PRACTICAL RECORD BOOK FOR BDS STUDENTS
BIOCHEMISTRY
INDEX
|
Expt. No. |
Name
of the Experiment |
Page
No. |
Date |
Remarks |
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Identification
of Carbohydrates |
|
|
|
|
1) |
Monosaccharides: Glucose and Fructose --- |
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|
2) |
Disaccharides: Lactose, Matltose & Sucrose --- |
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|
3) |
Polysaccharides: Starch --- |
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4) |
Identification of a reducing sugar by preparing --- osazone |
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5) |
Identification of Unknown Carbohydrates --- |
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Reactions of Proteins --- |
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6) |
Color reactions of protein --- |
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7) |
Precipitation reaction of proteins --- |
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8) |
Properties of albumin, casein, Gelatin pentone --- |
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9) |
Identification of unknown proteins --- |
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10) |
Reactions of NPN substances |
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11) |
General scheme for identification of physiologically --- important substances |
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12) |
Qualitative analysis of normal urine --- |
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13) |
Qualitative analysis of abnormal urine --- |
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14) |
Determination of Titrable Acidity --- |
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15) |
Estimation of urine creatinine --- |
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16) |
Estimation of blood suar --- |
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Expt. No. 1
Date………….
CHEMISTRY OF CARBOHYDRATES
REACTIONS OF MONOSACCHARIDES,
Carbohyrates
are polyhydroxy aldehydes or ketones. Monosaccharides are the basic compounds
in this series having one single carbon chain. The common monosaccharides that
are used in the biochemistry laboratory are Glucose & fructose. Both
Glucose and fructose are present in honey and fruits glucose is also the sugar
of blood.
Perform
the following tests with solution (1) 1% Glucose and (ii) 1% fructose
MOLISCH TEST:
In a clean and dry test tube
take 2 ml of the given solution and add 2 drops of molisch reagent mix and add
carefully down the sides of the test tube 2 ml of concentrated Sulphuric acid,
so that the acid & aqueous solution do not mix but form a separate layer.
Formation of the purple colour indicates the presence of carbohydrate.
EXPLANATION:
Molisch reagent consists of 1%
solution of alpha naphthol in ethanol. This reaction is due to the formation of
furfural and furfural derivatives. The furfural formed by the dehydration of
the acid condenses with the alpha naphthol, given a purple rings. This test is
general for all carbohydrates it is not specific for carbohydrates since all
furfural yielding substances answer this test. Appearance of a black, brown or
green coloured ring is not a positive reaction.
SELIWANOFF’S TEST:
Take 3 ml of cherry’s reagent
and .1ml of the given solution and heat for 30sec boiling. The rapid
development of red colour indicates the presence of ketoses like fructose, glucose
does not give the colour or gives it on prolonged boiling.
EXPLANATION:
Seliwanoff’s reagent contains
resorcinol in concentrated HC1 acts on Ketosugar to form a derivative of
furfural-dehyde which gives a red coloured compound with rescorcinol. Glucose
also gives red colour due to the transformation into fructose by the catalytic
action of HCl but after prolonged heating. This test distinguishes between
fructose and glucose.
Barfoed’s Test:
Take 2ml of the given solution
and 2ml of the Barfoed’s reagent. Mix and keep in boiling water bath for 3 minutes.
A red precipitate of cuprous oxide indicates the presence of monosaccharide. If
the precipitate does not appear after boiling allow the tube to stand for about
15 minutes and examine.
Explanation:
Barfoed’s reagent is a solution
of copper acetate and Glacial acetic acid. Reduction of cupric salt to cuprous
oxide in the acid midium of the reagent gives red precipitate Monosaccharides
react very fast where as the rection with the reducing disaccharides is slow.
This test is used to distinguish monosaccharides from reducing diaccharides,
but prolonged boiling produce hydrolysis of disaccharides and thus will also answer
this test.
Benedict’s Test:
Take 5 ml of Benedict’s reagent
add 8 drops of the given solution, boil vigorously for 2 minutes and allow to
stand till the precipitate settles, Greenish, Yellow, Yellow red or reddish
brown precipitate of cuprous oxide is obtained depending upon the concentration
of reducing sugar.
Explanation:
Benedict’s qualitative reagent
contains copper sulphate, sodium citrate and mild alkali sodium carbonate all
in one solution. Thus it has an advantage over the Fehling’s reagent which has
two solutions.
This test like the Fehlings test
is based on the reducing property of carbohydrates. Reducing sugars under alkaline
conditions tantomerise to form enodiols. The enodiols are unstable and
decompose to yield a variety of products. The chain reaction continues
to
produce short chain aldehydes which are powerfull reducing agents. They can
reduce cupric ions to form cuprous ion which precipitates as yellow cuprous
hydroxide or red cuprous oxide this is the basis for the Benedict’s &
Fehling’s reaction. The cupric hydroxide formed is not easily soluble. In order
to keep the hydroxide in solution a metal chelator like citrate (or titrate) is
include in the solution. This test is employed as a routine for examination of
urine for sugar. Detection of glucose in urine by this test is of clinical
significance in the diagnosis and control of diabetes mellitus. Depending upon
the amount of sugar present the following coloured precipitate will be
obtained.
|
Colour |
Blue |
Green |
Yellow |
Orange |
Brick Red |
|
Amount
of Sugar |
Nil …….. |
0.5% + |
1% ++ |
1.5% +++ |
2% ++++ |
Question:
1)
What is the general test of carbohydrates?
2)
How will you differentiate glucose from fructose?
3)
How do you test for the presence of sugar in urine?
4)
What are the advantages of Benedict’s test over the Fehling’s test?
5)
Name the reduction tests. What is the basis of tests?
6)
What is the significance of Barfoed’s test?
Expt. No. 2
Date………….
REACTIONS OF
DISACCHARIDES
Disaccharides are obtained by
the combination of two mono saccharides by a glycosidic linkage. On treatment
with hot acids or with enzymes the disaccharides can be hydrolysed to the
constituents monosaccharides, examples of commonly used disaccharides and their
constituent monosaccharides are given below.
Sucrose
: Sweetening
sugar (Cane Sugar) - Glucose + fructose
(Aldehydic group
of glucose is linked with keto group of fructose.)
Lactose
: Milk
sugar = Glucose + Galactose
(Aldehydic group
of galactose is linked with alcoholic group of glucose(4c).
Maltose
: Malt sugar = Glucose + Glucose. Aldehydic group of
glucose is
linked with
alcoholic group of glucose (4c).
Maltose
is obtained in hydrolysis of starch and other polysaceharides.
Maltose
and lactose are reducing sugars, where as sucrose is non redcuing sugar.
Perform
the following tests with lactose, maltose and sucrose.
(1)
Molisch test (2) Seliwanoff’s test (3) Barfoed’s test (4) Fehling’s test. (5)
Benedict’s test (6) Inversion test, (For sucrose only).
Acid Hydrolysis:
To 5ml of sucrose solution add 5
drops of conc HCl boil for a minute and cool. Neutralize the hydrolysed sample
with 40% sodium hydroxide and perform Fehling’s test on it. Write your
inference
Questions:
1) Name the sources of lactose maltose and sucrose?
2) What is the basis of Barfoed’s test?
3) What is inversion and invert sugar?
4) Why sucrose is not a reducing sugar?
5) How will you distinguish fructose from sucrose and other
monosaccharides?
Expt. No. 3
Date………….
