I.

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:
Monosaccharides
Glucose Aldehyde Hexose Reducing
Fructose Ketone Hexose Reducing
Galactose Aldehyde Hexose Reducing
Xylose Aldehyde Pentose Reducing
Procedure:
Disaccharides
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
Complications:
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
Condition:
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
carbohydrates
o Glucose must not be present in amounts exceeding
The reaction with a pentose and a hexose are
2%
represented in these equations:
The proposed reaction is:

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:
o 2mL test solution + 2mL conc HNO3
o Boiling water bath for one hour
o Scratch inner wall to hasten formation of crystals
(fine particles of glass will facilitate crystal
formation)
o Refrigerate test tube for 30-45 mins, if no crystals
o Examine crystals under LPO
D. Hydrolysis of Carbohydrates
Starch
o Used as the substrate in the demonstration of
hydrolysis of carbohydrates
o Storage form of carbohydrates in plants
o Contains an outer layer of amylopectin and an
inner layer of amylose
Can be brought into colloidal solution by heating
with boiling water
Both react with iodine (differ in color intensity):
Amylose: deep blue color
Amylopectin: light violet color
The color is formed on the surface of the celloid
particles when iodine is adsorbed and discharged
(iodine is reduced to iodide ion)
Principle: Upon hydrolysis, the acetal linkages of the
polysaccharide/dissacharide molecules take on a
molecule of water and break down into smaller
units/monosaccharides
Concept: (on acetal linkages)
o Relatively stable in basic media
o Easily hydrolyze in acid media, under enzymic
conditions, and by bacterial action
1. Acid Hydrolysis of Starch
Principle:
o Polysaccharides (starch glycogen, inulin, or
disaccharides sucrose, maltose, and lactose) are
heated with weak acids (about 10%)
o The sugars are broken down to their constituent
monosaccharides
Concept: Acidic hydrolysis of starch yields a succession
of polysaccharides of gradually diminishing size with the
ultimate production of glucose
Course of Hydrolysis (may be followed by the gradual
change in color produced by iodine and by the Benedicts
test from most complex (top) to simplest (bottom):
o Starch
o Soluble starch
o Amylodextrins
o Erythrodextrins
o Achroodetrins
o Maltose
o Glucose
The formula for the hydrolysis of starch is shown below:
Procedure:
o 7 test tubes with 2mL Benedicts reagent
o Hydrosylate: In a 50mL Erlenmeyer flask, 20mL 1%
starch solution + 0.5mL conc HCl or H2SO4 mix
and boil gently
o After 5 mins boiling, test the hydrosylate every 1
min interval (on a spot plate)
o Add 2 drops iodine in KI solution (stoppered since
I2 is volatile and will produce a false negative result)
o If after 5 min, iodine test is still positive, continue
testing the hydrolysate at 5 min intervals until iodine
color remains unchanged
o 3 drops of digest + 2mL Benedicts reagent (tubes
1 to 5)
o Boil (3 mins) all test tubes containing Benedicts
solution then cool
o Results:
Iodine:
Blue: starch is present (starch-iodine
complex formed)
Orange or yellow: starch has been broken
down (original color)
Benedicts:
Blue: starch is still present (original color)
Red: starch has been broken down
2. Enzymatic Hydrolysis of Starch
Course of Hydrolysis: ends at maltose with traces of
dextrins
Human salivary -amylase or ptyalin
o Contains 1 mole of calcium ions per mole of
enzyme protein
Bound calcium is required for enzymatic activity
o Requires the presence of anions for activity
Chloride is the most effective activator (bromide
and iodide are also activating)
Method of dialysis
o To demonstrate the diffusibility of carbohydrates
and the effect of digestion on starchy foods
o Entails the use of a membrane such as parchment,
collodion, cellophane, or an animal bladder
Selectively allows the passage of crystalloids
(particles which diffuse readily through the
membrane)
Prevents colloids (larger particles than
crystalloids that dont pass through the
membrane)
Principle: If a mixture of crystalloids and colloids is
placed in a dialyzing bag which is then immersed in
distilled water, the colloids will remain behind while the
crystalloids dialyze out
Condition:
o A preservative such as toluene, mercuric iodide or
1% boric acid is added to the starch solution and
distilled water
o To inhibit bacterial action which causes
decomposition and makes the solution ineffective
as an indicator
Procedure:
o 2-3mL filtered saliva + 20mL 1% starch paste
o Allow to stand at room temp for an hour (note
change in viscosity)
o Introduce to a dialyzing bag, string, and suspend it
in 100mL distilled water (let stand for 45 mins)
o Test contents of dialyzing bag and solution in
beaker with iodine and Benedicts test
o Results (Benedicts):
Blue: starch is still present (original color)
Red: starch has been broken down
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
Proteins may be identified by three types of reactions:
o Reactions which are due to the presence of specific
chemical groups or linkages in the protein molecule and
the chemical reagent used in any given test (e.g. biuret,
ninhydrin, xanthoproteic, etc.)
