INDEX

S.NO DATE CONTENTS Pg.No

1
Estimation of creatinine in urine



2

Estimation of phosphorus in urine

3

Estimation of calcium in urine

4

Estimation of ascorbic acid in urine

5

Estimation of urea in urine

6

Estimation of nitrogen in urine

7
Estimation of glucose in Blood
(FOLIN WU method)

8
Estimation of Haemoglobin by
(CYNMET HAEMOGLOBIN method)

9

Estimation of Cholesterol by ZAK'S method

10

Estimation of Phospholipids in serum

11
Estimation of iron and Haemoglobin
(WONGS method)

12

Estimation of Bilirubin

13
Estimation of Total Proteins- BIURET
method & Albumin- Globulin Ratio

14
Determination of serum Alkaline
phosphatase

15
Estimation of Serum Glutamic pyruvate
transaminase

16
Estimation of Serum Glutamic oxalo acetate
transaminase

17

Estimation of Vitamin A






EXP.NO:
DATE:
ESTIMATION OF CREATININE IN URINE
AIM:
To estimate the amount oI creatinine in urine.

PRINCIPLE:
This method makes use oI JaIIe's reaction the production oI mahogany red colour
with an alkaline picrate solution. The intensity oI the colour developed is compared in a
colorimeter against a reagent blank at 500 millimicron m\x)

REAGENTS:

1) 1 PICRIC ACID: 0.4M solution
9.16g oI crystalline picric acid is dissolved in 100ml oI water
2) 0.75 N SODIUM HYDROXIDE:
3gm oI sodium hydroxide in 100ml oI water.
3) N/10Hcl V
1
N
1
V
2
N
2

X * 10 1500 x 0.1
1485 ml water, 15ml Hcl
4) STOCK CREATINE
Dissolved lOOmg oI creatinine in N/10 Hcl and made up the solution to
100ml with the same
5) WORKING STANDARD
Diluted 1ml oI the stock solution to 50ml with water. This contains 20 gamma
oI creatinine per ml.




ESTIMATION OF CREATININE IN URINE
Volume oI
solution (ml)
Concentration
(Y)
Volume OI
Water
Volume oI
picric acid
(ml)
Volume oI
Noah(ml)
Klett
Reading
(O.D)
Blank 0 3.0 1.0 1.0
standard
0.5 10 2.5 1.0 1.0
1.0 20 2.0 1.0 1.0
1.5 30 1.5 1.0 1.0
2.0 40 1.0 1.0 1.0
2.5 50 0.5 1.0 1.0
Urine 1.0 1.0
1.0 - 2.0 1.0 1.0
1.0 - 2.0 1.0 1.0


CALCULATION:

1 nil oI the diluted urine contains
(50ml oI the diluted urine contain in mg

This 50ml was diluted Irom l ml oI urine
1ml oI urine contains
(100ml oI urine contains
(1500ml oI urine contains


PROCEDURE:
Test tubes containing 0.5, 1.0, 1.5, 2.0 and 2.5ml oI the standard solution
corresponding to the gamma values oI 10,20,30,40 and 50 were taken. The volume was made
up to 3ml with water in all the tubes. 3ml oI water was taken as blank.

1ml oI urine was taken and diluted to 50ml with water. 1ml oI the diluted urine was
taken in the test tube and diluted to 3ml. To all the tubes added 1ml oI 0.4M picric acid
solution and then 1ml oI 0.75N sodium hydroxide solution and allowed to stand Ior 20
minutes Ior the colour to develop was read in a colorimeter at 500 ma against a reagent
blank.

RESULT:
The amount oI creatinine in 1500ml oI urine is






















EXP.NO:
DATE:
ESTIMATION OF PHOSPHORUS IN URINE

AIM:
To estimate the amount oI phosphorus in urine sample.

PRINCIPLE:
When the solution is treated with acid ammonium moIybdate , Phosphomolybdic acid
is Iormed. This is reduced by the addition oI 1,2,4 amino napthol sulphonic acid reagent, to
produce a blue colour which is apparently a mixture oI oxides oI molybdenum. The intensity
oI the colour developed is the measure oI phosphorus present.

REAGENTS:
1) Molybdate solution - I :
Dissolved 25g oI the reagent grade ammonium molybdate in about 200ml oI water
and transIerred into a 1 litre TOlumetxic Ilask containing 500ml oI ION sulphuric acid and
then made up to the mark with water and mixed . The solution is stable indeIinitely.

