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Lab Expts 5-7 Review

The document provides details on various chemical tests used to analyze carbohydrates, lipids, proteins, and vitamins. It describes tests such as the Molisch test, Benedict's test, iodine test, emulsification test, and Lieberman-Burchard test. Specific tests are used to identify the presence of sugars, fats, phospholipids, cholesterol, vitamins A and E, and proteins like casein. The results of the tests can be used to distinguish between different classes of biomolecules and sometimes specific biomolecules based on color changes or precipitate formation.

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Kyra Bianca R. Famacion
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195 views

Lab Expts 5-7 Review

The document provides details on various chemical tests used to analyze carbohydrates, lipids, proteins, and vitamins. It describes tests such as the Molisch test, Benedict's test, iodine test, emulsification test, and Lieberman-Burchard test. Specific tests are used to identify the presence of sugars, fats, phospholipids, cholesterol, vitamins A and E, and proteins like casein. The results of the tests can be used to distinguish between different classes of biomolecules and sometimes specific biomolecules based on color changes or precipitate formation.

Uploaded by

Kyra Bianca R. Famacion
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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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

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