o Reactions which are due to the acidity or basicity of the
protein molecule (e.g. precipitation by strong mineral
acids, salts of heavy metals, or alkaloidal reagents)
o Reactions which are due to the colloidal nature of the
proteins (e.g. heat coagulation and salting out)
Test Solutions:
o Egg Albumin
Water-soluble, moderately soluble in concentrated salt
solutions, and experience heat denaturation
o Casein
Main protein in milk and cheese
Contains a fairly high number of proline residues,
which do not interact
No disulfide bridges
3
 o Gelatin
Mixture of peptides and proteins produced by partial
hydrolysis of collagen
o Peptone
A soluble protein formed in the early stage of protein
breakdown during digestion.
o Tryptophan
Essential, aromatic amino acid
o Tyrosine
Non-essential, aromatic amino acid
o Cystine
Oxidized dimer of cysteine
Non-essential, sulfur-containing
A. Color Reactions of Proteins and Amino Acids
General Principle: Due to a reaction between one or
more of the constituent radicals (chemical groups or
specific linkages) of the complex protein molecule and
the chemical reagents used in any given test
Complications:
o Not all proteins contain the same amino acids
therefore various color tests will yield reactions of
varying intensity of color (according to the nature
and amount of the groups contained in the certain
protein)
o Various substances (not protein) respond to certain
of these color reactions
Condition: Submit the material under examination to
several tests before concluding definitely regarding its
nature
Uses of color tests:
o Basis for the quantitative estimation of proteins and
amino acids
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
Product: Pinkish-violet biuret complex in an alkaline
solution
Concept:
o The intensity of the purple color produced is
proportional to the number of peptide bonds
present and hence, to the amount of protein
o The color also depends upon the nature of the
protein
Proteoses and peptones give a decided pink
color
With gelatin, the color produced is not far
removed from blue
Positive for:
o All substances with 2 or more peptide bonds
o Soluble proteins (negative for coagulated)
o Non-protein in character but which contain in piece
of the CONH2 group such as: CSNH2, CH2NH2,
or C(NH)NH2
Complications:
o The presence of magnesium sulfate in considerable
amounts in solutions containing protein interferes
with the test, forming a magnesium hydroxide
precipitate
o Excess ammonium salts (e.g. (NH4)SO4) interfere
by salting out proteins from their solutions (a
positive test is not obtained with high
concentrations of ammonium salt)
Condition:
o Sodium hydroxide in excess of that required to
decompose the ammonium salts must be present
(biuret test requires the presence of a strong base)
o The basic pH prevents protonation of the peptide
nitrogen
Procedure:
o 1mL test solution + 1mL 10% NaOH mix
o Add 5 drops 5% CuSO4
o Pink to violet solution
2. Ninhydrin Test or Triketohydrindenehydrate Reaction
Most general and one of the most sensitive reactions