REACTIONS OF
POLYSACCHARIDES
Polysaceharides are made up of
many number of same or different monosaccharides linked by glycosidic linkage.
The most commonly available polysaccharide is starch which is a mixture of
amylose and amylopectin. The individual glucose units in amylose are linked by
alpha 1, 4 glycosidic linkages. Amylopectin has branching points contributed by
alpha 1, 6 glycosidic bonds. Starch is insoluble in cold water but forms a co1loidol
solution in hot water which remains so even after cooling. Starch has no
detectable reducing activity. Starch gives a blue colour with iodine solution.
Glycogen is also called animal starch. It is present in liver and muscle. It is
highly branched than starch.
REACTION OF
STARCH
Perform the following tests:-
1) Molisch’s test: Purple coloured ring is observed.
2) Iodine test:
Principle: The amylose component
of starch has a helical structure when it is treated with iodine
solution, iodine is trapped inside the coil and the complex has an intense blue
colour, when amylose solution is heated, the helical conformation is
disrupted and it loses capacity to bind
iodine on cooling, the original conformation is regained and the capacity to
bind iodine is also recovered. Amylopectin and glycogen because of branched
structure gives light purple or red colour with iodine.
To 1 ml of starch add 2 or 3
drops dilute iodine solution. A blue colour is formed from starch – iodine
complex. This is general test for identifying starch.
3) Effect of Alkaline and Acidic medium
on Iodine test:
Add 1ml of 5% of NaOH to 2ml
starch to make it alkaline, now add 3 drops of iodine. There is no blue colour,
acidify this solution with dropwise addition of glacial acetic acid the blue
colour reappears.
Explanation:
In the presence of NaOH free
iodine is not available to form a starch iodine complex, hence no blue colour
develops. When acid is added it neutralises the NaOH and releases the iodine
for combining with starch.
4) Benedict’s test:
Starch gives a negative test
5) Hydrolysis of Starch by Acid:
To 3 ml of starch in a test tube
add 3 drops of conc. HCl and boil for 2 minutes, cool, neutralise with 20%
NaOH. Now perform Benedict test with one portion of the hydrolystate, the test
will be positive because of hydrolysis of starch to glucose. Take the other
portion of hydrolystate, add a drop of glacial acetic acid and two drops of
iodine. No blue colour is noticed indicating that starch is completely
hydrolysed by acid to reducing sugar namely glucose.
Expt.
No.4
Date……………….
IDENTIFICATION
OF REDUCING SUGARS BY PREPARING OSAZONE
All carbohydrates contain either
aldehyde or a ketonic group. This group can react with phenlyhydrazine to form
solid derivatives. These solid dervatives have characteristic crystalline forms
which can be easily identified under the microscope. These derivatives are
called osazones etc. The important feature about these osazones is that
different sugar give osazones with different crystalline forms.
Only reducing sugars form
osazones because they have free aldehyde or ketonic group. Sucrose which is a non
reducing sugar has no free aldehyde or ketonic group, and does not form an
osazone.
Hexoses which differ only in
carbon atoms 1 & 2 in their structure give the same osazone, Glucose,
Fructose & mannose form identical osazones.
Procedure for Osazone Formation:
Note:
Since osazone formation takes time this expt. should be started as early as
possible in the work period, so before doing anything else start to boil some
water in a large beaker or in a water bath.
Take 3 ml of 1% solution of
glucose, lactose and maltose in different test tubes and label them as G; L and
M. To each test tube add 10 drops of glacial acetic acid and add one measure of
phenylhydrazine hydrochloride followed by twice the amount of sodium apetate.
Mix well and place in a boiling water bath for 30 minutes. If any crystals are
formed remove the tube and study the shape of the crystals under the
microscope.
If no crystals are formed, after
30 minutes, remove the flame and allow the tubes to cool in the hot water
itself.
Glucose & fructose osazones
separate at boiling temperature but maltose and lactose osazones separate only
on cooling.
Draw the figures of these Osazones.
Note:
Osazones of Glucose and fructose are identical and are yellow broom stick or
needle like in shape.
Lactose
osazone crystals resemble cotton puff or hedge-hog and will be formed only on cooling.
Maltose
osazone crystals are yellow and Sunflower shaped and will be formed only on
cooling.
Questions:
1) Name the hexoses which form the same osazone and give
the reason?
2) What is the clinical significance of osazone formation?
3) Name the osazones which soluble in hot water?
4) What is the basis for osazones formation? Name the
characteristic osazone crystals for different sugars?
Expt.
No.5 Date………………
IDENTIFICATION OF AN UNKNOWN
CARBOHYDRATE
The following scheme givens the
various test to be followed in the sequential order.
Given solution
Molisch test
Purple ring
(Carbohydrates Confirmed)
Iodine reaction
|
Blue Colour (Starch) Benedicts test after hydrolysis
Red PPt Starch
Postive (Reducing Sugar) Glucose, Fructose, Lactose Maltose
|
No change in colour (Mono or disaccharides)
Benedicts test
Negative (Non reducing Sugar) (Sacrose)
Benedicts test After hydrolysis Red PPt (sucrose) |
|
Positive Glucose, Fructose |
Negative Lactose, Maltose |
|
|
Selwinoff’s Test |
Osazone Test |
Positive (Sucrose Confirmed) |
|
Positive Fructose |
Negative Glucose |
|
Sunflower Shape (Maltose) |
Cotton Puff (Lactose) |
Expt. No. 6
Date.
CHEMISTRY OF PROTENS
COLOUR
REACTION OF PROTEIN
Proteins are complex nitrogen
containing organic compounds formed by the condensation of large numbers of
alpha amino acid molecules by peptide linkages.
Proteins give characteristic
colours with certain reagents due to an amino acid or a class of amino acids
having a characteristic group or due to certain grouping in the protein
molecules. These rections are useful in the identification, characterisation
and estimation of these aminoacids or the proteins containing them.
EXPERIMENTS
Egg
white is the source of protein used for the following experiments.
(1) BIURET test:
To 1 ml of protein solution add
an equal volume of 5% NaOH and 2 drops of 1% copper sulphate, mix, purple or
violet colour is produced due to the presence of more than 2 peptide linkage.
Tripeptide give +ve test.
Explanation:
Cupric ion in alkaline medium
forms a violet coloured complex with peptide bond, nitrogens of peptides and
proteins. This reactions is so named, since biuret formed by the condensation
of 2 molecules of urea when heated to 1800C also answers this test.
The minimum requirement for a +ve test is the presence of 2 peptides bonds in
the molecule. This is a general test for proteins.
Note:
a) Proteoses and peptones give rose red or pink colour,
other proteins give violet colour.
b) Excess copper sulphate will impart its own blue colour
to solution and mask the violet or pink colour of the test.
2) Ninhydrine test
To 1 ml of protein solution add
10 drops of ninhydrine reagent, boil and cool. A bluish purple colour is
produced.
This test is positive for all
alpha amino acids. The test is used in the detection and estimation of amino
acids in chromatographic analysis. Proline and hydroxy proline give yellow
colour due to imino group (- NH group). This is one of the most sensitive test
for amino acids.
Ninhydrin
reactions with amino acid is as follows:
Alpha
amino acid + Ninhydrin à Aldehyde + CO2 + NH3 + Hydrindantin
Hydrindantin
+ NH3 + Ninhydrin à Blue complex / purple colour complex.
