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CARBOHYDRATES o Purple ring at the junction

Have structural as well as metabolic functions in both animal 2. Bials Orcinol Test
and plant tissues Test for pentoses (not absolutely specific for pentoses)
o Animal tissues: glycose and glycogen Principle:
o Plant tissues: starch, sugars, and cellulose o Pentoses are decomposed when heated with
Chemically, they are: concentrated HCl, forming a furfural
o Aldehyde or ketone derivatives of the polyhydric alcohols o The furfural condenses with orcinol to form blue to
o Compounds which can be converted into such aldehydes green colored compounds
or ketones by hydrolysis Concept: Furfural is more rapidly formed from pentoses
A number of reactions are used in the characterization of than hexoses under the influence of acid and heat
carbohydrates. Complications: Prolonged heating of some hexoses
Grouped according to the chemical reaction involved: yields hyroxymethylfurfural which also reacts with orcinol
o Fragmentation of carbohydrates by strong mineral acids to give a colored complex
o Reduction of certain metallic ions by sugars The proposed reaction is:
o Oxidation of carbohydrates by concentrated mineral acids
o Hydrolysis of carbohydrates by weak acids and enzymes
Test Solutions:
Glucose Aldehyde Hexose Reducing
Fructose Ketone Hexose Reducing
Galactose Aldehyde Hexose Reducing
Xylose Aldehyde Pentose Reducing
o 1mL Bials reagent + 1mL test solution
Maltose Glucose + glucose Reducing o Boil until bubbles come to the surface
Sucrose Glucose + fructose Non-reducing o Blue green color
A. Fragmentation with Strong Acids 3. Taubers Benzidine Test
Carbohydrates (particularly monosaccharides) are More specific test for pentoses (can detect as little as
subjected to strong solutions of HCl or H2SO4, at 0.01mg of a pentose 1 drop of 0.02% solution, either
elevated temperatures free or combined as in the various nucleotides)
The sugar is dehydrated and furfural or furfural Concept: The presence of a high concentration of
derivatives are formed hexoses does not interfere with the test
The furfural condenses with various aromatic Product: Cherry red color
compounds like phenol, alpha naphthol, and aromatic Condition: In testing for pentoses in urine, protein, if
amines to give colored derivatives present, must be removed first
Concept: Ketoses are more readily fragmented than Procedure:
aldoses o 0.5mL Taubers reagent + 5 drops test solution
1. Molisch Alpha Naphthol Reaction o Boil over open flame for 1-2 minutes
General test for carbohydrates in free or combined form o Cherry red solution
(but not specific for carbohydrates, see Complications) 4. Seliwanoff Test
Principle: Test for ketose sugars
o The presence of concentrated H2SO4 o Depends upon the more rapid formation of furfural
o Glycosidic bonds are hydrolyzed, giving from ketoses as compared to aldoses in the
monosaccharides presence of heat and HCl
o The monosaccharides are dehydrated to furfural, Principle: The furfural derivative condenses with
hydroxymethyl- furfural and other decomposition resorcinol (m-dihydroxybenzene)
products Product: Red compound with or without the separation
o These react with the alpha-naphthol forming purple- of a brown-red complex
colored condensation products at the zone of
Concept: This test is not specific for fructose but it is
junction of the two liquids
given by all ketose sugars
Complication: On prolonged heating, glucose or other
o Concentrated solutions of organic compounds may
aldoses, sucrose and maltose may also give a red color
give a red instead of a violet color due to the
with the reagent
charring action of H2SO4 (repeat reaction on a more
dilute solution of the specimen)
o Furfural and other furfural-yielding substances may o The concentration of HCl must not be more than
test positive 12%
o The reaction must be observed after not more than
A negative result is good evidence of the absence of
20-30 seconds of boiling
o Glucose must not be present in amounts exceeding
The reaction with a pentose and a hexose are
represented in these equations:
The proposed reaction is:

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
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)
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:

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
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
Principle: Copper reduction test carried out in an acid
o Allow the test rube to cool slowly to room temp
o Examine the crystals under LPO
Product: Brick red precipitate 2. Mucic Acid Test for Galactose
Test for galactose
o Sugars are less reactive in acid than in alkaline
Three principle derivatives of aldoses may be obtained
by appropriate oxidation to carboxylic acids:
o Disaccharides are less reactive than
o Aldonic acids (alcoholic acids): aldehyde is
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
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:

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
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
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

