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Procedure:
o 3mL Seliwanoffs reagent + 0.5mL test solution
o Heat mixture in boiling water bath for 10-15 minutes
Procedure: o Red color or brownish-red precipitate
o Test solution + 2 drops Molisch reagent mix
o Incline the test tube and + let 1mL conc. H2SO4 flow
down the wall of the tube
1
B. Reduction of Metallic Ions by Sugars o Time: The time formation for each osazone is quite
The reducing properties of sugars are dependent on the definite
presence of an actual or potential aldehyde or ketone o Solubility: Some osazones are relatively insoluble
group in the molecule in hot water whole others are soluble
Principle: When solutions of such sugars o Melting point: Each osazone, if carefully purified by
(monosaccharides and disaccharides- maltose and recrystallization and dried, has a definite melting
lactose) are heated in the presence of certain metallic point
ions, the latter are reduced to a lower valence state o Form: Osazones crystallize in characteristic forms
Reagents: which could be appreciated by microscopic
o Solutions of metallic salts of copper, mercury, examination and reference to standard osazones
bismuth, iron, gold, or silver (Cu: most widely used) Concept: Each individual sugar should give rise to an
o Alkaline medium osazone typical for that sugar
o Organic compounds containing alcoholic groups Exception: Glucose, glucosamine, fructose, and
1. Benedicts Test mannose yield the same osazone because of similarities
Test for ketoses (reduces the reagent more rapidly than in their molecular structure
aldoses) for detection of sugar in the urine The reaction with glucose and maltose resulting in the
Benedicts reagent: CuSO4, Na2CO3, formation of osazones are represented thus:
Ca3(C6H5O7)24H2O (calcium citrate)
Principle:
o The cupric ion is reduced
o The resulting cuprous oxide precipitates out of the
alkaline solution brick red solid
The reactions are shown in the ff. equations:
Procedure:
o 3mL Benedicts solution boil for 1 minute (must
remain clear blue)
o Add 8 drops of test solution continue: boiling
o Blue (di/poly/oligosaccharide) or Brick red
Procedure:
(monosaccharide)
o 1mL phenylhydrazine + 5 mL test solution shake
2. Barfoeds Test
Test to differentiate monosaccharides from and stopper boiling water bath (record time)
disaccharides o Remove if there is a distinct precipitate (yellow
o Not a specific test for glucose, to detect crystals)
monosaccharides o If there is no precipitate after 40 mins, add 4 mL
o Unsuited for the detection of sugar in urine or in any distilled water (cooling effect of added water usually
fluid containing chlorides causes an immediate recipitation of the osazone)
o Return to the boiling water bath until osazone
Barfoeds reagent: CuSO4 and HOAc
redissolves
Principle: Copper reduction test carried out in an acid
o Allow the test rube to cool slowly to room temp
solution
o Examine the crystals under LPO
Product: Brick red precipitate 2. Mucic Acid Test for Galactose
Concepts:
Test for galactose
o Sugars are less reactive in acid than in alkaline
Three principle derivatives of aldoses may be obtained
media
by appropriate oxidation to carboxylic acids:
o Disaccharides are less reactive than
o Aldonic acids (alcoholic acids): aldehyde is
monosaccharides
oxidized
Complications: o Uronic acids (aldehyde and ketone acids):
o Disaccharides will also respond to the test with primary alcohol group is oxidized
prolonged heating o Dicarboxylic sugar acids (saccharic acids): both
o Under proper conditions of acidity, the aldehyde and primary alcohol groups are oxidized
disaccharides may be hydrolyzed
Principle:
Procedure: o Galactose reacts with the oxidizing agent HNO3
o 3mL Barfoeds reagent + 10 drops test solution
o The aldehyde and primary alcohol groups are
o Boiling water bath for 5 minutes cool oxidized to carboxyl groups
o Brick red precipitate o Mucic acid is insoluble and separates out as
C. Oxidation of Carbohydrates colorless crystals in the cold
1. Osazone Test for Phenylhydrazine Reaction
Concept: Mucic acid is the saccharic acid formed from
Test for reducing sugars with free aldehyde or a ketone free or combined galactose
Principle: Complication: HNO3 could not be used in the
o Reducing sugars which have either a free aldehyde fragmentation and the formation of furfural derivatives
or a ketone group will react with phenylhydrazine to from sugars
form osazones o Has strong oxidizing properties
o With an excess of phenylhydrazine, the carbon o Capable of converting sugars to saccharic acids
adjacent to the aldehyde or ketone group is