3) Xanthoprotein test
To 3ml of the protein solution
add 1ml of conc. HNO3 and boil, solution turns yellow, cool under tap water.
Add 40% NaOH drop by drop till it turns alkaline to litmus. Deep yellow or
orange colour is developed.
This test is positive for
aromatic amino acids like phenyl alanine, tyrosine and tryprophan. Nitration of
phenyl ring present in these amino acids give yellow colour characteristic of
nitro compounds. In alkaline medium these nitro compounds ionize and produce
deep yellow or orange colour.
4) Millon’s test
To 1 ml of protein solution add
1.0 ml of million’s reagent. A white precipitate is formed, boil A brick red
ppt is formed.
This test is indicative of
phenolic aminoacids like - tyrosine, which contains hydroxy phenyl group.
Million’s reagent consists of a
solution of nitrate and nitrite of mercury in acid solution, which means
nitrous and nitric acid are also
present. Phenolic groups of tyrosine combines with mercury from millons reagent
to form mercuric – tyrosine complex which nitration develops red colour.
5) Hopkins Cole test: (Aldehyde test): Tryptophan
Mix 1ml of protein solution with
2 drops of 1 in 500 formalin solution and 1 drop of 10% mercuric sulphate. Add
gently through the sides of the test tube about 1 ml of Conc. H2SO4.
A purple colour ring develops at the junction of 2 liquids. This indicates presence
of tryptophan.
Explanation:
This reaction is due to the
presence of Indole ring amino acid tryptophan in the protein molecule. Several
aldehdes react with oxidised product of the indole nucleus of tryptophan to
give purple coloured complexes (Sulphuric acid with mercuric sulphate is used
as oxidizing in this reaction).
6) Sakaguchi test:
To 3 ml of the protein solution
add 2 drops of 40% NaOH and 4 drops of Molisch’s reagent mix. Add 2 ml of fresh
bromine water. A bright red colour is obtained.
Substances containing “Ganaidin
group” give this colour. Arginine is the only aminoacid in proteins that
contains this group. Hence this test is specific for arginine in protein.
Free arginine or arginyl
residues in proteins react with a-naphthol and alkaline hypobronite, to give a bright
red coloured complex.
7) Sulphur test:
To 1 ml of the protein solution
add 1 ml of 40% NaOH and boil for 2 minutes. Cool and add 2, to 3 drops of lead
acetate solution. The formation of black or brown precipitate indicates the
presence of sulphur containing aminoacids like cystine and cysteine.
Here the hot sod. hydroxide
converts-SH or -S.S group present in cystine or cysteine to sod. Sulphide which
gives a black precipitate of lead sulphidead acetate, Methionine does not
answer this test since the sulfur in methionine is in which is not split with
alkali.
8) Test For Carbohydrate Moiety in
Proteins:
Perform
Molish’s test: Note the formation of violet ring. Proteins containing
carbohydrates groups Glycoprotein and eg. albumin which contains bound
carbohydrates answer this test.
TABLE: Amino acids and reacting groups in protein colour reactions.
|
|
Test |
Responsible aminoacids |
Reaction Group |
|
1) |
Biuret reaction |
No. single aminoacid |
Peptide
linkage [dipeptides
are exception] Violet
colour |
|
2) |
Ninhydrin reaction |
Alpha aminoacids |
Alpha
amino group give purple
colour bluisMmino group
gives yellow colour, |
|
3) |
Xanthoproteic reaction |
Aromatic aminoacids |
Benzene
ring [which is nitrated] |
|
4) |
Millon’s |
Tyrosine |
Phenolic
group |
|
5) |
Aldehyde reaction |
Tryptophan |
Indole
ring |
|
6) |
Sakaguchi reaction |
Arginine |
Guanidin
group |
|
7) |
Sulphur reaction |
Cystine, Cysteine |
-S-S-and
SH group |
|
8) |
Molisch’s reaction |
|
Glycoprotein
moiety |
Questions:
1) What is the importance of colour reactions of proteins?
2) What is the general test for proteins? What is its
mechanism?
3) Define alpha amino acid?
4) What are amino acids? How will you detect them?
5) Name the aromatic amino acids? How will you detect
them?
6) Name the phenolic amino acid. And how will you detect?
7) Name the amino acid containing indole ring?
8) What groups are identified by following tests?
a) Sakaguchi test b)
Sulphur test.
9) Why methionine will not answer sulphur test?
Expt. No. 7
Date……………..
PRECIPITATION
REACTIONS OF PROTEINS
Proteins have either a positive
or a negative charge depending on the pH of the solution. If they are treated
with either a metallic salt or strong acid, these charges are neutralised and
the proteins precipitate out. Proteins form colloidal solution in which a shell
or colour of water molecules surround the proteins molecule. If salt solutions
of suitable concentration and charge are added, these ions destroy the shell of
hydration, neutralise the charge on the protein molecule and the proteins
precipitate out. Because of these properties proteins are preciitated out from
their solution.
EXPERIMENT
Egg:
white is the sources of protein used for the following experiments:-
I. Precipitation By Neutralisation and
Dehydration:
Effect
of concentration neutral salt solution:
(i) Half Saturation:
To 3ml of protein solution add
an equal volume of saturated ammonium sulphate solution, a white ppt is formed.
This is precipitation of proteins by half saturation with ammonium sulphate.
(ii) Full Saturation:
To 3ml of protein solution add
solid ammonium sulphate with mixing untill the solution is saturated (i.e.
there should be some undissolved salt in the bottom of the test tube). Let this
stand for 5 minutes. Write your inference.
Albumin being more hydrated than
glubulin, is precipitated by full saturation while globulin is precipitated by
half saturation. This property can be used for separating albumins from
globulins. Higher the molecular weight of a protein, lower the concentration of
salt required for precipitation from solution.
II. Precipitation By Heavy Metal Ions:
When solutions of lead acetate,
mercuric nitrate zinc sulphate & silver nitrate are added to protein
solutions, the cations interact with negatively charged groups on proteins
causing precipitation as metal-Proteinate and in many cases denaturation.
Note:- Raw eg: white is used as an immediate antidote in mercury poisoning
and then an emetic to remove Hg4 ions which are held by albumin.
To 1 ml of protein solution add
5-10 drops of lead acetate solution and observe the protein being precipitated.
Repeat the experiment with solutions of 5% mercuric nitrate, zinc sulphate and
silver nitrate solutions.
III. Precipitation by Alkaloidal
Reagents:
These alkaloidal reagents are
used to get protein free filtrate in the clinical investigation.
To 1ml of protein solution add
few drops 20% sulph salicylic acid. The protein is precipitated. Repeat the
experiment with 1% picric acid, 10% Trichloroacetic acid, phos photungstic acid
and Esbach’s reagent (1gm of picric acid. 2gm of citric acid in 10m1 water).
Esbach’s reagent is used for quantitative determination of albumin in urine.
IV. Precipitation by Strong Mineral
Acid:
This is used for detection of
protein in urine.
Heller’s test
To 3ml of strong HNO3 add drop
by drop of the given protein carefully along the side of the tube. A white ring
of precipitate due to the formation of acid meta-protein is observed.
V. Precipitation by Alcohol:
To 1ml of protein solution add 2ml
of ethanol, precipitation occurs, due to the denaturation of protein.
VI. Heat Coagulation Test:
Take 3/4 test tube of protein.
