CHEMICAL EXAMINATION OF URINE
Analyte
GLUCOSE
A. Fehling's Method
B. Trommer's Method
C. Nylander's Method
D. Moore Heller's Method
E. Benedict's (qualitative) test
Principle
Theblue alkalinecupric hydroxide
presentin Fehlings solution,when
heatedinthe presenceof reducing
sugars,gets reducedto yelloworred
cuprousoxide anditgets precipitate.
Hence, formationof thecoloured
precipitation indicatesthe presenceof
reducingsugars
Ability to reduce copper
In hot alkaline soln, reducing sugars
reduce bismuth salt to metallic salt
Conformational change of glucose
with 10% KOH to caramel
Cupric ions + reducing sugar (Blue)
(eg. glucose) ------alkali&heat------
>Cuprous ions + Oxidized sugar
(Cu2O)(e.g. gluconic acid) (Orange-
red )
Reagent (+)Result and Interpretation
1ml Fehling's A (+) yellow precipitate
1ml Fehling's B (-) absence of yellow precipitate
10% KOH (+) yellow red precipitate
10% Copper Sulfate (-) absence of yellow precipitate
0.5 Nylander's reagent (+) black color
TRACE- brown color
(-) absence or black or brown color
10% KOH 1% or less- camary yellow
1-2%- wine yellow
2-3%- cherry yellow
3-4%- rum color
>4%- dark brown or black color
Benedict's reagent: (-)-no change from original blue color
-Copper Sulfate-17.3 gms TRACE-green solution without any precipitate
-Sodium Citrate-173 gms (+)-greenish yellow solution with yellow
-Sodium Carbonate- 100 gms precipitate
-Distilled H2O (++)-yellowish green soution with yellow
precipitate
(+++)-yellowish orange solution with orange
precipitate
(++++)-orange red solution with brick red

ALBUMIN(Protein)
A. Heller's Nitric Acid Test
B.Picric Acid Test
C. Robert's Test
D. Heat and Acetic Acid Test
QUANTITATIVE TESTS
GLUCOSE
Benedicts's Method-Test Tube
Method
ALBUMIN
Esbach's Method
Sulfosalicylic Acid Test
The same as Robert`s Test
The principle of this test is based on
the precipitation of protein and
formation of flocculence using picric
acid
The principle of this test is based on
the precipitation of protein and
formation of white compact ring
using concentrated Nitric acid
(HNO3)
The test is based on the
precipitation of protein by heat.
Ability of the glucose to reduce copper
Precipitation of albumin in an acidic
medium thru increased sedimenation
by BaSO4
This test is based on the
precipitation of protein (particularly
albumin ) by sulphosalicylic acid.
0.5ml pure Nitric acid
Saturated Picric acid
1ml Robert's reagent
10% Acetic acid
5 ml Benedict's Quanti. Rgt.
1-2 g anhydrous Na2CO3
10% HAc
BaSO4
Esbach's reagent
3 ml of 3% SAA
precipitate
(+) albumin- white ring at the point of contact
of the 2 fluids
If albumin is present, a precipitate forms
varying from cloud to a heavy flocculence,
depending on the quality of albumin present
White ring at the point of contact of the two
fluids indicates albumin. The larger the ring,
the greater is the amount of albumim present
(-)- absence f cloudiness
TRACE-cloudiness is barely visible
(+)-cloudiness is distinct but not granular
(++)-cloudiness is distinct and granular
(+++)-cloudiness is heavy with distinct flocculi
(++++)-cloudiness is dense with large flocculi
0.01-0.03 gm/100ml
0.05-1.5 gm/24 hours
White ppt
White ppt
BENCE JONES PROTEIN
Heat and Acetic Method BJP precipitates at 40-60 C and
dissolves at boiling point.
REDUCING SUGARS
QUANTITATION
Fructose
Selivanoff's Method Hot HCl converts fructose to
oxymethylfurfural in hot acidic
medium which gives a red color when
condensed with resorcinol
Pentose
Tauber's Method Pentose in a saturated soln of
benzidine in glacial HAc which is
heated then cooled rapidly will give
pink result
Bial Orcinol Heating pentose with mineral acids
lead to formation of furfural which
combines with orcinol to produce
colored copmpound
LACTOSE
Rubner's Method When lactose is treated with lead
acetate and heated in the presence of
ammonium OH, gives red color
Woehlk's Method In an alkaline pH, Lactose reduce KOH,
produce red color
KETONE BODIES
If urine is not acidic, + 10% NEG-no increase in turbidity (>6)
Hac TRACE- noticeable turbidity (6-30)
1+ = distinct turbidity with no granulation
(30-100)
2+= turbidity with granulation with no
flocculation (100-200)
3+=turbidity with granulation and flocculation
(200-400)
4+=clumps of protein (>400)
3ml Selivanoff's rgt (+)intense orange-red color and dark ppt
w/cdissolves with ethanol
(-) absence of red orange color and dark ppt
2ml Tauber's rgt (+)pink to red color (cherry red)
(-) absence of pink to red color
other sugars=brown color after 9-10 min
0.2 g (orcinol) (+) olive green sol'n in amyl alcohol
1ml (conc. HCL) (-) no olive green sol'n
5 drops (10% KOH
2ml ammonia (+) red color
8 drops 10%KOH (-) absence of red color
2.5ml NH3 (+) red color
5 drops (15%KOH) (-) absence of red color
 Gerhardts Test : Diacetic Acid Ferric iron chelate with enol(OH) 10% FeCl3 (+) Bordeaux red color
group of diacetic acid

