part i lab manual 2013

7/27/2019 Part I Lab Manual 2013

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Biochemistry Laboratory ManualAtal Behari Bajpayee Medical College

Delhi, 110007


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Department of Biochemistry

Preface

This edition is laboratory manual for first MBBS Biochemistry course. It provides

medical students with a laboratory manual of basic biochemistry with a better

understanding of the clinical aspect of diseases. Clinical biochemistry is an clinical

chemistry with its clinical interpretion in disease detection, understanding cause of

disease, decision making in therapeutic monitoring and diagnostics to facilitate the

disease treatment and management. The Part I involves the chemistry and

determination of chemical analyte levels in body fluids. The Part II involvesestimations of metabolites, enzymes and examination of the results to use them in the

diagnosis of diseases, screening for susceptibility to diseases and for monitoring the

progress of treatment. This manual will enable the student to obtain a better

understanding of the analytical techniques, instruments and reagents used in

biochemistry and the clinical applications thereof.

We are grateful to all the faculty members and staff of the department of Biochemistry

for their valuable support and suggestions in the development of this practical manual.


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CONTENTS

S. No. TopicPage

No.Teacher

0 Brief review of Practical Biochemistry 5

1.Basic Biochemistry laboratory: Pipetting, Safety

principles9 Himani

2. Hydrogen ion concentration & preparation of buffers 17 Mukti

3. Properties of carbohydrate 22 Himani

4. Tests for proteins -I 25

5. Tests for proteins -II 29

6. Chromatography 32

7. Thin layer chromatography (TLC) 35

8. Electrophoresis of serum proteins 429. Enzyme kinetics 44

Appendix:


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INTRODUCTION TO BIOCHEMISTRY PRACTICAL

1.BRIEF REVIEW OF ANALYTICAL CHEMISTRY

A. Methods of expressing concentration

Concentration may be defined as weight per unit volume. The commonest expression ofconcentration is (1) Percent (%) (2) Molar (M) (3) Normal (N).

1. Percent: There are three ways of expression of percentage composition of a solution.

a) Weight per unit weight (W/W). A 10% (W/W) solution contains 10 g of solute in 90gof solvent.

b) Weight per unit volume (W/V). A10% (W/V) contains of 10 g of solute dissolved per100 ml of final volume of solution (NOT SOLVENT).

c) Volume per unit volume (V/V). A10% (V/V) solution contains 10 ml of concentrateper 100 ml of final volume of solution (NOT SOLVENT).

2. Molarity: A molar solution (1M) contains 1 gm mol wt. (mole) of solute in 1 liter of solution.The molarity of solution are commonly indicated as 1M, 0.5 M etc. 1 Molar solution of H

2SO

4

contains 98.08 gm H2SO

4/L (Mol .Wt of H

2SO

4= 98.08).

A millimole is 1/1000 of a mole =1 formula wt. in mg. 1 millimolar (mM) solution contains one

millimole of the substance per 1 liter solution.

For making concentration other than 1M and volume other than 1 liter:

1. Multiplying the desired molarity of the solution by the gm. Mol wt. of the solute givesthe number of gms necessary to make 1 liter of solution of desired molarity e.g.Prepare 1 liter of 0.5 M NaCI.

0.5 x 58.5=29.25g. of NaCI in 1 liter of solution.

2. When a volume of solution other than 1 liter is desired the following formula may beemployed.

W = wt. of substance necessary to make 1 liter of desired molarity.V = 1 liter.W1= Wt of substance needed to make the desired volume of some molarityV1= Desired volume in ml.

For Example,

Make 1500 ml of 2 M NaCI;


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To make 1 liter of 2 M NaCI, weight of NaCI (W) required is 2x58.5=117g.

W1 = 175.5g of NaCI.

3. Normality : A normal solution contains 1 gram equivalent weight of the solute in one litre ofsolution. 1 mole HCl, 0.5 mole H2SO4, 0.333 mole H3 PO4 in 1000 ml of solution in water

are one Normal solutions.

No. of moles x valency = No. of equivalents

Molarity x valency = Normality

The following equations define the expression of concentrations.

Since Eq. Wt. = Mol. WtValency

1. In case of monovalent compounds of elements M=N

2. When valencey is other than one. It must be multiplied by molarity to give thenormality.N=M x valency

e.g. Normality of 0.05 M H2SO4N= 0.05 x 2 = 0.1

One milli equivalent (mEq) is 1/1000 of equivalentMg

mEq = ---------------Gm. Eq.wt.

To convert mg per 100 ml to mEq/liter:

Since,

So,


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or

Example: If serum sodium is 322 mg/100 ml [3220 mg/L].

Atomic wt. of Na = 23, Valency = 1

m Eq/L = (322 x 10 x 1) / 23 = 140

Sodium concentration in plasma is also expressed as mmol/L.

B. Dilution Problems

All the volumetric solutions contain a definite amount of solute in a fixed volume of solution.Whenever a solution is diluted its volume is increased and its concentration is decreased buttotal amount of solute remains unchanged.

Dilutions are usually expressed as one unit of the original solution per total units of finalsolution. For eg 1:10 dilution required that one unit of concentrated solution be diluted to atotal volume of 10 units.

To calculate the conc. of a solution multiply by the dilution ratio. If several dilutions are made,multiply them together to arrive at final concentration.

e.g. A 10% solution is diluted 1:5 (twice) then conc. of the diluted solution =10%x1/5x1/5=0.4%

Large dilutions can be carried out by doing series of dilutions.

C. Specific Gravity

Specific Gravity is useful in preparing normal solution of liquid. The specific gravity of asolution multiplied by its volume and again by the percentage of material in solution equals theweight of solute in solution.

To prepare a normal solution of a liquid

mL/liter for 1N= GMWValency x Sp. gr x conc. (W/V)

To make stronger than 1 N, multiply by appropriate factor to make weaker than 1N diluteappropriately.


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D. Some common acids and alkalies

--------------------------------------------------------------------------Substance Normality

--------------------------------------------------------------------------Ammonium Hydroxide 15.1H2SO4 (conc) 35.0HNO3 (conc) 16.6HCI (conc) 11.7Acetic acid glacial 11.6---------------------------------------------------------------------------

E. Problems

1. Make 200ml of 70% (V/V) alcohol form 95% alcohol.2. How many gms. Of a salt would be required to make each of the following

solution.a) 100ml of 10% V/V solutionb) 500ml of 5 % (W/V) c) 50ml of 1% (W/V)

3. If 40 g. NaOH is diluted in 1 liter what is conc in terms of :a) Molarityb) Normalityc) Percent (V/V)

4. A 1N solution of NaOH is diluted 5:25 then rediluted 3:100 what is the finalnormality? How many gms of NaOH are present in 100ml of the final solution.

5. Calculate the molarity of conc. HNO3 , Sp.gr. 1.42, conc. 70%


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EXPERIMENT 1

BASIC LABORATORY PRINCIPLES

Experiment: Pipetting, Concentration units, Solutions

Procedure:

1. Volumetric Equipment

Most clinical chemistry procedure requires accurate measurement of volume. Volumetricequipment should be used with solutions equilibrated at room temp. For accurate work withpipettes and burettes, they should be rinsed out and drained well with some of the liquid to bemeasured. The filling of pipettes with poisonous corrosive of volatile liquids should be doneusing a rubber/other pipette aids.

Pipettes:Defination: device used for the transfere of a fixed volume of liquid from one container to other

They are caliberated to deliver the volume specified.Caliberated glass pipettsareavailable in various volumes ranging from 1 to 25 ml.

TC (To Contain)to contain pipettes are used for volumes lesser than 0.5ml

Types

1) Glass pipette are two types

A) To Contain (TC)these are used for small volumes(less than 0.5ml).Eg RBC pipettes,WBC pipettes. These calibarations are such that they contain the specific volumes.

B) To Deliver (TD)all these pipettes are caliberated to deliver a specific volume.

The difference between TC and TD types of pipettes is that the former must be rinsed out aftercomplete delivery in order to wash all the fluids contained in the pipette in to diluents.

To Deliver (TD) are two types(1) Blowoutthese pipettes have volume graduation marking extend to the delivery tip ofpipettes (Eg: serological pipettes) .Also called direct pipettes.

(2) Non blowouthere graduations are not up to the tip. Also called indirect pipettes

Othere types of pipettes:2) Semiautomatic pipettes: These are available in fixed or variable volumes.

A) Fixed volumes: These aspirate a fixed volume only.

B) Variable volumes: Here we set a fixed volume, then aspirate the sample and deliver thevolume.

