Biochemistry for Medical Laboratory Science

Lab // Carbohydrates: Test Based on the Formation of Furfural and its Derivatives

Introduction Principle
 A carbohydrate is a polyhydroxy aldehyde,
a polyhydroxy ketone, or a compound that
yields polyhydroxy aldehydes or polydroxy
ketones upon hydrolysis.

General Tests for Carbohydrates

Molisch Test
Classification of Carbohydrates

 Is the general test for carbohydrates.

 The sugars are mixed with a-naphthol. The
Monosaccharides test tube is inclined, and concentrated H2SO4
 “Simple sugars”, are highly soluble in is added along the side of the tube, causing the
water, less soluble in ethanol, and insoluble in formation of a lower layer of acid. The
ether. concentrated H2SO4 will dehydrate the sugar
 They are either aldoses or ketoses. allowing it to react with the alcohol forming
 They may also be classified into furfural or hydroxymethyl-furfural.
tetroses, pentoses, or hexoses.
 Free monosaccharides are all reducing Procedure
sugars. 1 Mix 4 ml of distilled water and two drops
 They also exhibit mutarotation. of the Molisch’s reagent in a test tube. This
tube will serve as the control.
2 Place 4 ml of 3% solution of glucose in a
Disaccharides
second test tube. Add two drops of the
 Are formed by two molecules of
Molisch’s reagent and mix the contents by
monosaccharides.
gently shaking the test tube.
 Examples:
3 Incline the test tube and cautiously add
 Maltose – which are abundant in
about 5 ml of concentrated sulfuric acid,
germinating barley.
allowing the acid to run down the side of
 Sucrose – also known as cane sugar or the tube. Sulfuric acid is denser than water
beet sugar. and will form a lower layer. Note the color
 Lactose or milk sugar – which does of the ring formed at the junction if the two
not taste very sweet and is not liquids.
fermented by yeast. 4 In the same manner of adding acid, add
sulfuric acid to the control tube.
Polysaccharides 5 Repeat the above test with 3% sample
 Found in nature function either as structural solutions of glucose, xylose, lactose, and
units (e.g. cellulose), or for storage such as starch.
starch, dextrin, glycogen, and insulin. 6 Record all results.

David, M.A 7
Biochemistry for Medical Laboratory Science
Lab // Carbohydrates: Test Based on the Formation of Furfural and its Derivatives

Bial’s Orcinol Test 3 Place the test tubes in a water bath filled
 Used to determine the presence of pentoses with boiling water and allow them to stay
and nucleotides and nucleotides that contain there for exactly 1 minute.
pentose sugars. 4 Note the changes and record which test
 When pentoses are treated with orcinol, tube gives a positive result in the shortest
furfurals are formed and they will yield a blue- time.
green compound in the presence of ferric ions. 5 Continue heating and observe the color
 The reaction is not specific for pentoses changes at 1 minute intervals.
because other compounds like trioses, uronic 6 Record the time required for a positive test
acids, and certain heptoses will also give blue for each sample
or green products. Hydroxymethyl furfural is
formed from hexoses to give yellow-brown
condensation products.

Procedure
1 Place 1 ml of each 3% solution of xylose,
glucose, and starch in separately labeled
test tubes.
2 Add 3 ml of Bial’s reagent to each test
tube.
3 Carefully heat each tube over a Bunsen
flasme until the solution begins to boil.
Add one to two drops of 10% FeCl3
solution.
4 Note the color of the product formed.
5 Record your results in the table.

Seliwanoff’s Test

 This test is used to differentiate

ketohexoses from aldohexoses. Ketohexoses
react faster with the solution containing
hydrochloric acid and resorcinol than
aldohexoses.
 The dehydrated ketohexoses form a bright
cherry red condensation product, while the
aldohexose yields only a pale pink coloration,
a negative result. In this test, prolonged
heating of samples should be avoided.

Procedure
1 Place 1 ml each of 3% xylose, glucose,
fructose, and sucrose in separately labeled
test tubes.
2 Add 4 ml of Seliwanoff’s reagent to each
test tube.

David, M.A 8
Introduction: Tests Based on the
Formation of Furfural and its Derivatives
Sugars are stable to hot dilute mineral acids, But hot sulfuric acid (H2So4) will dehydrate them
into furfural and its derivatives. Pentoses, when heated with concentrated sulfuric acid, will form
furfuraldehyde, while hexoses will form 5-hydroxymethyl furfural.
In the presence of acid, phenolic compounds like a-naphthol, orcinol, and resorcinol will react
with furfural or hydroxymethyl furfural to form colored condensation products. The formation of
these colored compounds constitutes a positive test for carbohydrates.

Test Based on the Formation of Furfural and its Derivatives

I. General Tests for Carbohydrates

Molisch Test
The Molisch test is the general test for carbohydrates. The sugars are mixed with
a-naphthol. The test tube is inclined, and concentrated H2SO4 is added along the side of the
tube, causing the formation of a lower layer of acid. The concentrated H2SO4 will dehydrate the
sugar allowing it to react with the alcohol forming furfural or hydroxymethul-furfural. Formation of
a purple ring at the interface of the two liquids will indicated the presence of a carbohydrate.

Bial’s Orcinol Test

Bial’s test is used to determine the presence of pentoses and nucleotides and
nucleotides that contain pentose sugars. When pentoses are treated with orcinol, furfurals are
formed and they will yield a blue-green compound in the presence of ferric ions. The reaction is
not specific for pentoses because other compounds like trioses, uronic acids, and certain
heptoses will also give blue or green products. Hydroxymethyl furfural is formed from hexoses to
give yellow-brown condensation products.

Seliwanoff’s Test
This test is used to differentiate ketohexoses from aldohexoses. Ketohexoses react
faster with the solution containing hydrochloric acid and resorcinol than aldohexoses. The
dehydrated ketohexoses form a bright cherry red condensation product, while the aldohexose
yields only a pale pink coloration, a negative result. In this test, prolonged heating of samples
should be avoided.

OBJECTIVE: To be able to identify the different types of carbohydrates using the different
specific chemical tests
REDUCING SUGAR:
A reducing sugar is any sugar that, in a solution, has an aldehyde or a ketone group. The
enolization of sugars under alkaline conditions is an important consideration in reduction tests.
The ability of a sugar to reduce alkaline test reagents depends on the availability of an aldehyde
or keto group for reduction reactions. A number of sugars especially disaccharides or
polysaccharides have glycosidic linkages which involve bonding a carbohydrate (sugar)
molecule to another one, and hence there is no reducing group on the sugar; like in the case of
sucrose, glycogen, starch and dextrin. In the case of reducing sugars, the presence of alkali
causes extensive enolization especially at high pH and temperature. This leads to a higher
susceptibility to oxidation reactions than at neutral or acidic pH. These sugars, therefore,
become potential agents capable of reducing Cu+2 to Cu+, Ag+ to Ag and so fort. Most
commonly used tests for detection of reducing sugars are Benedict’s Test, Barfoed’s Test and
Tollen's Test.

PDF
● It must have a free aldehyde or ketone group
● Common dietary monosaccharides: glucose, galactose and fructose
1. Benedict’s Test
2. Barfoed’s Test
3. Tollen’s Test

BENEDICT’S TEST
• It is a very sensitive test done under mildly alkaline conditions
• Benedict’s Reagent: Composed of CuSo4, Na2CO3, sodium citrate
• Positive result: Change of color of Benedict’s reagent
• Also used to test for the presence of glucose in urine
• When Benedict’s solution and simple carbohydrates are heated, the solution changes to
orange red/ brick red. This reaction is caused by the reducing property of simple carbohydrates.
The copper (II) ions in the Benedict’s solution are reduced to Copper (I) ions, which causes the
color change.
• benedicts solution: blue solution
-> carboxylate anion -> brick red precipitate

BENEDICT’S TEST
• Sample: Urine
• Aim: To detect the presence of reducing sugar in urine
• PROCEDURE:
1. Using a pipette, accurately take 5 mL of Benedict’s
reagent and slowly transfer it to the test tube.
2. Take 5 mL of freshly collected urine by pipette and add it to the test tube with Benedict’s
reagent.
3. Place the test tube in boiling water bath for 2-3 minutes
4. Observe the color of the solution and note whether a precipitate was formed
5. Avoid prolonged heating
6. Record your results

BARFOED’S TEST
• Barfoed’s reagent – contains cupric acetate in dilute acetic acid
• It oxidizes monosaccharides but not oligosaccharides
• Disaccharides are less oxidized but are oxidized if they undergo prolonged heatingàcausing
hydrolysis of the disaccharides into
monosaccharidesàproduces a positive result
• It is used to distinguish between monosaccharide, disaccharides, and
oligosaccharides
• Positive result: Brick red precipitate
• Unlike Benedict’s test, this test is carried out under acidic rather than basic medium