known for qualitative detection of proteins and their
hydrolysis products: proteoses, peptones, peptides, and
amino acids
Principle:
o A neutral solution with free carboxyl group, amino
acids, and proteins are made to react with ninhydrin
(triketohydrindenehydrate), a powerful oxidizing
agent
o Once the solution has been heated, a blue/purple
color will appear (Ruhemanns blue/purple)
Reaction:
o An alphadecarboxylation by ninhydrin produces
CO2, NH3, and an aldehyde with one less carbon
atom than the parent amino acid
o The reduced ninhydrin then reacts with the
liberated NH3, forming a blue complex
Basis for several different types of quantitative methods
for the determination of alpha amino acids:
o Color change which results from the reaction
o Determination of ammonia produced
o Measurement by gasometric means of the carbon
dioxide evolved (Van Slyke Method)
Conditions:
o Freshly prepared ninhydrin must be used
o The reaction proceeds best in neutral solution (pH
between 5 and 7)
o A few crystals of sodium acetate may be used to
adjust the pH
Procedure:
o 1mL test solution + 0.25mL freshly prepared 0.1%
ninhydrin solution heat then cool
o Compare intensity of blue solution
3. Xanthoproteic Test
General reaction of proteins containing amino acids
which have a benzene ring structure
Principle: Phenyl group (-C6H5) is nitrated upon heating
with nitric acid (HNO3)
Product: Nitroderivatives of benzene which are initially
yellow (xanthine) but become orange when alkalinized
4
 Indicates the presence of:
o Aromatic amino acids
o Tyrosine
o Tryptophan
Condition: H2SO4 is required as a catalyst
Complication: Phenylalanine is not as readily nitrated
does not/weakly responds to this test
Procedure:
o 0.5mL test solution + 0.5mL conc HNO3 heavy
white precipitate must form
o Heat precipitate turns yellow then dissolves
o Cool then add 2mL 10% NaOH til alkaline to litmus
o Orange solution
4. Millon-Nasse Test
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)
Positive for: Any phenolic compound which is
unsubstituted in the 3,5 positions (e.g. tyrosine, phenol,
and thymol)
Complications:
o For egg white solution, the initial white precipitate
(after mercuric sulfate reagent) is not a positive test
but rather the appearance of the color of the
precipitate (after nitrate treatment) indicates the
presence of tyrosine
o For hydrolytic products of protein (e.g. peptone or
peptides which contain tyrosine), a colored solution,
not a precipitate, will be obtained
Procedure:
o 1.5mL test solution + 0.5mL 15% HgSO4 in 6N
H2SO4 heat
o Cool for 3 mins then add 0.5mL 1% NaNO2
o Old rose solution
5. Acree-Rosenheim Reaction
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
Characterized by: Reddish-violet color at the interphase
between H2SO4 and formaldehyde solution
Positive for: Compounds with the indole ring (e.g.
tryptophan, nearly all proteins; except gelatin and
insulin)
Conditions:
o Chemically pure H2SO4 must be used since
excessive amounts of oxidizing substances (e.g.