Automatedpipettes: It has a digital display where aspiration and delivery of specific volume bymeans of motar delivery syrings.

Pipetting techique1. Pipette must be held in vertical position during adjustment of liquid levelto caliberation mark and during delivery


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2. for colourless liquid lower meniseus to be read3. for opaque liquid such as blood top of meniseus to be read.

They are principally used for measurement of reagents.

2. LABORATORY REGULATIONS AND SAFETY

It is expected that you will take care in handling apparatus and reagents and you will co-operate in keeping equipment and chemicals in good condition. Cleanliness is essential in allbiochemical work. Breakage of glassware must be promptly reported.

Biochemistry is a laboratory science and such student should possess an observationnotebook to record data immediately (and NOT in scraps of paper etc.) and for rough work.He will record for each experiment he performs both the detail of the experimental proceduresand the results, discussion etc. This must be promptly submitted for correction to the teacherconcerned every following week.

Before attempting to carry out any experiment study the direction carefully, familiarize andplan your work in advance.


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TUTORIAL 1

WASTE DISPOSAL

Biomedical waste means any waste, which is genarated during diagnosis, treatment orimmunization of human beings or animals or in the research activities. Waste should besegregated at the point of generation and disposed in bags with correct coding.

BLACK BAGS: (municipal dump) paper, peels, wrappers, kitchen waste.

YELLOW BAGS: (for Incinerator) swabs and item contaminated with blood and body fluids,discarded medicines, gloves etc.

BLUE BAGS: (for shredder) syringes, needles and sharps are destroyed in needle destrroyeror discarded in sharp disposal unit containg 1% bleach.

All bags and containers must be labelled by using water proof ink or by self adhesive labels.

Exercise: RECORD IN YOUR LAB MANNUALS

CONCENTRATION AND INTREPRETATION OF RESULTS

------------------------------------------------------------------------------------------------------------Tube No. Blank Std1 Std2 Std3 Std4 Std5 Test------------------------------------------------------------------------------------------------------------Optical Density

Corrected OD------------------------------------------------------------------------------------------------------------

Calculate the concentration in the test by using graph or formula.

Interpretation of results; Record in Lab mannuals

Values obtained with a particular parameter is interpreted as: increased, decreased or withinnormal (reference) range.

Reference values: Values obtained from individuals who are in good health as judged byother clinical and laboratory parameters, after suitable standardization and statistical analysis,under definite laboratory conditions.

Normal (Reference) Range:Values within which 95% normal healthy persons fall. The cutoff values are set as mean reference value +/- N times standard deviation, of a normal healthypopulation; where N varies between 1, 2 and 3.

Causes/ Etiology of your findings

Based on the result give diagnosis/differential diagnosis

Any further investigation you would like to do to confirm diagnosis


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A. COLLECTION AND HANDLING OF SPECIMENS

The different body fluids that are used for biochemical investigations are given below:

Body Fluid InvestigationPerformed

Method of Collection

WHOLE BLOOD Blood gasesGlucoseUrea

Obtained by arterial or venipuncture;collected with anticoagulants likeheparin;

PLASMA EnzymesMetabolitesElectrolyte

Blood with anticoagulantscentrifuged at 2000 rpm, thesupernatant is plasma

SERUM EnzymesMetabolitesElectrolyte

Blood collected in plain glasscontainer, without any anticoagulant,centrifuged at 2000 rpm afterclotting, the supernatant is serum

URINE SugarProteinsBile saltsPigmentsBlood steroids

Directly passed into a glasscontainer, sometimes a catheter isintroduced in the bladder

CEREBRO- SPINALFLUID

SugarProteinChloride

Lumbar puncture from Subarachnoidspace

GASTRIC JUICE HClBlood

Aspiration by Ryles tube

SEROUS FLUIDS ProteinsSerous space.

Needle puncture to the(e.g. Pleural,Peritoneal)

SWEAT Chloride Soaked into a filter paper

Anticoagulants

Chemical agents that prevent coagulation are routinely used when whole blood or plasma isrequired. Some of the commonly used anticoagulants are:(1) Heparin (2) Salts of Ethylene diamine tetra acetic acid (EDTA)(3) Oxalates (4) Sodium Fluoride

Heparin: It may be considered to be a natural anticoagulant because it is already present inthe blood, but in concentrations less than that required to prevent clotting in freshly drawnblood. Heparin prevents coagulation by increasing the activity of antithrombin III, an inhibitorof thrombin. This anticoagulant is used in a concentration of 0.2 mg / ml of blood and since itsmolecular weight is large, it produces no change in erythrocyte volume.

Salts of Ethylene diamine tetracetic acid (EDTA): It is an anticoagulant which acts byvirtue of removing calcium ions by chelation. A concentration of 2 mg of the disodium salt/mlof blood is sufficient. Concentrations even greater than this produce no detectable change inerythrocyte volume.

Oxalates: Lithium, sodium and potassium oxalates act as anticoagulants by removing calciumions essential for blood coagulation. Potassium oxalate (K2C204.H20) is commonly used. 1-2mg of salt / ml of blood is required.


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The disadvantage of the use of oxalate is the alteration of concentrations of plasmacomponents. Shrinkage of erythrocytes results from a water shift from the erythrocytes toplasma. This shift increases with increasing anticoagulant concentration, and if used in thesame concentration on a weight basis, all anticoagulants will have this effect inverselyproportional to their molecular weight. Aside from the water shift there may be alteration oferythrocyte permeability, which may explain the varied and inconsistent effects of oxalatesand other salt anticoagulants on certain plasma constituents. Because of the difficulty, attimes, in obtaining satisfactory preparation of heparin commercially, Heller and Paulintroduced in 1934, a balanced oxalate mixture for use in hematocrit and sedimentation ratedeterminations. It consists of three parts by weight of ammonium oxalate, which causesswelling of the erythrocytes, balanced by two parts of potassium oxalate which causesshrinkage. NH4

+ & K+ oxalate mixture in the ratio of 3:2, and 2 mg / ml of blood is the requiredamount.

Sodium Fluoride: It is used when blood is collected for glucose estimations. In theerythrocytes (RBC), it specifically inhibits the enzyme enolase of the glycolytic pathway,preventing the consumption of glucose by the RBCs if blood is left standing at room

temperature. Though it has a weak anticoagulant action, it is usually combined with anotheranticoagulant such as potassium oxalate.

Preservation of samples

Alteration in the concentration of a constituent in a stored specimen can result from variousprocesses such as

Adsorption on to the glass container

Evaporation if the constituent is volatile

Water shift due to the addition of anticoagulants

Metabolic activities of the erythrocytes & leucocytes (accelerated by haemolysis)Inducing O2 consumption and CO2 production, hydrolysis, glycolysis and finallydegradation.

Microbial (fungal / bacterial) growthChanges in concentration of volatile substances such as O2 and CO2 are prevented or

hindered by collection and storage of samples under anaerobic conditions.The problem of microbial growth appears when the sample is stored for longer than one

day either at room or refrigerator temperature. This can be solved by four alternative coursesof action:

Collection and storage under sterile conditions

Freezing of the sample

Extreme alteration of pH

Addition of an antibacterial agent.

Lyophilized samples are stable with respect to many constituents for periods of at least aslong as ten years.

Samples can be stored at room temperature 18-37oC, refrigerator temperature (4oC) andfrozen state (-10oC or lower). Except a few, the lower the temperature, greater is the stability.Further, microbial growth is considerably less at refrigerator temperature than at roomtemperature and is completely inhibited in the frozen state. Even in the frozen state, however,some components of plasma deteriorate.

Chemical Preservatives:They can be classified into two groups:

Prevention of chemical changes such as glycolysis

Prevention of microbial growth.


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Combination of 10 mg Sodium fluoride + 1 mg Thymol / ml of blood. The presence ofThymol effectively controls microbial growth so that non-sterile specimens were stable for alldeterminations (except non-protein nitrogen) for at least two weeks.

Monochlorobenzene and monobromobenzene have also been coupled with fluoride andhave been claimed to be superior to thymol.

Antibiotics can be used to prevent bacterial growth 1 mg of streptomycin base / 10 ml ofblood has been used for preservation of blood for Haemoglobin and Urea determinations.The common preservatives for urine specimen are formaldehyde, thymol, toluene andchloroform. All these act primarily as antimicrobial agents.

B. NORMAL VALUES AND INTERPRETATION OF RESULTS

Interpretation of resultsValues obtained with a particular parameter is interpreted as: increased, decreased or withinnormal (reference) range.