Product: Pinkish-violet biuret complex in an alkaline

Concept: Basis for several different types of quantitative methods
o The intensity of the purple color produced is for the determination of alpha amino acids:
proportional to the number of peptide bonds o Color change which results from the reaction
present and hence, to the amount of protein o Determination of ammonia produced
o The color also depends upon the nature of the o Measurement by gasometric means of the carbon
protein dioxide evolved (Van Slyke Method)
Proteoses and peptones give a decided pink Conditions:
color o Freshly prepared ninhydrin must be used
With gelatin, the color produced is not far o The reaction proceeds best in neutral solution (pH
removed from blue between 5 and 7)
Positive for: o A few crystals of sodium acetate may be used to
o All substances with 2 or more peptide bonds adjust the pH
o Soluble proteins (negative for coagulated) Procedure:
o Non-protein in character but which contain in piece o 1mL test solution + 0.25mL freshly prepared 0.1%
of the CONH2 group such as: CSNH2, CH2NH2, ninhydrin solution heat then cool
or C(NH)NH2 o Compare intensity of blue solution
Complications: 3. Xanthoproteic Test
o The presence of magnesium sulfate in considerable General reaction of proteins containing amino acids
amounts in solutions containing protein interferes which have a benzene ring structure
with the test, forming a magnesium hydroxide Principle: Phenyl group (-C6H5) is nitrated upon heating
precipitate with nitric acid (HNO3)
Product: Nitroderivatives of benzene which are initially
yellow (xanthine) but become orange when alkalinized
Indicates the presence of: Procedure:
o Aromatic amino acids o 1mL test solution + 1 drop 40% formaldehyde
o Tyrosine (1/500 commercial formaldehyde) + 1 drop 15
o Tryptophan HgSO4 in 6N H2SO4 mix
Condition: H2SO4 is required as a catalyst o Incline and slowly add 2mL conc H2SO4 2 distinct
Complication: Phenylalanine is not as readily nitrated layers
does not/weakly responds to this test o Purple ring at interphase
6. Bromine Water Test
To detect the presence of free tryptophan (does not
respond to tryptophan combined in the protein molecule)
Principle: Reaction of bromine water, amyl alcohol, and
tryptophan (bromination of the pyrrole ring)
Product: Brominated tryptophan
Characterized by: Pink or lavender color in the upper
o 0.5mL test solution + 0.5mL conc HNO3 heavy
alcohol layer
white precipitate must form
o Heat precipitate turns yellow then dissolves Complication: The pink color may disappear due to
o Cool then add 2mL 10% NaOH til alkaline to litmus excess bromine water and it may be masked by the color
o Orange solution of the reagent
4. Millon-Nasse Test The bromination (or halogenation) of the pyrrole ring is
shown below:
Principle: Hydroxyphenyl group (-C6H4OH) reacts with
the Millon-Nasse reagent (HgSO4 in H2SO4)
Product: Mercury salts of the phenolic compound which
are typically colored old rose to red (either in solids or
solutions) Procedure:
Positive for: Any phenolic compound which is o 1mL test solution + 5 drops freshly prepared brome
unsubstituted in the 3,5 positions (e.g. tyrosine, phenol, water + 1mL conc amyl alcohol/ethyl acetate
and thymol) o Pink alcohol layer (upper)
Complications: 7. Lead Acetate Reaction or Unoxidized Sulfur Test
o For egg white solution, the initial white precipitate Principle: Liberation of sulfur when proteins in strongly
(after mercuric sulfate reagent) is not a positive test alkaline solutions are boiled
but rather the appearance of the color of the Product: Darkening or browning of the color or a black
precipitate (after nitrate treatment) indicates the precipitate of PbS
presence of tyrosine Positive for: Sulfur-containing proteins (e.g. cysteine,
o For hydrolytic products of protein (e.g. peptone or cysteine, methionine)
peptides which contain tyrosine), a colored solution, Complication: Methionine does not give a positive test
not a precipitate, will be obtained o The thiomethyl group of methionine is not readily
removed by alkali
o Requires fusion with an oxidizing mixture
o Cystine and cysteine sulfur was formerly termed
Procedure: unoxidized, loosely-combined, mercaptan, or lead-
o 1.5mL test solution + 0.5mL 15% HgSO4 in 6N blackening sulfur
H2SO4 heat o Methionine sulfur was called oxidized or acid sulfur
o Cool for 3 mins then add 0.5mL 1% NaNO2 o Majority of proteins contain more methionine sulfur
o Old rose solution than cysteine and cysteine sulfur (exceptions:
5. Acree-Rosenheim Reaction keratins, insulins, and certain serum albumins)
Principle: Condensation of tryptophan with aldehydes in
the presence of strong sulfuric acid and a trace of
oxidizing substance
o Aldehyde couples with two molecules of the indole
derivative by a loss of one water molecule
o One hydrogen ion from the carbon adjacent to the
nitrogen in the ring is lost by each indole unit
o With glyoxylic acid, the reaction with tryptophan is
shown in this equation:

