Metabolic screening test

Qualitative test for screening of carbohydrates

• Molish test
• Benedict test
• Mucic acid test
• Barford’s test
• Seliwanoff test
 Schematic Diagram for Analysis of Unknown Carbohydrates

Molisch Test

If violet ring present- POSITIVE

Iodine Test

Blue: Starch If Negative, perform

Purple: Dextrin Benedict’s test

Green, Yellow or Red If no precipitate forms,

Precipitate: perform Seliwanoff’s test
Presence of Reducing Confirm by hydrolysis of
sugars (Glucose, Fructose, original solution and apply
Lactose or Maltose) Benedict’s test with
neutralized hydrolysate
Positive test indicates
Barfoed’s test
Sucrose

If Positive, Glucose or If Negative, Lactose or

Fructose Maltose
Perform Seliwanoff’s test Confirm by Osazone test
and Foulger’s test

Positive: Fructose Negative: Glucose

 Molisch’s test:

Principle
Procedure:

• 2ml of Sample is taken in a test tube.

• 2 drops of Molisch’s reagent is added and mix it.
• Add slowly conc.H2SO4 in the test tube.
• If Positive: purple color ring are present
 Benedict test
principle

.
 Benedict’s reagent: composition
CuSO4
Sodium carbonate
sodium citrate

Procedures
• Take 5ml of benedicts reagent in a test tube

• Add 8 drops of urine in a test tube

• Boil the solution for 2min

• Positive: green, yellow, orange and brick red ppt

• Negative : no change in blue color

Result :

Color observed Sugar % Result

Blue Nil Absent of suagr
Green color 0.5% +
Green ppt 0.5-1% ++
Yellow ppt 1-1.5% +++
Orange ppt 1.5-2% ++++
Brick red ppt >2% +++++
 Seliwanoff’s test
Principle:
• Reagents:
Seliwanoff’s reagent (0.5% resorcinol in 3N HCl)

Procedures
• Take 1ml of sample in test tube
• Add 3ml of Seliwanoff’s reagents in test tube
• Heat for 30 sec.
• Positive: development of cherry red color.
 Barfoed’s test:
Principle:
• Monosaccharides usually react very fast while the
reducing disaccharides very slow in acidic conditions.
Brick red color is obtained in this test which is due to
formation of cuprous oxide.

• Reagents for Barfoed’s test: cupric acetate in 1% acetic acid

Procedure:
a. Add 2-3 drops of Barfoed’s reagent (0.33 M Cupric acid and 1% acetic acid) to 1mL
sample.
b. Boil for 2 minutes

Positive: Red scum at the bottom and sides of the test tube
 Mucic acid test
Procedure:
• Take 6 ml of sample in a test tube

• Add 1 ml of Mucic acid reagent (concentrated nitric acid)

• Heat the test tubes in the water bath for 1-1/2 hours until the
volume of the solution is reduced to 2/3rd .

• Then put the test tube overnight before collecting the results.

• positive: galactose or its derivatives.

Mucic acid crystal observed under hpf of microscope

Mucic acid crystal

 Qualitative test for aminoacid metabolism disorders
For Presence of protein biuret test can be performed

• Ninhydrin test
• Millon’s test
• DNPH Test
• Fecl3
• Xanthroproteic test
• Sodium Nitroprusside test
• Sakaguchi’s test
• Sulphur Test
 Biuret test
principle

Procedure:

•To 3 ml of protein solution add equal volume of 10% NaOH

•Mix and add few drops of 0.5% copper sulfate solution

• If the solution turns from blue to violet (deep purple):Proteins are
present
• The solution turns from blue to pink: (Peptides or peptones are short
chains of amino acid residues which contain less number of peptide
bonds are present
 Ninhydrin test:

Procedure :

• Take 3ml of sample in a test tube

• add 3 drops of 0.2% ninhydrin

solution

• Boil for 1-2 minutes and allow to

cool.

• A blue (Ruhemann’s purple) color

indicates positive test while

• proline and hydroxyproline give

yellow color
 Millon’s test:
•To 5ml of dilute solution of protein add 1 ml of 15% mercuric sulfate prepared in
6N H2SO4

•Boil gently for 30 sec

• Add 1 ml of 1% sodium nitrite

• proteins containing tyrosine will give a red color or precipitate which is due to
formation of mercury phenolate ion with sulfonated phenol radical in tyrosine
 Dinitrophenylhydrazine test
• 2,4-Dinitrophenylhydrazine reacts with both aldehydes and ketones to form a 2,4-
dinitrophenylhydrazone. For example with ethanal;

Aldehydes and Ketones

 Dinitrophenylhydrazine test:

• Take 2ml ethanol in test tube

• Add 3drops of sample
• 3 drops of 2,4-dinitrophenylhydrazine reagent
• Then allow to stand for 10-15 minutes
Positive: yellow color developed
 Ferric Chloride test:
• Add 3 drops of FeCl3 to 20 drops of urine sample

Color Compound Disorder

Blue green Phenylpyruvic acid Phenylketonuria
Imidazolepyruvic acid Histidinemia
Homogentisic acid Alkaptonuria

Greenish gray BCAAs MSUD

Green Hydroxyphenylpyruvate Tyrosinemia
Cherry red Acetoacetic acid 3-oxothiolase deficiency
Diabetic ketoacidosis

Purple Ketones 3-oxothiolase deficiency

Diabetic ketoacidosis
Sulphur test

• To 2 ml solution, add 2 ml 40% NaOH and 10 drops of 2% lead acetate solution

• Boil for a minute and cool
or
• To 2 ml of protein solution add an equal volume of 40% NaOH and boil 2 minutes
• Cool and acidify with dilute HCl
• Add 1 ml 5% lead acetate solution and warm
Sakaguchi’s test

•To 5 ml of protein solution add 5 drops 5% NaOH and 4 drops of 1% ethanolic α-

naphthol (Molisch’s reagent)

•Add 10 drops of bromine water (fresh) or sodium hypochlorite solution

Positive: caramine red color develop

Xanthroproteic test

•To 3 ml of protein solution add 1 ml of conc. HNO3

•A white precipitate forms due to denaturation of proteins

•heat the solution for one minute and cool under tap water

•Yellow color appears due to nitration of benzene ring, with excess of 40% NaOH,
the color turns deep orange
 Watson–Schwartz test
For the diagnosis of acute intermittent porphyria
Procedure:
a. Mix 500 microlitres urine and 500 microlitres Kovac’s reagent
b. If the color of the solution changes into pink, add chloroform.
• Pink or red color indicates presence of porphobilinogen or
urobilinogen
• Positive for porphobilinogen if the pink layer migrates on
the top as supernatant with chloroform at the bottom.
• If the pink layer is on the bottom, this is indicative or
urobilinogen instead of porphobilinogen and the test is
negative