Reference values: Values obtained from individuals who are in good health as judged byother clinical and laboratory parameters, after suitable standardization and statistical analysis,under definite laboratory conditions.Normal (Reference) Range:Values within which 95% normal healthy persons fall. The cutoff values are set as mean reference value +/- N times standard deviation, of a normal healthypopulation; where N varies between 1, 2 and 3.

C. QUALITY CONTROL

A major role of the clinical laboratory is the measurement of substances in body fluids ortissues for the purpose of diagnosis, treatment or prevention of disease, and for greaterunderstanding of the disease process. To fulfill these aims the data generated has to be

reliable for which strict quality control has to be maintained. Quality control is defined briefly asthe study of those sources of variation, which are the responsibility of the laboratory, and theprocedures used to recognize and minimize them.

Accuracy has to do with how close the mean of a sufficiently large number of determinationson a sample is to the actual amount of substance present and is dependent on themethodology used.Precision refers to the extent to which repeated determination on an individual specimen varyusing a particular technique and is dependent on how rigorously the methodology is followed.

D. SAFETY

Safety is each persons responsibility even in a small clinical laboratory. Even then everyclinical laboratory must have a formal safety program. It is a good practice to assign a specificperson the title of safety officer with the duties of administering the safety program andkeeping it current.

It should be ensured that laboratory environment meets the accepted safety standards.Which should include, but not be limited to attention to such items as:

1. Proper labelling of chemicals2. Types and location of fire extinguishers3. Hoods that are in good working condition4. Proper working and grounding of electrical equipment

5. Providing means for proper handling and disposal of bio-hazardous materials includingall patient specimen.


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Safety measures to avoid hazardsTo prevent chemical, electrical and biological hazards following precautions should befollowed:

1. Proper storage and use of chemicals is necessary to avoid chemical hazards. Thusknowledge of the properties of chemicals in use and of proper handling proceduresgreatly reduces dangerous situations.

2. All the electrical equipment should be grounded using three-point plugs and use of theextension cord should be prohibited.

3. Every laboratory should have the necessary equipment to put out a fire in thelaboratory, as well as to put out a fire on the clothing of an individual. Easy access tosafety showers should be made.

4. Biological Hazards can be avoided by following precautions called universalprecautions.

All specimens should be treated as if they are potentially infectious.

i. Avoid performing mouth pipetting and never blow out pipettes that contain potentiallyinfectious material, for example serum.

ii. Do not mix potentially infectious material by bubbling air through the liquid, whichleads to aerosol formation.

iii. Barrier protection such as gloves, mask and protective eyewear and gowns must beavailable and used when drawing blood from a patient. This includes removal andhandling of all patient specimens. Disposable, non-sterile latex or vinyl glovesprovide adequate protection.

iv. Wash hands whenever gloves are changed.v. Facial barrier protection should be used if there is a significant potential for the

spattering of the blood or body fluid.vi. Avoid re-using syringes and dispose off needles in rigid containers without touching

these, using one-handed technique.vii. Dispose off all sharp objects appropriately.viii. Wear protective clothing, which serves as an effective barrier against potentially

infective materials. When leaving the laboratory, protective clothing should beremoved.

ix. Make a habit of keeping your hands away from your mouth, nose, eye and any othermucous membranes. This will reduce the possibility of infection.

x. Minimize spills and spatters.xi. Decontaminate all surfaces and reusable devices after use with appropriate

registered hospital disinfectant.xii. No warning labels are to be used on patient specimens.

xiii. Before centrifuging tubes, inspect them for cracks. Inspect the inside of caps for signsof erosion or adhering matter. Be sure that rubber cushions are free from all bits ofglass.

xiv. Never leave a discarded tube or infected material unattended or unlabelled.xv. Periodically clean out freezer and dry ice chests to remove broken ampoules and

tubes of biological samples. Use rubber gloves and respiratory protection during thiscleaning.


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3. REFERENCE INTERVAL

Normal serum values of some common constituents both in conventional and SI units aregiven below:

Blood Constituents Conventional Units SI Units

1. Protein (total) 6.7-8.6 g/dl 6.0-7.8 g/dl2. Albumin 4.0-5.0 g/dl 3.5-5.0 g/dl

3. Bilirubin (conjugated) 0.1-0.4 mg/dl upto 3.4mol/dl4. Bilirubin (total) 0.3-1.4mg/dl upto 0.2 mmol/l

5. Urea 20.0-35.0 mg/dl 3.0-36 mmol/l6. Creatinine 0.2-2.0 mg/dl 17.0-117.0 mol/l

7. Uric acid (male) 3.1-7.0 mg/dl 0.18-0.42 mmol/l

(female) 2.5-5.6 mg/dl 0.15-0.37 mmol/l8. Cholesterol (total) 150.0-250.0 mg/dl 4.0-6.0. mmol/l9. HDL-Cholesterol 30.0-66.0 mg/dl 4.0-6.0 mmol/l10. Triglycerides 30-200 mg/dl 0.4-1.75 g/l

11. Sodium 136.0-146.0 mEq/l 136.0-142.0 nnol/l12. Potassium 3.5-5.0 mEq/l 3.0-4.8 mmol/l

13. Glucose (fasting) 75.0-100.0 mg/dl 4.0-5.5 mmol/l

14. Calcium (total) 8.7-10.2 mg/dl 2.0-2.5 mmol/l15. Phosphorous 2.5-4.3 mg/dl 0.8-1.4 mmol/l

16. ALT (SGPT) - 7-44 IU/L17. AST (SGOT) - 12-38 IU/L

18. Phosphatase (alkaline) 4.0-13.0 KA Units/dl 33 - 96 U/l19. Phosphatase (acid) 1.0-4.0 KA Units/dl upto 7.4 U/l

20. Amylase 60.0-150.0 Somogyi Units./dl 20-96U/l

******************************************


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Experiment 2

Experiment: HYDROGEN ION CONCENTRATION & PREPARATION OF

BUFFERS

Principle;pH & ITS SIGNIFICANCE

All biochemical reactions in vivo and in vitro are greatly influenced by the hydrogen ionconcentration of the surrounding medium. The convenient way of expressing hydrogen ionconcentrations is by the term pH which is defined as the negative logarithm of hydrogenion concentration. Enzymes are optimally active at a particular H+ ion concentration. Water(H2O) is dissociated to H

+ and OH- ions.

pH = - Log [H+]

E.g. pH of a solution having hydrogen ion concentration 0.00000001 (10-7) is 7.

The pH values of some of the important biological fluids are as follow.

Fluid pH fluid pH

Pancreatic juice 8.8 Saliva and human milk 6.7Bile 7.6 Urine (Mean) 6.0

Blood (at 38 C) 7.35 Gastric Juice 1.77

Buffer: The H+ ion concentration varies very little in any biochemical fluid or environment. Thisvariation is arrested by some bases or acids which respectively absorb or donate H+ ionsdepending on the situation. This phenomenon is called buffering.A bufferhas the capacity ofresisting the changes in pH (hydrogen ion concentration) of a solution after the addition ofsmall amounts of an acid or an alkali. All weak acids or bases, in the presence of their saltswith strong base or strong acid respectively form buffer systems, e.g. carbonicacid/bicarbonate, dihydrogen phosphate/ monohydrogen phosphate, proteins/proteinate.

The capacity of the buffer decreases as the ratio deviates from 1. In general buffers should beused at a pH 1 from the pK. If the ratio is beyond 50:1 or 1:50, the system is considered to

have lost its buffering capacity. The above mentioned buffers are present in different bodyfluids which help in the regulation of pH. Buffers are also used in clinical chemistry forenzymatic estimation.

In aqueous solutions the pH ranges from 0 to 14. Molar concentration of H+ or OH- ion in purewater is 1 x I0-7mol/L. The ionic product is 1x10-14 mol2/L2.

With water, at neutral point:

[H+] = [OH-] = 10-7 mol/L.

So pH at neutral point is, -log [10-7mol/L], So neutral pH= 7

pH more then 7 indicates that the solution is alkaline

pH less than 7 is acidic.pH 0 would be given by 1M HCL.


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pH 14 would be given by a 1M NaOH.

Many biochemical substances possess functional groups that are weak acids or weak basese.g, -COOH, -NH2, phosphates present on proteins, nucleic acids, coenzymes, intermediarymetabolites. Their dissociation behaviour influences pH and the structure and function of the

entire molecule.