PROCEDURE:
1. Place 1 mL each of 3% solutions of glucose, xylose, fructose, lactose and starch in
separately labeled test tubes
2. Add 3 mL of Barfoed's reagent in each test tube
3. Prepare a control tube using distilled water instead of sugar solution
4. Place all the tubes in a boiling water bath for 10 minutes
5. Record your observations
TOLLEN’S TEST (SILVER MIRROR TEST)
• Sugars with aldehyde groups are capable reducing Tollen’s reagent
• Tollen’s reagent: Ammoniacal solution of silver
• Positive result: Gray to black precipitate (silver mirror)
PROCEDURE:
1. Place 5 drops of 3% solutions of glucose,
xylose, and sucrose in separate test tubes
2. Add 2 mL of Tollen’s reagent into each tube
Preparation: Add 1 drop of NaOH solution to 6 mL of 5% AgNO2. Add dilute NH4O4 (1 mL
concentrated NH4OH + 5 mL water) until the brown precipitate of silver oxide that forms just
dissolves
This reagent must be prepared fresh and not stored since it decomposed when left standing and
yields an explosive decomposition product. Discard all leftover materials.
3. Boil for about 5 minutes. Note and record your observations.
 Proteins: Color Test for Amino Acids Ninhydrin Test Procedure
• Ninhydrin reagent
• Ninhydrin Test • Put the test tube in a boiling water bath for 5 minutes.
• Xanthoproteic Test
• If the solution turns blue, it indicates the presence of alpha
• Pauly’s Diazo Test amino acids.
• Millon’s Test • If the solution turns yellow, it indicates the presence of
• Histidine Test imino acids (Proline)
• Hopkin’s Cole Test • In the pH range of 4-8, all α- amino acids react with
• Sakaguchi Test ninhydrin (triketohydrindene hydrate), a powerful oxidizing
• Lead Acetate Test agent to give a purple colored product (diketohydrin)
termed Rhuemann’s purple.
• Follins McCarthy Sullivan’s Test
• All primary amines and ammonia react similarly but
• Isatin Test without the liberation of carbon dioxide.
• Biuret Test

Ninhydrin Test Procedure Ninhydrin Test

• When amino acids are sprayed with ninhydrin, it will give
a blue to violet colored result. The presence of amino
group including those found in amines and ammonia will
also give the same result except for proline and
hydroxyproline that will give a yellow colored result.
• The imino acids proline and hydroxyproline also react
with ninhydrin, but they give a yellow colored complex
instead of a purple one.
• Besides amino acids, other complex structures such as
peptides, peptones and proteins also react positively
when subjected to the ninhydrin reaction.

Xanthoproteic Test Procedure

• Few drops of nitric acid
Pauly’s Diazo Test Procedure
• Place some marble chips in the solution to avoid bumping of • Unknown sample then place it in a small ice bucket
the solution while boiling. Heat the solution using Bunsen
burner and cool it by placing under the running tap water. • Take pre-chilled sulphanilic acid then add few drops to
the same test tube with sample
• Add few drops of 40% NaOH, if the solution changes to
orange it indicates the presence of aromatic amino acids. • Add pre-chilled sodium nitrate
• Aromatic amino acids, such as Phenylalanine, tyrosine and • Add dropwise of sodium carbonate solution
tryptophan, respond to this test. • Appearance of red color during the addition of sodium
• In the presence of concentrated nitric acid, the aromatic carbonate solution indicates the presence of histidine
phenyl ring is nitrated to give yellow colored nitro- and tyrosine.
derivatives. • Is based on the principle of coupling between the amino
• At alkaline pH, the color changes to orange due to the acids and the diazonium ion formed in the reagent.
ionization of the phenolic group.
• Collagen and gelatin do not give positive reaction
 Pauly’s Diazo Test Procedure Hopkin’s Cole Test Procedure
• Add 1 ml acetic acid-glyoxylic acid reagent
• Pauly’s reagent consists of sulphanilic acid dissolved in • Mix the solution the carefully, add few drops of conc.
concentrated hydrochloric acid. The sulphanilic acid
undergoes diazotization in the presence of sodium nitrate sulfuric acid running on the side of the test tube.
and hydrochloric acid. • The tube must be inclined
• The result of the diazotization reaction is the diazonium • Purple-violet ring indicates the presence of tryptophan.
salt (p-phenyldiazosulphonate). • This test is specific test for detecting tryptophan.
• The diazonium salt thus formed couples with histidine • The indole moiety of tryptophan reacts with glyoxylic acid
molecule in an alkaline condition to form a dark red or
cherry colored compound. The color might decrease in in the presence of concentrated sulfuric acid to give a
intensity and changes into an orange color when the purple colored product.
solution is made acidic. • Glyoxylic acid is prepared from glacial acetic acid by being
exposed to sunlight.

• The hopkins-cole test is used to determine the presence Lead Acetate Test Procedure
of the amino acid tryptophan, which has an indole ring • Add few drops of NaOH and marble chips
which is responsible for the purple - violet ring found at • Heat using a Bunsen burner for 5-10 minutes
the junction between the two layers by it's reaction with • Cool it down using a running tap water
glyoxylic acid in the presence of H2SO4.
• Add few drops of 10% lead acetate solution (lead sulfide)
• Gelatin, collagen and tyrosine fail to give a positive
• Black ppt indicates presence of cysteine
result with this test indicating the absence of
tryptophan in these proteins. • Sulfur containing amino acids, such as cysteine and
methionine.
• Upon boiling with sodium hydroxide (hot alkali), yield
*the reason why tryptophan is the only acid that yields
sodium sulfide. This reaction is due to partial conversion
positive result is because of the indole in it
of the organic sulfur to inorganic sulfide, which can
* Sulfuric acid (H2SO4)causes the separation of the two detected by precipitating it to lead sulfide, using lead
liquids acetate solution.

Isatin Test Procedure

Lead Acetate Test • Apply a drop of unknown amino acid solution on a filter
paper strip then dry the spot
S. (Proteins) + 2NaOH  Na2S • Apply isatin reagent onto the dried spot and dry it again
NaS + (CH3COO)2 Pb  PbS + 2CH3COONa • When blue colored spot is seen in the filter paper it
indicates the presence of imino acid (Proline &
The sulfahydryl or disulfide groups are converted to Hydroxyproline)
inorganic sulfide, Na2S, in strongly alkaline solution. This • Isatin reagent used in the test works as a visualizing agent
will react further with lead acetate to form a brownish- that provides different colors with different amino acids in
black ppt. of lead sulfide. the chromatography technique.
• The reaction between imino acids like proline and
hydroxyproline and isatin yields a blue-colored adduct.
 Folins McCarthy Sullivan’s Test Procedure
• Add few drops of NaOH followed by few drops of glycine Folins McCarthy Sullivan’s Test Principle
and sodium nitroprusside solution
• Thus, acid is added to the sample solution in order to
• Place the test tubes in a water bath maintained at 40oC,
for 15 minutes. destroy any tryptophan present by the process of acid
hydrolysis.
• Add 0.5ml 6N HCl into the same test tube taken from
water bath • Similarly, it has been stated that the addition of glycine
• On adding HCl, the content of the tube changes to red eliminates the detection of histidine.
color indicating the presence of methionine • The red color is obtained after the addition of sodium
• The Sullivan and McCarthy’s test is based on the reaction nitroprusside to an alkaline solution of methionine
between nitroprusside and alkaline solution of followed by the acidification of the reaction.
methionine under acidification.
• The test was developed when it was discovered that
tryptophan and histidine both produce a red color with
nitroprusside under similar conditions.

Sakaguchi Test Procedure

Sakaguchi Test Principle
• Transfer pre-chilled unknown amino acid into a test tube
• Add few drops of 40% NaOH, alpha-naphthol solution, 5% • Sakaguchi test is based on the principle of reaction
urea, hypobromite solution respectively between 1-naphthol and the guanidinium groups in
arginine, in the presence of an oxidizing agent.
• The color of the solution changes to red indicating the
presence of arginine • The Sakaguchi reagent consists of sodium hypobromite
and 1-naphthol. The sodium hypobromite acts as an
• Sakaguchi test is a biochemical test consisting of
oxidizing agent that facilitates the hydrogen bonding
colorimetric reaction for the detection and quantification
between two arginine molecules.
of guanidinium groups, used as a qualitative test for
arginine that is either free or in protein. • The reaction results in the formation of a red-colored
complex due to the formation of an indole-like
• The test is a specific test for arginine where the
structure.
guanidinium group of arginine reacts with 1-naphthol or
α-naphthol to produce a colored product.

Millon’s Test Procedure

Histidine Test • Add few drops of Millon’s reagent and mix.
• Place 2ml of unknown sample in a test tube • Add few drops of conc. nitric acid
• Add few drops of 5% bromine in 33% acetic acid solution • solution turns red it indicates the presence of tyrosine
• Keep the test tube at room temp for 10 minutes • Phenolic amino acids such as Tyrosine and its derivatives
respond to this test.
• Then add 2ml of ammonium carbonate solution to the
• Compounds with a hydroxybenzene radical react with Millon’s
test tube and keep it boiling water bath for 5 minutes. reagent to form a red colored complex.
• After 5 minutes, the color of the solution turns blue • Millon’s reagent is a solution of mercuric sulfate in sulfuric acid.
indicating the presence of histidine.
• The presence of phenol group in amino acid like tyrosine is
nitrated by a solution of mercuric and mercurous nitrates in
conc. Nitric acid. A white ppt. will start to form, turning brick
red on prolonged heating due to formation of a mercury
complex of nitrophenyl derivatives. Addition of NaNO2 turns
the ppt. to darker pink or red.
 PROTEINS
Biuret Test Protein Denaturation
• Coagulation • Precipitation
• This test give a positive result for compounds that
- Heat - Heavy Metal Salts
contain two or more peptide linkages. It will give a
- Inorganic Acids - Alkaloidal Reagents
distinctive purple color which is due to the formation
of complex cupric ions with the amino groups except - Alcohol
serine and threonine.
*Egg albumin contains peptide linkage
Heat
- The result was coagulation of the albumin solution.
- Egg-white is alkaline. Complete precipitation takes
place only in acid solution.