Hold the test tube in a slanting position and only boil the top layer, observe
for coagulum. The lower half serves as a control. Add 1-3 drops of acetic acid,
persistence of coagulum confirm the presence of protein.
Denaturation is a change in the native
character of the protein brought about by various chemical and physical agents.
A denatured protein becomes less viscous, less soluble and looses its chemical
property. All proteins do not coagulate on heating. Only albumin & globulin
coagulate on heating.
Precipitation reactions are
useful in the separation of serum proteins Deproteinisation of blood or body
fluids for chemical analysis is brought by precipitation reactions.
Questions:
1)
What is the principle for precipitation of proteins?
2)
What is denaturation of proteins? What changes takes place during denaturation?
3)
What is the importance of precipitations in clinical chemistry?
4)
What are heat coagulable proteins?
5)
Name the common protein precipitating reagents.
Expt.
No. 8A
Date……………..
CHEMISTRY OF
PROTEINS
Proteins are classified into
various groups depending upon their solubility. Chemical composition. molecular
complexity etc. They are classified as
i.
Simple proteins:
Albumin and Globulin of egg.
ii.
Conjugated
protein: Casein of Milk.
iii. Derived proteins: Gelatin, Proteoses and Peptone.
We will take one member from
each group and study their reactions like the presence or absence of some
particular aminoacids and precipitation reactions.
REACTIONS OF
ALBUMIN
Albumin is a simple protein
which is present in serum, milk, egg and muscles. Albumin has molecular weight
of about 69,000. Albumin is the fast moving protein in electrophoresis. It is a
glycoprotein. Human serum albumin makes upto about 60% serum protein and
contribute 80% of colloidal osmotic pressure.
Albumin is soluble in water,
heat coagulable and is precipitated only by full saturation with ammonium
sulphate. It is not precipitated by half saturation with ammonium sulphate.
(1) Heat Coagulation Test:
Take 3/4 of the given albumin in
test tube, boil the top portion of the solution. The lower portion of the
solution which is not heated serves as a control for comparison. A cloudy
white
precipitate will be observed in the heated portion.
(2) Half Saturation Test:
To 3ml of albumin solution add
an equal volume of saturated ammonium sulphate solution write your inference.
(3) Full Saturation Test:
To 3ml of albumin add solid
ammonium sulphate until the solution is saturated. A precipitate is formed.
(4) Heller’s Test:
To 3ml of cone. nitric acid in a
test tube add drop by drop albumin solution along the side of tube. A white
ring of precipitate is formed.
(5)
Perform all the colour reactions of proteins and write your conclusions.
Question:
(1)
What are the sources of albumin?
(2)
Name the fast moving protein in serum electrophoresis.
(3)
What is meant by full saturation and half saturation?
(4)
Does albumin give Molisch’s test + ve, If so why?
Expt. No.8B
Date ……………….
REACTIONS OF CASEIN
Casein is the chief protein of
milk. It is a phosphoprotein. It is insoluble in water but soluble in dilute
alkali. It gets precipitated at its iso-electric pH 4.6. It is precipitated by
half saturation with ammonium sulphate. It is not a heat coagulable protein.
Casein being an animal protein
is rich in most of the essential amino acids and is therefore a protein of high
biological value. Sulphur containing amino acids are absent in casein.
(1) Precipitation at Iso-electric pH
4.6:
To 5ml of casein solution add 3
drops of bromocresol green and adjust the pH to 4.6, according to the procedure
given below. A thick precipitate will be observed proving that casein is
precipitated at its isoelectric pH.
a) BCG gives blue colour above pH 4.6 a green colour
below pH 4.6 a pale green colour at pH 4.6.
b) Add to the given solution few drops of BCG ,If a blue
colour is formed the pH is above 4.6 and bring it down by adding dil acetic
acid drop by drop till a pale green colour is observed.
c) On adding the BCG If you get a green colour the
solution is below pH 4.6 and bring up pH, by adding drop by drop and with
shaking dilute sodium carbonate till the colour is pale green.
d) If on adding BCG a pale green colour is got, the
solution is below pH 4.6.
(2) Half Saturation Test:
Perform half saturation test
with ammonium sulphate. Draw your conclusion.
(3) Neumann’s Test:
This test is for organically
combined phosphorus. Principle of this test to convert the organic phosphorus
into inorganic phosphate by heating with hot cone. nitric acid. Inorganic
phosphate thus formed is detected by ammonium molybdate.
In this test you are using hot conc. acids, so be
careful while heating and water for dilution.
PROCEDURE:
Take 5ml of casein + 1ml of
conc. Nitric acid warm cool add 1ml of ammonium molybdate solution, formation
of canary yellow color of ammonium phosphor molybdate confirm that casein is a
phsophoprotein.
(4)
Perform all the colour reactions of proteins and draw your conclusions.
Note:
While
performing the ninhydrin test add few drops of 1% aceticacid to neutralise the
NaOH and then boil with ninhydrin reagent. (Casein in dissolved in NaOH).
Questions:
(1)
What do you mean by iso electric pH.?
(2)
What is the chief protein present in milk, to which type of protein does it
belong?
Expt. No. 8 C
Date…………..
CHEMISTRY OF
GELATIN AND PEPTONE
Gelatin and peptone are derived
proteins. Gelatin is a rich source of glyoine, but contains only traces of
tyrosine and lacks tryptophan, cystine and cystieine. Gelatin is a derived
protein obtained from collagen present in bones and white fibrous tissues. It
has a poor biological value, it is not heat coagulable and is precipitated by
half saturation with ammonium sulphate.
Peptones are the hydrolytic
products of proteoses. They are not heat coagulable, cannot be precipitated
either by half saturation or by full saturation with ammonium sulphate. If any
precipitate is formed it shows the contamination of primary or secondary proteoses.
Perform that following test
separately with gelatin and peptone and draw the proper inference.
1) Half Saturation test:
2) Full Saturation test:
3) Heat Coagulation test:
4) Colour reactions of protein:
Question:
1) Mention the amino acids absent in
gelatin?
2) What is the precursor of gelatin?
3) What type of protein are gelatin and
peptone?
Expt.
No.9 Date ………………
IDENTIFICATION
OF AN UNKNOWN PROTEIN
Given solution
Biuret Test
Violet Colour
(Protein
Confirmed)
|
Palegreen precipitate (May be casein)
|
No precipitate (Other protein) Heat and Acetic acid Test or Heat coagulation test |
|
Canary yellow PPT Casein confirmed |
|
|
Coagulum
|
No coagulum Gelatin or peptone. Half Saturation with Ammonium sulphate solution Biuret test with filtrate |
|
Half Saturation PPT (Globulin) |
Full Saturation PPt (Albumin) |
Half
Saturation |
|
PPt (Gelatin) No violet colour (Gelatin) test of performance sulphur &
Tryptophan test (-ve) Perform sulphur test & hopkin cole test (Gelatin confirmed) |
No PPt (Peptone) Violet colour Peptone Full saturation (Peptone) |
Expt.
No. 10 Date …………..
REACTION OF
NON PROTEIN NITROGENOUS SUBSTANCES (NPN)
NPN Substances include all
nitrogenous substances other than proteins. The NPN substances comprises Urea,
Creatinine and Uric acid. Other nitrogenous substances of biological interest
are ammonia, aminoacids, heme, purine nucleotides, pyrimidine nucleotides and
small peptides. Urea creatinine and uric acid are included in this experiment.