nitrites, nitrates, chlorides, and chlorates) may
interfere with the test
o HgSO4 acts as an oxidant
Procedure:
o 1mL test solution + 1 drop 40% formaldehyde
(1/500 commercial formaldehyde) + 1 drop 15
HgSO4 in 6N H2SO4 mix
o Incline and slowly add 2mL conc H2SO4 2 distinct
layers
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
alcohol layer
Complication: The pink color may disappear due to
excess bromine water and it may be masked by the color
of the reagent
The bromination (or halogenation) of the pyrrole ring is
shown below:
Procedure:
o 1mL test solution + 5 drops freshly prepared brome
water + 1mL conc amyl alcohol/ethyl acetate
o Pink alcohol layer (upper)
7. Lead Acetate Reaction or Unoxidized Sulfur Test
Principle: Liberation of sulfur when proteins in strongly
alkaline solutions are boiled
Product: Darkening or browning of the color or a black
precipitate of PbS
Positive for: Sulfur-containing proteins (e.g. cysteine,
cysteine, methionine)
Complication: Methionine does not give a positive test
o The thiomethyl group of methionine is not readily
removed by alkali
o Requires fusion with an oxidizing mixture
Concept:
o Cystine and cysteine sulfur was formerly termed
unoxidized, loosely-combined, mercaptan, or lead-
blackening sulfur
o Methionine sulfur was called oxidized or acid sulfur
o Majority of proteins contain more methionine sulfur
than cysteine and cysteine sulfur (exceptions:
keratins, insulins, and certain serum albumins)
Procedure:
o 1mL test solution + 1mL 10% NaOH + 1mL
saturated lead acetate heat
o Brown solution or black precipitate
B. Precipitation Tests for Proteins
No definite evidence concerning the nature of the
mechanisms involved
Precipitation reactions are probably due to:
o The formation of insoluble complexes of the protein
and the precipitating agents
o The colloidal character of protein both acids and
bases to form ionizable salts
NOTE: Although proteins generally precipitate from their
solutions in an amorphous condition, many of them have
5
 been isolated from animal and vegetable sources as
characteristic crystals.
The proteins are denatured and precipitated from their
solution by:
o Chemical means when reacted with mineral
acids, alkalies, salts of heavy metals, alkaloidal
reagents, organic solvents, dyes, detergents,
neutral salts, high concentrations of urea, guanidine
salts, and formamide
o Physical means heating, violent shaking, stirring,
grinding, or use of ultrasonic waves
The denaturation of proteins is characterized by:
o Changes in the physical conformation, e.g.
Change in particle size
Increase in surface tension
o Changes in chemical properties, e.g.
Decrease in solubility at the isoelectric point of
the original protein (pI)
Increased reactivity of several of the side-chain
groups
Change in optical rotation in the direction of
increased levorotation
o Loss of biological properties of the protein
Proteins precipitate readily at their isoelectric point
o At this point, the net charge of the proteins
dissociable groups is equal to zero
o Since the electrostatic repulsive forces are at a
minimum, the solubility of most proteins is also at a
minimum at their pI (e.g. for egg albumin it is at pH
4.6)
Precipitation reactions of proteins have been used:
o For the isolation, purification, and characterization
of proteins
o For the deproteinization of biological fluids as blood
and urine for physiological clinical analysis
o As antidote (e.g. white egg in heavy metal
poisoning with mercury of lead)
o In the preparation of useful protein derivatives
1. Precipitation with Mineral Acids
Mineral acids: concentrated HNO3, H2SO4, HCl
Principle: Acids alter the ionization of the carboxyl group
of proteins, disrupting the hydrogen bonds and salt
bridges
o The protein will have excessive electrostatic charge
of one kind
o If the repulsion among like charges is too great, the
polypeptide chain will unfold and seek an expanded
configuration
o The repulsion is minimized and this leads to their
denaturation
Product: Denatured protein insoluble in the strong acid
medium
Complications: If enough acid is added, or if the
precipitate remains in contact with the acid for a period
of time, the precipitate may redissolve due to the
hydrolysis of the protein
Condition: Resolution in HNO3 is less rapid than in HCl
or H2SO4
Procedure:
o 2mL conc HNO3 (or HCl/H2SO4), incline then add
