 1 litre.

A 1 M solution of glucose contains 180 g/l, so 10 g/l represents a molarity of

10/180 = 0.056 M (or 56 mM).
B. Dilutions

This is another source of great confusion. Most experiments require you to make
dilutions of reagents, either before use or as a consequence of the actual assay.
When you are asked to make a ten-fold dilution of a reagent, the objective is to
produce a solution that has a reagent concentration one-tenth of the original. It
follows that the molecules of reagent that occupied a volume of "x" before must
now occupy a volume of "10x". This is obtained by adding to one volume of
reagent to nine volumes of diluent (sometimes referred to as a “1 plus 9” dilution
or, more commonly, a "1 in 10" dilution).

When a solution of known concentration is diluted, it is obvious that the

concentration will fall. Less obvious is the amount by which it falls! Take a
typical example:
A solution of a compound is maintained as a stock solution at 5 mM. What is the final
concentration of the compond in which 0.15 ml of stock solution is mixed with 0.4 ml of
buffer and 0.2 ml of water?

There are many ways to derive this answer. Here are a few different approaches — all
equally acceptable. Take the one that seems most logical to you!

1. The final volume is 0.75 ml, therefore the concentration of the substrate will be
0.15/ 0.75 x 5 mM = 1 mM

2. 5 mM is 5 mmol/l, or 5 µmol/ml. Therefore 0.15 ml of stock solution contains 0.15

x 5 = 0.75 µmol. This is expanded into a volume of 0.75 ml and therefore there are
now 0.75 µmol/0.75 ml, or 1 µmol/ml, or 1 mmol/l, or 1 mM

3. The formula for a dilution is v1 x c1 = v2 x c2 (where c1 and c2 are the

concentrations before and after dilution, and v1 and v2 are the volumes before and
after dilution). Therefore, c2 = v1/v2 x c1, which works out to be c2 = 0.15 / 0.75 x
5 mM , i.e. 1 mM
Note that the last and first are identical, except that the latter defines the problem
as a formula. It may be valuable for dilutions when a solution of known
concentration must be diluted to a new concentration. The formula will give the
new volume, but remember that part of that volume will come from the sample
before dilution! Method 2 seems tortuous with this example, but it suits some to
work this way.

Lastly, remember to think about the answer — it’s not unusual to see erroneous
calculations giving serum concentrations of metabolites in the tens or hundreds of
molar - try to imagine “10 M glucose” in the blood? (the molecular weight of
glucose is 180 — imagine a cross between a Mars bar and black pudding!). You
should always do rough calculations to ensure that your answers make biological
(and logical!) sense.
Carbohydrates

In this experiment, you will perform tests that distinguish among various
carbohydrates. The carbohydrates included and the classes they represent are as
follows:

● Aldopentoses: xylose and arabinose

● Aldohexoses: glucose and galactose

● Ketohexoses: fructose

● Disaccharides: lactose and sucrose

● Polysaccharides: starch and glycogen

The structures of these carbohydrates can be found in your lecture textbook.

The tests are classified in the following groups:

A. Tests based on the production of furfural or a furfural derivative:

1. Molisch’s Test for Carbohydrate
2. Bial’s Test for Pentoses
3. Seliwanoff’s Test for Ketohexoses.

B. Tests based on the reducing property of a carbohydrate (sugar):

1. Benedict’s Test For Reducing Sugars
2. Barfoed’s Test for Reducing Monosaccharides

C. Iodine Test for Starch.

D. Hydrolysis of Sucrose.

E. Tests on Unknowns
REQUIRED READING

● Read the sections in your lecture textbook that give the structures and describe the
chemistry of aldopentoses, aldohexoses, ketohexoses, disaccharides, and
polysaccharides.

SPECIAL INSTRUCTIONS

● All of the procedures in this experiment involve simple test tube reactions.

● You will need a minimum of 10 test tubes (15 mm x 125 mm) numbered in
order. Clean them carefully each time they are used.

● The laboratory instructor will prepare the 1% solutions of carbohydrates and the
reagents needed for the tests in advance. Be sure to shake the starch solution
before using it.
Part A. Tests Based on Production of Furfural or a Furfural Derivative

● Under acidic conditions, aldopentoses and ketopentoses rapidly undergo

dehydration to give furfural (see equation 1).
● Ketohexoses rapidly yield 5-hydroxymethylfurfural (see equation 2).