Inorganic Acids Inorganic Acids

• Strong acid will often denature (unfold) proteins.
• In the denaturation test, strong acid is used to test
Proteins that are properly folded into their normal
a solution for the presence of protein. When
3-dimensional shape tend to be soluble in
strong acid is added, the formation of a white
aqueous solution. However, proteins that have
precipitate (composed of denatured protein
been denatures tend to clump together and come
molecules) is considered a positive denaturation
out of solution as a precipitate because they are
test and indicates the presence of protein in the
no longer soluble when they are denatured and
solution.
clumped together.
• If no precipitate forms, the denaturation test is
negative, and indicates that no protein is present
in the solution.

Alcohol
• Denatures proteins by disrupting the side chain Alkaloidal Reagents
intramolecular hydrogen bonding. New hydrogen bonds
are formed instead between the new alcohol molecule
• Alkaloidal reagents (e.g. tannic acid &
and the protein side chains.
trichloroacetic acid) are high molecular weight
Heavy Metal Salts anions. The negative charge of these anions
• Proteins are precipitated by salts of heavy metals, such as counteracts the positive charge of the amino
mercuric chloride, zinc sulfate, lead acetate etc. in weak group in proteins giving a precipitate.
alkaline solutions, protein molecules carry negative • Orange ppt. and yellow ppt.
charges and combine with positively charged metal ions
to form insoluble salts which precipitate (white) from
the solution. The precipitated proteins are denatured
this process is irreversible
BIOCHEMISTRY
BIOCHEMISTRY FOR MEDICAL LABORATORY SCIENCE (CHEM123) | LABORATORY
BS Medical Laboratory Science | PROF. Kamille Samson | SEM 1 2022

1) Ability to form furfural and its derivatives.

Midterm Coverage 2) Ability to reduce and form characteristic compounds
o Carbohydrates (Part 1) within the agents like Barfoed’s Test or Barfoed’s
o Carbohydrates (Part 2) Reagent or Benedict’s Solution.
o Proteins
o Lipids (Emulsification) General Tests for Carbohydrates
o Lipids (Saponification) Based on the formation of furfural and its derivatives
Molisch Test
• General test for carbohydrates.
CARBOHYDRATES
• Sugars are mixed with alpha naphthol and then the test
Part 1: Carbohydrates
tube is inclined, and concentrated sulfuric acid is added
• Sugars are stable to hot dilute mineral acids, but hot
along the side of the tube, causing the formation of the
sulfuric acid (H2So4) will dehydrate them into furfural and
lower layer of acid.
its derivatives.
• The concentrated sulfuric acid with dehydrate the sugar
• Pentoses, when heated with concentrated sulfuric acid, will
allowing it to react with the alcohol and then it will form a
form furfuraldehyde, while hexoses will form 5-
furfural or hydroxymethyl furfural.
hydroxymethyl furfural.
• The formation of purple ring in the test tube at the interface
• In the presence of acid, phenolic compounds like a-
of two liquids will indicate the presence of carbohydrates.
naphthol, orcinol, and resorcinol will react with furfural or
hydroxymethyl furfural to form colored condensation
products. The formation of these colored compounds Molisch’s Test Procedure
constitutes a positive test for carbohydrates. 1) Gather the materials that are needed for the experiment.
• Biochemistry usually has qualitative tests. We only have 2) Label the four different test tubes (glucose, lactose,
changes in color or cloudiness in our tests. fructose, and sucrose) to avoid the confusion when
• Carbohydrates in theory includes polyhydroxy aldehydes transferring the carbohydrate sample.
and polyhydroxy ketones and their byproducts. 3) Drop 2 mL of each carbohydrate solution in the four test
• Carbohydrates make up most of our diet and serve as the tubes.
major source of our energy. 4) Add 10 drops of Molisch reagent to each carbohydrate
• As Asians, we love rice. It’s our number 1 source of energy. sample then mix thoroughly.
• Carbohydrates play an important part in our metabolic 5) Add 15 to 20 drops of concentrated sulfuric acid and allow it
processes by furnishing the carbon chain for compound to flow down on the side of the tube. Do not stir! The
synthesis by our living organism. sulfuric acid will form a bottom layer.
• Carbohydrates can be categorized in three parts: 6) Note the color that is formed at the zone in between the two
o Monosaccharides liquids.
o Disaccharides
o Polysaccharides Seliwanoff’s Test
• This test is used to differentiate the ketohexoses from
Categories of Carbohydrates aldohexoses.
Monosaccharides • Ketohexoses reacts faster with the solution containing
• Monosaccharides are known as simple sugars. hydrochloric acid and resorcinol than aldohexoses.
• Highly soluble in water, less soluble in ethanol, and • Dehydrated ketohexoses will form a bright cherry red
insoluble in ether. condensation product.
• They cannot be further hydrolyzed to simpler units. • Aldohexoses yields only a pale pink coloration.
• They can be either aldoses or ketoses depending on the • If it formed a bright cherry red condensation product, it
type of functional group present. is a positive result.
• They may be classified into tetroses, pentoses, or hexoses • A pale pink coloration indicates a negative result.
depending on the number of carbon atoms that they • In this test, prolonged heating of the sample should be
possess. avoided to avoid a false positive or false negative result.
• Free monosaccharides are all reducing sugars.
• They also exhibit multi-rotation or muter rotation which Seliwanoff’s Test Procedure
means that they can exist in alpha and beta forms. NOTE: Overheating of the solution should be avoided for the
reason of continuous boiling may cause false positive result.
Disaccharides 1) Add 2 mL of the sugar solution to 3 mL to seliwanoff's
• Formed by two molecules of monosaccharides. reagent.
o Maltose: abundant in germinating our barley. 2) Immerse the solutions in a boiling water bath. Observe the
o Sucrose: also known as sugar cane or beet sugar. color changes during the first 10 minutes of boiling.
o Lactose: known as milk sugar which does not taste 3) Remove the tubes from the water bath and put it on the test
very sweet, and it is not fermented by yeast. tube rack. Observe and write the final results.

Polysaccharides CARBOHYDRATES
• Polysaccharides are found in our nature function either as Part II: Carbohydrates
structural units or for storage such as starch, dextrin, • A reducing sugar is any sugar that, in a solution, has an
glycogen, and inulin. aldehyde or a ketone group. Therefore, most of
monosaccharides, and some of disaccharides, can reduce
We have tests to determine the presence of carbohydrates the oxidizing reagents such as cupric ion, dinitro salicylic
based on their abilities: acid, and picric acid.
BIOCHEMISTRY LABORATORY | BS Medical Laboratory Science

• The enolization of sugars under alkaline conditions is an • Barfoed’s Reagent actually oxidizes monosaccharides but
important consideration in reduction tests. cannot oxidize oligosaccharides.
• The ability of a sugar to reduce alkaline test reagents • Disaccharides are less easily oxidized but if they undergo
depends on the availability of an aldehyde or keto group for prolonged heating, that will cause hydrolysis of the
reduction reactions. disaccharides into monosaccharides, which will then give a
• A number of sugars especially disaccharides or positive result to the test.
polysaccharides have glycosidic linkages which involve • The concentration of sugar solutions can be used in this
bonding a carbohydrate (sugar) molecule to another one, test, and it should be approximately the same because the
and hence there is no reducing group on the sugar; like in use of a more concentrated disaccharide solution may give
the case of sucrose, glycogen, starch and dextrin. a much faster reaction than the relatively more dilute
• In the case of reducing sugars, the presence of alkali monosaccharide solution.
causes extensive enolization especially at high pH and • Unlike Benedict’s Test, Barfoed’s Test is carried out under
temperature. This leads to a higher susceptibility to acidic rather than basic medium.
oxidation reactions than at neutral or acidic pH.
• These sugars, therefore, become potential agents capable Barfoed’s Test (from YouTube)
of reducing Cu+2 to Cu+, Ag+ to Ag and so forth.
1) Measure 3 mL of freshly prepared Barfoed’s reagent into
• Most commonly used tests for detection of reducing sugars labelled test tubes.
are Benedict’s Test, Barfoed’s Test and Tollen's Test. 2) Add 1ml of the test solution to Barfoed’s reagent.
3) Add glucose to Barfoed’s reagent.
Test Based on the Reducing Property of Sugars 4) Place the test tubes into a boiling water bath and heat for 3
Benedict’s Test minutes. Cool and observe.
• Benedict’s Test is a very sensitive test that is done under a 5) Formation of red or yellow to orange precipitate is a positive
mildly alkaline condition. test for reducing monosaccharides.
• The reagent (Benedict’s) contains copper sulfate, sodium
carbonate and sodium citrate. From “Qualitative Tests for Carbohydrates”
• The formation of a brick red precipitate of cuprous oxide 1) Add 2 mL of Barfoed’s reagent in 4 test tubes.
(Cu+) is considered a positive result. 2) To each of the test tubes, add 10 drops of each of the
• Most aldehydes have the ability to reduce benedict’s sugar solution.
reagent. 3) Place the test tubes into the boiling water bath for 5
• Other compounds, like formic acid, hydrazobenzene, minutes. Let it cool and observe the first 15 minutes.
phenols, phenyl hydrazine, pyrogallol, and uric acid will also
give positive result. Tollen’s Test
• In Tollen’s Test, sugars with other aldehyde groups, are
Benedict’s Test for Reducing Sugars (from YouTube) capable of reducing Tollen’s Reagent to form a grey to
1) Using a pipette, accurately take 5 mL of Benedict’s reagent black precipitate, indicating a positive result.
and slowly transfer it to the test tube. • Tollen’s Reagent is an ammoniacal solution of silver.
2) Take 5ml of freshly collected urine by pipette and add it to • If the reaction vessel is clean and the rate of the position is
test tube with Benedict’s reagent. low enough, there will be silver deposits as a silver mirror.
3) The test tube should be held securely with the test tube
holder to heat it on the burner for 2 minutes. Tollen’s Test (from YouTube)
4) Observe the changes. 1) Take 1 mL of aqueous silver nitrate solution in a test tube.
5) On heating the sample, the presence of sugar is indicated 2) Add sodium hydroxide solution drop wise to get the
in the sample of urine when a green precipitate (traces of precipitate of silver oxide.
reducing sugar) is apparent. 3) Now, add 2 drops of ammonium hydroxide solution while
6) Different precipitates are formed depending upon the sugar shaking the mixture, previously formed precipitate
concentration in urine. dissolves. This is called Tollen’s reagent.
• Blue: none/no sugar 4) Take 2 mL of sugar solution in another test tube.
• Green: traces of reducing sugar 5) Take 2 mL of Tollen’s reagent to it.
• Orange red: moderate amount of reducing sugars 6) Formation of silver mirror on the walls of test tube indicates
• Brick red: large amount of reducing sugar presence of reducing sugar.