The oxidation reaction is shown in this equation:
oxidized to an aldehyde or a ketone
o The aldehyde or ketone condenses with
phenylhydrazine to form a yellow crystalline
compound or osazone
These yellow compounds are valuable for the
identification of sugars for the following reasons:
2
Procedure: Iodine:
o 2mL test solution + 2mL conc HNO3 Blue: starch is present (starch-iodine
o Boiling water bath for one hour complex formed)
o Scratch inner wall to hasten formation of crystals Orange or yellow: starch has been broken
(fine particles of glass will facilitate crystal down (original color)
formation) Benedicts:
o Refrigerate test tube for 30-45 mins, if no crystals Blue: starch is still present (original color)
o Examine crystals under LPO Red: starch has been broken down
D. Hydrolysis of Carbohydrates 2. Enzymatic Hydrolysis of Starch
Starch Course of Hydrolysis: ends at maltose with traces of
o Used as the substrate in the demonstration of dextrins
hydrolysis of carbohydrates Human salivary -amylase or ptyalin
o Storage form of carbohydrates in plants o Contains 1 mole of calcium ions per mole of
o Contains an outer layer of amylopectin and an enzyme protein
inner layer of amylose Bound calcium is required for enzymatic activity
Can be brought into colloidal solution by heating o Requires the presence of anions for activity
with boiling water Chloride is the most effective activator (bromide
Both react with iodine (differ in color intensity): and iodide are also activating)
Amylose: deep blue color Method of dialysis
Amylopectin: light violet color o To demonstrate the diffusibility of carbohydrates
The color is formed on the surface of the celloid and the effect of digestion on starchy foods
particles when iodine is adsorbed and discharged o Entails the use of a membrane such as parchment,
(iodine is reduced to iodide ion) collodion, cellophane, or an animal bladder
Principle: Upon hydrolysis, the acetal linkages of the Selectively allows the passage of crystalloids
polysaccharide/dissacharide molecules take on a (particles which diffuse readily through the
molecule of water and break down into smaller membrane)
units/monosaccharides Prevents colloids (larger particles than
Concept: (on acetal linkages) crystalloids that dont pass through the
o Relatively stable in basic media membrane)
o Easily hydrolyze in acid media, under enzymic Principle: If a mixture of crystalloids and colloids is
conditions, and by bacterial action placed in a dialyzing bag which is then immersed in
1. Acid Hydrolysis of Starch distilled water, the colloids will remain behind while the
Principle: crystalloids dialyze out
o Polysaccharides (starch glycogen, inulin, or Condition:
disaccharides sucrose, maltose, and lactose) are o A preservative such as toluene, mercuric iodide or
heated with weak acids (about 10%) 1% boric acid is added to the starch solution and
o The sugars are broken down to their constituent distilled water
monosaccharides o To inhibit bacterial action which causes
Concept: Acidic hydrolysis of starch yields a succession decomposition and makes the solution ineffective
of polysaccharides of gradually diminishing size with the as an indicator
ultimate production of glucose Procedure:
Course of Hydrolysis (may be followed by the gradual o 2-3mL filtered saliva + 20mL 1% starch paste
change in color produced by iodine and by the Benedicts o Allow to stand at room temp for an hour (note
test from most complex (top) to simplest (bottom): change in viscosity)
o Starch o Introduce to a dialyzing bag, string, and suspend it
o Soluble starch in 100mL distilled water (let stand for 45 mins)
o Amylodextrins o Test contents of dialyzing bag and solution in
o Erythrodextrins beaker with iodine and Benedicts test
o Achroodetrins o Results (Benedicts):
o Maltose Blue: starch is still present (original color)
o Glucose Red: starch has been broken down
The formula for the hydrolysis of starch is shown below: II. PROTEINS
Comprise the greater part of the organic matter of the
protoplasm of plant or animal cells
Contain the elements: N, C, H, O, and most also contain S, P
Amino acids: structural units of proteins
Procedure: Proteins may be identified by three types of reactions:
o 7 test tubes with 2mL Benedicts reagent o Reactions which are due to the presence of specific
o Hydrosylate: In a 50mL Erlenmeyer flask, 20mL 1% chemical groups or linkages in the protein molecule and
starch solution + 0.5mL conc HCl or H2SO4 mix the chemical reagent used in any given test (e.g. biuret,
and boil gently ninhydrin, xanthoproteic, etc.)