● Disaccharides and polysaccharides can first be hydrolyzed in an acid medium to

produce monosaccharides, which then react to give furfural or 5-
hydroxymethylfurfural.
● Aldohexoses are slowly dehydrated to 5-hydroxymethylfurfural. One possible
mechanism is shown in equation 3.

● The mechanism is different from that given in equations 1 and 2 in that

dehydration occurs at an early step and the rearrangement step is absent.
● Once furfural or 5-hydroxymethylfurfural is produced by equations 1, 2, or
3, either will then react with a phenol to produce a colored condensation
product.

● The substance α-naphthol is used in Molisch’s test, orcinol in Bial’s test,

and resorcinol in Seliwanoff’s test.
● The colors and the rates of formation of these colors are used to differentiate
between the carbohydrates.

● The various color tests are discussed in Sections 1, 2, and 3.

● A typical colored product formed from furfural and α-naphthol (Molisch’s test) is
the following (equation 4):
 Experiment No.01

Aim: To Detect The Presence Of Carbohydrate In The Given Samples By Molisch’s Test.

Principle
Molisch’s test is a general test for carbohydrates. Most carbohydrates are dehydrated with
concentrated sulfuric acid to form furfural or 5-hydroxymethylfurfural. These furfurals
react with the α-naphthol in the test reagent to give a purple product. Compounds other
than carbohydrates may react with the reagent to give a positive test. A negative test
usually indicates that there is no carbohydrate.

Reagents
1) Molisch’s reagent: Dissolve 2.5 g of α-naphthol in 50 mL of 95% ethanol
2) Concentrated H2SO4
3) 1% solution of different carbohydrates.
Procedure for Molisch’s Test

1. Place 1 mL of each of the following 1% carbohydrate solutions in nine separate

test tubes: xylose, arabinose, glucose, galactose, fructose, lactose, sucrose, starch
(shake it), and glycogen. Also add 1 mL of distilled water to another tube to serve
as a control.

2. Add two drops of Molisch’s reagent to each test tube and thoroughly mix the
contents of the tube.

3. Tilt each test tube slightly and cautiously add 1 mL of concentrated sulfuric acid
down the sides of the tubes.

4. An acid layer forms at the bottom of the tubes.

5. Note and record the color at the interface between the two layers in each tube.

6. A purple color constitutes a positive test.

Observation Table
Tube No. Sample Observation Interpretation

1 1% Xylose

2 1% Arabinose

3 1% Glucose

4 1% Galactose

5 1% Fructose

6 1% Lactose

7 1% Sucrose

8 1% Starch

9 1% Glycogen

10 Water
 Experiment No.02

Aim: To Detect The Presence Of Pentose Sugar In The Given Samples By Bial’s Test.

Principle
Bial’s test is used to differentiate pentose sugars from hexose sugars. Pentose sugars
yield furfural on dehydration in acidic solution. Furfural reacts with orcinol and ferric
chloride to give a blue-green condensation product. Hexose sugars give 5-hydroxy-
methylfurfural, which reacts with the reagent to yield colors such as green, brown, and
reddish-brown.

Reagents

1) Bial’s reagent: Dissolve 3 g of orcinol in 1 L of concentrated hydrochloric acid, and

add 3 mL of 10% aqueous ferric chloride.

2) 1% solution of different carbohydrates.

Procedure for Bial’s Test

1. Place 1 mL of each of the following 1% carbohydrate solutions in separate test

tubes: xylose, arabinose, glucose, galactose, fructose, lactose, sucrose, starch
(shake it), and glycogen. Also add 1 mL of distilled water to another tube to serve
as a control.

2. Add 1 mL of Bial’s reagent to each test tube.

3. Carefully heat each tube over a Bunsen burner flame until the mixture just begins
to boil. Note and record the color produced in each test tube.

4. If the color is not distinct, add 2.5 mL of water and 0.5 mL of 1-pentanol to the test
tube.

5. After shaking the test tubes, again observe and record the color.

6. The colored condensation product will be concentrated in the 1-pentanol layer.

Observation Table
Tube No. Sample Observation Interpretation

1 1% Xylose

2 1% Arabinose

3 1% Glucose

4 1% Galactose

5 1% Fructose

6 1% Lactose

7 1% Sucrose

8 1% Starch

9 1% Glycogen

10 Water
 Experiment No.03

Aim: To Detect The Presence Of Ketose Sugars In The Given Samples By Seliwanoff’s
Test.

