ILOILO DOCTORS’ COLLEGE

COLLEGE OF MEDICAL LABORATORY SCIENCE

ESMAYA, JESSALYN T. DEC. 18, 2022

BSMLS II-B
Laboratory Activity 8: Identification of Amino Acids through Colorimetric Tests

NINHYDRIN TEST
Describe a positive ninhydrin test Paste a picture below a positive result
- A positive result shows by the
appearance of a complex with a purple
color.

The reagent ninhydrin will react at

what pH?

- 5.5

XANTHOPROTEIC TEST
Describe a positive xanthoproteic test Paste a picture below a positive result

- A positive xanthoproteic test yield a

yellow, dark-yellow or orange color.

What is the reason upon the addition of

alkali the precipitates turn orange?

- The benzene ring in the amino acids is

nitrated by heating with nitric acid and
forms yellow nitro-compounds which
turns to orange color with alkali.
HOPKINS-COLE TEST
Describe a positive Hopkins-Cole test Paste a picture below a positive result

- A positive Hopkins-cole test yield a

violet color.

What is the other name for this test?

- Glyoxylic acid reaction

What is the purpose of adding concentrated

sulfuric acid to the test?

- helps to stabilize the glyoxylic acid

and prevent its decomposition and
the release of carbon dioxide.

MILLON’S TEST
Describe a positive Millon’s test Paste a picture below a positive result

- A positive Million’s test yield a red

color.

This test is specific for proteins

containing which amino acid?

- A specific test for tyrosine, the

only amino acid containing a
phenol group, a hydroxyl group
attached to a benzene ring.

SAKAGUCHI TEST
Describe a positive Sakaguchi test Paste a picture below a positive result

- A positive Sakaguchi test yield a

red color.

The test is specific for which amino

acid?

- Arginine

What is the purpose of adding sodium

hypochlorite to the test?

- To give a red-colored complex.

NITROPRUSSIDE TEST
Describe a positive nitroprusside test Paste a picture below a positive result

- A positive Nitroprusside test yield a

red color complex.

What is the reason behind many

proteins giving a positive result after
heat coagulation or denaturation?

- When a solution of a protein is

boiled, the protein frequently
becomes insoluble. It is denatured
and remains insoluble even when
the solution is cooled.

LEAD ACETATE TEST

Describe a positive lead acetate test Paste a picture below a positive result

- A positive Lead acetate test yield a

brownish-black color.

The test is specific for which amino

acid?

- Cysteine and methionine

PAULY’S TEST
Describe a positive Pauly’s test Paste a picture below a positive result

- A positive Pauly’s test yield a

cherry-red color

With tyrosine,
Is formed.

- Form a cherry-red diazotized

product.
SULLIVAN TEST
Describe a positive Sullivan test Paste a picture below a positive result

- A positive Sullivan test yield a

chromogen color.

What is the purpose of adding sodium

dithionite to the test?

- Detect the presence of methionine

in peptides and protein.

Experiment Questions
1. An unknown protein yielded the following test results using the procedure in this experiment. Based on
these results, what amino acids must be present in the protein?

Test Observation Conclusion

Ninhydrin Yellow Solution α-amino acids

Xanthoproteic (+) yellow solution tyrosine and tryptophan

Millon’s (+) red precipitate tyrosine

Hopkins Cole (-) colorless tryptophan

Nitroprusside (+) red solution cysteine

2. Amino acid sequencing of vasopressin revealed the following primary structure:

Gly-Arg-Pro-Cys-Asn-Gln-Phe-Tyr-Cys

Describe the results you would expect when vasopressin is tested with the following reagents:

Test Observation
Xanthoproteic -when the reaction occurs in basic solution the yellow color deepens.
Millon’s -some proteins that contain tyrosine initially form white participate
that turns into red when heated.
Hopkins Cole -a positive result can be observed by the appearance of a purple-
colored ring near the interactions between two layers
Nitroprusside - Nitroprusside then binds, causing a purple hue in 2-10 minutes.
Laboratory Activity 10: Physical and Chemical Denaturation of Proteins
COAGULATION TESTS
BY HEAT
Describe a positive coagulation test by Paste a picture below of a positive result
heat

- A positive result of the heat

coagulation test is represented
by the formation of a dense
coagulum at the upper part of
the solution.
Is it soluble in water?

- No

BY ALCOHOL
Describe a positive coagulation test by
heat

Is it soluble in water?

- Yes

PRECIPITATION TESTS
HELLER’S RING TEST
Describe a positive Heller’s ring test Paste a picture below of a positive result

- A white ring appears between

the two layers
What does the formation of the ring
indicate?