I. Test for urea:
i. Sodium hypobromite test:
ii. Specific Urease test
II. Test for Creatinine:
i. Jaffe’s test
III. Test for Uric acid:
i. Phosphotungstic acid reduction test:,
I. TEST FOR UREA:
i) Sodium hypobromite Test:
To 2ml of given solution add a
few drops of freshly prepared sodium hypo-bromite solution. A brisk
effervescence due to the evolution of gaseous nitrogen is observed.
ii) Urease Test:
This test depends on the fact
the enzyme urease hydrolyses urea into ammonia which makes the solution
alkaline.
To 2ml of given solution add a
few drops of phenolphthalein indicator and a few drops of a freshly prepared
urease solution wait for 15 minutes. Solution becomes pink due to the formation
of ammonia indicating the presence of urea.
Test for creatinine:
Jaffe’s
Test,
Take 5ml of given solution in a
test tube add 1ml of picric acid and make the mixture alkaline with NaOH
solution (10%). Orange colour is due to formation of tautomer of creatinine
picrate.
Test for Uric Acid:
Folin
and Dennis Test (Phosphotungstic acid reduction test)
To 2ml of given solution add 1ml
of 10% Sodium carbonate and 1ml of phosphotungstic acid. Development of blue
color indicates the presence or uric acid.
Expt.
No. 11 Date
………………..
GENERAL SCHEME
FOR INDENTIFICATION OF
PHYSIOLOGICALLY
IMPORTANT SUBSTANCES
Identification of an unknown
substance is possible only when the systematic procedure is followed. The
following scheme given the various tests to be followed in their sequential
order. Only one substance among the following compounds of physiological
importance will be dissolved in water and given as an unknown solution.
Protein: Casein, Albumin, Gelatin and Peptone.
Carbohydrates: Starch, Maltose, Lactose, Sucrose, Glucose and Fructose.
NPN Substances: Urea, Uric acid and creatinine.
Unknown solution
(i) Biuret test
|
Violet
colour Protein
is present Follow
the scheme for identification
of proteins |
No Violet colour (may be carbohydrates or NPN substance) (II) Molisch’s test |
|
Purple
ring Carbohydrate is present Follow
the scheme for indentification of
carbohydrates. |
No purple ring (may be NPN substance) (III) Sodium hypobromite test |
|
Effervescence (May
be Urea)
Confirm
by Urease test
Pink
colour (urea)
Blue
oIour (Uric acid). |
No effervescence (May be uric acid or creatinine)
(IV) Folin and Dennis test
No blue colour may creatinine (V) Jaffe’s test
Orange colour (Creatinine) |
Report:
This
given solution is …………………………………………………………………..
Expt.
No. 12
Date………….
QUALITATIVE
ANALYSIS OF NORMAL URINE
Physical Characteristics:
1) APPEARANCE: Normal fresh urine is clear. Turbidity may develop on
standing on the pH increases and the phosphates precipitate.
2) Colour: Fresh normal urine is pale yellow or amber colour.
The colour may be light or dark depending on the volume. The colouring pigment
is urochrome a compound of the Urobilin or Urobilnogen and a peptide of unknown
structure.
3) Volume: The quantity of urine excreted by normal subject
range from 1000-2000 ml per day. The volume is influenced by intake of fluid,
protein and sodium chloride. Excessive perspiration and strenuous exercise
decrease the volume of urine. A high protein diet causes a physiological
oliguria due to the diuretic effect of urea formed.
4) Specific Gravity: The specific gravity of normal urine varies in the
range of 1.012-1.024. It can be as low as 1.001 when water intake is high and
as high as 1.040 when volume of urine is restricted. The specific gravity is
directly proportional to the concentration of solutes excreted.
5) Reaction: The normal pH of the urine is ranges from 48-7.5 with
an average of pH 6.0 High protein diet gives rise to a more acidic urine because
more phosphates and sulphates are eliminated from the protein catabolism. Urine
on standing becomes alkaline by bacterial action on urea and formation of
ammonia. After meals the HCl secretion in the stomach makes the urine alkaline
during the “alkaline tide”. Urine becomes slightly alkaline when the diet is
rich in vegetables and fruits.
Chemical Constituents:
Normal urine contains both
inorganic and organic constituents. The inorganic constituents include Na+, K+,
Ca + +, Mg + + , NH4 +, Cl-, H2PO4, HPO2-4, SO2-4,
and traces of HCO3 ions. The normal non protein nitrogenous constituents are
urea, uric acid and creatinine. The total non protein nitrogen varies from
10-15 gm./day depending mainly on the protein intake. In addition to these
major organic constituents detoxified products like indican and ethereal sulphates
are found in urine.
Since the composition of normal
urine in a healthy individual varies considerably in the course of a day, for
accurate analysis, it is necessary to use a 24 hours sample, the volume of
which averages 1.5 to 2 litres. Since urine contains a number of organic
components which are easily decomposed by bacteria, it is necessary to preserve
urine samples by covering the surface with a thin layer of toluene.
EXPERIMENTS
Collect
a fresh sample of urine. Note the appearance and colour of the sample.
1. Specific Gravity: Full 3/4 of urinometer cylinder with the urine. Float
the urinometer in it. It should not touch the sides of cylinder. Note the
reading on the urinometer scale that is the depth to which it is immersed.
2. Reaction: Test the reaction of the urine with litmus paper.
Test For Inorganic Constituents: (Any 3)
(1) Chloride: To a 5ml urine add 5 drops of conc. HNO3
and 3ml of silver nitrate soln.
A
white curdy precipitate of AgCl confirms chloride.
If
the chloride concentration is small, the solution nearly becomes milky opalescent.
On an average diet 8-15 gm chloride as NaCl is excreted per day.
(2) Inorganic Sulphate
To 5 ml of urine add few drops
of conc. HC1 and 3ml of barium chloride. A white
precipitate
indicates inorganic sulphate. Here the HCI is necessary to prevent the
precipitation of phosphates.
This is derived from the sulphur
containing amino acids. On an average diet about 1gm of sulphur is excreted per
day. 85-95% of sulphur in excreted as inorganic sulphate.
(3) Calcium & Phosphate
To a test tube full of urine add
a few drops of strong ammonia and warm. A thin gelatinous precipitate of
phosphate of Ca & Mg is formed. Filter and reject the filtrate. Wash the
precipitate once with water and then add 5 ml of dilute acetic acid to the
precipitate to dissolve it. Collect the solution and divide into two parts. To
one part add a few drops of potassium oxalate. A white precipitate of
calciumoxalate indicates calcium. To the other portion add equal volume of conc.
HNO3 & 5 ml of ammonium molybdate, warm yellow precipitate of
ammonium phosphomolybdate shows the presence of phosphate.
(4) Ammonia:
To 5 ml of urine sample add 2 ml
of 5% NaOH and boil. Hold the glass rod dipped in phenophthalein near the
fumes, it turns pink. When urine is made alkaline ammonia is liberated.
Urinary ammonia is derive form
glutamine and other amino acids in kidney. The normal daily output of ammonia
is about 0.5-0.8gm. There is an increase in ammonia excretion when acid forming
foods are taken.
Test for Organic Constituents: (any 3)
a) Urea:
Urea is formed in liver as the
end product of protein metabolism and so its excretion depends on protein
intake.
