BCH practical

what’s Rothera’s test about

it’s a test for ketone or acetone bodies in urine. A purple ring shows the presence of ketone or acetone bodies in urine

Reaction of Rothera’s test

Add (NH4)2SO4 crystals+ conc. NH4+ drops of sodium nitroprusside soln to urine

Functions of the stuff added in Rothera’s test

Ammonium sulphate = saturated the solution so that it can be sensitive

Sodium nitroprusside = reacts with ketone bodies directly

NH4 = alkaline medium for purple ring formation

What’s Hay’s test about

Presence of Bile salts.

Bile salts reduce surface tension of urine making sulphur sink

Reaction of Hay’s test

Sprinkle some sulphur on urine in a beaker. Bile salt precipitates the particle.

If it sinks, there is bile salt. If it floats, there is no bile salt

What’s Harrison and Gmelin’s test about

Bile pigments

Add BaCl2 to urine, filter= BaCl2 precipitates bile pigments onto filter paper. Add Fouchet’s reagent. Green colour signifies bile pigments

Conc. HNO3 down side of urine tube, green ring formed

mention some albumin tests

Coagulation test, Heller’s test

What’s coagulation test about

urine+HCl+heat

If there’s a precipitate, there’s albumin

Why do we add HCl in coagulation test

To make the urine faintly acidic

Albumin coagulates best near its isoelectric point which is acidic. Heat+acid=denaturation=coagulation

What else can precipitate on heating urine and in what condition

Phosphates, in alkaline condition.

Acid dissolves them

What’s Heller’s test

Urine+HNO3=white ring

This signifies presence of albumin

In why test for abnormality of urine do we use HNO3 and how do we use it?

Heller’s test for albumin

We pour it down the side so it can form a separate layer below. Protein gets denatured and coagulates to form a white ring at the junction

Clinical significance of Albumin measurement

Renal function:protein iris is an early marker of kidney diseases

Liver function: it is produced only in the liver so reduced levels equal chronic liver disease

Malnutrition: low levels indicate protein deficiency

Fluid balance: albumin helps regulate inciting pressure

Principles of chromatography

Absorption and capillary action

Coloured complex formed in uric acid estimation using uricase method

Allantoin

Why does bromocresol green change color when it binds albumin at pH 4.2

BCG opposes charges with albumin and binds electrostatically to it. BCG structure changes and this changes absorbance. Higher absorbance equals higher colour shift from yellow green to green blue

Alkaline picrate is made by what and why is it important

Colour formed is

What reaction is this

Mixing picric acid and NaOH

It’s important because creatinine reacts with picrate in alkaline conditions so the NaOH activates the picrate to be used by Creatinine

Forms an orange colour

Jade’s reaction

In Jaffe’s reaction, why is the color only stable for a short time and why does temperature matter

It’s an unstable intermediate that breaks down over time(could change color). So 15 minutes is when it should be read

Heat speeds it up. So a controlled temperature will give consistent values

To concentrate conc for Jaffe

C unknown= Abs sample/Abs standard)* C standard

Determination of ammonia in urine Urease-Berthelot method

Urease breaks down only urea into ammonia and CO2

Ammonia reacts with hypochlorite and phenol- green complex

Sodium determination

Colorimetric kit method

Retention factor

Distance travelled by solute/distance travelled by solvent

What’s liver extract and sodium arsenide for

Liver extract is where the aminotransferase is located

Sodium arsenide inhibits alpha keto acid oxidation( stops metabolism of keto acid product so the accumulate and get detected)

paper chromatography

Differential partitioning of solutes between stationary and mobile phase