Analysis of Carbohydrates

A. General Test for Carbohydrates

Samples (1%): Glucose (mono, aldohexose), Sucrose (di), Arabinose (mono,

aldopentose), Starch (poly), cotton or filter paper suspension (poly, cellulose)

 Molisch Test for Carbohydrates

- Positive Test: Purple Color at the interface of the sugar and acid (ALL)
- Dehydration of pentoses and hexoses through test reagent and form
furfural derivatives, then furfurals further react with a-naphthol to form
purple product.
- The test reagent dehydrates pentoses to form furfural and dehydrates
hexoses to form 5-hydroxymethyl furfural. The furfurals further react with
a-naphthol present in the test reagent to produce a purple product.
- Carbohydrates + H2SO4 undergo dehydration to give furfural derivatives
- Condense with alpha-naphthol or thymol to form colored products
- Pentoses yield Furfural
- Hexoses yield 5-hydroxymethylfurfurals or HMF
- Oligosaccharides and Polysaccharides need to be hydrolysed to
monosaccharides and be dehydrated to give a + test
 Anthrone Test for Estimation of Mono and Poly (Quali and Quanti)
- Positive Test: Deep green or blue color
- Dehydration of monosaccharides to furfural derivatives
- 50% H2SO4 used to dilute to dissolve cloudy ppt
 Iodine Test for Polysaccharides (Presence of Helical Poly)
- Positive Test: Blue-black color Negative Test: Brown-blue color
- Starch (+) Dextrin (+) Glycogen ( - )
- More iodine atoms in helices=more intense color
- Starch-iodine complex and lycogen-iodine complex

B. Qualitative Tests for Sugars

Test Solutions(5%): Glucose (hexoses), Fructose (ketohexose), Arabinose (mono,

aldopentose), Maltose (di), Sucrose (di, ketohexoses), Galactose (aldohexose),
Lactose (di)

Reducing Sugar = there is a free OR group

 Benedict’s Test for Reducing Carbohydrates

- Reagent contains Cu2+ ions in Alkaline sol. with Sodium Citrate added to
keep the ions in the solution, Sol. has been used for testing urine
- semi-quantitative test, Alkaline sol. of Cu are reduced by sugars having
free aldehyde or ketone group with the formation of Cu2O
- Positive Test: Deep-red Cu2O ppt Negative Test: Blue
 - Glucose ( + ) Fructose ( + ) Sucrose ( - )

 Barfoe
d’s Test for Reducing Monosaccharide
- Reduce monosaccharides very fast, reduce disaccharides very slow
- Use Cu(II) ions in a slightly acidic medium
- Reducing mono are oxidized by the Cu ion in sol. to form a carboxylic acid
and a red ppt of Cu (I) oxide
- Positive Test: Red Cu2O ppt within 2-3 mins, green or yellow ppt
- Maltose ( - ) Lactose ( - ) Sucrose ( - )

 Seliwanoff’s Test for Ketose

- Used to differentiate ketose from aldose
- Aldoses converted to ketoses
- Reagent uses 6M HCl as the dehydrating acid and resorcinol as the
condensation reagemt
- Reagent dehydrates ketohexoses to form 5-hydroxymethylfurfural then this
further reacts with resorcinol in reagent to produce the color
- Positive Test: Cherry-red condensation product within 2 mins Negative:
no
- Ketohexoses and Di are positive
- Glucose, Galactose ( - ) Fructose ( + ) Hydrolysate from Inulin ( + )
 Orcinol Test for Pentose
- Used to differentiate pentose from hexose
- Pentoses are converted to furfural by reagent and form color with orcinol
- Bial’s reagent contains orcinol (5-methylresorcinol in concentrated HCl
with a small amount of FeCl3 catalyst
- Decomposition of sugars when heated with concentrated HCl to a furfural
which then condenses with orcinol to form colored compounds
- Positive Test: Blue-green color Negative Test: Green, Red, Brown
- Arabinose ( + ) Hydrolysate from Gum Arabic ( + )