o After 5 mins boiling, test the hydrosylate every 1 o Reactions which are due to the acidity or basicity of the
min interval (on a spot plate) protein molecule (e.g. precipitation by strong mineral
o Add 2 drops iodine in KI solution (stoppered since acids, salts of heavy metals, or alkaloidal reagents)
I2 is volatile and will produce a false negative result) o Reactions which are due to the colloidal nature of the
o If after 5 min, iodine test is still positive, continue proteins (e.g. heat coagulation and salting out)
testing the hydrolysate at 5 min intervals until iodine Test Solutions:
color remains unchanged o Egg Albumin
o 3 drops of digest + 2mL Benedicts reagent (tubes Water-soluble, moderately soluble in concentrated salt
1 to 5) solutions, and experience heat denaturation
o Boil (3 mins) all test tubes containing Benedicts o Casein
solution then cool Main protein in milk and cheese
o Results: Contains a fairly high number of proline residues,
which do not interact
No disulfide bridges
3
o Gelatin o Excess ammonium salts (e.g. (NH4)SO4) interfere
Mixture of peptides and proteins produced by partial by salting out proteins from their solutions (a
hydrolysis of collagen positive test is not obtained with high
o Peptone concentrations of ammonium salt)
A soluble protein formed in the early stage of protein Condition:
breakdown during digestion. o Sodium hydroxide in excess of that required to
o Tryptophan decompose the ammonium salts must be present
Essential, aromatic amino acid (biuret test requires the presence of a strong base)
o Tyrosine o The basic pH prevents protonation of the peptide
Non-essential, aromatic amino acid nitrogen
o Cystine Procedure:
Oxidized dimer of cysteine o 1mL test solution + 1mL 10% NaOH mix
Non-essential, sulfur-containing o Add 5 drops 5% CuSO4
A. Color Reactions of Proteins and Amino Acids o Pink to violet solution
General Principle: Due to a reaction between one or 2. Ninhydrin Test or Triketohydrindenehydrate Reaction
more of the constituent radicals (chemical groups or Most general and one of the most sensitive reactions
specific linkages) of the complex protein molecule and known for qualitative detection of proteins and their
the chemical reagents used in any given test hydrolysis products: proteoses, peptones, peptides, and
Complications: amino acids
o Not all proteins contain the same amino acids Principle:
therefore various color tests will yield reactions of o A neutral solution with free carboxyl group, amino
varying intensity of color (according to the nature acids, and proteins are made to react with ninhydrin
and amount of the groups contained in the certain (triketohydrindenehydrate), a powerful oxidizing
protein) agent
o Various substances (not protein) respond to certain o Once the solution has been heated, a blue/purple
of these color reactions color will appear (Ruhemanns blue/purple)
Condition: Submit the material under examination to Reaction:
several tests before concluding definitely regarding its o An alphadecarboxylation by ninhydrin produces
nature CO2, NH3, and an aldehyde with one less carbon
Uses of color tests: atom than the parent amino acid
o Basis for the quantitative estimation of proteins and o The reduced ninhydrin then reacts with the
amino acids liberated NH3, forming a blue complex
o May aid in the determination, identification, and
estimation of constituent amino acids in proteins
1. Biuret Test
General test for proteins
Biuret:
o Non-physiological substance
o Formed on heating urea to 180 Co
Principle: Coordination of cupric ions with the unshared
electron pairs of four nitrogen atoms, two from each of
two peptide chains, as shown below
Bials Orcinol - - - - - +
Yellow soln Yellow soln Brown soln Yellow soln Brown soln Blue green soln
Taubers +
- - - - -
Benzidine Brown soln
Seliwanoff - - + - + -
Dark yellow soln Dark yellow soln
+ + + + - +
Benedicts Orange soln,, brown Orange soln and
Orange soln and ppt Orange soln and ppt Blue soln Orange soln and ppt
ppt brown ppt
Barfoeds + + + - - +
Brick red ppt Brick red ppt Brick red ppt No ppt No ppt Brick red ppt
Osazone + + + + + +
Orange ppt Yellow ppt Orange ppt Yellow ppt Yellow ppt Dark brown ppt
Mucic Acid + - - - - -
White crystals
Enzymatic Hydrolysis[1]
Solution Iodine Test Benedicts Test
Concentrate (solution inside the bag) + -
Dialyzing Bag Hydrolysate (solution outside
- +
the bag)
Acid Hydrolysis
Time in minutes Iodine Test Benedicts Test
1 + (purple) - (blue green)
2 + (purple) - (blue green)
3 + (purple) - (blue green)
4 + (purple) + (yellow)
5 + (purple) + (yellow)
10 - (light purple) + (yellow orange)
15 - (yellow + (orange)
20 - (yellow) + (dark orange)
25 - (yellow) + (red)
Glucosazone (needle-like) Maltosazone (sun-flower) Galactosazone (rhombic) Mucic acid crystals (galactose)
[1]
The dialyzing bag which contains the saliva and starch paste was submerged into the beaker with distilled water. Ideally, some of the starch would have
been broken down, therefore some maltose/glucose (dialyzing bag hydrolysate) would have diffused into the solution outside the bag, giving it a negative
result for iodine, and positive result for Benedicts. As for the concentrate, some starch would not have been broken down, giving a positive result for
iodine, and negative result for Benedicts.