2} Molybdate solution- II:
Dissolved 25g oI the reagent grade ammonium molybdate in about 200ml oI water
and transIerred into a 1 litre volumetric Ilask containing 300ml oI 10N sulphuric acid and
then made up with water to 1 litre. This solution is stable indeIinitely.

3)1, 2, 4 Aminonapthol Sulphonic Acid Reagent (ANSA)
Placed 195ml oI 15J sodium bisulphate solution in a volumetric Ilask and adder!
0.5g oI 1, 2, 4 aminonapthol sulphonic acid was added and then 5ml oI 20 sodium sulphite
was added. Put the stopper and shook until the powder was dissolved. II the solution was not
complete, added more 20 sodium sulphite (1ml at a time) with shaking but avoid excess.
This solution was transIerred to a brown glass bottle and stored n a reIrigerator.



ESTIMATION OF PHOSPHORUS IN URINE

Volume oI
solution (ml)
Concentration
(Y)
Ammonium
Molydate
Volume oI
Water (ml)
Volume oI
ANSA(ml)
Klett
Reading
(O.D) I II
Blank 0 1.0 - 8.6 1.0
standard
1.0 8 1.0 - 7.6 1.0
20. 16 1.0 - 6.6 1.0
3.0 24 1.0 - 5.6 1.0
4.0 32 1.0 - 4.6 1.0
5.0 40 1.0 - 3.6 1.0
Urine
0.5 - 1.0 8.1 1.0
0.5 - 1.0 8.1 1.0

CALCULATION:
Klett reading corresponds to oI phosphorus
(0.5 ml oI the diluted urine contain
50ml oI diluted urine will contain

50 ml was made up Irom 2.5ml oI urine
( 1500ml oI urine contains


4) Stock Standard Phosphorus Solution:
35.1mg oI pure potassium dihydrogen phosphate was weighed and dissolved in water.
Added 1ml oI ION sulphuric aod and made up to 100ml with water.

51 Working Standards:
Working standard was prepared by diluting 10ml oI standard to 100ml. One ml oI this
solution contains 8jag oI phosphorus.
PROCEURE:
2.5ml oI the urine was taken and diluted to 50ml with water. 0.5ml oI this solution
was taken in 2 test tubes. 1ml - D and 0.4ml oI 1,2,4 amino napthol sulphonic acid was added
and the volume was made up to 10ml with distilled water. For standard 1,2,3,4 & 5ml oI
standard solution and add 1ml oI ammonium molybdate- I & 0.4ml oI amino napthol
sulphonic acid was taken and made up to 10ml. All the tubes containing 10ml oI the solution
was mixed well and allowed to stand Ior 15 min. Simultaneously a blank was prepared by
mixing 8.6ml oI water, lml oI molybdate - I and 0 4ml oI amino napthol sulphonic acid. The
colour developed read into a colorimeter using a red Iilter oI wavelength 620

RESULT:
The amount oI phosphorus present in 1500ml oI urine is
















EXP.NO:
DATE:
ESTIMATION OF CALCIUM IN URINE
AIM:
To estimate the amount oI calcium present in urine.
PRINCIPLE:
Calcium is precipitated Irom urine as oxalate. The ium is not co- precipitated as the
conditions are selected increase the solubility oI magnesium oxalate. The precipitate is
dissolved in acid and the oxalate ion is determined ritrtiDetricaDv by titrating against
potassium permanganate.

REAGENTS:
1) AmmounIum oxalate -4 solution:
4 grams oI ammonium oxalte in 100ml oI water.
2) Ammonia- 10 solution:
Diluted 10 ml oI ammonia in 90 ml oI water.
) Potassium Permanagnate: 0.0IN
Prepared Ireshly beIore use by diluting stock 0.1N potassium permanganate.
Dissolved 316mg in 100ml and made up to the mark (0. IN)
4) Approximately normal Sulphuric acid: 2N H
2
SO
4
.
5)0.01N Oxalic acid:
Dissolved 0.063gm (63mg) oI oxalic acid crystals in 100ml oI distilled water.