Principle
Seliwanoff’s test depends on the relative rates of dehydration of carbohydrates.
Aketohexose reacts rapidly by equation 2 to give 5-hydroxymethylfurfural, whereas an
aldohexose reacts more slowly by equation 3 to give the same product. Once 5-hydroxy-
methylfurfural is produced, it reacts with resorcinol to give a dark red condensation
product. If the reaction is followed for some time, you will observe that sucrose
hydrolyzes to give fructose, which eventually reacts to produce a dark red color.

Reagents
1) Seliwanoff’s reagent: Dissolve 0.5 g of resorcinol in 1 L of dilute hydrochloric acid
(one volume of concentrated hydrochloric acid and two volumes of distilled water).
2) 1% solution of different carbohydrates.
Procedure for Seliwanoff’s Test.

1. Prepare a boiling-water bath for this experiment.

2. Place 0.5 mL of each of the following 1% carbohydrate solutions in separate test

tubes: xylose, arabinose, glucose, galactose, fructose, lactose, sucrose, starch
(shake it), and glycogen. Add 0.5 mL of distilled water to another tube to act as a
control.

3. Add 2 mL of Seliwanoff’s reagent to each test tube.

4. Place all 10 tubes in a beaker of boiling-water for 60 seconds.

5. Remove them and note the results in a notebook.

6. A cherry red condensation product will be observed indicating the presence of

ketoses in the test sample.

7. There will be no significant change in colour produced for aldose sugar.

Observation Table
Tube No. Sample Observation Interpretation

1 1% Xylose

2 1% Arabinose

3 1% Glucose

4 1% Galactose

5 1% Fructose

6 1% Lactose

7 1% Sucrose

8 1% Starch

9 1% Glycogen

10 Water
Part B. Tests Based on the Reducing Property of a Carbohydrate (Sugar)

● Monosaccharides and those disaccharides that have a potential aldehyde group will
reduce reagents such as Benedict’s solution to produce a red precipitate of
copper(I) oxide:

● Glucose, for example, is a typical aldohexose, showing reducing properties.

● The two diastereomic α- and β-D-glucoses are in equilibrium with each other in
aqueous solution.
● The α-D-glucose opens at the anomeric carbon atom (hemiacetal) to produce the free
aldehyde.

● This aldehyde rapidly closes to give β-D-glucose, and a new hemiacetal is produced.

● It is the presence of this free aldehyde that makes glucose a reducing carbohydrate
(sugar).

● It reacts with Benedict’s reagent to produce a red precipitate, the basis of the test.

● Carbohydrates that have the hemiacetal functional group show reducing properties.

● If the hemiacetal is converted to an acetal by methylation, the carbohydrate (sugar)

will no longer reduce Benedict’s reagent.
With disaccharides, two situations
may arise.

● If the anomeric carbon atoms are

bonded (head to head) to give an
acetal, then the sugar will not
reduce Benedict’s reagent.

● If, however, the sugar molecules

are joined head to tail, then one
end will still be able to
equilibrate through the free
aldehyde form (hemiacetal).

● Examples of a reducing and a

nonreducing disaccharide follow.
 Experiment No.04

Aim: To Detect The Presence Of Reducing Sugar In The Given Samples By Benedict’s
Test.

Principle

Benedict’s test is performed under mildly basic conditions. The reagent reacts with all
reducing sugars to produce the red precipitate copper(I) oxide, as shown in the reaction
below. It also reacts with water-soluble aldehydes that are not sugars. Ketoses, such as
fructose, also react with Benedict’s reagent. Benedict’s test is considered one of the
classic tests for determining the presence of an aldehyde functional group.
Reagents

1) Benedict’s reagent: Dissolve 173 g of hydrated sodium citrate and 100 g of

anhydrous sodium carbonate in 800 mL of distilled water, while heating.
Filter the solution. Add to it a solution of 17.3 g of copper(II) sulfate (CuSO 4.
5H2O) dissolved in 100 mL of distilled water. Dilute the combined solutions
to 1 L.

2) Sugar solution: 1% solution of different carbohydrates.

Procedure for Benedict’s Test

1. Prepare a boiling-water bath for this experiment.

2. Place 0.5 mL of each of the following 1% carbohydrate solutions in separate test

tubes: xylose, arabinose, glucose, galactose, fructose, lactose, sucrose, starch
(shake it), and glycogen. Add 0.5 mL of distilled water to another tube to serve as a
control.

