Identification of Unknown Sample
- monosaccharides/disaccharides
- Amino acids
Open Journal & Notes
Individual samples
Experiment 1: Carbohydrates
I. Qualitative Test and Analysis of
Unknown
1. Osazone Test
● 10 drops of each test sol’n
(glucose, sucrose, fructose
etc) and unknown sample
● 30 drops phenylhydrazine
reagent
● Put in boiling water bath
● Observation: Yellow crystals
● Put in ice bath if none
● Expected Result: ??
○ (+) All reducing
sugars form
osazones
○ Sucrose,
non-reducing (-)
2. Molisch Test
● 1 drop Molisch reagent
● 30 drops test sol’n and
unknown
● Incline and pour H2SO4
● Observation: Purple Ring
● Expected Result:
○ (+) All carbohydrates
3. Fehling’s Test
● 10 drops test solution
● 30 drops Fehling’s reagent
○ (5 mL Fehling’s sol’n
A + 5 mL of Fehling’s
sol’n B + 5 mL
distilled H20)
● Shake and immerse in
boiling water (2-3 mins)
● Observation: Brick Red
precipitate
● Expected Result:
○ (+) Aldose
monosaccharides
(Aldehydes)
4. Barfoed’s
● 30 drops Barfoed’s
● 20 drops test sol’n
● Place in boiling water bath
○ Remove the test tube
once cloudy or
changes color
● If no changes within 10 mins,
continue heating for another
5 mins
● Observation: Brick-red
precipitate
● Expected Result:
○ (+) Monosaccharides
5. Seliwanoff’s Test
● 5 drops of each test sol’n
● 20 drops Seliwanoff’s
reagent
● Place in Boiling water bath
for 1 minute
● Observation: Red color/ Faint
pink
● Expected Result:
○ Aldose vs ketose
○ Red - ketose
○ Faint pink - aldose
6. Iodine Test
● 5 drops of test sol’n on
separate wells on a spot
plate
● Add 2 drops I2 in KI sol’n
● Observation: Blue/black color
● Expected Result:
○ Only positive in starch
○ The ring concept ni
Noel
II. Isolation of Glycogen from Chicken Liver
 ● Rinse liver with water ● 10 drops of sample
● Dry with filter paper and weigh ● 10 drops of 10% NaOH
○ Not necessary to weigh ● 1 drop 0.1% CuSO4
● Mince into a 250 mL beaker ● Observation: Purple color
containing 100 mL of boiling distilled ● Expected Result:
H20 ○ (+) Presence of
● Cover with watch glass and boil for peptide bonds
30 mins
● Remove from hot plate then add 2. Ninhydrin Test
10% acetic acid until solution is ● 10 drops 0.1% ninhydrin
acidic to litmus ● 20 drops of sample
● Filter while hot ● Heat in water bath for 2 mins
● Appearance of Filtrate: ? ● Observation: Purple color
● Perform the following tests: ● Expected Result:
○ Molisch ○ (+) Presence of
○ Fehling’s amino acid
○ Make slightly alkaline 3. Xanthoproteic Test
■ 2 ml A + 2ml B + 2ml ● 10 drops sample
H20 ● 5 drops HNO3
○ Iodine Test ● Heat in water bath for 5
● Expected Result: ?? minutes
● Cool the solution
III. Hydrolysis of Polysaccharides ● Add 20% NaOH until Alkaline
● 2 mL of 1% starch solution in test ● Expected Result:
tube ○ Yellow ​based sa
● 1 ML HCl google lol
● Heat in water bath and place 2 drops ○ (+) Presence
of the rxn mixture one a spot plate Aromatic amino acid
at: ○ Usually Tyrosine,
○ 0, 5, 10, 15, 20 and 25 mins Tryptophan but not
○ Expected Result: ?? phenylalanine (lacks
● Add 1 drop I2 in KI sol’n an OH group on its
● Cool and add 1 drop phenolphtalein phenyl ring)
in mixture 4. Millon-Nasse Test
● Neutralize with 10% NaOH ● 2 drops Millon Nasse reagent