PROCEDURE:
Take 2ml oI urine, 2ml oI distilled water, lml oI 4 ammonium oxalate in centriIuge
tubes. AIter calcium was precipitated, centriIuged and removed the supernatant Iluid without
disturbing the precipitate, then 3ml oI 2 ammonia was run down inside the tube. Mixed the
precipitate well and centriIuged again. Poured oII the supernatant Iluid. Repeated this until
the precipitate is completely washed and the


ESTIMATION OF CALCIUM IN URINE
Oxalic acid Vs Potassium Permanganate
Volume oI
Oxalic acid (ml)

Burette Reading(ml)
Volume oI
KMnO
4
(ml)
Indicator
Initial Final



Urine Sample Vs KMnO
4
Volume oI
Urine (ml)

Burette Reading(ml)
Volume oI
KMnO
4
(ml)
Indicator
Initial Final



AL&LATIONS:

Siandardizaiion of KMnO4 V1N1 V2N2

1 ml oI 0.01NKMnO
4
is equivalent to mg oI calcium
( 1 ml oI NKMnO
4
Contains

( oI NKMnO
4
Contains

( ml oI urine contains mg oI calcium
( 1500 ml oI urine contains




Supernatant gave no precipitate with calcium chloride solution discarded the
supernatant Iinally and added 2 ml oI normal sulphuric acid mixed and dissolved the
precipitate with the acid then kept in a boiling water bath, in the hot condition, titrated against
N/10 potassium permanganet to pale pink colour which persisted Ior about a minute. As a
blank, titrated 2 ml oI 2N sulphuric acid against N/100 postassium permanganet to same end
point.

RESULT;
Amount oI calcium present in 1500ml oI urine is


















EXP.NO:
DATE:
ESTIMATION OF ASCORBIC ACID IN URINE
AIM:
To estimate the amount oI ascorbic acid in urine.
PRINCIPLE:
Vitamin C is a good reducing agent and it reduces the dye 2, 6 dichlorophenol
indophenol. In this reaction the ascorbic acid itselI is oxidized to dehydro ascorbic acid in the
absence oI an interIering substance. The capacity oI an extract oI the sample to reduce a
standard solution oI the dye is determined by titration, a is directly proportional to the vitamin
C content. Oxalic acid is not only used to reduce the pH oI the existing medium andalso, Ior
complexes with metallic ion e.g. copper, thereby preventing the catalytic oxidation oI
Vitamin C.
REAGENTS:
1. Glacial acetic acid
2.10 Oxalicacid
.Solution of 2,6 dichlorophenal Indophenol (dye)
Weighed accurately 40mg oI the dye and dissolved in l00 ml oI distilled water,
1ml oI solution is equivalent to L2mg oI vitamin C.

PROCEDURE:
Dissolved 400mg oI pure, dry ascorbic acid in 100ml 10 oxalic acid. Diluted 5ml oI this to
100ml with 10 icacid. Pipetted out 0.5ml oI dye solution in a test tube and added 1ml oI
glacial acetic acid titrated this against ascorbic acid in the burette. About 5ml should be
required to decolourize

TECHNIQUE:
Pipetted out 0.5ml oI the dye into the test tube and added 1ml oI glacial acetic acid.
Run in the urine Irom the tie slowly with constant shaking until the red colour has been
disappeared. Noted the amount oI urine required.

ESTIMATION OF ASCORBIC ACID IN URINE

2, 6 Dichlorophenol indophenols Dye Vs Ascrbic acid
Volume oI
Dye (ml)
Burette Reading (ml)
Volume oI
Ascorbic Acid
(ml)
initial Final
0.5
0.5

2, 6 Dichlorophenol indophenols Dye Vs Ascrbic acid
Volume oI
Dye (ml)
Burette Reading (ml)
Volume oI
Urine(ml) initial Final
0.5
0.5

CALCULATIONS:
100ml oI ascorbic acid solution contains 40g oI ascorbic acid
( 5ml oI the solution contains
5 ml is diluted to 100ml
(100ml oI the made up solution contains mg oI ascorbic acid
(1 ml oI the made up solution contains mg oI ascorbic acid
0.5 ml oI the dye is equivalent to
1ml oI the urine was consumed by 0.5ml oI the dye
1.0 ml oI the urine contains oI ascorbic acid
(100 ml oI the urine contains
50 ml was made up Irom 2.5ml oI urine

(1500 ml oI urine contains
RESULT:
The amount oI ascorbic acid present in 1500 ml oI urine





















EXP.NO:
DATE:
ESTIMATION OF UREA IN URINE
AIM:
To estimate the amount oI urea in urine.
PRINCIPLE:
Urea reacts directly with diacetyl monoxime in the oI thiosemicarbazide to Iorm a red
coloured product, i is measured colorimetrically at 540nm.