3. Add 2 mL of Benedict’s reagent to each test tube.

4. Place the tests tubes in a boiling water bath for 2–3 minutes.

5. Remove the tubes and note the results in a notebook.

6. A red, brown, or yellow precipitate indicates a positive test for a reducing sugar.

7. Ignore a change in the color of the solution.

8. A precipitate must form for the test to be positive.

Observation Table
Tube No. Sample Observation Interpretation

1 1% Xylose

2 1% Arabinose

3 1% Glucose

4 1% Galactose

5 1% Fructose

6 1% Lactose

7 1% Sucrose

8 1% Starch

9 1% Glycogen

10 Water
 Experiment No.05

Aim: To Differentiate Between Monosaccharides & Reducing Disaccharides By

Barfoed’s Test.

Principle

Barfoed’s test distinguishes reducing monosaccharides and reducing disaccharides by

a difference in the rate of reaction. The reagent consists of copper(II) ions, like
Benedict’s reagent. In this test, however, Barfoed’s reagent reacts with reducing
monosaccharides to produce copper(II) oxide faster than with reducing disaccharides.
Reagents

1) For Barfoed’s reagent, Dissolve 66.6 g of copper(II) acetate in 1 L of distilled

water. Filter the solution, if necessary, and add 9 mL of glacial acetic acid.

2) Sugar solution: 1% solution of different carbohydrates.

Procedure for Barfoed’s Test.

1. Place 0.5 mL of each of the following 1% carbohydrate solutions in separate test

tubes: xylose, arabinose, glucose, galactose, fructose, lactose, sucrose, starch
(shake it), and glycogen. Add 0.5 mL of distilled water to another tube to function
as a control.

2. Add 2 mL of Barfoed’s reagent to each test tube.

3. Place the tubes in a boiling-water bath for 10 minutes.

4. Remove the tubes and note the results in a notebook.

Comment:

● This test is not specific for glucose or any other monosaccharides but simply
used to detect reducing sugars.

● Disaccharides also respond to this test.

● This test is copper reduction test but it differs from Fehling’s or Benedict’s
test in that reduction is brought about in acid solution.

● Chloride interferes in this test and therefore unsuitable for detection of sugar
in urine or any other body fluid containing Cl.
Observation Table
Tube No. Sample Observation Interpretation

1 1% Xylose

2 1% Arabinose

3 1% Glucose

4 1% Galactose

5 1% Fructose

6 1% Lactose

7 1% Sucrose

8 1% Starch

9 1% Glycogen

10 Water
 Experiment No.06

Aim: To Detect The Presence Of Polysaccharides (Starch & Glycogen) In The Given
Samples By Iodine Test.

Principle
Starch forms a typical blue color with iodine. This color is due to the absorption of iodine
into the open spaces of the amylose molecules (helices) present in starch. Amylopectins,
which are the other types of molecules present in starch, form a red to purple color with
iodine.

Reagents
1. The iodine solution is prepared as follows. Dissolve 1 g of potassium iodide in 25 mL
of distilled water. Add 0.5 g of iodine, and shake the solution until the iodine dissolves.
Dilute the solution to 50 mL.

2. The sodium thiosulfate solution is prepared by dissolving 1.25 g of sodium thiosulfate

in 50 mL of water.

3. Sugar solution: 1% solution of different carbohydrates.

Procedure for the Iodine Test

1. Place 1 mL of each of the following 1 % carbohydrate solutions in three separate

test tubes: glucose, starch (shake it), and glycogen. Add 1 mL of distilled water
to another tube to act as a control.

2. Add one drop of iodine solution to each test tube and observe the results.

3. Add a few drops of sodium thiosulfate to the solutions and note the results.
Observation Table
Tube No. Sample Observation Interpretation

1 1% Xylose

2 1% Arabinose

3 1% Glucose

4 1% Galactose

5 1% Fructose

6 1% Lactose

7 1% Sucrose

8 1% Starch

9 1% Glycogen

10 Water
 Experiment No.07

Aim: Hydrolysis of Sucrose

Principle
Sucrose can be hydrolyzed in acid solution to its component parts, fructose and glucose.
The component parts can then be tested with Benedict’s reagent.

Reagents

1. 1 % solution of sucrose

2. Concentrated hydrochloric acid

3. 10% sodium hydroxide solution

4. Benedict’s reagent
Procedure for the Hydrolysis of Sucrose.

1. Place 1 mL of a 1 % solution of sucrose in a test tube.

2. Add 2 drops of concentrated hydrochloric acid, and heat the tube in a boiling-
water bath for 10 minutes.

3. Cool the tube and neutralize the contents with 10% sodium hydroxide solution
until the mixture is just basic to litmus (about 12 drops are needed).