2mL test solution (let it run down the side to form a
layer)
o 2 layers, brownish solution
2. Precipitation with Alkaloidal Reagents
Alkaloidal reagents: picric acid, trichloroacetic acid,
phosphotungstic acid, tannic acid, phosphomolybdic
acid, tungstic acid, ferrocyanic acid, and sulfosalicylic
acid
Principle: Alkaloidal reagents lower the pH of proteins,
favoring the protonation of the carboxyl groups
o Hydrogen bonds and salt bridges are disrupted and
polypeptide chains unfold
o The protein is left with net positive charges
o The positively charged protein combines with the
anion radical of the alkaloidal reagent
o Insoluble salts are formed
Product: Insoluble salts (e.g. protein picrate, protein
trichloroacetate)
Uses of alkaloidal reagents:
o Trichloroacetic acid and phosphotungstic acid: for
the preparation of protein free filtrates of blood and
other biological materials preliminary to analysis
o Picric and tannic acid: for treatment of burns
o Tannic acid: for treatment of hides for its conversion
to leather; it reacts with the proteins to form
insoluble tannates
Procedure:
o 2 test tubes: 2mL test solution, add
T1 10 drops saturated picric acid solution
Yellow solution (picrate)
T2 10 drops 10% TCA
Turbid (trichloroacetate)
3. Precipitation with Salts of Heavy Metals
Salts of heavy metals: HgCl2, AgNO3, Pb(CH3COO2),
FeCl3
Principle: Salts of heavy metals react with proteins to
form precipitates on the alkaline side of the isoelectric
point
o The precipitating ion is a cation
o The protein must bear a negative charge
Products: Hg-proteinate or Ag-proteinate (if albumin is
used, mercuric albuminate and silver albuminate)
Complication: Alkaloidal reagents may disrupt salt
bridges by forming new salt bridges to themselves
On poison treatment:
o Basis: Certain heavy-metal salts denature and
coagulate proteins
o Mercuric, silver, and lead salts are poisons
o Metallic ions wreak havoc among important body
proteins once they enter general circulation if they
are coagulated in the stomach by the administration
of the handiest protein available (e.g. raw egg
white)
o If the coagulated poison-albumin mixture remains
too long in the stomach, the process of digestion
will eventually dissolve the protein and the heavy
metal ions will be liberated
o An emetic must be given right away
Procedure:
o 2 test tubes: 2mL test solution, add
T1 1% HgCl2 until excess is added
T2 1% AgNO3 until excess is added
o Unless the reagent is added very gradually, the
formation of the precipitate may not be noted due
to its solubility in excess of the reagent
o Turbid
4. Precipitation with Organic Solvents and Dehydrating
Agents
Organic solvents and dehydrating agents: ethanol,
methanol, isopropyl alcohol, acetone
Principle: Organic solvents that are miscible with water
lower the isoelectric constant of the system and
decreases the activity of water
o The shielding of charges on the protein surface will
be diminished
o The interaction of protein molecules with water will
be reduced
o The proteins in solution are converted into
suspensoids which flocculate upon the addition of a
few drops of salt solution
Product: Suspensoids
Condition: Precipitation by alcohol is most effective at the
isoelectric point of the protein
Complication: Prolonged contact with alcohol produces
an irreversible coagulation of the protein
Concept: The fixing of tissues for histological
examination and the disinfectant action of alcohol
against protein-rich bacteria are based on the
coagulating action of alcohol on proteins
6
 Procedure:
o 2mL test solution + 1 drop 1% HOAc + 4mL 95%
alcohol
o White precipitate
5. Salting Out with Neutral Salts
Neutral salts: (NH4)2SO4, MgSO4, NaCl, Na2SO4
Principle:
o Neutralization and dehydration of the protein
molecules
Ascribed to the ability of the salt ions to bind water
or become hydrated
Salt ions compete with the protein molecule for
water
o Direct ionic interaction of the salt with protein
Concept:
o Precipitation of a protein by salting out is most
complete at or near the isoelectric point of the
protein
o Protein precipitated by this method is unaltered and
usually redissolves when treated with fresh portions
of the original solvent
Condition: The concentration of salt required for
precipitation depends upon the particular protein and on
the pH of the solution that is on the charge of the protein
complex.