From “Qualitative Tests for Carbohydrates” PROTEINS

1) Add 2 mL of Benedict’s solution to 4 empty tubes and then Proteins
placed into the water bath for 30 seconds. • Proteins comes from the greek word “proteis” meaning first
2) If the solution remains clear blue, we can continue with the rank of importance.
test which means the reagent is not contaminated. • These are macromolecules composed of polymers of
3) Allow the four test tubes to cool down until they are just covalently linked amino acids that are involved in every
warm to touch. Then add 5 drops of the carbohydrate cellular processes.
solutions. • Because of their acid and basic amino acid compositions,
4) Place the four test tubes containing Benedict’s Solution and proteins can bear positive and negative charges
carbohydrate samples into a boiling water bath for 2 amphoteric.
minutes. Cool and observe what happens. • Synthesized in the liver and secreted by the hepatocyte into
the circulation.
Barfoed’s Test
• Barfoed’s Test contains cupric acetate in dilute acetic acid, Testing of Urine Albumin
and it is used to distinguish the difference between some • We aim to perform this test to detect the presence of
monosaccharides, disaccharides, and oligosaccharides. albumin in the given sample of urine.
BIOCHEMISTRY LABORATORY | BS Medical Laboratory Science

• Usually, for us to be able to know our protein levels or if • It can also be caused by kidney damage due to heart
there is a presence of protein in the urine, we use chemistry failure, high blood pressure, lupus, and cirrhosis.
(multi-sticks).
• Chemical Strips (kind of multi-stick) Detection of Albumin in Urine
- chemically produced Sulphosalicylic Acid Test
- dipped in urine; changes in color will occur • Formation of coagulation resulting in white cloudiness in the
- has a specific parameter for protein; has a designated solution.
number • The appearance of a whitish or cloudy turbid solution will
- the easiest and most convenient way to test the indicate the albumin’s presence in the sample.
presence of urine albumin
Sulphosalicylic Acid Test
Albumin 1) Take 2 ml urine sample in a measuring cylinder from the
• A protein that is produced by the liver. urine sample bottle.
• It is a typical constituent of the blood that is filtered by the 2) Take a test tube and pour the urine in the test tube.
kidney. 3) Using a dropper, take some sulphosalicylic acid.
• Significance: It maintains the intravascular oncotic pressure 4) Add few drops of sulphosalicylic acid in the test tube
by checking the stability of the fluid pressure inside the containing urine. A whitish color appears in the solution.
blood vessels. 5) Using a test tube holder, hold the test tube firmly and heat it
• Oncotic Pressure (Colloid Osmotic Pressure) gently upon a burner.
- Form of osmotic pressure that is exerted by proteins, 6) A whitish or cloudy turbid solution indicates the presence of
notably by albumin, in the blood vessel plasma. albumin in the urine sample.
- Usually tends to pull water into the circulatory system.
- The opposing force to capillary filtration pressure and Heller’s Test
interstitial colloidal osmotic pressure. • Presence of white ring that is caused by albumin
- Checks fluid stability to make sure it meets the precipitation.
equilibrium. • There will be changes that will take place in the test tube.
• Significance: Acts as a carrier protein for fatty acids, thyroid At the intersection of the two layers in the test tube, a white
hormones, and steroids in our blood. ring will appear, which indicates that albumin is present in
the urine sample.
What is Albuminuria?
Heller’s Test
1) Take 5 ml concentrated nitric acid in a measuring cylinder
from the reagent bottle.
2) Pour the concentrated nitric acid in a test tube from the
measuring cylinder.
3) Using a dropper, take some urine from the urine sample
bottle.
4) Now incline the test tube and add sample of urine by
means of a dropper along the inner side of the test tube so
that it forms a layer over the nitric acid.
5) The presence of white ring at the junction of two layers
indicates the presence of albumin in the urine.

In a healthy kidney, albumin cannot pass through the filter. (right) Other Informations
In a damaged kidney, albumin can pass through the filter since it cannot withstand the filtration
process due to damage.
What type of sample is preferred for the urine protein
examination?
• Usually seen in patients with damaged kidneys. • Usually, an early morning urine sample is preferred
• A normal functioning kidney has very less to no traces of because it is more concentrated.
albumin in the urine.
• About 250 mg of proteins in a normal urine in a day. What are the grades or levels of proteinuria?
• Any damage to the kidney will cause an unusual range of • Mild Proteinuria: less than 500 mg per dL (<500 mg/dL)
albumin that is way above the normal range or normal level • Moderate Proteinuria: up to 4 g per day (4 g/day)
that will pass through in the urine. This condition is known • Heavy Proteinuria: more than 4 g per day (>4 g/day)
as albuminuria.
• If the albumin level is very little and persists to stay What are the different types of protein found in urine?
abnormal, this condition is called microalbuminuria. • Albumin: the most common protein that can be detected in
the urine.
Causes of Albuminuria • Others: Globulin, mucin, blood proteins like hemoglobin,
• It can be caused due to kidney damage from the condition Bence-Jones protein.
of diabetes.
- Kidney produces a type of hormone called What are the different tests for the detection of proteins in
erythropoietin, that helps make RBCs. RBCs carry urine?
oxygenated blood all throughout the body. • Qualitative Test: Heller’s Test, Sulphosalicylic Acid Test
- Once a person’s sugar level is high, the blood • Quantitative Test: Esbach’s Salbuminometre Test
becomes viscous (malapot). This causes amputation • Other Tests: Protein Reagent Strip Test, Biuret Test, Urine
for diabetic patients to avoid the further spread of Protein Electrophoresis
infection because blood no longer flows properly (hindi
na gumagaling ang mga sugat).
BIOCHEMISTRY LABORATORY | BS Medical Laboratory Science

What are the cases/what causes proteinuria?

• Physiological Causes (based on actions): Elevation of
protein that is exercise induced or triggered by exercising,
pregnancy, orthostatic or standing for a very long time.
• Pathological Causes: with the presence of diseases.
o Prerenal Causes: vomiting, severe diarrhea, intestinal
obstruction, diabetic coma, Addison’s disease, fever,
Ascites with intraabdominal tumor, severe anemia.
o Renal Causes: causes of glomerulonephritis
glomerular and tubular diseases, nephrotic syndrome.
o Postrenal Causes: lesions of prostate, lesions of
urethra, severe urinary tract infection (UTI), lesions of
renal pelvis and urinary bladder.
Dialysis is the process of separating to lower concentration until equilibrium is
molecules in a solution by the difference in reached.
their rates of diffusion through a
● In Dialysis, the migration of molecules
semipermeable membrane.
occurs through a semipermeable
Dialysis is a common laboratory technique membrane, which allow only small
widely used for removing contaminants from molecules to pass through restricting
solution. To retain macromolecules. the movement of large molecules like
proteins.
Dialysis technique is commonly used to
remove small unwanted molecules such as o Semipermeable membrane –
salts, reducing agents, preservatives, etc. means not all molecules or particles
could enter and exit the membrane.
Formalin – preservatives used when storing
urine. common preservative, if refrigeration is The duration of the dialysis depends on the
not possible. buffer, the volume and characteristics of the
sample.
Refrigeration – using refrigerated
temperature, which is usually in 2 – 5 o C. Dialysis Procedure
Common preservation technique if we
cannot use reagents. ● Pre-wet the Membrane
● Load the sample on dialysis tubing
Dialysis works by selective and passive ● Dialysis for two hours
diffusion through a semipermeable ● Change dialysis buffer and dialysis for
membrane. 2 hours
● Change dialysis buffer and dialysis
Dialysis can also be used for buffer
overnight
exchange.
Dialysis Procedure could depend on what
Buffer – substances that can regulate the pH
substance you’re going to undergo dialysis.
of a substances or solution. Substances that
Time of dialysis, change buffer.
is responsible in balancing the pH in our
body, or certain substances/solutions. There Pre-wet the Membrane
are substances integrity is being destroyed if
the pH has changed. • Pre-wet the membrane by using
Deionized water, to make sure that it will
• Bicarbonate Carbonic Acid – our not affect the diffusion if use water or wet
body’s buffer system. the membrane before the loading of the
• Dialysate – is the buffer used for sample.
dialysis. • The purpose on why we wet the
- Phosphate buffer solution (PBS) is one of membrane is to make the diffusion fast
the examples of dialysate in dialysis. ones the solution loaded.
Principles of Dialysis Dialysis for two hours
● Dialysis works by the principle of ● The duration of the dialysis will depend
diffusion. on the buffer that is being used, what
substances that will be dialyze and how
Diffusion is the migration of molecules
many the sample is.
randomly from areas of higher concentration
 ● Example is Dialysis for two hours, III. Properties of Water Solutions
meaning if we load the sample there will
Dialysis
be a dialysate that is present in the set
up which is the buffer. a. Obtain a dialysis bag about 20 to 25 cm
long and soak in clean water for about 10
PROCEDURES:
minutes.
I. Water: A Universal Solvent
● The purpose of the water is to make
a. Put about 0.5 grams of the following sure the membrane can penetrate the
substances into six separate test tubes: unwanted molecules when we put it
NaCl, sugar, gelatin, CuSO 4, lard, and in the membrane.
ethanol. Add 1 ml of water to each test tube
b. Fill with 30 ml of 1% starch-NaCl mixture,
and shake vigorously to dissolve the
tie the bag (dialysis bag) and rinse
substances. For substances that did not
thoroughly with water. Put the bag in a
dissolve, add another 1 ml of water and
beaker containing deionized water.
shake again. For the solids that still did not
dissolve, add another 1 ml water and shake. ● Deionized water – it doesn’t have
b. On a six separate test tube, Repeat the electrolytes or ions, meaning it
solubility test using CCl 4 , instead of water. doesn’t have sodium, chlorine,
c. Describe solubility in both solvents as potassium, or any minerals that might
soluble, slightly soluble, and insoluble. interfere the dialysis procedure. The
Record observations in the table. reason is because in the procedure
we have NaCl that is present in the
SUBSTANCES SOLUBILTIY SOLUBILITY
sample
IN WATER IN CCl4
c. Adjust the setup such that the levels of
NaCl Soluble Insoluble
fluids inside and outside the bag are the
Sugar Soluble Insoluble same.