4. Test the mixture with Benedict’s reagent (Part B).

5. Note the results, and compare them with the results obtained for sucrose that has
not been hydrolyzed.
 Experiment No.08

Aim: Tests on Unknowns

Procedure.

1. Obtain an unknown solid carbohydrate from the laboratory instructor or assistant.

2. The unknown will be one of the following carbohydrates: xylose, arabinose, glucose,
galactose, fructose, lactose, sucrose, starch, or glycogen.

3. Carefully dissolve part of the unknown in distilled water to prepare a 1% solution

(0.060 g carbohydrate in 6 mL water).

4. Also prepare a 10% solution by dissolving 0.1 g of carbohydrate in 1 mL of water.

5. Save the remainder of the solid for the mucic acid test.

6. Apply whichever tests are necessary to identify the unknown.

QUESTIONS

1. Find the structures for the following carbohydrates (sugars) in a reference work or
a textbook, and decide whether they are reducing or nonreducing carbohydrates
(sugars): sorbose, mannose, ribose, maltose, raffinose, and cellulose.

2. Predict the results of the following tests with the carbohydrates listed in question
1: Molisch, Bial, Seliwanoff (after 1 minute and 6 minutes), Barfoed, and mucic
acid tests.

3. Give a mechanism for the hydrolysis of the acetal linkage in sucrose.

 Experiment No.09 Isolation and Identification of Casein
Objectives

1. To isolate the casein from milk under isoelectric conditions.

2. To perform some chemical tests to identify proteins.

Principle
There are three kinds of proteins in milk: caseins, lactalbumins, and lactoglobulins.
Casein is a phosphoprotein, which has phosphate groups are attached to some of the amino
acid side chains. These are attached mainly to the hydroxyl groups of the serine and
threonine moieties. Actually, casein is a mixture of three similar proteins, which differ
primarily in molecular weight and amount of phosphorus they contain (number of
phosphate groups).
Casein exists in milk as the calcium salt, calcium caseinate. This salt has a complex
structure. It is composed of α, β, and κ caseins which form a micelle, or a solubilized
unit. Calcium caseinate has its isoelectric (neutrality) point at pH 4.6. Therefore, it is
insoluble in solutions of pH less than 4.6. The pH of milk is about 6.6; therefore casein
has a negative charge at this pH and is solubilized as a salt. If acid is added to milk, the
negative charges on the outer surface of the micelle are neutralized (the phosphate groups
are protonated) and the neutral protein precipitates:

In the first part of the experiment, you will be isolating casein by lowering milk pH to
4.6. The fat that precipitates along with casein can be removed by dissolving it in alcohol.

In the second part of the experiment, you are going to prove the precipitated product is a
protein. The identification will be achieved by performing some important chemical tests.
1. The Biuret Test. This is one of the most general tests for proteins. When a
protein reacts with copper(II) sulfate, a positive test is the formation of a copper
complex which has a violet color.
2. The Ninhydrin Test. Amino acids with a free –NH2 group and proteins
containing free amino groups react with ninhydrin to give a purple-blue
complex.
3. The Million Test. Heavy metal ions precipitate proteins from solution.
Although many di- and tri-valent ions can cause precipitation, Hg 2+, Cd2+ and
Pb2+ are infamous toxins to human. They can cause serious damage to
proteins by denaturing them. This can result in death. The precipitation is a
result of proteins becoming cross-linked by the heavy metals. The Million’s
test (shown below) is specific for the hydroxy-phenyl group of tyrosine and
will result in a red precipitate.
4. The Xanthoproteic Test. This is a characteristic reaction of proteins that
contain phenyl rings. Concentrated nitric acid reacts with the phenyl ring to
give a yellow-colored aromatic nitro compound (ortho position). Additional
alkali at this point will deepen the color to orange.
Chemicals and Equipment
1. Hot plate
12. Concentrated nitric acid
2. Büchner funnel in a no. 7 one-hole
rubber stopper 13. 2% albumin

3. 500-mL filter flask 14. 2% gelatin

4. Filter paper (Whatman no. 2, 7 cm) 15. 2% glycine

5. Cheese cloth 16. 5% copper(II) sulfate
6. Rubber band 17. 5% lead(II) nitrate
7. Boiling chips 18. 5% mercury(II) nitrate
8. 95% ethanol 19. Ninhydrin reagent
9. Diethyl ether–ethanol mixture 20. 10% sodium hydroxide
10. Regular milk 21. 1% tyrosine
11. Glacial acetic acid 22. 5% sodium nitrate