Legals Test: Acetone Acetone and diacetic acid react with NAOH/ KOH (+) Purple or violet-red color
Na nitroprusside in alkali medium Sodium nitroprusside
Conc. Acetic acid

Harts Test : B-hydroxybutyric B-hydrobutyric acid is converted to Distilled H2O (+) Red Ring at the point of contact
acid acetone thru addition of H2O2. Acetic acid
Acetone is tested thru Na 0.5 ml H2O2
nitroprusside

BLOOD
Benzidine Test Pseudoperoxidase activity of 1ml Benzidine rgt Trace faint green
hemoglobin or myoglobin 1ml 3% H2O2 (+) green
(++) greenish blue
(+++) blue
(++++) deep blue

BILIRUBIN
Rosenbachs Modification of 2ml HNO3 (+) play of colors green towards the urine
Gmelins Test layer

Harrison Spot Test 5ml of 10% BaCl2 (+) blue to green color
Fouchets reagent

UROBILINOGEN
Wallace-Diamond Method Colorless urobilinogen is changed to a 0.5 ml of Ehrlichs rgt (+) rose red color
(Ehrlichs Benzaldehyde Test) colored compound by Ehrlichs rgt

Semi-quantitative Test

UROBILIN
Schlesingers Method After removal of bile pigments from 5 drops Lugol Soln (+) greenish fluorescence
urine by barium chloride and oxidation 5ml Schlesingers rgt
of urobilinogen to urobilin by addition
of alcoholic Schlesingers rgt to form a
complex
BILE ACIDS
 Hays Test Bile acids have the ability to reduce Sulfur powder 0.01 % or more
surface tension of liquids -The powder sinks once
0.0025 % or more
- The powder sinks with agitation
Absence
-The powder remains floating

ELECTROLYTES
Sulkowitch method: Calcium
Calcium reacts with ammonium Sulkowitch rgt (-) no ppt
oxalate to form insoluble Ca2C2O4. (+) Slight cloudiness
The degree of precipitation is roughly (++) Definite cloudiness
proportional to the amount of Ca in (+++) Not total opaque cloudiness
the specimen. Sulkowitch rgt prevents (++++) Opaque ppt
the precipitation of phosphates and
other urinary substances

Fantus Test: Chlorine Silver nitrate reacts with Cl in urine to 1 drop Potassium (+) Reddish brown
precipitate AgCl. Excess silver nitate dichromate
racts with K chromate 2.9% Silver nitrate

ALKAPTONES
Alkali Test If the urine is allowed to stand it will 10% NaOH Black ring which goes down
slowly oxidize and darken at the
surface. Urine at and acid pH is not
colored; the addition of alkali will
hasten darkening when homogentisic
acid is present

Ferric chloride To determine the presence of phenols. 10% FeCl3 Transient blue color
The OH which is attached directly to
an aromatic nucleus is detected by
FeCl3 and yields color

Benedicts Qualitative Test
MELANIN
Homogentisic acid reduces the copper 5 ml Benedicts rgt Brown to black color
reagent in test; due to alkali presence,
it will also darken to produce a yellow
precipitate in an orange to yellow
solution
 Thormahlen method Na nitroprusside is reduced to Na nitroprusside Addition of NaOH red color
ferrocyanide by reducing action of the 10% NaOH Addition of Acetic a blue green color
melanogen Glacial acetic acid

INDICAN
Obermayers Test Obermayers reagent will oxidize Obermayers rgt Blue color in chloroform
indican to indigo-blue or occasionally Chloroform Pink color in chloroform indicates iodine
indigo-red ingestion