Gelatin Slightly Insoluble ● The purpose of the diffusion is to

soluble reach the equilibrium.

CuSO4 Slightly Insoluble d. After 1 hour, test 1 ml of dialyzate (water

soluble in the beaker) with a few drops of 0.1M
AgNO3. Formation of a white precipitate of
Lard Insoluble Soluble AgCl confirms the presence of chloride ions
in dialyzate.
Ethanol Soluble Soluble
● The purpose of silver nitrate is to
check whether the chloride ions was
II. Water: A Good Medium for Biochemical able to penetrate the membrane or
Reactions exit the membrane.
a. Mix 0.1 gram of dry, powdered citric acid, ● If there is a formation of white
and sodium bicarbonate (NaHCO3) in a dry precipitate it means that the chloride
test tube. Observe if a chemical reaction ion is present in the dialysate (water
occurs. in the beaker).
b. Add about 10 ml of water to the mixture
and note what happens.
Procedure in Dialysis Video ● Depending on the dialyzer it is how many
tubes it can full like the example it can
process 10, 25 and 100.
● The sample volumes depends on the
tube that is being used on how much the
volume it can cater.
● The dialyzer usually includes floating
racks for dialysis, floating racks use to
put the dialysis tube to make it float on
the set up.
● Supporting trays is used when we do
electro illusion, electro illusion is used
when we process to extract like nitric
● KDa – Kilo Dalton, unit measure use acid, proteins or macromolecules.
for the particles that can be filter in ● The reason why it is electro illusion, it is
dialysis set up. because of the electrophoresis gel that
● MWCO – Molecular Weight Cut Off, we used.
refer to the lowest molecular weight ● The electrophoresis gel is used in
solute that is depend on the electro illusion, we used a current for
membrane. electro illusion. It’s simple and
inexpensive.

● Most of the time this are the

characteristics of a Dialyzer, which is Notes
supposed to be free in metals. Specific Refrigeration – 2-5 %, common
free from heavy metals for example preservation used in dialysis
sulfur. And they’re usually EDTA-treated
membrane also use as anticoagulant. Formalin – example of preservatives that is
● Even during the dialysis, the membrane most common use for preserving body fluids.
that being use in this procedure they
Kidneys – are the most responsible for
could be treated with EDTA.
natural dialysis, it has filtration of unwanted
● Usually, the purpose of EDTA is to
molecules that being eliminated as urine.
chelate the calcium.
● The MWCO changing is depends on the Buffer – substance that are responsible to
substance that you want to test. balance the pH level, it can regulate the pH
of the certain substance.
KDa – Kilo Dalton, unit measure use for - Pipette out the sample carefully from the
dialysis dialyzer tube to a clean tube and use for
downstream applications
MWCO – Molecular Weight Cut Off.
Note: if sample volume increase during the
Permate – being eliminated.
dialysis, the tube can be left on the bench to
Retentate – being retain on the set up that evaporate volume without fully drying the
being concentrated. sample

STEP 1 You have to wet the membrane Broad Application

- Make sure that when you do, do not ● Dialysis, buffer exchange, electro-
touch the membrane. You add 2-3 ml elution
dH2O. No dH2O should leak from the
- For using gel or electrophoresis gel for
tube. Because it could affect the diffusion
extracting the nucleic acid and the
or the dialysis. Then wait for 5 minutes to
protein.
make sure that membrane is already wet.
● Mass spectrometry sample clean-up
STEP 2 Load the sample
- The buffer exchange this is remove for
- Depend to the direct tube that we used. salts on the sample then exchange for
The amount of the sample that is the present to the buffer dialysis.
available. Add sample (0.1-3ml) into the
dialyzer tube and seal with the cap. ● Sample concentration

STEP 3 Set up dialysis - You just want to extract the nucleic

acid for example protein that you will
- Place the dialyzer tube in the supplied use. Let’s say for the research to
floating rack in a stirred beaker asses the property.
containing desired buffer 100-1000x
sample volume. You could adjust the stir ● Peptide dialysis
bar speed.
- Your peptide is used to form amino
Dialysis Time Guideline acid is bond to another amino acid.
The peptides is made up to one
- Allow minimum 30 minutes per 0.1 ml proteins. Your polypeptides could
sample made up your proteins. Peptide
- Low MW salts and buffer (e.g., Tris-HCl dialysis sometimes called protein
and KPO4): - 3 hours dialysis. Remove the unwanted
- Viscous samples: > 3 hours molecules and you will only take the
Note: the user must determine equilibration peptide or protein.
times for complete dialysis and change the ● Virus purification
dialysis buffer as necessary.
- Viruses they are made up of nucleic
Step 4 Collect Dialyze Sample acid they are actually categorize or
- You can now collect the dialyze, you can classified based on the nucleic acid
see the difference the intensity of the are present to their structure. Lets say
color sample. It’s lighter after the dialysis. the DNA or RNA single stranded. For
Take out the Dialyzer Tube from the virus purification the dialysis is part of
sample. the technique.
CONTINUOUS DIALYSIS IN THE amount needed for buffer will still be
LABORATORY achieve. Also we removed water or buffer
during the procedure.
- The dialysis technique is used to
purify large molecules (proteins, DNA, 🡪 After such time the only left behind the
Polysaccharides…) macro molecules or substance that you
want to concentrate
- Polysaccharides which are
carbohydrates your DNA and your COMPLETE SYSTEM
proteins so basically our
macromolecules. Using the dialysis to
purify the macromolecules.

Dialysis
Load sample into dialysis column. You will
notice that the tube using is different, you will
also notice the floating rack is different
Mixture of products in aqueous solution
- Product to purify
- Smaller molecules

🡪 Place the column into dialysis buffer (1x

PBS)
🡪 The amount of the water the we put is - Phosphate buffered saline usually used
depend on the sample. in the laboratory. The reason because
🡪 Now it’s continuous, because as you can the pH that we need to regulate is near to
see on the video, continuous of putting the phosphate buffered.
water. While adding water the molecules
that eliminated by unwanted molecules
or eliminated during the process of 🡪 Dialysis at 40C overnight (change dialysis
dialysis and it’s getting missing to the buffer for 3-4 times
buffer that we used.
- Have a stir bar for regulate for removing
🡪 So once they escape to the membrane, unwanted molecules.
they also they coming out to the set up.
🡪 Dialysis column with appropriate MWCO
Because adding water to make sure the
(Molecular Weight Cut-Off)
- It could hold the larger molecules. Means
its higher molecular weight to the cut off,
eliminating smaller particles.

❖ Permeate

- Smaller than MWCO (ion, Alt..,)

- The ones we are eliminated is called

permeate. Smaller than molecular weight
cut-off they will eliminated or escape the
membrane.
Example: salts, detergent, preservatives

❖ Retentate

- From the word itself being retained on the

set up. This is concentrated to
macromolecules
Example: protein, nucleic acid, or
carbohydrates
Protein- the color blue has smaller size
particles, smaller molecular to the cut off so
this called permeate the red ones is
retentate.