About 20-30 gm urea is excreted
in 24 hours.
i) Sodium Hypobromite Test
To 2ml of urine add a few drops
of *freshly* prepared sodium hypobromite solution
A
brisk effervescence due to the evolution of gaseous nitrogen is observed.
ii) Urease Test:
This test depends on the fact
that the enzyme urease hydrolyses urea into ammonia which makes the solution
alkaline.
To 2ml of urine add few drops of
phenolphthalein indicator and a few drops of a freshly prepared Urease
solution. Wait for 15 minutes, solution becomes pink due to the formation of ammonia,
indicating the presence of urea.
b) Uric Acid
Uric acid is the end product of
purine metobolism. The daily output of uric acid varies in the range of 0.6 to
1 gm.
Alkalies dissolve uric acid in
the form of urates. It is insoluble in water. In gout the uric acid level in bi
blood increased. Uric acid is greatly excreted in urine in conditions like
leukemia, polycythemia – uric acid output decrease in renal failure.
Folin and Dennis Test:
To 2ml of urine add 1ml 10%
sodium carbonate and 1ml of phosphotungstic acid. Development of blue colour
indicates the presence of uric acid.
c) Ethereal Sulphates
Ethereal sulphates in urine are
the conjugated phenols, like phenol sulphuric acid and indican. These ethereal
sulphates result from phenols produced during purification of protein material
in the intestine. This is conjugated in the liver to form ethereal sulphates.
About 100 mg of ethereal sulphates are excreted per day.
To 5ml of urine add equal volume of baryta mixture
(barium hydroxide + Barium nitrate). A precipitate of barium sulphate and
phosphate is formed. Filter, reject the precipitate and use the filtrate. The
filtrate contains the ethereal sulphates as the barium salts. Now to the
filtrate add 3ml of cone. HCI and boil for few minutes. A faint white cloud of
barium sulphate (formed by the decomposition of ethereal sulphate by the HCl)
is formed confirming the presence of ethereal sulphate.
d) Creatinine:
Urinary creatinine is derived
from muscle creatine and is not influenced by protein intake. Excretion in
adults ranges from 1-2gm/day. In women and elderly people the values are
lowered.
Jaffe’s Test
Place 5ml of urine in a test
tube, add 3ml of solution of picric acid, and make the mixture alkaline with
NaOH solution (10% 1ml). Orange colours observed shows the presence of
creatinine, Orange colour is due to formation of tautomer of creatinine
picrate.
e) Test For Urobilinogen
Normal urine contains traces of
urobilinogen in fresh urine. Its concentration in urine increases in the diseases
of liver and obstructive jaundice.
To 5ml of freshly avoided urine,
add 1ml of Ehrlich reagent. Mix Let stand for 5 minutes. A red colour is seen.
Note: Upon .standing urobilinogen of urine is oxidised to urobilin which
does not answer the test. Urobilinogen reacts with p-dimethylaminobenzol dehyde
to give a red colour. Absence of urobilinogen in urine is indicative of
obstructive jaundice. Excess urinary urobilinogen to suggestive of hemolytic
jaundice.
Expt.
No. 13 Date……………
ANALYSIS OF
ABNORMAL CONSTITUENTS IN URINE
Metabolic disorders in most
common diseases are readily reflected in the composition of the urine of such
patients. So analysis of the urine for such abnormal constituents is of great
diagnostic value. Examples are the presence of protein, glucose, blood ketone
bodies, bile pigments, bile salts etc.
Many of the pathological constituents
are present in trace amounts in normal urine, but they escape detection due to
the low sensitivity of the methods employed. The concentration of these constituents
in urine are marked, increased in different pathological condition. Usua1ly the
analysis is carried out in properly preserved 24hours urine specimens. When
this is not possible the early morning specimen can be used. On standing urine
undergoes bacterial fermentation, it can be preserved under refrigeration or
using chemicals such as toluene or chloroform.
A. Physical characteristics in
pathological condition:
1) Volume:
An increase in urinary output
(polyuria) occurs in diabetes mellitus and diabetes insipidus and after administration
of drugs like digitalis and salicylates etc. A diminished urinary excretion (Oliguria)
occur in nephritis, fever and during diarrhea and vomiting. A total suppression
or urine formation (anuria) may occur during shock, acute nephritis,
incompatible blood transfusion, mercury poisoning and bilateral renal stone
formation.
2) Colour:
The urine becomes smoky brown
when blood is present. When bilirubin is present the colour will be yellow,
melanin makes the urine black. The urine is milky in appearance due to the
presence of bacterial or epithelial cells and lipids.
3) Reaction:
Acidic urine is voided in fever
and diabetes. Alkali therapy and urinary retention make urine alkaline.
4) Specific gravity:
Specific gravity is increased in
acute nephritis and fever and decreased in diabetes in-sipidus.
b) Chemical constituents:
The abnormal constituents which
are routinely looked for in urine are albumin, glucose, ketone bodies, bile
salts, bilirubin and blood.
1) Test for Protein:
The presence of detectable
amounts of protein (Albuminuria or proteinuria) is characteristic of kidney
diseases.
Bence jones proteins
(immunoglobulin) appear in urine in cases of multiple myeloma. This coagulates
between 40 to 600C.
a) Heat coagulation Test:
Filter urine if it is not clear.
Take about 3/4 test tube of urine and heat the top layer of urine to boiling.
If a coagulum is found add 1 or 2 drops of dilute acetic acid. A precipitate
which becomes easily coagulable upon addition of the acid indicates the
presence of proteins commonly albumin and globulin.
b) Sulphosalicylic Acid Test:
To 2ml of clear urine add 1ml of
the sulphosalicylic acid solution. A turbidity or precipitate is formed if
proteins are present in urine. You may have to wait a few minutes for the
precipitate to develop.
2) Test for Blood:
Hematuria (presence of RBCs in Urine)
occurs due to bleeding in the urinary tract or due to trauma caused by
introducing catheter through urethra. When hemolysed blood is found in urine
the condition is known as hemoglobinuria, this occurs in seveitburns, chemical
poisoning and incompatible blood transfusion.
Benzidine Test:
3ml of benzidine in glacial
acetic acid, add 3ml of H2O2 & mix well. Divide the
solution into 2 parts. To one part add 3ml of urine. If the solution develop
bluish/greenish tint blood pigments are present. To the remaining part add
water and observe the colour control is done for comparison.
Principle:
The peroxidase activity of
hemoglobin decomposes hydrogen peroxide releasing nascent oxygen which in turn
oxidizes benzidine to give blue colour.
Heme part of hemoglobin has
peroxidase like activity which releases the nascent oxygen from H2 02 this
nascent oxygen oxidises O-tolidine which gives green colour.
Note: The test is also positive when pus cells are present. These cells
contain peroxidase which is responsible for the positive reaction. However, the
test is negative which fluid is heated before testing, with blood the test is
positive even after heat treatment.
3) Bile in urine:
Bile salts appear in urine in
obstructive jaundice and bilirubin is found in urine in obstructive jaundice
and in any form of hepatitis involving destruction of liver cells. For bile
salts carry out the hay’s sulphur test, for bile pigments carry out the
Fouchet’s test.
A) Hey’s test for Bile Salts:
Principle:
Bile salts cover the surface tension allowing the sulphur powder to sink.
Procedure:
Fill half the test tube with urine and another test tube with distilled water
as control. Now sprinkle a little of fine sulphur powder in both test tubes.
Observe without mixing. If bile salt is present in urine, sulphur sinks to the
bottom and in absence it will float. Sulphur floats in the control tube.