The pH of a buffer solution can be calculated by Handerson Hasselbalch equation.(Salt)

pH = pKa + Log ..(Acid)

where pK = negative logarithm of the dissociation constant K for acid.

Determination of pH

A. By using indicator:

Indicators are compounds which change in colour with changes in the pH of the solution towhich they are added. They are weak organic acids of weak bases. In unionized forms, theindicators show one colour while in their ionized forms (anions or cations) they have adifferent colour. The colour of a solution in presence of an indicator depends upon the relativeproportions of ionized and the unionized forms of the indicator which in turn depends on thehydrogen ion concentration. For such indicator there is a definite pH range in which it ispresent as mixture of the ionized and unionized forms. In this specific range variations in pH ofthe solution will bring about visible change in the color of indicator. It is necessary that theeffective pH range of the indicator includes the pH of the unknown sample.Do not let the pHpaper dry before comparing with indicator.

B. By using pH paper:The indicator paper given to you is accompanied by a color chart which shows different colorswhich the indicator exhibits at different pH values. Take a strip of indicator paper (Wide range)and moisten it with small drop of the solution whose ph is to be determined. Remove theexcess fluid adhering to the indicator paper and compare with the color chart of the indicatorand thus determine the pH of the solution. Now take a narrow range paper of suitable rangeand repeat the procedure to get the pH.

C. By using universal indicator solution:Place 5.0 ml of the solution in a test tube and 0.1 ml of the universal indicator Mix well andfind out the pH by referring to the color chart of the indicator. Determine the pH of biological

fluids viz. urine, saliva. Record your observations.

B. By using pH meter:

The most accurate method for the routine measurement of pH is the pH meter in which achange in [H+] is measured as a change in electrical potential.

If a metal rod is placed in a solution of its salts, it acquires potential. If two dissimilar metalsare dipped into the solutions of their own salts, the difference in potential can be measured orcalculated from the two separate potentials. A standard electrode is thus required againstwhich the potential of all other electrodes can be compared. This is the standard hydrogenelectrode, consisting of a platinum rod dipped in an aqueous solution with a given H + activityin which Hydrogen gas is bubbled continuously at 1 atmosphere pressure. But as this is toocumbersome to be used as a reference electrode for routine use, othersecondary referenceelectrodes of known potential in relation to the standard hydrogen electrodes are used.


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The most commonly used secondary reference electrode is the calomel electrode consistingof mercury-mercurous chloride in contact with a saturated solution of KCl. Its potential is pHindependent.

In the pH meter the most commonly used pH dependent unit is the glass electrode. Certaintypes of borosilicate glass are permeable to H+ but not to other cations and anions. Therefore,

if a thin glass membrane separates two solutions of different pH, a potential difference isgenerated across the membrane, the magnitude of which is given by the equation

log

Where E = potential, R = gas constant, T = absolute temperature

F = Faraday constant, [H+]i = concentration on the inside which is fixed (0.1N HCl),and [H+]o = concentration on the outside (test sample).

The voltage measured by such a system is primarily the difference between that of the glassand the reference electrodes (other fixed potentials in the circuit also contribute) and it islinearly related to the pH of the test solution, i.e. [H+]o. The system therefore consists of theglass electrode in contact with the solution to be measured, the calomel reference electrode, aKCl bridge (the KCl should flow slowly into the sample) and the measuring device (meter).These are designed so that pH 7 gives a zero potential. High resistances are used so thatvery little current is drawn from the circuit (A large current flow could change the ionconcentration).

Certain precautions have to be observed while using a pH meter. The glass electrode isfragile and must be handled with care. It must not be left to dry. It is usually kept dipped in 3MKCl. The temperature compensation dial must be set before it is calibrated as potentialproduced is dependent on temperature. The meter must be calibrated first with a standardbuffer pH 7, and then with a standard of pH 4 (if the test sample is expected to be acidic ) orwith a standard of pH 9 or 10 ( if test is expected to be basic).

PH METER

Setting of instrument:

1) Keep the SELECTOR in zero position.


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2) Switch on the instrument and wait for 10 minutes.3) Connect the cleaned electrode the proper terminals.4) Adjust ZERO control to the pointer read 7 pH.

pH measurement1. Dip the electrode in a buffer of known pH value (usually 4 or 7) and set the TEMP

COMPENSATE to the temperature of the Buffer.2. Keep the SELECTOR in the proper pH range and adjust SET BUFFER to get buffer

pH value.3. Keep the SELECTOR in zero position, clean the electrodes and keep them in the

sample.4. Keep the SELECTOR in the proper pH range and read the value.5. The electrode should be carefully washed after each pH determination.


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PRACTICAL EXERCISE 2:Experiment: Preparation of Buffers

Procedure: Prepare the following buffer solution:

a) In acid range: (Acetate Buffer)Mix 9.2 ml 0.1 N acetic Acid +0.8 ml of 0.1 N. Sodium acetate and check the pH.

b) In neutral range : (phosphate buffer)Mix 6 ml of 0.1 M Na2HPO4 + 4ml of 0.1 KH2 PO4

c) In alkaline range: (carbonate buffer) Mix 1.4 ml of 0.2 M sodium carbonate, 1.1 ml 0.2M Sodium bicarbonate and 7.5 ml of water to get pH of 10. Determine the pH of thesolution by one or more of the methods described below.

Observation :

pH with wide rangepH paper

pH with narrow rangepH paper

pH with pH meter

Acetate buffer

Phosphate buffer

Carbonate buffer


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Experiment 3

PROPERTIES OF CARBOHYDRATES

Theory: Carbohydrates are wide spread in nature both in plant and animal kingdom and are

classified ad monosaccharides (glucose and fructose) disaccharides (lactose, maltose andsucrose) polysaccharides (starch and glycogen)

1. Molish Test: It is a general test for carbhohydrate. A positive Molish test indicates thepresence of carbohydrate in a test solution. Carbohydrates undergo dehydration when treatedwith conc. H2SO4 to form furfural derivatives which on condensation with -nephthol form aviolet coloured complex. All carbohydrates except amino sugars give this text.

Procedure:To 2ml of sugar solution add 2drops of 1% ethanolic -nephthol and mix. Add 2mlof conc. H2SO4 by the side of the tube slowly. Note the colour of the ring formaton at thejunction of two solution.

2.Benedicts Test: Reducing sugars under alkaline conditions tautomerise and formenediols. Enediols are powerful reducing agents. They can reduce cupric ions to cuprous formwhich is the basis of Benedicts reaction. The cupric hydroxide formed is not easily soluble. Inorder to keep the hydroxide in solution, a metal chelator like citrate is included in the solution.Benedicts solution contains milder alkali Na2CO3 Cuprous hydroxide, during the process ofheating is converted to red cuprous oxide.

Procedure: To 5 ml of benedicts qualitative reagent (cupric sulphate, sod citrate, sods.Carbonate) add 8 drop of sugar solution. Heat it for 2 minutes.

Depending upon the concentration of reducing sugar following coloured precipitates may be

obtained. Green precipitate (=upto 0.5%), yellow precipitate (=0.5% to1%), orange precipitate(=1%to1.5%), brick red precipitate (=upto 2% or above).

3. Barfoeds Test: This test is used to distinguish reducing monosaccharide from a reducingdisaccharide by controlling pH and time of heating. This is also a copper reduction test inacidic conditions. Aldoses and ketoses can reduce cupric ions even in disaccharide is slow.However, if heating is prolonged disaccharide may by hydrolysed by the acid and the resultingmonosaccharides will give the test positive.

Procedure: To 2 ml of dilute carbohydrate solution add 2 ml of Barfoeds reagent (cupricacctate in acetic acid). Mix and heat in a boiling water bath for 3 minutes, cool. An appearanceof brick red precipitate of cuprous oxide indicates the presence of monosaccharides.

4. Seliwanoffs Test: This test is positive for ketohexoses only and so is answered byfructose, sucrose and other fructose containing carbohydrates. Ketohexoses on treatment withhydrochloric acid from 5 hydroxymethlyl furfural which on condensation with resorcinol gives achery nred coloured complex. It can be used to distinguish between fructose and glucose.Sucrose will also give Seliwanoffs test positive because the acidity of reagent is sufficientenough to hydrolyse sucrose to glucose and fructose but benedicts test will be negative.

Procedure: To 1 ml of sugar solution add 3 ml of Seliwanoffs reagent. (Resoreinol in dil HCI).Heat to boil (do not boil the solution for more than 1 minute). Cool it. An appearance of cherryred colour indicates the presence of fructose / sucrose.