Positive for:
o All proteins (except peptones) are precipitated by
saturation with ammonium sulfate or full saturation
with NaCl
o Polyvalent ions are more effective than univalent
ions because polyvalent ions:
Can neutralize certain proteins more effectively
Interact with charges on two or more different
protein molecules to form intermolecular cross-
links which enhances the tendency of neutralized
protein molecules to aggregate
Procedure:
o 2mL test solution + solid (NH4)2SO4 until saturated
precipitates
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
agitation
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
Procedure:
o Heat 2mL test solution observe if coagulation
occurs
o Add 2 drops 1% HOAc
o White precipitate
C. Hydrolysis of Proteins
Most commonly employed in the investigation of the
protein molecule
Function and importance:
o Yields the individual units from which the protein is
formed
o Tells the percentage of composition of the
substance under examination without indicating the
atomic arrangement within the molecule
o Reveals the quantities of the various constituent
amino acids without giving much indication as to
their spatial arrangement within the giant molecule
Hydrolysis may be effected by:
o Boiling with mineral acids (e.g. HCl H2SO4)
o Boiling with strong alkalies (e.g. NaOH or hot
saturated Ba(OH)2
o Treatment with certain long-chain sulfonic acids as
cetylsulfonic acid or diphenylbenzenesulfonic acid
o Organic acids as hydroiodic acid, oxalic acid, or a
mixture of ormic and HCl
o Digestion with proteolytic enzymes
Principle:
o Chemically: Any of the above methods leads to the
formation of a series of ill-defined fragments of
decreasing complexity known as proteins,
metaproteins, proteoses, peptones, polypeptides,
peptides, and amino acids.
Certain amino acids are split off early in the
hydrolytic process
This liberation of amino acids continues until the
large intermediate fragments have all been
reduced to these simpler compounds
o Physically: Hydrolysis consists in a breaking down
of the large, colloidal, nondiffusable complexes into
a series of fragments in which the colloidal
character becomes less and less pronounced until
finally only in the simple, crystalloidal and diffusible
amino acids remain
Types of Hydrolysis:
o Acid Hydrolysis (especially if the protein contains
carbohydrate) results in the destruction of certain
amino acids (e.g. serine, cysteine, threonine)
o Alkaline Hydrolysis (prolonged heating by strong
alkalies) does not affect tryptophan, but results in
the partial or complete destruction of loss of optical
activity of all the amino acids
o Enzymatic Hydrolysis no disadvantage but very
time consuming and is seldom complete
Procedure:
o Concept: Hydrolysis destroys the primary level of
protein structure
o Demonstrated by:
Examination of protein hydrolyzates with time
Biuret test there is an increase in the number of
broken peptide bonds as shown by a decreasing
intensity of the purple color
The hydrolysis of a peptide bond in the presence of a
strong acid or at low pH at high temperature may be
depicted in the following manner:
Reaction:
o An extreme change in pH results in the electron pair
sharing of the H+ with the oxygen of the carbonyl
carbon
o The peptide bond is destabilized
o A molecule of water attaches itself to the carbonyl
carbon
o The carbonyl to nitrogen bond is rupture
o The H+ from the oxygen of the carbonyl group and
from the water molecule are cleaved off
o The H+ from H2O attaches itself to the N
Procedure:
o 4 test tubes (hydrolyzed): 1mL 1% gelatin + 1mL
dilute (1:1) HCl boil and remove one tube every
15 minutes
o Once removed, neutralize the HCl with 2mL 12%
NaOH + 5mL distilled water
o Reference test tubes (6): 5mL distilled water + 1.0,
0.8, 0.6, 0.4, 0.2, 0 mL 1% gelatin
o To hydrolyzed and reference tubes, add 5mL 3%
NaOH + 0.25mL 20% CuSO4 mix by inversion
o Compare the color of the hydrolyzates
o Longer time in water bath, higher percent hydrolysis
7
CARBOHYDRATES
SUMMARY OF QUALITATIVE TESTS
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
RESULTS FOR QUALITATIVE TESTS
Test Solution (1% W/V)
Test
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)

[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.

8
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.
9