- Indicates the presence of

proteins in the given sample. A
white ring is caused due to
albumin precipitation.

ROBERT’S TEST
Describe a positive Robert’s test Paste a picture below of a positive result

- It is similar to Heller's test, that

has white ring.

What does the formation of the ring

indicate?

- A white ring develops at the

junction of the two liquids
which indicates the presence
of albumin in the urine sample.

PRECIPITATION TESTS
SALT OF HEAVY METALS
Paste a picture of a positive result
Silver nitrate Lead acetate Ferric chloride Barium chloride

Are these precipitate soluble in water?

- Yes
Why do you call these metallic precipitates?

- Transforms dissolved contaminants into an insoluble solid, facilitating the contaminant's subsequent
removal from the liquid phase by sedimentation and / or filtration.

ALKALOIDAL REAGENTS
Paste a picture of a positive result
Esbach’s reagent TCA

Describe a positive test with Describe a positive test with TCA

Esbach’s reagent
- A positive test of TCA yield a colorless.
- A positive Esbach’s reagent
yield a yellow color.

ISOELECTRIC PRECIPITATION TEST

 What does the curdy white Paste a picture below of a positive result
precipitate indicate?

- contains hard water, tube well

water, produces curdy white
precipitates.
What does the absence of a
precipitate indicate?

- Precipitation reactions occur

when cations and anions in
aqueous solution combine to
form an insoluble ionic solid
called a precipitate.
HEAT AND ACETIC ACID TEST
Describe a positive heat and acetic Paste a picture below of positive result
acid test

- A positive heat and acetic acid

test yield a cloudiness or
turbidity.
What does the presence of
cloudiness in the upper notion of the
solution indicate?

- cloudiness may be caused by

excessive cellular material or
protein in the urine. If
cloudiness is still present,
protein is present in urine. If
cloudiness disappears, that is
due to phosphates or
carbonates present in urine.

What does the disappearance of

cloudiness upon the addition of
acetic acid acetate?

Experiment Questions
1. What are zwitterions? Do they interfere with the precipitation tests?
- A zwitterion is an ion that contains two functional groups. They do not interfere with precipitation
because they are polar molecules such that it can act as an acid as well as a base. Zwitterions are
molecules with a minimum of 2 functional groups. One of the groups has a positive charge and the other
a negative charge. The overall charge on the zwitterion is zero.
2. What is isoelectric point? Is it related to the full and half saturation points of proteins?
- The isoelectric or isoionic point of a protein is the pH at which a protein carries no net electrical charge
and hence is considered neutral
Laboratory Activity 11: Chemical Tests for Proteins in Solutions
BIURET TEST
Describe a positive Biuret test Paste a picture below of a positive result

- A positive Biuret test yield a

violet color due to the copper
ion complex with the amide
group of the protein.
What is the minimum requirement for a
positive reaction?

- at least two peptide linkage

Experiment Questions
1. Do all proteins react positively with the Biuret test? Explain your answer.
- Yes, because all proteins and peptides give positive results and because the bonds in biuret molecules
([H2N-CO]2NH) are similar to peptide bonds, a positive result for this test is also received when the
analyte contains them. Proteins bind copper at higher pH, generating the biuret complex in particular.
2. What are peptides? State the physiological significance of breaking down proteins into peptides.
- Peptides are short chains of amino acids linked by peptide bonds. Long chains of amino acids are n
called proteins. It is necessary to break down proteins into small peptides (tripeptides and dipeptides) and amino
acids so they can be absorbed by the intestines, and the absorbed tripeptides and dipeptides are also further
broken into amino acids intracellularly before they enter the bloodstream.
3. What are salting in and salting out processes? Are they involved in the formation of precipitates?
- Salting in refers to the effect where increasing the ionic strength of a solution increases the solubility
of a solute, such as a protein and the salting out is a purification method that utilizes the reduced solubility of
certain molecules in a solution of very high ionic strength. The salting out process is a purification method that
relies on the basis protein that’s why this process are involved.

Laboratory Activity 13: Chemical Tests for the Identification of Carbohydrates

MOLISCH TEST
Describe a positive Molisch test

- A purple ring appears at the

interface between the acid and
test layers which confirms the
presence of carbohydrates.
What is the reason why Molisch test is
considered a general test for
carbohydrates?

- Because Molisch's test for

carbohydrates is very useful in
the detection of the presence of
carbohydrates in a substance.
I2/KI TEST
When do you perform the iodine test? Paste a picture below a positive result

- mainly performed to test the

presence of carbohydrates

What substances are responsible for

the different colors of the solution
after the iodine test?