5. Iodine Test: It is a test for polysachharides which adsorb iodine and form colouredcomplex.Starch gives reddish brown colour. On heating, starch loses blue colour because


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starch iodine complex dissociate. On cooling, colour again appears due to re-formation ofcomplex.

Procedure: To 1ml of sugar solution, add a drop of dil. HCL to acdifiy the solution. Add a fewdrops of iodine solution. Mis and observe the colour. Gently warm the solution and then coolit. Note the change.

Results:

Tests of Carbohydrate: Carry out the above tests with given solution of carbohydrate andrecord your observations in the tabular form.

Carbohydrate Test Observation Inference

A). Molish Test

B). Benedicts Test

C). Barfoeds Test

D). Seliwanoffs Test

E). Iodine Test

Carbohydrate Test Observation Inference

A). Molish Test

B). Benedicts Test

C). Barfoeds Test

D). Saliwanoffs Test

E). Iodine Test

Carbohydrate:

Test Observation Inference

A). Molish Test

B). Benedicts Test

C). Barfoeds Test

D). Saliwanoffs Test

E). Iodine Test


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Experiment4

Experiment: COLOUR TESTS FOR PROTEINS -1

Principle:

1. Colour reactions of Proteins

(I) Biuret test: This test is given by substances containing two or more peptide linkages in themolecule. Proteins respond positively since there are pairs of CO-NH groups in the molecule.This is a general test for the detection of proteins.

When urea is heated to 180oC, it forms biuret, which in the presence of strong alkalireacts with dilute solutions of copper sulphate to form a violet coloured complex. Proteins andpeptides give this reaction. Presence of at least two peptide bonds is essential for the

reaction. Proteins give violet or purple colour while proteoses and peptones give light pinkcolour. Histidine gives a positive Biuret reaction. Two or more CO-NH groups joined directlyor through a carbon or nitrogen atom gives this reaction.

Absorption maximum of the colored complex is 540 nm. Since the method is based onreaction with peptide bonds, it is an absolute one. The main disadvantage is its lack ofsensitivity. It cannot be used to estimate protein less than 1 mg/ml. Amino acids anddipeptides do not give this reaction.

Procedure: Take 2 to 3 ml of protein solution in a test tube and add an equal volume of 10%sodium hydroxide. Mix thoroughly; add a few drops of 0.5% copper sulphate solution. Apurplish violet colour is obtained.

(II) Ninhydrin reaction: This test is given by all compound having free -amino groups e.g.,

peptide, protein and free amino acids ( except proline and hydroxy proline which are notamino acids but imino acids). Ninhydrins osidases an- amino acid to an aldehyde liberatingNH3 and CO2 and is itself reduced to hydrindantin which then reacts with ammonia andanother molecule of ninhydrin to form a purple coloured complex called Ruhemanns purple.Failure of purple colour development is indication of absence of -amino acid of group.

Procedure: To 1ml protein solution and add 1ml of 0.02% freshly prepared ninhydrin solution.mix and boil.

(III) Xanthoproteic reaction: Compounds containing benzenoid ring (e.g. Phenyl Group,C6H5) react with nitric acid to form yellow nitro derivatives which turn deeply coloured onaddition of alkali. Only tyrosine and tryptophan respond to the test. Phenylalanine does notrespond to the test since it is difficult to nitrate it under the above conditions.

Procedure: To 2 to 3 ml of protein solution add 1 ml of concentrated nitric acid and heat.White precipitate is obtained which turns yellow and becomes orange on adding 40% sodiumhydroxide (excess).

(IV) Millon Test: This reaction is given by substances containing hydroxyl phenyl groups C6H4OH e.g. tyrosine.

Procedure: To 5 ml of a dilute solution of protein, add 1 ml of 15% solution of mercuricsulphate in 6N H2SO4. Place the tube in boiling water bath for 10 minutes. Cool the contentsin water for 5 to 10 minutes and add 1 ml of 1% sodium nitrite. A deep red colour indicates apositive test. Tyrosine answers this test.


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(V) Sakaguchis arginine reaction: Guanido group of arginine react with alpha napthol andalkali hypobromite to give a red coloured complex.

Procedure: To 5 ml of protein solution, add 5 drops of 10% sodium hydroxide and 4 drops ofMolischs reagent. Then add 10 drops of bromine water. A carmine red colour is obtained.Compare with blank.

(VI) Hopkin-Cole reaction for tryptophan: This reaction is due to the presence of indole ringof the amino acid tryptophan in the protein molecule. Several aldehydes(e.g. formaldehyde)react with the oxidized product of the indole nucleus of trypotophan to give violet colouredcomplexes (Sulphuric acid with mercuric sulphate is used as oxidizing agent in this reaction).

Procedure: To 1 ml of protein solution, add 1 drop of 1:500 formalin and then I drop of 10%mercuric sulphate in 10% suphuric acid. Mix well and gently add 3 ml of concentratedsulphuric acid along the sides of the test tube. Tap gently at the junction of the two layers. Aviolet ring develops at the junction.

(VII) Sulphur test: Cysteine and Cystine amino acids contain sulphur and proteinscontaining these amino acids make precipitate with lead acetate in alkaline medium.

To 3 ml of the protein solution, add an equal volume of 40% sodium hydroxide and heat for 2minutes. Cool, and acidify with dilute HCI. Then add 1 ml of 5% lead acetate solution andwarm. A grey or black precipitate indicates a positive test. Cysteine and cystine give positivetest while methionine does not.

Perform the colour reaction and precipitation with the given solution of protein and record yourobservation.

Results: Date:

Test Observation Inference

I) Biuret test

II) Ninhydrin reaction

III) Xanthoproteic test

IV) Millon test

V) Sakaguchis test

VI) Hopkin-Cole test

VII) Precipitation by heavy metals

VIII) Precipitation by TCA


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Experiment 5

Experiment: PRECIPITATION TEST FOR PROTEINS II

Principle:

1. Precipitation of Protein: Most of the proteins are lyophilic and hence can be precipitatedby dehydration and neutralization of the electrical charges, which they carry to bring them totheir iso-electric point. Proteins like casein and metaprotein practically being lyophobiccolloids, require only neutralization of their charges for their precipitation.

The proteins can be precipitated from their solutions by salts of heavy metals (e.g. mercuricchloride, silver nitrate lead acetate, Zinc sulphate etc.), certain acids some of which are calledalkaloidal reagents (e.g. picric acid, phosphotungstic acid, trichloroacetic acid, salicylic acid),concentrated solutions of salts such as ammonium sulphate, sodium sulphate, etc. and byethyl and methyl alcohol.

(I) Precipitation by heavy metals: Proteins are precipitated as metal proteinate.

To 3 ml of the protein solution, add 5% zinc sulphate solution drop by drop till there is amaximum precipitation.

(II) Precipitation by alkaloidal reagents: To 3 ml of the protein solution, add a few drops of10% trichloroacetic acid. A white precipitate is obtained.

II. Detection and separation of albumin and globulin

Albumins: Some of the native proteins present in serum, milk, egg white, etc., belong to this

class. They are insoluble in water but soluble in weak salt solution. They are also heatcoagulable and can be precipitated by full saturation with ammonium sulphate.

Globulins: These are also native proteins present in serum, milk, egg white, pulses etc. Theyare insoluble in water but soluble in weak salt solutions. They are also heat coagulable andcan be precipitated even by half saturation with ammonium sulphate, being less hydrated thanthe albumins.

(I) Heat Coagulation Test: Take 5 ml of the protein solution in a serum tube. If the solution isalkaline, make it slightly acidic by adding 1-2 drops of 2% acetic acid and heat upper portionof the liquid. Coagulation takes place. The lower portion serves as control.

(II) Hellers Test: Take 2 ml of concentrated nitric acid in a test tube and gently add 2 ml ofprotein solution along the sides, a white colored ring is seen due to the formation of acidmetaprotein. This is the most sensitive test for albumin and globulin.

(III) Separation of albumin and globulin: Fractional precipitation with ammonium sulphate.To 5 ml of egg-white solution, add an equal volume of saturated ammonium sulphate solution(half saturation). Globulin is precipitated. Filter. To the filtrate, add solid ammonium sulphatetill full saturation occurs. Albumin is now precipitated.


