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Biochemistry Practical Techniques Guide

The document outlines a biochemistry practical summary that includes essential laboratory equipment, solution preparation methods, and various tests for detecting carbohydrates, lipids, and proteins. It details specific procedures for tests such as Molisch's, Benedict's, and Biuret tests, as well as solubility and emulsification tests. Additionally, it covers concepts like molarity, normality, and the isoelectric point relevant to protein solubility.

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10 views5 pages

Biochemistry Practical Techniques Guide

The document outlines a biochemistry practical summary that includes essential laboratory equipment, solution preparation methods, and various tests for detecting carbohydrates, lipids, and proteins. It details specific procedures for tests such as Molisch's, Benedict's, and Biuret tests, as well as solubility and emulsification tests. Additionally, it covers concepts like molarity, normality, and the isoelectric point relevant to protein solubility.

Uploaded by

70182766
Copyright
© All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as DOCX, PDF, TXT or read online on Scribd

Biochemistry Practical Summary (Foundation Module)

1) Introduction
 Beaker
 Tongs
 Bunsen burner
 Stirrer
 Test tube
 Water bath
 Spectrophotometer
2) Solutions
 w/w = How much solute(g) in every 100 g of sol.
 w/v = How much solute(g) per 100 ml of sol
 v/v = How much liquid solute(ml) per 100 ml of sol.

o Molality: Number of moles of solute per 1000g/1kg solvent. (w/w)


o Molarity: number of moles of solute per 1000ml/1L solution
o Normality: Gram equivalent weight of a solute per liter of solution
o Equivalent Weight: Molecular weight/ valency

Practicals:
1) Solution preparation
 Calculate solute required
Eg. 0.9% NaCl
0.9 g per 100ml so for 1000ml 9g NaCl required
 Weigh the required amount on WEIGHT BALANCE
 Pour it in to the BEAKER, add water and STIR to dissolve
 Transfer solution to VOLUMETRIC FLASK using a FUNNEL.
 Close flask and shake it.
 Add water till LOWER MENISCUS touches 1000ml.

Habiba’s Notes
2) Molisch’s Test: carb detection if positive
3) Iodine test: polysaccharide detection if positive
4) Benedict's test: reducing carbs test

o Reagent:
 Benedict's Reagent
 Given sol
o Procedure:
 3ml Benedict’s in test tube
 8 drops given sol
 Heat for 3 mins
 Record results

5) Barfoed’s test: positive for monosaccharide


6) Seliwanoff test: positive for fructose
7) Grease Spot: Identification of lipids

o Reagents:
 Ether
 Oil
 Filter paper
o Procedure:
 3ml of ether in test tube
 Add 5 drops of oil
 Put one drop of sol on filter paper
 Let it dry, grease spot seen

8) Solubility test: tests Solubility in diff solvents at diff temp


o Reagents:
 Oil
 Ether
 Chloroform
 Water
 Alcohol

Habiba’s Notes
o Procedure:
 Take 3 ml of diff solvent in test tubes and label them.
 Add 5 drops of oil
 Shake and allow to stand
 Observe solubility
 Ether: clear solution
 Chloroform: clear solution
 Water: insoluble hot or cold, oil layer formed
 Alcohol: insoluble
9) Emulsification test: mixture of 2 immiscible solvents.
o Reagents:
 Na2Co3 sodium carbonate
 Bile
 Soap sol
 Oil
o Procedure:
 5ml of each solution in test tube and label
 3-5 drops of oil in each test tube
 Shake and allow to stand 10 mins (all dissolve separation time tells how stable
they were)
 Oil and water separate quickly (unstable emulsion)
 Na2Co3 separation after some time(more stable)
 Soap sol separation after longer time (fairly stable)
 Bile Salt no separation (most stable)

10) Biuret test: identifies protein presence


o Reagents:
 NaOH
 CuSO4
 Sample sol
o Procedure:
 2ml of sample solution in test tube
 2ml of naoh
 3-4 drops of cuso4
 Wait 2 mins (violet= protein present)

11) Heat coagulation test: To detect coagulable proteins


o Reagents:
 Sample sol
 Acetic acid
 Chlorophenol red
o Procedure:

Habiba’s Notes
 Fill 2/3 test tube with given sol
 4-5 drops chlorophenol red
 Add 1% acetic acid drop by drop till faint pink colour formed
 Hold test tube at bottom and HEAT UPPER PORTION OF FLUID
 Coagulation: albumin and globulin
 No Coagulation: gelatinous proteases histamines and protamines

12) Iso-electric Point: pH at which a molecule has a net zero charge


Proteins lowest solubility at isoelectric point
o Reagents:
 Sample sol
 Acetic acid
 Bromocresol green
o Procedure:
 3ml given sol in test tube
 1 drop bromocresol green
 1% acetic acid drop by drop till colour change to green (4.6pH)
 Thick ppt = Casein present
 No ppt = casein absent

13) Salt Saturations: to identify and separate proteins based on their solubility in
different salt concentrations
o Reagents:
 Solid ammonium sulphate
 Saturated sol of ammonium sulphate
 40% sodium hydroxide
 1% copper sulphate
 Sample sol
Half Salt Saturation:
o Procedure
 7 ml of given sol and ammonium sulphate in test tube
 Shake and wait 5 mins
 Filter out the filtrate
 Preform biuret test on filtrate using equal volume of naoh and cuso4
(Equal vol of given sol and (NH₄)₂SO₄ = solution is half saturated)
 White ppt = globulin &casein
 No ppt = albumin
Full Salt Saturation:
o Procedure:
 5ml of given solution and add ammonium sulphate (solid form)
 Mix until saturates, shake and allow to stand for 5 mins
 Filter out filtrate
 Preform biuret test on filtrate using equal volume of NaOH and CuSO4
 White ppt =albumin present

Habiba’s Notes
 No ppt = polypeptides and peptones present
* Even if solution gives positive biuret test peptides and
peptones are present.

Habiba’s Notes

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