 Osazone Test to Distinguish Sugars

- Sugars form osazones with phenylhydrazine
- Osazones have characteristic crystalline forms, quite definite time of
formation, and differ in their relative solubility in water
- Osazone Crystals = Yellow crystals with diff shapes under microscope
- Glucose (needle, 2 mins), Fructose (needle, 2mins), Galactose (rhombic
plate, 15-19 mins), Maltose (sunflower, ppted when cooled), Lactose
(powderpuff/Hedgehog, ppted when cooled), Arabinose (10 mins)
- Sucrose ( - ) no reaction/crystals

 Mucic Acid Test for Galactose and Lactose

- Aldohexoses are converted to their corresponding dicarboxylic acids in the
presence of strong oxidizing agents like conc. HNO3
- The dicarboxylic acid produced from galactose (mucic acid) is relatively
insoluble and separates out as colorless crystals
- Positive Test: Colorless needle-like crystals (Mucic acid crystals)
- Galactose ( + ) Lactose ( + )
 Lipids

A. Acrolein Test for Glycerin or Fats

- Positive Test: Colorless to yellow liquid, irritating (burnt grease or candle

wax) odor
- Glycerol ( + ) Coconut Oil ( + ) Oleic Acid ( + ) Lecithin ( - )

B. Test for Unsaturation

- Addition of Hubl’s sol. (I2 in HgCl2) across the double bond

- I2 saturation
- All double bonds are converted to single bonds when the sample solution
already has the same color as Hubl’s sol.
- Positive Test: Red-orange
-

C. Test for Phosphate

- Phosphate residues of phospholipids will react with ammonium molybdate

(NH4)2MoO4, in HNo3
- Positive Test: Yellow ppt Lecithin ( + )
D. Emulsification Test

- Suspension of liquid within another liquid

- Formation of pseudo solutions by lipids in water as micelles
- Pseudo Sol. are many bubbles oil, good emulsifying agent to put oil to the
bottom
- Positive Test: bubbles suspended in water
- 1mL coco oil + 5 drops 0.1% bile salt = small bubbles suspended in water
- 1 mL coco oil + 5 drops H2O + tiny crystal of chol. = large bubbles “
- 1 mL olive oil + 5 drops 1% aq. sol’n of lecithin = small bubbles “

E. Lieberman-Burchard or Acetic Anhydride Reaction (estimate Cholesterol)

- Chol. Will react with acetic anhydride in acidic medium (H2SO4)

- The hydroxyl groups of chol. Will react with the reagents
- Unsaturated sterols react more rapidly than the saturated ones
- Positive Test: Deep blue-green
Red or pink Purple Blue Bluish-green

F. Carr-Price Reaction for Vitamin A and related Carotenoids

- Vit A will react with antimony trichloride, SbCl3, to yield a Brilliant blue colored
product.
- Positive Test: Color change
Blue Green Gray Pink
- Cod Liver Oil ( + ) Vit A ( + ) Coco oil ( - ) Olive oil ( - )

G. Modified Furter-Meyer for Vitamin E and related Tocopherols

- Vit. E will react with n-amyl alcohol in HNO3 to yield bronze-red color
- Carotenoids and certain sterols will yield Yellow or Brown, and when present
in sufficient amounts, may mask the bronze-red color of small amount of
tocopherols
- Positive test: Bronze-red a-tocopherol ( + ) vit E ( + )

Isolation and Purification of Proteins

- Casein is soluble at pH 6.6 so it cannot be seen in milk, but solubilized as a

salt: Calcium Caseinate
- Lowering pH will lead to Isoelectric point (4.6) which makes casein become
insoluble and be seen
- Negative charges on the outer surface of the micelle are neutralized,
phosphate groups are protonated, neutral protein precipitates
- Denurturation= opens up 3D/globular form of Casein protein
- Anionic Form = pH is raised Cationic form = pH is lowered
- 1% AgNO3 = remove Cl so that casein will not have/react with HCl when
isolated
- Acetic Acid = used instead of HCl to be Cl free
Ca2+¿ caseinate +2 HCl→ Casein+CaCl ¿
2