● Perform Fehling’s Test ● 10 drops sample
● Expected Result: ?? ● Heat for 5 mins
● Cool
● Add 2 drops of 0.1% NaNO2
Experiment 3: Proteins and Amino acids ● Expected Result:
I. Qualitative Test and Analysis of ○ (+) Amino acids
Unknown having hydroxy
1. Biuret Test benzene (tyrosine)
 ○ Brick red color
5. Hopkins-Cole Reaction
● 10 drops glycoxylic acid
● 10 drops sample
● 2 mL concentrated H2SO4
● Allow to stand for 10 minutes
● Expected Result:
○ (+) Presence of
imidazole ring
○ tryptophan
○ Purple
6. Sakaguchi Reaction
● 10 drops sample
● 5 drops 10% NaOH to
ensure alkaline medium
● Add 2 drops of dilute
a-naphthol in alcohol and mix
● Add 3 drops of NaOBr
● Expected Result:
○ (+) Presence of
guanidine/arginine
○ Red color
7. Lead Acetate Reaction
● 10 drops sample
● 3 strands of hair
● 10 drops of 20% NaOH
● 1 drop 10% Pb(OAc)2
● Heat for 5 mins
● Expected Result:
○ (+) Presence of sulfur
in protein (cysteine)
○ Black precipitate
II. Precipitation Reaction
1. Heat and Acid
● 10 drops of 10% albumin in
two tubes
● 1 drop 5M HOAc to the
second tube
● Gently boil
● Expected Result:
2. Alcohol
● 10 drops ethanol
● 10 drops 10% alumin sol’n
● Expected Result:
○ White precipitate,
cloudy
3. Alkaloidal Reagents
● 10 drops of 10% albumin
● 2 drops picric acid
● Repeat using 2 drops of
trichloroacetic acid
● Expected Result:
○ With precipitate
○ Yellow
4. Heavy Metal Salts
● 10 drops of 10% albumin
● 2% CuSO4 dropwise until
precipitate forms
● Repeat using 2% FeCl3
● Expected Result:
○ 10% albumin +
CuSO4: cloudy, with
precipitate
○ Albumin + 2% FeCl3:
without precipitate,
yellow color
5. Salting-Out
● 10 drops albumin in two
separate tubes
● Tube 1
○ Add solid (NH4)2SO4
until no more salt
dissolves
● Tube 2:
○ Add solid NaCl until
no more salt
dissolves
● Expected Result:
○ Albumin +
(NH4)2SO4: white
precipitate, dissolved
○ Albumin + NaCl: no
precipitate, did not
dissolve
 6. Precipitation at the Isoelectric Point ● Cover with watch glass
● 20 ml of skimmed milk ● Remove paper from the bath when
● Pour in 50 mL beaker solvent is 2cm from the upper edge
● Check initial pH of skimmed ● Mark the solvent front using pencil
milk ● Dry the paper then spray with 0.1%
● Add 0.05 HCl dropwise until ninhydrin solution
pH is 4.5 ● Blow dry and encircle spots
● Transfer the mixture into 2 ● Calculate Rf value of each amino
small centrifuge tubes acid:
● Centrifuge for 3 mins ○ Distance travelled by amino
● Discards the supernate acid / Distance travelled by
● Add 10 drops HCl to Test solvent
tube 1
● Add 10 drops NaoH to Test
tube 2
● 10 drops albumin in two
separate tubes
● Expected Result:
○ Initial pH with
skimmed milk: 7
○ Casein + 1.0 M HCl:
precipitate did not
dissolve
○ Casein + 1.0 M
NaOH: Precipitate
dissolved

III. Amino Acid Analysis

● 5 by 4 square inches filter paper
● Draw a line using pencil on one side
of the paper 1 cm from the edge
● Mark 6 points along this line
○ Amino acids and unknown
● Using capillary tubes, place a drop
of the amino acid solution on the
designated spot
● Allow samples to dry
● Form a cylinder by connecting one
end of the paper with the other
● Place the paper in a jar containing
solvent (n-butanol:HOAc:H2O
100:22:50) Make sure this line is not
submerged in the solvent