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Results: Date

Precipitation Tests Observation Inference

1.Precipitation of proteins:

(A) By zinc sulphate

(B) By TCA

2. Detection of albumin

(A) Heat coagulation test

(B) Hellers test

3. Seperation of Albumin & Globulin


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SPOTING, DEMONSTRATIONS, TUTORIALS

Experiment: DEMONSTRATION OF CHROMATOGRAPHY

Principle: This technique was originally used to separate chlorophyll from plant extracts onsilica, hence the name chromatography, which means separation of colored compounds. It isthe name given to any technique in which the members of a group of similar substances areseparated by a continuous redistribution between two phases. One is the stationary phase,which may be solid, liquid, gel or solid/liquid mixture which is immobilised. The second mobilephase may be liquid or gaseous and flows over or through the stationary phase. The choice ofstationary or mobile phases is made so that the compounds to be separated have differentdistribution coefficients.

The movement of any substance being chromatographed is the outcome of:

a. Forces resulting from its interaction with the mobile phase which tend to pull it alongwith the mobile phase

b. Forces resulting from its attraction to the stationary phase, which tend to retard itsmovement.

Since the forces are different for different substances chromatography will be able to separateout the components of a mixture of substances.

Based on the properties of the stationary phase and the acting forces, several different typesof chromatographic procedures have been described.

1. Adsorption chromatography: Adsorption equilibrium between a stationary solid and amobile liquid phase. This is used to separate the non ionic, water insoluble compounds

such as vitamins, triglycerides, drugs etc.2. Partition chromatography: Partition equilibrium between a stationary liquid and a mobile

liquid phase. This is used to analyze drugs, insecticides, pestisides,amino acids .

3. Gas-Liquid chromatography: Partition equilibrium between a stationary liquid and amobile gaseous phase. The mobile phase is generally an inert gas used for steroidseparation.

4. Ion-exchange chromatography: Ion exchange equilibrium between an ion-exchangestationary resin phase and a mobile electrolyte phase. So amino acids proteins whichhave ionized groups can be separated on the basis of this.

5. Gel permeation chromatography: Equilibrium between a liquid phase inside and outsidea porous molecular sieve depending on molecular size - preparative procedure.

6. Affinity chromatography: Equilibrium between a macromolecule and a small molecule(Ligand) for which it has high affinity - monoclonal antibody purification.

A.PAPER CHROMATOGAPHY

Paper chromatography is very simple and convenient method of separating a mixture of anumber of constituents of similar nature but having different physical properties. Theseparation is carried out on pieces of filter paper and therefore, the technique is referred to aspaper chromatography.

In case of paper chromatography, a small quantity (2-25 ml) of the mixture to beanalyzed is placed as a spot on a piece of filter paper and the solvent is allowed to travel upthe paper (which is called ascending chromatography) or down the paper (in which case it is


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called descending chromatography). In this process, the mixture is separated into its individualcomponents, which occupy different positions on the chromatogram depending on thedistance traveled by each component. If the constituents are colored, the separation isimmediately apparent. But in most of the cases, the chromatogram will have to be sprayedwith or dipped in a reagent, which reacts with the compounds to be separated, to give color.Amino acids can be sprayed with Ninhydrin. The chromatogram on warming shows purplespots.

In principle, the paper serves as an inert support for an aqueous stationary phase(which saturates the paper) over which flow a water immiscible organic solvent (the mobilephase). In such a system, the solutes undergo a continuous redistribution between the twophases. The position to which, a given solute moves up the paper, is therefore, largelydependent upon its partition co-efficient.

Under standard conditions of solvent composition, temperature and type of paper, thedistance traveled by each individual component of the mixture applied to the paper, ischaracteristic of that component, and is expressed by R f (relative fraction)

Distance traveled by a solute (center of spot)Rf = ---------------------------------------------------------------Distance traveled by solvent (front)

In practice, known standards are run simultaneously on adjacent lanes so that by comparingthe Rf values, the unknown spots can be identified. Difficulty of identification arises if the twocomponents have the same Rf value. This can be solved by doing a two dimensionalchromatography (separating along one direction and then perpendicularly).


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Experiment: IDENTIFICATION OF AMINO ACIDS BY PAPER CHROMATOGRAPHY

MATERIALS REQUIRED:

1) Whatman filter paper (no. 1).2) Standard amino acid solutions: (A) Cysteine (B) Glutamic acid (C) Leucine

(D) A mixture of unknown amino acids.3) Development solvent: N-butanol: Acetic acid: Water = 4:1:2.4) Spraying reagent: 0.1% Ninhydrin in acetone or N-Butanol.

PROCEDURE:

Take a piece of Whatman filter paper (No. 1; size about 5 x 15cm) and draw ahorizontal line (using a pencil) about 2 cms away from the edge of the paper. Do not touchthe paper with bare hands or make it dirty or wet. Keep the paper on a piece of ordinary filterpaper or clean brown paper. Put 4 pencil marks along the line, about 1 cms, apart from eachother.

Apply one tiny drop (1-2l) of each of the amino acid solutions (at each point) with the

help of a capillary tube at the first three points (only one amino acid on one point). Then applythe solution containing mixture of amino acids at the fourth point. Let the spots dry. Put thepaper (chromatogram) in the chromatography chamber, which is already equilibrated withthe solvent. Then place the solvent (mixture of n-butanol, acetic acid and water) in thechromatography chamber. Leave the chamber closed and allow the chromatogram to run,taking care to see that the solvent does not run off the paper. Take the paper chromatogramout of the chamber and allow it to dry in air, after marking with a pencil, the position uptowhich the solvent front has traveled. Spray the dried paper with ninhydrin solution and let it dryfor few minutes. Keep the paper in the oven at 45oC for 20 minutes. Each amino acid will givea purple color. Spot mark each spot with pencil and compare the Rfvalues of standard aminoacids and thereby identify the amino acids in the given mixture.

INTERPRETATION:

Paper chromatography is particularly well suited for identification of reducing sugars oramino acids excreted in urine. Paper chromatography also permits excretory products such ascatecholamines and tryptophan metabolites to be separated from interfering substances sothat they can be identified by specific color reactions.

The renal threshold for amino acid is lowered in pregnancy, increasing the number ofamino acids appearing in the chromatogram. Histidine is frequently seen; phenylalanine,lysine and tyrosine may also occur. Newborn babies have greater excretion of amino acidsthan adults and premature babies show marked renal aminoaciduria. The adult pattern isreached by six months.

The pathological aminoacidurias have been classified into renal aminoaciduria andoverflow aminoaciduria resulting from increased plasma levels. Typical of renalaminoacidurias are cystinuria in which a congenital defect leads to excretion of increasedamount of the basic amino acids, cystine, lysine, ornithine and arginine, Fanconis syndrome,in which nearly all the common amino acids are excreted in excess, and the aminoaciduriadue to tubule cell poisons such as heavy metals or Lysol.

In overflow aminoaciduria, the plasma amino acid levels are high or normal dependingon their thresholds, without primary disease. These may be acquired, secondary to otherconditions. In cirrhosis and other chronic liver diseases, generalized aminoaciduria occurs. Inmalignant disease of bone and bone growth, hydroxy prolinuria occurs. They may also becaused by an inborn error of metabolism, congenital disorders due to enzyme defects e.g.

phenylketonuria, alkaptonuria, maple syrup urine disease, etc.


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RESULTS: OBSERVATION, CALCULATION AND INTERPRETATION OF RESULTS

Paste here the chromatography paper


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B.THIN LAYER CHROMATOGRAPHY (TLC)

Principle: The principle involved in the separation is similar to paper chromatography but TLChas many advantages over it. The basic technique involves use of thin films of adsorbingmaterial spread evenly over the surface of a glass or metal plate. After application of the

sample, the plate is immersed in a suitable solvent, run and developed by ascending /descending flow as in paper chromatography.

So the basic procedure of TLC involves the following 4 steps:

(1) Preparation of appropriate thin layers.(2) Spotting of the substance.(3) Proper developing system.(4) Detection or visualization and identification.

Advantages of TLC: Greater speed, better resolution, greater sensitivity and separation ofhydrophobic substances viz. lipids, which are difficult to separate on paper. Compared to thevarious advantages, a few minor disadvantages have been found with TLC (1) Difficulty in

recording and preserving (2) Greater cost of plates compared to paper.

Experiment: SEPARATION OF SERUM LIPIDS BY TLC:

PROCEDURE:

(1) Glass plates are coated uniformly with a thin layer of absorbent (silcagel-G). The platesare generally dried and activated for a short time at 105 to 120oC. The sample is appliedas a single spot with a micropipette in the form of a solution in a non-polar solvent.Selection of proper solvent depends on the type of separation desired.