🡪 Collect clarified sample in a new tube

- The sample that being collect is could

now use other applications could be in
peptide, virus purification, for
electroelution and so on. That’s the
purpose of the dialysis.
Osmosis and Diffusion Module structure it’s not moving away the
concentration radiant, those molecules or
➢ (The difference between Osmosis and
particles during the process they just go
Diffusion. On osmosis we are more focus
along with the concentration radiant.
on the solvent, movement of the solvent
or specifically the solvent is usually water Pre-Lab Discussions
wherein the movement of the solvent or
• Osmosis is defined as the
water molecules are from region of lower
movement of water from higher
solute concentration to region of higher
water concentration to lower water
solute concentration. While for diffusion
concentration through a semi-
it’s the movement of the solute from an
permeable membrane.
area of higher concentration to area of
lower concentration) Take Note:
Take Note: The reason why it is vice versa ❖ The movement of water is affected on the
from lower to higher and higher to lower it’s amount of substance dissolve to it.
because in osmosis we are referring to the
movement wherein most of the time it’s you ❖ In osmosis the tonicity is quite related to
water while for your diffusion it is the it but the difference between osmosis
movement of solute from an area of higher to and tonicity is in osmosis it just go
lower concentration. depending on the substance dissolved
but in tonicity it access whether the size
Just a recap: What type of transport of the cell will be affected base on the
mechanism or cell transport mechanism movement of the water or solvent due to
is osmosis and diffusion? the solute that are dissolved inside and
outside the cell.
➔ Osmosis and Diffusion are both under the
passive transport. • Interesting to know about osmosis
is that the movement of water is
When you say passive transport what
affected by the amount of
makes it different from active transport?
substance dissolved to it.
Take Note:
• Diffusion is the movement of
➔ The difference between passive and particles from an area of higher
active transport is whether the transport concentration to lower
mechanism requires or consumes energy concentration. The overall effect is
during the process. Passive Transport no to equalize concentration
net energy require, no energy consumed throughout the medium.
during the process. Active Transport
❖ The goal of diffusion is to reach the
energy is needed or required.
equilibrium.
➔ The most commonly used energy for the
Examples of Osmosis and Diffusion
transport mechanism is the ATP.
Examples of Osmosis: Examples of
➔ The reason why it does require energy for
osmosis include red blood cells swelling up
passive transport is because the
when exposed to fresh water and plant root
molecules they move towards the
hairs taking up water. To see an easy
concentration radiant; it doesn’t need to
demonstration of osmosis, soak gummy
spend energy because there is no need to
change the shape. For example, the
candies in water. The gel of the candies acts juice or a 10% sucrose solution until the
as a semipermeable membrane. solution reaches up to the base of the
tube.
❖ As I said a while ago, in osmosis its
related to tonicity hence example ❖ The laboratory apparatus that we
swelling up the red blood cells when use for osmosis is the thistle tube,
exposed to fresh water is example of here we put the sample for the
osmosis but it’s related also to tonicity. osmosis.
Normally the red blood cell should not
❖ The first thing you need to do is to
swell because of the exposure to the
prepare the laboratory apparatus.
fresh water the concentration of solute
on the fresh water is different to the ❖ To stop the thistle use the index
concentration of the solute that could finger or the thumb, stop the
be found inside the cell will make the narrow end of a thistle tube and
red blood cell swell up. then the typical solution that we
use for the osmosis could be
❖ Another example of osmosis is that
sugar cane juice or a 10%
when the plant root hairs is taking up
sucrose solution.
water.
2. Cover the mouth of the thistle tube with
❖ To visualize the osmosis just get a
a cellophane membrane and keep it in
gummies and soak it with water the
place with a rubber band.
gel of the gummies it will act as
semipermeable membrane. ❖ For the set up you have to make
sure that the solution will reach
Examples of Diffusion: Examples of
the end or base of the tube and
diffusion include perfume filling a whole room
then the mouth of the thistle tube
and the movement of small molecules across
it has a large opening which is
a cell membrane. One of the simplest
considered as the mouth of the
demonstrations of diffusion is adding a drop
thistle tube (like a bell shape)at
of food coloring to water. Although other
the end of the thistle tube.
transport processes do occur, diffusion is the
key player. ❖ Cover it using a cellophane
membrane. The cellophane
Key Point:
membrane will primarily act as the
Diffusion and osmosis are both passive semipermeable membrane and to
transport processes that act to equalize the avoid it from moving and to cover
concentration of a solution. properly the mouth of the thistle
tube with a rubber band.
❖ In osmosis it focuses more on the
movement. 3. Suspend the thistle tube in a beaker of
water, making sure that the levels of
❖ In diffusion if focuses on the
liquids inside and outside of the tube
movement of solute.
are equal.
Experiment Discussion:
❖ It also requires solvent that
Procedure could be found on your beaker.
In this case, we have water
1. Stop the narrow end of a thistle tube making sure that the levels of
with a finger and fill it with sugar cane
 the liquids inside and outside of So, I’m using red food coloring to
the tube is equal. demonstrate the hot beaker and blue food
coloring to demonstrate the cold beaker.
4. Observe the difference in solution levels
after minutes. So, we’re going to be looking to see how
quickly the food coloring spreads out in the
YT VIDEO 1
two different beakers.
Diffusion and Osmosis Experiment
➢ (This one is the experiment set up for
osmosis and diffusion. The example of
diffusion is adding the food coloring you
will see how the diffusion work and then
the osmosis on the part of utilizing the
thistle tube but again here it will not use
sugar cane or it can use different sample
for the experiment.)

• When we think about cell biology and

we’re looking at diffusion and osmosis,
it’s really helpful to zoom down to the
level of the cell which really is the
molecular level.

Cells are made of molecules and they’re

surrounded by molecules. They are a part of
their molecular environment and what that
means is that the dynamics in the forces that Now, as you can see, the red food coloring is
effect molecules also have a big effect on moving through the beaker of hot water much
cells and on cell biology. more rapidly than the blue food coloring is
moving through the beaker of cold water and
Diffusion is the movement of molecules from
an area of high concentration to an area of that’s because diffusion is based on
molecular movement.
low concentration.
Molecules will tend to speed up at a higher
To demonstrate how that works, we’re going
temperature which means that diffusion will
to do an experiment in which I’m going to
drop a drop of food coloring into a beaker of happen more rapidly.
hot water and another drop of food coloring
into a beaker of cold water at the same time.
Now, how is this important in the context • So here the iron III chloride goes into the
of the cell? beaker, and as you can see it’s a light
orange in color
- In the cell, diffusion can happen across
the cell membrane. The cell membrane is So, how are we going to know that
what we call a semipermeable diffusion has taken place?
membrane, and what that means is that
some molecules can flow freely across it - We’re going to know that diffusion has
or diffuse freely across it while others will taken place if there is a color change in
be blocked or stopped from crossing. the beaker, in the tube, or in both.

One of the most important criteria that we So while we wait for something to happen
use to determine whether a molecule will be there, I’m going to set up our second
able to cross is size. experiment.

So small molecules can cross freely across • Here I’m going to be adding distilled
the cell membrane whereas large molecules water, which is going to be diluting an
may be blocked from diffusing across the cell iodine solution, into the tube, the
membrane. dialysis tubing here, and then I’m
going to add to the beaker, a
So, we’re going to do an experiment that’s concentrated starch solution.
going to demonstrate the movement of • Now the iodine solution is a light
molecules, the diffusion of molecules across yellow in color. The starch solution,
a semipermeable membrane. which you can see here, is a cloudy
white and once again, we’ll know if
What I have right here is dialysis tubing.
diffusion has taken place because
Dialysis tubing, just like the cell membrane is
we’re going to see a color change in
a semipermeable membrane that allows
either the beaker or the tube or both.
some molecules to move through and
excludes others. • So, here I’m adding the starch
solution to the beaker.
What I’m going to do is that I’m actually going • Now, if we go back to our first
to pour some liquid into the tube and a experiment, we can see that there’s a
different solution into the beaker for each of marked color changes that has
these two experiments and we’re going to already happened.
see whether the solutions are able to cross • So here the liquid inside the tube has
the membrane or diffuse across the turns a dark red and there’s a dark
membrane in order to join each other. red color that’s also forming in the
liquid inside the beaker.
• So the first thing I’m going to do is I’m
• Now what that means is that the two
going to add a solution of potassium
substances have been able to diffuse
thiocyanate into the tube, pour it in
across the membrane and react with
carefully and hang it on the edge.
each other. And what they’ve done is
• So the potassium thiocyanate is a
they’ve actually formed that reaction
colorless liquid.
in both the tube and in the beaker.
• The next thing I’m going to be adding is a
solution of iron III chloride which I’m
going to pour into the beaker.
 The iodine solution that we have in the tube
is a very small molecules it’s easily able to
pass through the pores in the tube and intro
the beaker where it causes a color change
with the starch.
The starch, on the other hand, is a very large
molecule it isn’t able to penetrate through the
What that tells us is that 2-way diffusion took pores of the dialysis membrane and what
place in this example. Whereas, here, in our that means is that it demonstrates that this is
second experiment we can see that there is a semipermeable membrane. Even though
a color change that’s starting to happen but the starch would tend to want to diffuse into
that color change is only happening inside the tube, it can’t because of the size
the beaker. exclusion of the pores in that semipermeable
membrane. This is similar to what happens
in a cell membrane.
The next thing that we’re going to
demonstrate is osmosis.
Osmosis is similar to diffusion but osmosis is
the movement of water molecules from an
area of low concentration to an area of high
Our tube still has the same yellow-clear solute concentration.
color. We’re going to demonstrate that using an
experiment with what we call a thistle funnel
tube.
The thistle funnel tube, just like the tubes that
we looked at previously, has a length of
dialysis membrane stretched across the
bottom.
So, once again, this is a semipermeable
Inside the beaker, we’ve got our starchy membrane.
cloudy white solution but we’re seeing a dark
precipitate which is a purple-ly brown which What I’m actually going to do is that I’m going
is starting to form. to fill the thistle funnel tube with a very
concentrated sucrose solution, so we’re
Now that purple-ly brown color indicates that actually going to just add the sucrose
the molecules, the solution in the tube were solution directly to the thistle funnel tube and
able to diffuse into the beaker and cause a once we have added it to a high enough
color change. But there’s no color change degree we’re actually going to suspend this
happening in the tube. thistle funnel tube in a beaker of distilled
The reason for that is that, as I mentioned, water.
the dialysis tubing is a semipermeable Now, that would mean that the concentration
membrane. It allows some molecules to of solute in this thistle funnel is much higher
cross but not others. than the concentration that we would see in
the beaker of water and so the tendency ➔ In this course of this activity you will be
would be for the water to osmose across the using a three decimal place balance you
semipermeable membrane entering into the will become familiar with its operation
thistle funnel tube. and how to take accurate readings from
it.
So, we’ve added enough of our concentrated
sucrose solution, we’re going to suspend this ➔ Initially your balance may display a small
in a beaker of double distilled water and value in order to make accurate
we’re going to leave it because osmosis in observations you will need to zero the
this context takes a little bit of time in order to balance as shown in the video.
occur.
Selecting Potato
So, what we’re going to see is that water is
➔ The Potato tuber you are provided with
going to cross that semipermeable
has a cross-sectional area of 10
membrane through osmosis, and moving
millimeters by 10 millimeters.
from an area of low solute concentration to
an area of higher solute concentration. In this ➔ Choose only the potato tuber samples
case, the solute is sucrose and we’re actually which do not any potato skin on the
going to see the level of fluid in the tube rising outside.
over time.
➔ Nominal amounts of skin on the ends of
the sample can be trimmed away. As far
as possible this will mean that each
potato tuber sample will be able to be
affected by the concentration of the
sucrose solution evenly.
Trimming Potato Sections