Product: Violet colored compound of unknown nature Procedure:

Characterized by: Reddish-violet color at the interphase o 1mL test solution + 1mL 10% NaOH + 1mL
between H2SO4 and formaldehyde solution saturated lead acetate heat
Positive for: Compounds with the indole ring (e.g. o Brown solution or black precipitate
tryptophan, nearly all proteins; except gelatin and B. Precipitation Tests for Proteins
insulin) No definite evidence concerning the nature of the
Conditions: mechanisms involved
o Chemically pure H2SO4 must be used since Precipitation reactions are probably due to:
excessive amounts of oxidizing substances (e.g. o The formation of insoluble complexes of the protein
nitrites, nitrates, chlorides, and chlorates) may and the precipitating agents
interfere with the test o The colloidal character of protein both acids and
o HgSO4 acts as an oxidant bases to form ionizable salts
NOTE: Although proteins generally precipitate from their
solutions in an amorphous condition, many of them have
been isolated from animal and vegetable sources as o Insoluble salts are formed
characteristic crystals. Product: Insoluble salts (e.g. protein picrate, protein
The proteins are denatured and precipitated from their trichloroacetate)
solution by: Uses of alkaloidal reagents:
o Chemical means when reacted with mineral o Trichloroacetic acid and phosphotungstic acid: for
acids, alkalies, salts of heavy metals, alkaloidal the preparation of protein free filtrates of blood and
reagents, organic solvents, dyes, detergents, other biological materials preliminary to analysis
neutral salts, high concentrations of urea, guanidine o Picric and tannic acid: for treatment of burns
salts, and formamide o Tannic acid: for treatment of hides for its conversion
o Physical means heating, violent shaking, stirring, to leather; it reacts with the proteins to form
grinding, or use of ultrasonic waves insoluble tannates
The denaturation of proteins is characterized by: Procedure:
o Changes in the physical conformation, e.g. o 2 test tubes: 2mL test solution, add
Change in particle size T1 10 drops saturated picric acid solution
Increase in surface tension Yellow solution (picrate)
o Changes in chemical properties, e.g. T2 10 drops 10% TCA
Decrease in solubility at the isoelectric point of Turbid (trichloroacetate)
the original protein (pI) 3. Precipitation with Salts of Heavy Metals
Increased reactivity of several of the side-chain Salts of heavy metals: HgCl2, AgNO3, Pb(CH3COO2),
groups FeCl3
Change in optical rotation in the direction of Principle: Salts of heavy metals react with proteins to
increased levorotation form precipitates on the alkaline side of the isoelectric
o Loss of biological properties of the protein point
Proteins precipitate readily at their isoelectric point o The precipitating ion is a cation
o At this point, the net charge of the proteins o The protein must bear a negative charge
dissociable groups is equal to zero Products: Hg-proteinate or Ag-proteinate (if albumin is
o Since the electrostatic repulsive forces are at a used, mercuric albuminate and silver albuminate)
minimum, the solubility of most proteins is also at a Complication: Alkaloidal reagents may disrupt salt
minimum at their pI (e.g. for egg albumin it is at pH bridges by forming new salt bridges to themselves
4.6) On poison treatment:
Precipitation reactions of proteins have been used: o Basis: Certain heavy-metal salts denature and
o For the isolation, purification, and characterization coagulate proteins
of proteins o Mercuric, silver, and lead salts are poisons
o For the deproteinization of biological fluids as blood o Metallic ions wreak havoc among important body
and urine for physiological clinical analysis proteins once they enter general circulation if they
o As antidote (e.g. white egg in heavy metal are coagulated in the stomach by the administration
poisoning with mercury of lead) of the handiest protein available (e.g. raw egg
o In the preparation of useful protein derivatives white)
1. Precipitation with Mineral Acids o If the coagulated poison-albumin mixture remains
Mineral acids: concentrated HNO3, H2SO4, HCl too long in the stomach, the process of digestion
Principle: Acids alter the ionization of the carboxyl group will eventually dissolve the protein and the heavy
of proteins, disrupting the hydrogen bonds and salt metal ions will be liberated
bridges o An emetic must be given right away
o The protein will have excessive electrostatic charge Procedure:
of one kind o 2 test tubes: 2mL test solution, add