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PROTEINS
SUMMARY OF COLOR REACTIONS OF PROTEINS AND AMINO ACIDS
Color Test Group Involved Reagents Compound Produced Visible Result
1% NaOH Coordination complex
Biuret Test Peptide linkage Pinkish violet solution
5% CuSO4 between Cu and N atoms
Ninhydrin Test Free COOH Group and/or
(Triketohydrindenehydrate NH2 group to the COOH 0.1% Ninhydrin solution Ruhemanns blue/purple Blue or purple solution
Reaction) group
Concentrated HNO3
Xanthoproteic Test Benzene ring Nitroderivative of benzene Orange solution
10% NaOH
15% HgSO4 in 6N H2SO4 Mercury salt of phenolic
Millon-Nasse Test Phenol group Old rose to red solution
1% NaNO2 compound
40% Formaldehyde
Acree-Rosenheim Indole ring (benzene + Violet complex of unknown Reddish-violet ring at
15% HgSO4 in 6N H2SO4
Reaction pyrrole rings) nature interphase
Concentrated H2SO4
Bromine water
Pink/lavender alcohol layer
Bromine Water Test Free tryptophan Concentrated amyl alcohol Brominated tryptophan
(upper)
or ethyl acetate
Lead Acetate Reaction 10% NaOH Brown solution or black
Sulfur PbS
(Unoxidized Sulfur Test) Saturated lead acetate precipitate
RESULTS FOR COLOR REACTIONS AND OTHER TESTS
Test Solution
Color Test
Albumin Casein Gelatin Peptone Tryptophan Tyrosine Cystine
Biuret + - + + - - -
Purple soln Blue soln Purple soln Purple soln Blue soln Blue soln Blue soln
Ninhydrin + + + + + + +
Purple soln Blue soln Blue soln Dark violet soln Dark violet soln Purple soln Yellow soln
Xanthoproteic + + - + + + -
Yellow soln Yellow soln Clear soln Yellow soln Yellow soln Yellow soln Clear soln
Millon-Nasse + + + + - + -
Old rose soln Old rose soln Old rose soln Old rose soln Yellow soln Old rose soln White ppt
Acree- + + - + + - -
Rosenheim Purple ring Purple ring Clear soln Purple ring Purple ring Clear soln Clear soln
Bromine - - - - + - -
Yellow soln Yellow soln Yellow soln Yellow soln Pink soln Yellow soln Yellow soln
Lead Acetate - - - - - - +
White ppt White ppt White ppt White ppt White ppt White ppt Grey ppt
Tests Egg Albumin Gelatin Peptone
Strong Acids (H2SO4, HNO3, HCl) + (Cloudy white soln) - (Clear soln) + (Cloudy brown soln)
Alkaloidal Reagent: Picric Acid + (Turbid) + (Turbid) - (Clear soln)
Alkaloidal Reagent: 10% TCA + (Turbid) - (Clear soln) + (Turbid)
Salts of Heavy Metals: 1% HgCl2 + (Turbid) - (No ppt) + (Turbid)
Salts of Heavy Metals: 1% AgNO3 + (Turbid) - (No ppt) + (Turbid)
Ethanol + (Turbid) + (Turbid) - (Clear)
Salting out: Saturation with
+ (Ppt) + (Ppt) - (No ppt)
(NH4)2SO4
Salting out: with addition of H2O + (Reversible) + (Reversible) - (No ppt)
Heat: upon boiling - (No change) - (No change) - (No change)
Heat: after adding 1% HOAc + (White ppt) - (No change) - (No change)
Reference tube no. Gelatin concentration in mg Number of Tube Percent Hydrolysis
1 10 1 20%
2 8 2 40%
3 6 3 60%
4 4 4 80%
5 2
6 0
10
% = 100
10
Reference:
Laboratory Manual in Biochemistry. Department of Nutrition, College of Public Health.
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