YT VIDEO 2 ➔ Trim away any remaining skin from

the potato sections to create even
Osmosis using Potato tubers and known samples. Please adhere to the
concentrations of Sucrose solution health and safety guidelines when
using bladed or sharp equipment.
• Osmosis - investigating the water
potential of Potato tubers with known ➔ Trim all the potato sections to an
concentration of Sucrose solution even length so they are uniform. At
the end of this process you will
Basic Equipment
require six even potato samples.
➔ To carry out the investigation you will be
Making Dilutions
provided with the following items of basic
equipment. Boiling tubes, Marker pen, ➔ Using the one molar sucrose
Ruler, White tile, Forceps, Scalpel, and solution and distilled water by the
10 millimeter syringe. Other items will be means of the 10 milliliter syringe
a potato or potato chips. One molar make up the dilutions indicated on
sucrose solution, a sieve, some blotting the boiling tubes
paper, some cling film, and a test tube
rack.
Accurate Labelling
➔ Using the marker pen provided in
your kit accurately label the boiling
tubes with the dilutions that they
contain.
➔ Please ensure that you have made
up an appropriate volume of each
concentration to cover your potato or
sample. ➔ From the raw data shown on screen
tabulate the results using S.I units
➔ Cover the boiling tubes containing the
samples with the cling film provided this
will prevent any evaporation of the
sucrose solutions and any contamination
that may enter the tubes.
Time Passes
➔
➔ 24 hours later you will be able to re-weigh
➔ At this stage of the investigation your your sample.
equipment and samples should look
something like this. Final Weighing

Initial Weighing ➔ Following the same processes as the

initial weighing take the final weights of
➔ The process of weighing the potato the potato samples from each
sections can now begin. concentration as each raw data weight
appears there will be an on-screen
➔ Blot any excess liquid or starch from
graphic to associate with the correct
the surface of the potato section.
concentration.
➔ Ensure that the weight of each
➔ Draining the sucrose from the samples is
section is correctly associated with
done using a sieve. This can be done into
the concentration in which it will be
a beaker provided or into one of the
immersed.
laboratory sinks.
Good Laboratory Practice
➔ As good scientists we should take care
of our equipment. Please be tidy when
you work in the labs and return the
equipment to the basket.

➔
➔ As each weight is registered an on-
screen caption will associate it with
the concentration
 YT VIDEO 2
Osmosis (Red Blood Cells)
Before we take a look at how osmosis and
tonicity affect a cell, let’s review what each
of these terms means.
Osmosis represents the diffusion of water
across a semipermeable membrane.
The term tonicity refers to the relative In this case, the solute concentration is
solute concentration of two environments greater inside the cells than in the
separated by a semipermeable membrane. surrounding water.
In other words, the contents of the cells are
hypertonic in relation to the hypotonic
contents of the beaker, because of this,
osmotic pressure results in the diffusion of
water across the membrane and into the
cells.

In other words, by comparing the tonicity of

the solution, you can determine the
direction in which osmosis occur.

Over time, if enough water enters the cells

the cell membranes may burst. This is
called lysis.

You can determine the direction in which

osmosis will occur.
To demonstrate how tonicity affects a cell,
let’s place some red blood cells into a
beaker containing pure water.

Now let’s place the red blood cells in a

beaker containing a solution of salt, such as
sodium chloride. Since the contents of the
beaker are hypertonic in relation to the
interior of the cell.
 The water within the cell will diffuse across
the membrane and into the contents of the
beaker. This cause crenation or shrinking of
the cells.

In other words, water moves toward areas of

high salt or sugar concentration. This sample
process is used to drive the operation of our
kidneys and explains some of the
physiological consequences of diseases
such as diabetes.

➢ As mentioned a while ago, on the

osmosis and tonicity. The tonicity, it
assess the concentration of the solute
that to be found inside the cell and also
outside the cell. In tonicity it has to make
sure that there is an equal concentration
If the red blood cells are placed in a beaker on the solute on both sides which is
whose contents match the tonicity within the inside of the cell and outside of the cell
cells, then there is no net gain or loss of because it plays a role whether the cell
water. The environments within the beaker could change its size or it could swell up
and inside the cells are said to be isotonic, or or eventually burst or it could cause
the same. shrinkage because of the solutes or
solvent inside and outside the cell.
➢ On your osmosis a while ago, you have
to pay attention on how the potato is
prepared.

An important thing to remember, is that

osmotic pressure always causes water to
move from a hypotonic environment toward
a hypertonic environment.
Introduction: Tests Based on the furfural is formed from hexoses to give
Formation of Furfural and its Derivatives yellow-brown condensation products.

• Sugars are stable to hot dilute mineral 3. Seliwanoff’s Test

acids, But hot sulfuric acid (H2So4) will
This test is used to differentiate
dehydrate them into furfural and its
ketohexoses from aldohexoses.
derivatives. Pentoses, when heated with
Ketohexoses react faster with the solution
concentrated sulfuric acid, will form
containing hydrochloric acid and resorcinol
furfuraldehyde, while hexoses will form 5-
than aldohexoses. The dehydrated
hydroxymethyl furfural.
ketohexoses form a bright cherry red
• In the presence of acid, phenolic
condensation product, while the aldohexose
compounds like a-naphthol, orcinol, and
yields only a pale pink coloration, a negative
resorcinol will react with furfural or
result. In this test, prolonged heating of
hydroxymethyl furfural to form colored
samples should be avoided.
condensation products. The formation of
these colored compounds constitutes a
positive test for carbohydrates.
Test Based on the Formation of Furfural
and its Derivatives
I. General Tests for Carbohydrates
1. Molisch Test
OBJECTIVE: To be able to identify the
The Molisch test is the general test different types of carbohydrates using the
for carbohydrates. The sugars are mixed with different specific chemical tests
a-naphthol. The test tube is inclined, and
concentrated H2SO4 is added along the side MATERIALS:
of the tube, causing the formation of a lower 20 test tubes Dropper
layer of acid. The concentrated H2SO4 will Test tube rack Glass slides with cover
dehydrate the sugar allowing it to react with Test tube holder Microscope
the alcohol forming furfural or Test tube brush Distilled water
hydroxymethyl-furfural. Formation of a 3% solutions of
purple ring at the interface of the two liquids glucose, fructose,
Aspirator and pipet
will indicated the presence of a carbohydrate. lactose, sucrose,
xylose
2. Bial’s Orcinol Test Graduated cylinder Molisch’s reagent
Spatula Bial’s orcinol reagent
Bial’s test is used to determine the
Cotton balls Seliwanoff’s reagent
presence of pentoses and nucleotides and
Watch glass Concentrated H2SO4
nucleotides that contain pentose sugars. Water bath 10% FeCl solution
When pentoses are treated with orcinol, Tripod
furfurals are formed and they will yield a blue- Bunsen burner
green compound in the presence of ferric Wire gauze
ions. The reaction is not specific for pentoses Spot plate
because other compounds like trioses, 250 mL beaker
uronic acids, and certain heptoses will also
give blue or green products. Hydroxymethyl
PROCEDURES allow them to stay there for
exactly 1 minute
I. Molisch’s Test
d. Note the changes and record
1. Mix 4 ml of distilled water and two drops
which test tube gives a positive
of the Molisch’s reagent in a test tube.
result in the shortest time.
This tube will serve as the control.
2. Place 4 ml of 3% solution of glucose in e. Continue heating and observe
a second test tube. Add two drops of the the color changes at 1 minute
Molisch’s reagent and mix the contents intervals
by gently shaking the test tube.
f. Record the time required for a
3. Incline the test tube and cautiously add
positive test for each sample
about 5 ml of concentrated sulfuric acid,
allowing the acid to run down the side Carbohydrates – it is a polyhydroxy
of the tube. Sulfuric acid is denser than aldehyde, a polyhydroxy ketone, or a
water and will form a lower layer. Note compound that yields polyhydroxy aldehydes
the color of the ring formed at the or polyhydroxy ketones upon hydrolysis.
junction if the two liquids.
4. In the same manner of adding acid, add
sulfuric acid to the control tube. What do
you observe?
5. Repeat the above test with 3% sample
solutions of fructose, lactose, and
sucrose.
6. Record all results.
II. Bial’s Orcinol Test
Classification of Carbohydrates
1. Place 1 ml of each 3% solution of
1. Monosaccharides – “simple sugars”,
xylose, and glucose in separately
are highly soluble in water, less soluble in
labeled test tubes
ethanol, and insoluble in ether.
2. Add 3 ml of Bial’s reagent to each test
• They are either aldoses or ketoses
tube
• They may also be classified into
3. Carefully heat each tube over a Bunsen
tetroses, pentoses, or hexoses
flame until the solution begins to boil.
• Free monosaccharides are all
Add one to two drops of 10% FeCl3
reducing sugars.
solution.
4. Note the color of the product formed • They also exhibit mutarotation
5. Record your results in the table 2. Disaccharides – are formed by two
6. III. Seliwanoff’s Test molecules of monosaccharides.
Examples are maltose, which are
a. Place 1 ml each of 3% glucose, abundant in germinating barley; sucrose,
fructose, lactose and sucrose in also known as cane sugar or beet sugar;
separately labeled test tubes. and lactose or milk sugar, which does not
taste very sweet and is not fermented by
b. Add 4 ml of Seliwanoff’s reagent
yeast.
to each test tube
3. Polysaccharides – found in nature
c. Place the test tubes in a water function either as structural units (e.g.
bath filled with boiling water and
 cellulose), or for storage such as starch, Principle:
dextrin, glycogen, and inulin.
• When Benedict’s solution and simple
carbohydrates are heated, the
solution changes to orange red/ brick
red. This reaction is caused by the
reducing property of simple
carbohydrates. The copper (II) ions
in the Benedict’s solution are
reduced to Copper (I) ions, which
causes the color change.