o If the repulsion among like charges is too great, the T1 1% HgCl2 until excess is added
polypeptide chain will unfold and seek an expanded T2 1% AgNO3 until excess is added
configuration o Unless the reagent is added very gradually, the
o The repulsion is minimized and this leads to their formation of the precipitate may not be noted due
denaturation to its solubility in excess of the reagent
Product: Denatured protein insoluble in the strong acid o Turbid
medium 4. Precipitation with Organic Solvents and Dehydrating
Complications: If enough acid is added, or if the Agents
precipitate remains in contact with the acid for a period Organic solvents and dehydrating agents: ethanol,
of time, the precipitate may redissolve due to the methanol, isopropyl alcohol, acetone
hydrolysis of the protein Principle: Organic solvents that are miscible with water
Condition: Resolution in HNO3 is less rapid than in HCl lower the isoelectric constant of the system and
or H2SO4 decreases the activity of water
Procedure: o The shielding of charges on the protein surface will
o 2mL conc HNO3 (or HCl/H2SO4), incline then add be diminished
2mL test solution (let it run down the side to form a o The interaction of protein molecules with water will
layer) be reduced
o 2 layers, brownish solution o The proteins in solution are converted into
2. Precipitation with Alkaloidal Reagents suspensoids which flocculate upon the addition of a
Alkaloidal reagents: picric acid, trichloroacetic acid, few drops of salt solution
phosphotungstic acid, tannic acid, phosphomolybdic Product: Suspensoids
acid, tungstic acid, ferrocyanic acid, and sulfosalicylic Condition: Precipitation by alcohol is most effective at the
acid isoelectric point of the protein
Principle: Alkaloidal reagents lower the pH of proteins, Complication: Prolonged contact with alcohol produces
favoring the protonation of the carboxyl groups an irreversible coagulation of the protein
o Hydrogen bonds and salt bridges are disrupted and Concept: The fixing of tissues for histological
polypeptide chains unfold examination and the disinfectant action of alcohol
o The protein is left with net positive charges against protein-rich bacteria are based on the
o The positively charged protein combines with the coagulating action of alcohol on proteins
anion radical of the alkaloidal reagent
Procedure: Principle:
o 2mL test solution + 1 drop 1% HOAc + 4mL 95% o Chemically: Any of the above methods leads to the
alcohol formation of a series of ill-defined fragments of
o White precipitate decreasing complexity known as proteins,
5. Salting Out with Neutral Salts metaproteins, proteoses, peptones, polypeptides,
Neutral salts: (NH4)2SO4, MgSO4, NaCl, Na2SO4 peptides, and amino acids.
Principle: Certain amino acids are split off early in the
o Neutralization and dehydration of the protein hydrolytic process
molecules This liberation of amino acids continues until the
Ascribed to the ability of the salt ions to bind water large intermediate fragments have all been
or become hydrated reduced to these simpler compounds
Salt ions compete with the protein molecule for o Physically: Hydrolysis consists in a breaking down
water of the large, colloidal, nondiffusable complexes into
o Direct ionic interaction of the salt with protein a series of fragments in which the colloidal
Concept: character becomes less and less pronounced until
o Precipitation of a protein by salting out is most finally only in the simple, crystalloidal and diffusible
complete at or near the isoelectric point of the amino acids remain
protein Types of Hydrolysis:
o Protein precipitated by this method is unaltered and o Acid Hydrolysis (especially if the protein contains
usually redissolves when treated with fresh portions carbohydrate) results in the destruction of certain
of the original solvent amino acids (e.g. serine, cysteine, threonine)
Condition: The concentration of salt required for o Alkaline Hydrolysis (prolonged heating by strong
precipitation depends upon the particular protein and on alkalies) does not affect tryptophan, but results in
the pH of the solution that is on the charge of the protein the partial or complete destruction of loss of optical
complex. activity of all the amino acids
Positive for: o Enzymatic Hydrolysis no disadvantage but very
o All proteins (except peptones) are precipitated by time consuming and is seldom complete
saturation with ammonium sulfate or full saturation Procedure:
with NaCl o Concept: Hydrolysis destroys the primary level of
o Polyvalent ions are more effective than univalent protein structure