B) Fouchet’s test for Bile Pigments:
Principle:
When barium chloride is added to urine it combines with sulphate in urine and
precipitate of barium sulphate is formed. The bile pigments get absorbed to the
ppt of barium sulphate. Ferric chloride present in Fouchet reagent then oxidises
billirubin to biliverolin in presence of tricklors acetic acid.
Fouchet’s
Reagent: Trichloroacetic acid, 10% ferric chloride and distilled water.
Procedure:
To 5ml of urine add 5ml of 10% barium chloride and few crystals of magnesium
sulphate. Filter the content of tube, the filtrate may be discarded. The filter
paper is dried by placing another filter paper below. To the dried ppt add one
or two drops of fouchet’s reagent. A green colour is observed in presence of
bile pigments in urine.
4) Glucose in Urine:
Positive Benediet’s test is
usually taken for Glucose. Glycosuria occurs mainly in diabetes mellitus and in
a renal diabetes. Positive reaction will also be seen in lactosuria, galactosemia
and pentosuria. Identity of different sugars may be established by other
relevant test.
Benedict’s Test:
To 5ml of Benedict’s qualitative
reagent add exactly 8 drops of urine and boil for two minutes over a small
flame. A green 0.5, yellow 1.5, red 2.0, orange 1.5 precipitate indicate the presence
of glucose more correctly reducing sugar in urine blue-nosugar.
5) Ketone Bodies:
Ketone bodies are found in urine
when fat catabolism is excessive in the ease of diabetes and starvation. The
ketone bodies are acetoacetic acid, betahydroxy butyric acid and acetone.
A) Rothera’s Test:
To 5m1 of the urine add ammonium
sulphate till saturation, add 4 drops of freshly prepared 5% sodium
nitroprusside solution. Mix well and add 2ml, liquor ammonia (strong ammonia).
A permanganate colour is developed if either acetone or acetoacetic acid is
present.
B) Gerhardt’s Test:
To 5m1 of the urine add ferric
chloride drop wise until no more precipitate forms. If acetoacetic acid is
present a red colour is produced. This test is answered by acetoacetic acid and
not by acetone.
Expt.
No. 14
Date…………
DETERMINATION OF TITRABLE ACIDITY
AND AMMONIA CONTENT IN URINE
Principal: The titrable acidity of urine is mainly due to acid
phosphate and to a lesser extent due to weak organic acid. Titrable acidity can
be determined by titrating urine with standard alkali with phenolphthalem indicator.
NaH2PO4 +
NaOH Ã NaHPO4 + H2O
Since Calcium interferes during
titration due to the precipitation as calcium phosphate. It is removed
completely as calcium oxalate before estimation.
Ammonia in urine arises from
hydrolysis of glutamine (60%) and due to oxidative deamination (40%) in the
Kidney.
Ammonia is estimated by the
formol titration method when neutral formaldhyde is added to solution.
Containing ammonium salt’s H+ ions are librated which can be titrated with
standard alkali. Hexamethylen tetramine is the other product in this reaction.
Procedure: A: Titration for titrable acidity
Pipette 25ml urine into a 250 ml
conical flask and add 2 spatula of potassium oxalate to precipitate calcium. Add
2 drops of phenolphthalein. Rotate the flask for 1-2 minutes and titrate
against NaOH. Note the titer value (A ml) when a permanent pale pink colour
appears preserve the contents for ammonia estimation.
B: Titration for ammonia:
Note initial reading of burette,
Add 5 ml of neutralized formalin to the above flask, pink colour disappears as
a result of liberation of H+. Titrate against NaOH until the pale pink colour
appears. Record the titre value (Bml)repeat both titrations till concordant
value are obtained.
Tabulation of
Burette Readings
|
Sl. No. |
Burette
Readings |
Volume of
Alkali required for |
||
|
Initial |
Final |
Titrable
acidity |
Ammonia |
|
|
1 |
0 k |
K L |
K ml |
L – k ml |
|
2 |
0 k |
K L |
K ml |
L – k ml |
Calculation
Calculation for acidity
Volume
of 0.1 N NaOH required to neutralize= A ml
Titrable
acidity in 25 ml urine
Therefore
volume of Alkali required for } = A
x Kml
Titrable
acidity in 25ml urine
i.e.,
Titrable acidity of 100ml urine =
A x Kml of 0.1 N NaOH
Assuming
the out put of urine for 24 hour’s
to
be 1500ml Titrable acidity of urine =
A x 15ml/day
Calculation’s for ammonia
Volume
alkali required in this titration
Titrable
acidity in 25 ml of urine =
Bml
There
volume of alkali required for
100ml
of urine =
Bml
Volume
alkali required in this titration
Titrable
acidity in 25 ml of urine =
B x Kml
Since1ml
of 0.1 N NaOH =
1.7mg NH3
Ammonia
content of 100ml urine =
KB x 1.7 mg
Or
Ammonia content of 100 ml urine =
KB x 1.7 x 15 mg/day
Note: Titrable acidity in urine varies from 200-300ml (20 to 30 milli
equivalent). Ammonia excretion per day is in the range of 0.5 to 0.85 mg (30-50
milli equivalents). Titrable acidity is generally low on carbohydrate diet and
high on protein diet.
Expt. No. 15
Date………
ESTIMATION OR
URINE CREATININE
Creatinine is one of the
substances excreted in urine. Three aminoacids viz. Glutamic acid, glycine and cystine
are involved in the formation of creatinine. It is anhydride of creatine.
Principle:
Creatinine m urine is determined
by Jaffe’s reaction., i.e. reaction with picric acid in alkaline medium to form
orange coloured tautomer. Since creatinine picrate content of urine is high it
is suitably diluted. Equal volumes of diluted urine, standard creatinine
solution and blank are treated with picric acid and NaOH. The intensity of
orange colour is read using green filter (540nm). From the O.D. values the
concentration of creatinine per 100ml and per day is calculated.
PROCEDURE:
Dilute 5.0ml of urine to 50.Oml
in a volumetric flask. Label three tubes as B, S and T. Pipete 5.0ml distilled
water in to B and 5.0ml of standard creatinine solution in to S. Pipette 5.0 ml
dilute urine T. To each add 2.0ml of picric acid and 2.0ml of 0.75 M NaOH. Mix
and read O.D after 15 minutes at 520 nm at orange, green titler.
|
|
T |
B |
S |
|
Urine
|
5ml |
|
|
|
Distilled
Water |
|
5ml |
|
|
Std
Creatinine |
|
|
5ml |
|
0.4M.
Picric Acid |
2ml |
2ml |
2ml |
|
0.75M
NaOH |
2ml |
2ml |
2ml |
CALCULATION:
Mg
of Creatinine in 100ml Urine = 
If
1500ml is the days output urine, then the amount of creatinine excreted in a
day.
= 
NOTE: STANDARD CREATININE
10mg/100ml
10mg à 100ml 5ml à 50ml
1mg – 10ml 1ml ß 10ml
0.1mg ß 1ml 0.1ml ß 1ml
0.5mg ß 5ml
i.e.,
0.1mg/1ml.
So
5ml contains 0.5mg.
Calculation: Creatinine in 100ml urine:
= 
Considering
24hr. urine out put as 1500ml.