Principle

Sample: Urine
Aim: To detect the presence of reducing
sugar in urine
Procedure:
1. Using a pipette, accurately take 5 mL
of Benedict’s reagent and slowly
transfer it to the test tube.
Test Based on Reducing Property of 2. Take 5 mL of freshly collected urine by
Sugar pipette and add it to the test tube with
Benedict’s reagent.
Reducing Sugars – it must have a free 3. Place the test tube in boiling water
aldehyde or ketone group. Common dietary bath for 2-3 minutes
monosaccharides: glucose, galactose and 4. Observe the color of the solution and
fructose. note whether a precipitate was formed
1. Benedict’s Test 5. Avoid prolonged heating
2. Barfoed’s Test 6. Record your results
3. Tollen’s Test Barfoed’s Test
Benedict’s Test • Barfoed’s reagent – contains cupric
• It is a very sensitive test done under acetate in dilute acetic acid
mildly alkaline conditions ▪ It oxidizes monosaccharides but
• Benedict’s Reagent: Composed of not oligosaccharides
CuSo4, Na2CO3, sodium citrate
• Positive result: Change of color of ▪ Disaccharides are less oxidized
Benedict’s reagent but are oxidized if they undergo
• Also used to test for the presence of prolonged heating → causing
glucose in urine hydrolysis of the disaccharides
into monosaccharides →
produces a positive result
 • It is used to distinguish between Tollen’s Test (Silver Mirror Test)
monosaccharide, disaccharides, and
• Sugars with aldehyde groups are
oligosaccharides
capable reducing Tollen’s reagent
• Positive result: Brick red precipitate
• Tollen’s reagent: Ammoniacal
• Unlike Benedict’s test, this test is
solution of silver
carried out under acidic rather than
• Positive result: Gray to black
basic medium.
precipitate (silver mirror)

Procedure:
1. Place 5 drops of 3% solutions of
glucose, xylose, and sucrose in
separate test tubes
2. Add 2 mL of Tollen’s reagent into
each tube
❖ Preparation: Add 1 drop of NaOH
Procedure: solution to 6 mL of 5% AgNO2. Add
dilute NH4O4 (1 mL concentrated
1. Place 1 mL each of 3% solutions of
NH4OH + 5 mL water) until the brown
glucose, xylose, fructose, lactose and
precipitate of silver oxide that forms just
starch in separately labeled test tubes
dissolves
2. Add 3 mL of Barfoed’s reagent in each
❖ This reagent must be prepared fresh
test tube
and not stored since it decomposed
3. Prepare a control tube using distilled
when left standing and yields an
water instead of sugar solution
explosive decomposition product.
4. Place all the tubes in a boiling water
Discard all leftover materials.
bath for 10 minutes
3. Boil for about 5 minutes. Note and
5. Record your observations
record your observations.
Qualitative Tests for Carbohydrates
Materials:

• Test tubes
• droppers
• graduated cylinders
• Marker and paper tape
• Tube racks
 • reagents (Fehling’s A&B, Blue – if it remains as blue, it menas that the
Benedict’s, Seliwanoff’s, Molisch’s, sample is not contaminated.
Barfoed’s and Iodine Soln)
Step 2: Allow for the test tubes to cool down,
• The Carbohydrate solutions aree:
then add 5 drops of carbohydrate solution.
▪ Glucose
After that place the test tube in water bath for
▪ Fructose
2 minutes.
▪ Lactose
▪ Sucrose Step 3: Cool and observe what happened.
▪ Starch
Molich’s Test
- general test for carbohydrate
- concentrated sulfuric acid will also be
used in this test.
Procedures:
Step 1: label the test tubes
Step 2: Add 2ml carbohydrate solution in test
tubes and add 10 drops of Molich’s reagent
Fehling’s Test
to each carbohydrate sample and mix
thoroughly. - reduction test to determine the presence
of reducing sugar.
Step 3: Carefully add 15-20 drops of sulfuric
- it differs from benedict’s test, in that
acid, and allow it to flow down in the side of
Fehling’s rgt. contains sodium potassium
the tube. Do not stir.
in place with sodium citrate.
❖ The sulfuric acid will form a bottom - Fehling’s solutions consist of solution A
layer. Note the color form in the liquids. and solution B.
Procedure:
Step 1: In the procedure, we will mix 1 ml of
solution A and solution B and add 3 ml of
water.
Step 2: After that we will place them in a
boiling water bath for 1 minute.
If the solution remains clear blue, we will add
8 drops of each sugar solution to each of the
Benedict’s Test
test tube. Boil again for another 2 minutes.
- a test for reducing sugar.
Procedures:
Step 1: About 2ml of benedict solution is
added to 4 empty tubes and place to water
bath for 30 seconds.
Color Change:
 Seliwanoff’s Test
- distinguishes between fructose and
glucose. Over heating of the solution
should be avoided because continuous
boiling will give positive result.
Procedures:
Step 1: Add 2ml of sugar solution to 3ml
Seliwanoff rgt.
Step 2: Immerse the solution in boiling water
Glucose and Lactose – small brick red
bath. Observe the color change during the
precipitate
first 10 minutes of boiling.
Fructose – brick red
Sucrose – Blue
Barfoed’s Test
- distinguishes reducing monosaccharides
from reducing disaccharides by
controlling Ph and the time of heating.
- it is also a copper reduction test in acidic
condition.
Procedures: Iodine Test for Starch

Step 1: In each 4 tubes, add 2ml of Barfoed’s Procedure:

rgt.
Step 1: Add 2ml of starch and add a drop of
Step 2: Then, add 10 drops of sugar solution. iodine solution.
then place it in boiling water bath for 5
Step 2: Heat the solution and note the
minutes.
change.
Step 3: Let it cool and observe for 15
minutes.

Benedict’s Test
- its aim is to detect the presence of sugar
in the given sample of urine by benedict’s
reagent.
Color Change: conversion of cupric ions in the Barfoed’s
reagent to cuprous ions with the
• Blue – none formation of copper(I) oxide as seen as a
• Green – traces of reducing sugar brick red precipitate in the mixture. Unlike
• Orange Red – moderate amount of Benedict’s test, copper reduction in
reducing sugars Barfoed’s test occurs in acidic medium
• Brick Red – large amount of reducing (ph 4.6) rather than in alkaline medium.
sugar
Materials:
Material Required:
• Maltose
• Benedict’s solution serves as reagent in • Barfoed’s Reagent
this test. The solution contains copper, • Glucose
sodium citrate, sodium carbonate, and • Test tubes
copper II sulphate pentahydrate
• Measuring cylinder
(CuSO2.5H20)
• pipette
• Test Tube
• Test tube holder Procedure:
• Pipette
Step 1: Measure 3ml of freshly prepared
• Urine Sample
Barfoed’s reagent into labelled test tubes.
• Burner
Step 2: Add 1 ml of the test solution to
Procedure: Barfoed’s reagent.
Step 1: Using a pipette, accurately take 5ml Step 3: Place the test tubes into a boiling
of Benedict’s reagent and slowly transfer it to water bath and heat for 3 minutes
test tube.
❖ Formation of red and yellow to orange
Step 2: Take 5ml of freshly collected urine by precipitate is a positive test for reducing
pipette and add it to test tube with Benedict’s monosaccharides.
reagent.
Step 3: Test tube should be held securely
with the test tube holder to heat it on the
burner for 2 minutes.
➢ On heating the sample, the presence of
sugar is indicated in the sample of urine
when a green precipitate (traces of
reducing sugar) is apparent.
➢ Different precipitates are formed
depending upon the concentration in
urine.
Berfoed’s Test
- it distinguishes monosaccharides from
disaccharides.
- it is a copper reduction test in which
heating a mixture of a monosaccharide in
a given time interval results in the
 Tollen’s Test (Black/gray)
– its aim is to detect the presence of
reducing sugars in the given sample by
Tollen’s test.
Procedure:
Step 1: Take 1 ml of aqueous silver nitrate
solution in a test tube.
Step 2: Add sodium hydroxide solution drop
wise to get the precipitate of silver oxide.
Step 3: Now add 2 drops ammonium
hydroxide solution while shaking the
mixture. Previously performed precipitate
dissolves. This called Tollen’s reagent.
Step 4: Take 2 ml of sugar solution in
another test tube.
Step 5: Take 2 ml of Tollen’s reagent to it.
Step 6: Formation of silver mirror on the
walls of tube indicates presence of reducing
sugar.