ions because polyvalent ions: o Demonstrated by:
Can neutralize certain proteins more effectively Examination of protein hydrolyzates with time
Interact with charges on two or more different Biuret test there is an increase in the number of
protein molecules to form intermolecular cross- broken peptide bonds as shown by a decreasing
links which enhances the tendency of neutralized intensity of the purple color
protein molecules to aggregate The hydrolysis of a peptide bond in the presence of a
Procedure: strong acid or at low pH at high temperature may be
o 2mL test solution + solid (NH4)2SO4 until saturated depicted in the following manner:
o Add 1mL to one portion reversible
6. Precipitation with Heat
Principle: Heat disrupts hydrogen bonds and salt bridges
o The molecules vibrate too violently due to thermal
o When proteins are heated, the proteins separate in
an insoluble form as a coagulum (e.g. what
happens in frying/boiling of egg)
Application: Detection of protein by heating of a sample
of urine
o Heat 2mL test solution observe if coagulation
o Add 2 drops 1% HOAc Reaction:
o White precipitate o An extreme change in pH results in the electron pair
C. Hydrolysis of Proteins sharing of the H+ with the oxygen of the carbonyl
Most commonly employed in the investigation of the carbon
protein molecule o The peptide bond is destabilized
Function and importance: o A molecule of water attaches itself to the carbonyl
o Yields the individual units from which the protein is carbon
formed o The carbonyl to nitrogen bond is rupture
o Tells the percentage of composition of the o The H+ from the oxygen of the carbonyl group and
substance under examination without indicating the from the water molecule are cleaved off
atomic arrangement within the molecule o The H+ from H2O attaches itself to the N
o Reveals the quantities of the various constituent Procedure:
amino acids without giving much indication as to o 4 test tubes (hydrolyzed): 1mL 1% gelatin + 1mL
their spatial arrangement within the giant molecule dilute (1:1) HCl boil and remove one tube every
Hydrolysis may be effected by: 15 minutes
o Boiling with mineral acids (e.g. HCl H2SO4) o Once removed, neutralize the HCl with 2mL 12%
o Boiling with strong alkalies (e.g. NaOH or hot NaOH + 5mL distilled water
saturated Ba(OH)2 o Reference test tubes (6): 5mL distilled water + 1.0,
o Treatment with certain long-chain sulfonic acids as 0.8, 0.6, 0.4, 0.2, 0 mL 1% gelatin
cetylsulfonic acid or diphenylbenzenesulfonic acid o To hydrolyzed and reference tubes, add 5mL 3%
o Organic acids as hydroiodic acid, oxalic acid, or a NaOH + 0.25mL 20% CuSO4 mix by inversion
mixture of ormic and HCl o Compare the color of the hydrolyzates
o Digestion with proteolytic enzymes o Longer time in water bath, higher percent hydrolysis
Qualitative Test Test for Reagents Visible Result
Molisch reagent (5% -naphthol and
Molisch Alpha-Naphthol Carbohydrates in free/combined
95% alcohol) Purple ring
Reaction form
Concentrated H2SO4
Bials reagent (0.3% orcinol and
Bials Orcinol Test Pentoses Blue-green solution
0.02% FeCl3 in 10N HCl)
Taubers reagent (4% benzidine
Taubers Benzidine Test Pentoses (more specific) Cherry red solution
dihydrochloride in glacial HOAc)
Seliwanoffs reagent (5% resorcinol in
Seliwanoff Test Ketose sugars Red to brownish-red precipitate
12% HCl)
Ketoses > aldoses Benedicts solution (CuSO4, Na2CO3,
Benedicts Test Brick-red precipitate
Sugars in urine Ca3(C6H5O7)24H2O)
Barfoeds Test Disaccharides vs. monosaccharides Barfoeds reagent (CuSO4 and HOAc) Brick-red precipitate
Osazone Test Sugars with free aldehyde or ketone Phenylhydrazine mixture Orange to brown crystals
Mucic Acid Test Galactose Concentrated HNO3 White crystals
Test Solution (1% W/V)
Galactose Glucose Fructose Maltose Sucrose Xylose
Molisch - + + + + + +
naphthol Purple ring Purple ring Purple ring Purple ring Purple ring Purple ring

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)

Osazone Test Mucic Acid Test

Glucosazone (needle-like) Maltosazone (sun-flower) Galactosazone (rhombic) Mucic acid crystals (galactose)

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.

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
or ethyl acetate
Lead Acetate Reaction 10% NaOH Brown solution or black
Sulfur PbS
(Unoxidized Sulfur Test) Saturated lead acetate precipitate
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)
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

% = 100

Laboratory Manual in Biochemistry. Department of Nutrition, College of Public Health.