- Using an iodine solution, you

can test for the presence of
starch. When starch is present,
the iodine changes from brown
to blue-black or purple.
What is the significance of the
different colors produced during the
iodine test?

- A positive result for the iodine

test (starch is present) was a
colour change ranging from
violet to black; a negative result
(no starch) was the yellow
colour of the iodine solution.

BIAL’S TEST
Describe a positive Bial’s test

- A positive test is indicated by

the formation of a bluish
precipitate.
What substances give positive and
negative results with the Bial’s test?

- A positive test for pentoses is

indicated by the formation of a
bluish precipitate. A negative
test is indicated by the
formation of all other colors.

SELIWANOFF’S TEST
Describe a positive Seliwanoff’s test Paste a picture below of a positive result

- a red color is formed rapidly

indicating a positive test.
What is/are the reason/s why fructose
reacts more rapidly than glucose with
Seliwanoff’s reagent?

- Prolonged boiling can transform

glucose to fructose by the
catalytic action of acid and form
cherry red-complex giving a
false-positive result.
 BENEDICT’S TEST
Describe a positive Benedict’s test Paste a picture below of a positive result

- Benedict's reagent is shown by

a color change from clear blue
to brick-red with a precipitate.

Compare and contrast the color

changes of the different sugars
subjected to Benedict’s test.

- Benedict's solution to test for

simple carbohydrates like
glucose. Benedict's solution is
blue but, if simple
carbohydrates are present, it
will change color to
green/yellow if the amount is
low and red if it is high.

BARFOED’S TEST
Describe a positive Barfoed’s test Paste a picture below of a positive result

- A positive test is a dark red

precipitate and is evidence of a
reducing monosaccharide.
What is/are the reason/s why reducing
disaccharides react very slowly with
the Barfoed’s reagent?

- The reaction with disaccharides

is slower because disaccharides
have to get hydrolyzed first and
then react with the reagent
cupric acetate to produce
cuprous oxide.

Experiment Questions
1. When an aldose reacts with Barfoed’s reagent, what type of organic compound forms? What type of
chemical reaction is this?

- The aldehyde group of aldose sugar is oxidized to the carboxylic group known as, aldonic
acid. Barfoed's reagent is copper(II) acetate in acetic acid which on reduces to copper(I)
oxide forming a brick-red precipitate.

2. Explain why fructose, an α-hydroxyketose reacts with Benedict’s reagent. What structural
rearrangement is necessary for this reaction to occur?

- Both fructose and alpha-hydroxyketose are reducing sugars. They both contain a ketone group that will
react to form the red color of a positive Benedict's test. For the Benedict's test to successfully occur, the
sugar must have an aldehyde or ketone functional group.

3. Explain what happened when you mixed sucrose with Seliwanoff’s reagent. Was this the result you
expected? Explain your answer.

- In this test, sucrose is first hydrolyzed into fructose and glucose. Fructose is a ketose, which produces a
cherry-red product in Seliwanoff's test, which would explain the cherry-red solution.
4. An unknown carbohydrate tested positive with Molisch reagent, formed a red precipitate with
Barfoed’s reagent within 3 minutes, and turned blue-green when mixed with Bial’s reagent. What
carbohydrate is this? Why?

- Bial reagent is used to differentiate the presence of pentose and hexose sugar. Pentose sugar gives
green coloured precipitate and hexose sugar gives muddy brown precipitate. The unknown solution has
given muddy brown precipitate with Bial reagent. It means the unknown carbohydrate is a hexose.

5. Erythrose is a four-carbon aldose. Describe the results of testing it with the following reagents:
a. Molisch - The presence of carbohydrates is indicated if the test solution creates purple or purplish-red
tinted layers. As a result, the Molisch test results are positive.

b. Barfoed’s - If the color appears within the first few minutes, the sample contains reducing
monosaccharides. However, if the color appears later than the first 3 minutes, the sample is of reducing
disaccharides.

c. Benedict’s - In general, blue to blue-green or yellow-green is negative, yellowish to bright yellow is a

moderate positive, and bright orange is a very strong positive.

d. Seliwanoff’s - The formation of the cherry red-colored complex indicates a positive result which means
that the given sample contains ketoses. The absence of such color or the appearance of the color after a
prolonged period of time indicates a negative result which means that the test sample doesn't have
ketoses.

Results for Laboratory 14 (copy the tables below on your answer sheet)

TEST FOR UNSATURATION

Describe a positive test for unsaturation. Paste two pictures below showing the original color when
the KI or bromine water was immediately added and the final
color after mixing was done.

If the orange red color of bromine disappears

then the given organic compound is
unsaturated.

What is responsible for the color change in KI

or bromine water after mixing?