(2) After spotting the sample, the plate is placed vertically in the chromatographic tank, which

has been saturated with solvent mixture. Two solvent systems are employed solventsystem 1: n-Hexane; diethyl: ether: glacial acetic acid (60:4:1 vol/vol). Solvent 2: n-Hexane: Diethyl ether : Glacial acetic acid (90:10:1 vol/vol). The plates are first developed upto 7cm height in solvent system 1, air-dried and subsequently developed upto 15 cm insolvent system 2.

(3) After the run for a specific time (usually 2-4 hrs.), the plate is taken out, air-dried and spotsoutlined by exposure to iodine vapor.

(4) Draw a diagram of the pattern obtained. Usually 6 spots can be visualized for phospho-lipids, diglyceride, free cholesterol, free fatty acid, triglyceride and esterified cholesterol.


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C.GEL FILTRATION CHROMATOGRAPHY

Principle: Gel Filtration Chromatography, also known as size exclusion chromatography or

gel permeation chromatography, separates molecules on the basis of their differences inmolecular size and exploits the molecular sieve properties of a variety of porous materials.The most commonly used of such materials is a group of polymeric organic compounds,which possess a three dimensional network of pores, that confers gel properties upon them.e.g. Dextran, Agarose, Polyacrylamide.

A column of gel particles or porous glass granules is in equilibrium with a suitablemobile phase for the analytes to be separated. Large analytes that are completely excludedfrom the pores will pass through the interstitial spaces between the gel particles and willappear in the eluate first. Smaller analytes are distributed between the mobile phase insideand outside the gel particles and will pass at a slower rate, hence appearing last in the eluate.

Thus, the distribution of an analyte in a column of a gel is determined solely by thetotal volume of the mobile phase, both inside and outside the gel particles, that is available to

it.

`

Applications:

(i) Purification- The main application of gel filtration chromatography is in thepurification of biological macromolecules by facilitating their separation fromlarger and smaller molecules.

(ii) Relative molecular mass determination - Construction of a calibration curvewith proteins of a similar shape and known molecular mass, helps in

estimating the mass of other unknown proteins.

(iii) Solution concentration- Solution of high molecular mass can beconcentrated by the addition of dry sephadex beads.

(iv) Desalting Solutions of high molecular mass can be desalted using acolumn of sephadex. e.g. removal of phenol from nucleic acid preparationsand ammonium sulfate from protein preparation.

Procedure: In this experiment, a mixture of three different molecules ranging in molecular

size form 376 Da to 2000 KDa, is separated.

(i) The column is fixed vertically to a stand.(ii) The column is equilibrated with 4 ml of gel filtration buffer and thebuffer is allowed to drain completely.(iii) 0.2 ml of the sample is loaded on the column and allowed to sink in it.(iv) 0.2 ml of buffer is then added and allowed to flow out.(v) Buffer is then added to the column continuously to allow all the coloured

biomolecules to elute out.(vi) The coloured fractions are collected in different tubes.


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Observation and Results:

Interpretation:(i) The colored component is blue dextran, which has a molecular weight of

2000KDa. These are very large molecules that exit fast from the column and are collectedas the first fraction.

(ii) The brownish red colored component is hemoglobin and has a molecularweight of 64500 Da. They are of intermediate size and require an average time to exit.

(iii) The pink colored component is Vitamin B12 , having a molecular weight of376Da. These are very small molecules that permeate the pores of the beads in the

column and are retained for a longer time. They therefore, take a long time to exit thecolumn and are collected as the third fraction.


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D.AFFINITY CHROMATOGRAPHY

Principle: It exploits the unique property of extremely specific biological interaction toachieve separation and purification. Theoretically, it is capable of going absolute purification,

even from complex mixture in a single process. The performance of affinity chromatographyis determine by comparing the specific activity of protein before and after purification.

The material to be isolated is capable of binding reversibly to a specific ligand (exampleconcavlin A for HRP, Heparin for lipoproteins) i.e. attached to an insoluble matrix.

M + L ML Complex

Macromolecule Ligand

Applications

1. Purification of enzymes example Horse Radish Peroxidase2. Purification of Nucleotides, Nucleic acids example mRNA is routinely isolated by

selective hybridization on poly (U) sepharose by exploiting its poly (A) tail3. Isolation of membrane receptors4. Purification of Immunoglobins5. Separtion of whole cells, cell fragments

Procedure

1. Set up conventional chromatographic column2. A complex mixture containing the specific compound to be purified was added to the

immobilized ligand3. A compound will bind to the ligand, all other compound was washed away4. The require compound was recovered by the displacement from the ligand5. Estimation of proteins was done according to the any preferable method6. Calculate specific activity

Specific activity is defined as number of units of enzyme activity present per milligramof the protein.

= Enzyme Activity mol/ min/mgProtein concentration

7. Calculate enzyme activityOne unit of enzyme activity is that amount of enzyme which transforms 1micromole of substrate into product /min.


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E. ION-EXCHANGE CHROMATOGRAPHY

This form of chromatography relies on the attraction between oppositely charged particles. Itis frequently chosen for the separation and purification of proteins, peptides, nucleic acids,

polynucleotides and other charged molecules mainly because of its high resolution andcapacity.

Principle:The stationary phase consists of fixed charges on a solid support. These charges can beeither negative or positive. Hence there are two types of ion-exchangers: cation and anionexchangers.

Cation exchangers: possess negatively charged groups and will attract positively chargedcations. These are also known as acidic ion-exchangers because their negative charges resultfrom ionisation of their acidic groups. Strong cation exchange resins contaion sulphonic acidgroups SO3

-, while weak ones contain carboxylic acid groups COO-. Eg: Carboxymethyl

cellulose.

Anion exchangers: have positively charged groups that will attract negatively charged anions.They are also described as basic ion-exchangers as positive charges generally result from theassociation of protons with basic groups. Strong anion-exchange resins have N+(R1R2R3)and weak ones have N(R1R2). Eg: Diethylaminoethyl cellulose Ion-exchange mechanismconsists of five distinct steps:

1. Diffusion of the ion to the exchanger

2. Diffusion of the ion through the matrix structure of the exchanger

i) Dependent on the degree of cross-linkage of the exchanger and concentration ofthe solution.

ii) Controls the rate of the whole process.

3. Exchange of ions at the exchange site

4. Diffusion of the exchanged ion through the exchanger to the surface.

5. Selective desorption by the eluent and diffusion of the molecule into external eluent

i) Selective desorption of the bound ion is achieved by changes in pH and/or ionic

concentration.

Procedure:1) Solution containing the protein of interest (lysozyme in egg white) is applied to the

ion-exchanger.

2) Protein binding to the ion-exchanger is dependent on the net charge on the protein

which in turn is dependent on the pH of the solution and the ionic strength of the mobile

phase. Lysozyme has a pI of 10.5. Hence, it is positively charged at pH below 10.5. At pH

7.0 (Phosphate buffer) it binds to the negatively charged cation exchanger (CM-cellulose).

3) Wash buffer (pH 9.0) is used to remove proteins that have pI below 9.0.


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4) Bound protein is eluted out from the stationary phase by changing the pH or by

increasing the concentration of cations, which compete with positively charged groups of

lysozyme for binding sites on the column.

5) Extent of purification is determined by estimating its specific activity before and

after purification and quantitatively expressed as fold purification.

6) Enzyme activity of lysozyme is determined using the bacterium Micrococcus luteus

by estimating its specific activity. The initial suspension of bacteria is cloudy, absorbing

light strongly at 450nm. As lysozyme breaks down the bacterial cell wall due to osmotic

shock, the breakdown products dissolve and the solution becomes clearer.

One unit of lysozyme is defined as the amount of lysozyme that will produce adecrease in absorbance at 450nm of 0.001 absorbance units/minute.

7) Protein concentration of lysozyme is estimated by UV absorbance at 260nm and

280nm.

8) Estimation of Fold purification:

Fold purification= .This is a measure of efficiency of purification by ion-exchange chromatography.


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2. ELECTROPHORESIS OF SERUM PROTEINS DEMONSTRATION

PRINCIPLE: Electrophoresis is the movement of charged particles through an electrolyte inan electric field. The positively charged particles move towards the cathode and the negativeions to the anode. Since proteins differ in theirisoelectric points, they will generally bear net

charges of different magnitudes at any given pH. Serum proteins are generally separated atpH 8.6. At this pH, all the serum proteins are negatively charged. Passing of an electriccurrent through the solution will then cause the proteins to migrate towards the positivelycharged electrode (anode) at characteristically different rates. While molecular size and shapeare contributing factors, the net charge on the protein molecule is the most important factor indetermining the electrophoretic mobility. Albumin has the greatest magnitude of negativecharge and therefore moves the greatest distance in a given length of time. It is followed in

order by 1-2, and globulins.