Amount
of creatinine excreted per day : 
INTERPRETATION:
The normal daily excretion of
creatinine ranges from 1-2gm. This is little influenced by the diet. As
creatinine is related to amount of muscle mass and so of phosphocreatine in the
body. Its excretion in urine normally remains constant in an individual. For
this reason it can be used to check the reliability of 24 hour urine
collection. The creatinine coefficient (mg creatinine/Kg body weight/24 hour)
is 15-25, being higher in males, Creatinine clearance test is widely used as a
measure of the glomerular filtration rate and it is decreased in advanced renal
failure.
Expt.
No. 16 Date………………
ESTIMATION OF
BLOOD SUGAR BY FOLIN-WU METHOD
Principle: Proteins are precipitated with tungstic acid. The
protein free filtrate containing glucose is treated with alkaline copper
reagent. The cuprous oxide formed is in turn treated with phosphomolybdic acid.
The phosphomolybdic acid is reduced to phosphomolybdous acid (Molybdenum blue).
The intensity of blue coloured solution is measured in colorimeter against a
standard of pure glucose, similarly treated at 680nm using red filter.
METHOD:
Deproteinisation of blood:
In
a clean test tube take the following reagents.
- 7.0 ml. of distilled
water
- 1.0 ml. of oxalated
blood.
- 1.0 ml. of 10%
sodium tungstate.
- 1.0 ml. of 2/3 N H2SO4
Mix the contents well and allow
to stand for few minutes. The colour of the precipitate should be chocolate.
Filter the precipitate using whatman No. 1 filter paper to get protein free
filtrate.
Reduction of alkaline copper reagent:
Take 3 Folin-Wu tubes and mark
S, T and B for standard, test and blank and take solution as follows.
|
|
S. ml |
T. ml |
B. ml |
|
|
|
|
|
|
Glucose
working standard |
2.0 |
-- |
-- |
|
Protein
free filtrate |
-- |
2.0 |
-- |
|
Distilled
water |
-- |
-- |
2.0 |
|
Alkaline
copper reagent |
2.0 |
2.0 |
2.0 |
|
|
|
|
|
Mix
the contents of each tube and place in boiling waterbath for 10 minutes.
Colour development: After 10 minutes remove the tubes from the water bath
and quickly cool under tap water for 1 minute. Add 2 ml of phosphomolybdic acid
to each tube, blue colour is developed. Mix the contents till the effervescence
ceases make the solution up to 25ml. mark with distilled water and mix the
contents by inversion so that the colour is uniformly distributed.
Measurement of Colour: Set the blank to 100% 680nm (Red filter). Read the
O.D of both test and standard.
Calculation:
Glucose
in 100ml of blood (mg)
O.D
Test Concentration of Std. 100
= X
X
O.D Standard Volume of sample used 1
Note: Glucose
standard
Stock standard: 10mg per 100ml
i.e., 1ml
10.0ml of stock is diluted to
100ml contains 0.1mg of glucose
So, 2ml contains 0.2mg of
glucose
Clinical Significance: The fasting blood glucose level is 70-100/100ml in
normal person. It is called as true glucose. But in Folin-Wu method the normal
fasting blood sugar level is 80-120 mg/100ml. The value is slightly higher
(about 20mg) because it measures other reducing substance which are present in
the blood like fructose, vit C, glutathione. The level reaches upto 160 mg% at
1 hour after carbohydrate rich meal or oral ingestion of 75mg of glucose and
returns to <140mg% at 2 hours. Blood sugar estimation is useful in assessing
glucose tolerance. Hyperglycemia a common feature of diabetes mellitus,
hyperthyroidism, hypopituitarism, and hyperadrenalism. Hypoglycemia occurs in
hypothyroidism, hypopituitarism, hypoadrenalism or over dose of insulin in the
treatment of diabetes mellitus.
|
Sl. No. |
Normal Range |
|
Laboratory Parameters |
|
|
|
|
|
|
1 |
Blood Sugar (Fasting) |
: |
70-100mg/dl |
|
2 |
Blood Sugar (Post Prandial) |
: |
80-140mg/dl |
|
3 |
Serum Bilirumbin |
: |
|
|
|
Total |
: |
0.2-1.0mg % |
|
|
Conjugated |
: |
0 – 0.4 mg % |
|
|
Unconjugated |
: |
0.2 – 0.6mg% |
|
4 |
Serum Alkaline Phosphatase |
: |
3 – 13 K.A units/100ml |
|
5 |
Serum Alanine Transminase |
: |
6 – 30 IU/L |
|
|
(ALT, SGPT) |
: |
|
|
6 |
Serum Asparatate Transaminase |
: |
8-35 IU/L |
|
|
(AST, SGQT) |
: |
|
|
7 |
Serum gamma Gultamyl transferose |
: |
10-30 IU/L |
|
8 |
Serum Creatinine phosphotinose |
: |
10-50 IU/L |
|
9 |
Serum L.D.H Lactate Dehydrogenase |
: |
60-200 IU/L |
|
10 |
Serum Cholesterol |
: |
150-200 mg/dl |
|
11 |
Serum HDL Cholesterol |
: |
35-70mg/dl |
|
12 |
Serum LDL Cholesterol |
: |
60-150mg/dl |
|
13 |
Serum total protein |
: |
6-8gm/dl |
|
14 |
Serum Albumin |
: |
3.5-5gm/dl |
|
15 |
Serum Globuin |
: |
1.8-3.5gm/dl |
|
16 |
A/G Ratio |
: |
1.2:1 to 2.0:1 |
|
17 |
Blood urea |
: |
15-45mg/dl |
|
18 |
Serum Creatinine |
: |
0.7-1.4mg/dl |
|
19 |
Blood PH |
: |
7.35 to 7.5 mm Hg (7.4) |
|
20 |
PCO2 |
: |
35-45mm Hg (40) |
|
Sl. No. |
Normal Range |
|
Laboratory Parameters |
|
|
|
|
|
|
21 |
Plasma (HCO3)
Bicarbonate |
: |
22 – 28m Eq/L |
|
22 |
Plasma H2CO3 |
: |
1 – 15.5m Eq/L |
|
23 |
Serum Uric Acid |
: |
3 – 7mg/dl |
|
24 |
Serum Amylase |
: |
80-180 Somogyi units / dl |
|
25 |
Serum inorganic phosphate |
: |
2.5 – 5.0mg / dl |
|
26 |
Serum Calcium |
: |
9 – 11mg/dl |
|
27 |
Serum Electrolytes |
: |
|
|
|
i. Sodium |
: |
135 – 150m Eq/L |
|
|
ii. Potassium |
: |
3.5 – 5.0m Eq/L |
|
|
iii. Chloride |
|
90 – 106m Eq/L |
|
28 |
Serum Acid Phosphatase |
|
1 – 5 K.A unites/dl |
|
|
URINE |
|
|
|
|
1. Protein |
: |
150mg/day |
|
|
2. Bile Pigments |
: |
Negative |
|
|
3. Bile Salts |
: |
Negative |
|
|
4. Urobilinogen |
: |
Trace |
|
|
5. Ketone Bodies |
: |
Absent |
|
|
6. Sugar |
: |
Absent |
|
|
COLOUR WITH BENEDICT’S % OF SUGAR IN URINE |
||
|
REAGENT: |
|
|
|
|
|
1. Blue |
: |
Absent |
|
|
2. Green |
: |
Trace |
|
|
3. Greenish-Yellow |
: |
0.5% |
|
|
4. Yellow |
: |
1% |
|
|
5. Orange |
: |
1.5% |
|
|
6. Brick Red |
: |
2.0% and more |
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