- Bromine Water is a pale yellow/orange

color. When it is added to potassium
iodide solution it oxidise the iodide ion
to iodine, which is also colored -
forming a brown solution Hence one
might notice an intensification of color.
ACROLEIN TEST

Describe a positive acrolein test. Paste a picture below of the acrolein test set-up.
- A pungent irritating odor or smell of
acrolein confirms the presence of fat or
oil.

Name the major products formed.

- product formed involves the addition of
two acrolein molecules to the primary
amine of GPEtn lipids and subsequent
aldol condensation to form 1,2-diradyl-
sn-glycero-3-phosphoethanol-(3-formyl-
4-hydroxy)piperidine (FHP) lipids.

Analysis of Lipids
SAPONIFICATION

Describe the appearance of the soap formed. Paste a picture below of a positive result for each test tube
in the written procedure.
- Soap can be made from the base
hydrolysis of a fat or an oil. This
hydrolysis is called saponification, and
the reaction has been known for
centuries.

Based on the written procedure, what was

formed in the hydrolytic process in the first test
tube?
- Indicates that lipids is present in sample
A.

Based on the written procedure, what does the

scum on the top layer of the third test tube
indicate?
- the third test tube indicates the presence
of insoluble soap without any froth.

Based on the written procedure, what was

formed after the addition of HCl in the third test
tube?
- The solid formed when the products of
the reaction are passed through the
calcium hydroxide solution is calcium
carbonate.
LIEBERMANN-BURCHARDT REACTION

Describe a positive Liebermann-Burchardt test. Paste a picture below of a positive result

- The formation of a green or green-blue
color after a few minutes is positive.

What is the effect of the presence of water in

the test?
- The Liebermann–Burchard or acetic
anhydride test is used for the detection
of cholesterol

What is the purpose of running this test? What

does it test for?
- The purpose of this test is to determine
acceleration, maximum running speed
and speed endurance, depending on the
distance run.

SALKOWSKI TEST

Describe a positive Salkowski test. Paste a picture below of a positive result

- A solution that has tested positive on
this qualitative chemical test exhibits
two distinct layers in a test tube; the
upper layer (chloroform) gets blueish
red to violet color, while the layer of
sulfuric acid becomes yellow to green,
with greenish glow being visible.

To get a positive result, what would the liquid

layer at the lower portion of the solution
exhibit?
- It is essential that you know whether
the aqueous layer is above or below the
organic layer in the separatory funnel,
as it dictates which layer

The upper chloroform/carbon tetrachloride

layer develops _________.
- Carbon tetrachloride
Results for Laboratory Activity 15 (copy the tables below on your answer sheet)

AMYLASE

Test Tube 1 (boiled saliva) Test Tube 2 (unboiled saliva)

Expected observation
with Fehling’s solution
- The appearance of a
reddish-brown
precipitate indicates a
positive result and the
presence of reducing
sugars.

Expected observation
with iodine solution
- The appearance
of blue-black or
purple color
represents a
positive test,
indicating the
presence of
starch.

Write down the salivary amylase-catalyzed reaction in this experiment.

- Salivary amylase, formerly known as ptyalinis, a glucose-polymer cleavage enzyme that is
produced by the salivary glands, breaks down starch into maltose and isomaltose.

Conclusion
- In conclusion amylase, like other enzymes, works as a catalyst. All catalysts are enzymes,
but not all enzymes are catalysts.
 OXIDASE

Expected observation after Test Tube 1 (warm filtrate) Test Tube 2 (unheated filtrate)
addition of 0.5 mL phosphate
buffer with pH 6.0 and 2 mL of
freshly-prepared 0.2% catechol
solution

Write down the oxidase-catalyzed reaction in this experiment.

Conclusion

Analysis of Enzymes
CATALASE

Test Tube 1 (without vinegar) Test Tube 2 (with vinegar)

Expected observation
after addition of 3%
hydrogen

peroxide (H2O2)

Write down the catalase-catalyzed reaction in this experiment.

Conclusion
 PROTEASE (e.g., pepsin)

Test Tube A Test Tube B Test Tube C Test Tube D

(pepsin + (pepsin + (boiled (boiled
dilute HCl) distilled water) pepsin + pepsin +
dilute HCl) distilled
water)

Expected
observation of
egg white cube

Conclusion

RENNIN

Expected observation after Test Tube 1 Test Tube 2

incubation at 40°C for 20
[without (NH4)2C2O4] [with (NH4)2C2O4]
minutes

After full incubation, the

samples are assessed
either by visual
comparison of the
uninhibited growth of
the microorganism in the
dish and the growth on

Conclusion