Different rates of migration separate a complex mixture such as plasma proteins into anumber of fractions according to mobility. Electrophoresis is not used to purify proteinsbecause some alteration in protein structure and ultimately function may result from this

procedure. This is used as a analytical method. It permits to estimate number of proteins in amixture. This is also useful to determine isoelectric point and approximate molecular weight.

In zone electrophoresis the proteins are placed on a supporting medium such as filterpaper, cellulose acetate membrane, agar gel, starch gel, etc. After migration, the proteins arestained and examined.Cellulose acetate membrane electrophoresis; Cellulose acetate membraneelectrophoresis(CAME) is becoming more popular and is virtually replacing filter paper electrophoresis. Theadvantages include : shorter separation time (1-2 hr.), minimal absorption and easy & rapidwashing out of excess dye.

REAGENTS:

(1) Barbitone buffer: Barbitione-1.3 gms., Sodium Barbitone-9.1 gms in 1 liter of water pH isadjusted to 8.6.

(2) Dye solution: 1 g of amidoschwatrz 10 B per 100 ml of 2% acetic acid; 5% Acetic acid.

TECHNIQUE:

The CAM strips should be handled carefully as they are brittle and expensive. A CAMstrip is labeled. Forceps are used to handle the strip (do not touch with bare hands). The stripis floated on the surface of buffer solution so that the liquid soaks up from underneath. After afew seconds, the strip is immersed completely and left for 15 minutes. After blotting lightly witha clean filter paper, place it in the tank. 0.5 ml of serum per cm width of strip is applied using acapillary tube. 0.4 mA per cm with of strip current is delivered at a constant voltage of 200 V.

Run for 60 minutes. The current is turned off and the strip is placed in the dye, which containsTCA to fix the protein. Stain for 10 minutes. Then the strip is de-stained. When clearbackground is obtained, the stained, separated protein bands are visible.

INTERPRETATION:

Alteration in the normal electrophoretic pattern of proteins occurs in certain pathologicalconditions. Thus, this technique is a useful aid in diagnosing these conditions. The commonclinical conditions where alterations are seen are: (1) cirrhosis of liver (2) nephrotic syndrome.(3) multiple myeloma. (4) agammaglobulinemia.


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veelectrodeveelectrode

+ veelectrod

Cov

Bufferreservoir

Supporting

CAM / agarose gelwick

HORIZONTAL ELECTROPHORESIS APPARATUS

ELECTROPHOTETIC PATTERN IN DIFFERENT DISEASES CONDITONS


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3. ENZYME KINETICS GRAPHS

Enzyme Kinetics deals with the factors affecting the rates of enzyme-catalyzedreactions. Enzymes are biocatalysts and their activity depends upon pH, temperature,electrolyte and substrate concentration. When all these factors are analyzed properly, it is

possible to learn a great deal about the nature of the enzyme-catalyzed reaction. Forexample, by varying the substrate and product concentrations, it is possible to deduce thekinetic mechanism of the reaction, that is the order in which substrates add & products leaveand whether this order is obligate or random. Such studies can establish the kinds ofenzyme-substrate and enzyme-product complexes that can form. The kinetics of a reactionmay indicate the way in which the activity of the enzyme is regulated in vivo.

Enzyme activity is calculated in terms of the number of molecules of product formedper minute in the presence of a given concentration of the particular enzyme under standardassay conditions.

E + S E.S > E + P Eq.1

The above mentioned equation shows that as the reaction proceeds, substrateconcentration [S] will decrease and product concentration [P] will increase.

In general, enzyme catalyzed reactions can be thought to proceed in two steps. In thefirst step, enzyme (E) and substrate (S) bind, reversibly to form an enzyme-substrate complex(E.S). In the second step the product (P) forms and is released from the enzyme, usually thisstep is irreversible, and E and P do not combine to give E.S, at an appreciable rate. Thisprocess is depicted more concisely, in Eq.1 above.

MEASUREMENT OF ENZYME ACTIVITY

Enzyme Unit:1 unit of enzyme activity is that amount of enzyme which transforms one micro mole ofsubstrate into product per minute.

Turnover Number: It is the number of substrate molecules converted to product per moleculeof enzyme per minute.

Specific Activity:

It is the number of units of enzyme activity present/mg of protein.

The kinetics of many enzyme catalysed reactions is described by the Michaelis-Menten equation, which is as follows:

Vo = Initial velocity of the reaction,

Vmax = Maximum velocity of reaction.

Eq 2


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[S] = Concentration of the substrate expressed as moles/Litre.

Km = Michaelis-Menten constant for the particular enzyme.

Let us consider the reaction represented by Eq.1 again. According to the law of massaction, as the concentration of the substrate is increased the reaction will shift towards right.A stage is reached where all the enzyme molecules will get saturated with the substrate andthere will be no further increase in the velocity of the reaction on adding more and moresubstrate. The maximum velocity is then attained and is called the Vmax

When a graph is plotted between [S] and the velocity of the reaction a rectangularhyperbola is obtained as shown below ( Fig.1).

An important numerical relationship that can be developed from Michaelis-Menten equationwhen Vo is exactly one half of Vmax is;

Eq. 3

This represents a very useful and practical definition ofKm :

Km is defined as the substrate concentration at which the initial velocity (Vo) is

half of maximal velocity (Vmax). It has the units of substrate concentration.

We can rewrite the Michaelis-Menten equation to get a much more useful form of the

curve called the Double Reciprocal or Lineweaver Burke Plot in the following way:

Eq. 4

If 1/Vo is plotted on Y axis and 1/[S] on the X-axis then eq.4 is an equation of a straightline with Km/Vmax as its slope and 1/Vo as Y-intercept. Since both Vo and S are plotted as


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their reciprocals on the Y and x-axes, respectively, it is called Double Reciprocal Curve(Lineweaver-Burk plot).

When 1/[S] = 0, then 1/V0 = 1/ Vmax = AO in Fig 2.

and when, 1/V0 = 0, 1/[S] = - 1/Km = BO in Fig 2

Double reciprocal presentation of has the great advantage of allowing a more accuratedetermination of Vmax which can only be approximated from a simple plot of Vo versus [S].Double reciprocal plot offers an easy way of determining whether an enzyme inhibitor ofcompetitive, non-competitive or mixed.

KINETICS OF ENZYME INHIBITION

Competitive Inhibition:

Classical competitive inhibition occurs at the substrate binding site (catalytic site). Structure of

the inhibitor (I) is generally analogous to that of substrate (S). It can therefore combinereversibly with the enzyme, forming an EI Complex rather than ES Complex. When both thesubstrate and inhibitor are present, they compete for the same binding site on the enzymesurface. Again according to the law of mass action, at a very high substrate concentration theeffect of inhibitor will be negligible i.e. Vmax is attainable at a high substrate concentration orthe inhibition is surmountable or reversible. So the Vmax in such a case remainsunchanged while Km value is raised. A classical example of competitive inhibition isinhibition by Malonate of Succinate dehydrogenase which catalyses the formation of fumaratefrom succinate. The inhibitors which have lowest Ki value (Inhibitor Constant) for an enzymewill cause the greatest degree of inhibition.


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Mixed Inhibition:

Mixed inhibition refers to a combination of two different types of reversible enzyme inhibitioncompetitive inhibition and uncompetitive inhibition. The term 'mixed' is used when the inhibitorcan bind to either the free enzyme or the enzyme-substrate complex. In mixed inhibition, the

inhibitor binds to a site different from the active site where the substrate binds. Mixedinhibition results in a decrease in both Vmax and Km.A special kind of mixed inhibition where the affinity of the enzyme is same for both thesubstrate and the inhibitor is known as non competiive inhibition. In the special case wherenoncompetitive inhibition occurs, in which case Vmax is reduced but Km is unaffected. Thisis very unusual in practice.

Uncompetitive-inhibition:

Uncompetitive inhibition takes place when an enzyme inhibitor binds only to the complexformed between the enzyme and the substrate (the E-S complex) and inactivates it. Thisreduction in the effective concentration to the E-S complex increases the enzyme's apparentaffinity for the substrate (Km is lowered) and decreases the maximum enzyme activity(Vmax), as it takes longer for the substrate or product to leave the active site. Uncompetitiveinhibition works best when substrate concentration is high. An uncompetitive inhibitor need notresemble the substrate of the reaction it is inhibiting.


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part i lab manual 2013

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