vny seminar

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Prepared By:Vinay Prajapati.M. Pharm Sem-II

Roll no:13

Department of Quality Assurance

Guided By:Ms. Parula B. Patel(H.O.D. of Q.A. Dept.)

S.J.Thakkar Pharmacy College,

Rajkot.


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CONTENTS IntroductionPrinciple Stationary & mobile phase

DerivatizationAmino Acid AnalyserParameters affecting Amino acid separation

ApplicationReferences

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ION EXCHANGE CHROMATOGRAPHY

Each protein has overall net charge at aparticular pH

Some are negatively charged and somepositively charged

This property of protein is the basis for ionexchange chromatography

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Fine cellulose resins are used that are either

negatively(cation exchanger) or positivelycharged (anion exchanger).

Protein of opposite charge to the resin are

retained, as a solution of proteins passedthrough the coloumn.

The bound protein are then eluted by passinga solution of ions bearing a charge opposite tothat of the column.

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o E.g. Dowex-50 is a cation-exchange resin

o It has covalently attached sulfonic acid groupswhich, at pH 3, are deprotonated andcharged-balanced by associated sodium ions.

o Proteins with significant regions of opposingcharge will bind to this column material byionic attraction, displacing the sodium ions.


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The charges of the aspartic acid at different pH:

oAt pH=1 the molecule has one positive charge, but if thepH value is increasing, larger number of molecules situated

in the -carboxil group will have a negative charge up tothe limit of pH=2.8

owhen all of them disposes it. This is the isoelectric point ofthe aspartic acid.

o The carboxylic group in the side chains less acid than the-carboxilic acid, and the concentration of the hydrogenions is suffcient enough to prevent its ionization.

o

If the pH value rises to 6.6, the carboxylic group of the sidechain will be ionised, and the molecule will get two negativeand one positive charge

o if the pH rise to 11.0, the molecule will dispose only twonegative charges.


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PRINCIPLE OF SEPARATIONThe positively charged amino acids are bound

to the resin which is negatively charged.

The conditions are then altered to increase thepH, temperature and the concentration of thebuffer counter ion.

When the isoionic point of an amino acid isbeing reached, the ionic attraction to the resinis lost and the amino acid elutes from thecolumn.

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STATIONARY PHASES OF IEC1. FUNCTIONALGROUPCATION EXCHANGER ANIONEXCHANGER

Quaternary amine -N(CH3)3+ OH-

Quaternary amine -N(CH3)2(EtOH)+OH-

Tertiary amine -NH(CH3)2+ OH-

Secondary amine -NH2(CH3)2+ OH-

Primary amine -NH3+OH-

Sulfonic acid -SO3- H+

Carboxylic acid -COO- H+ Phosphonic acid PO3- H+

Phosphinic acid HPO2- H+

Phenolic -O- H+

Arsonic -HAsO3- H+

Selenonic -SeO3- H+


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2. MATRIXES1. Silica-based

Better chromatographic efficiency,stability and durability in high pressure

limited pH range : 2< pH


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MOBILE PHASES OF IEC

Properties of Mobile phases

compatibility with the detection mode nature of the competing ion concentration of the competing ion mobiles phases pH

buffering capacity of the mobile phase ability to complex the ionic samplecomponents

organic modifiers 12


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ELUENTS FOR ANIONS

Aromatic carboxylic acids and their salts mostly widely employed eluent

low conductances

ex) lithium hydroxide

Aliphatic carboxylic acid Aromatic and aliphatic sulfonic acids

Potassium hydroxide

Polyol-borate complexes Ethylenediaminetetraacetic acid -EDTA

Inorganic salts such as Cl-, SO42- or PO43-13


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ELUENTS FOR CATIONS

Inorganic acids such as nitric acidOrganic bases

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DERIVATIZATION1

Amino acids are colourless and most ofthem have very little absorption in the UVregion.

Problem in detecting amino acid

To overcome the difficulty, amino acids are

converted into its derivative by usingninhydrin

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Ninhydrin (2 mol) reacts with one mol ofANY amino acid to give the SAME bluecolored product.

This reaction is performed post-column, afterIon Exchange Chromatography separation ofa mixture of amino acids.

The area of each peak in the chromatogramis proportional to the relative molar amountof the amino acid of that retention time.

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H3N C CO

H O

R(any)

O

O

OH

OH

2 +

O

ON

O

O


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EXAMPLEA simple mixture of three amino acidshaving very different isoelectric points

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H3 C CO

OH

CH2CO2

N

A (pI=6.0)

+ H3N

CH3

H O

COC

D (pI=2.8)

+ H3

CH2CH2CH2CH2NH3

H O

COCN

K (pI= 9.7)

Mixture of:

buffered at pH 6.0

Aspartic acid Alanine Lysine


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SO3

SO3

SO3

A

D

K (strongly retained)

(unretained)

(slightly retained, &sulfonated

polystyrene

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D- elutes first, followed by A; K+ eluteslast, and only after pH of buffer isincreased and K+ is deprotonated


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A KD

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injection

Retention time

Increase pH of buffer

In the simple mixture D- elutes first, followed byA; K elutes last, and only after the pH of buffer

is increased and K+ is deprotonated


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AMINO ACID ANALYSER3


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AMINO ACID ANALYSER (AAA)Amino Acid Analyser is a specifically

configured system optimised for the analysis offree amino acids.

PRINCIPLE The system utilises ion-exchange

chromatography incorporating post column

reaction with ninhydrin and subsequentdetection in the visible region spectrum.

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1. Mixture Purification:

Colum Preparation Sample loading

Elution

2. Establishing Standard Rfs:

3. Identification:

WORKING PROCEDURE STEPS 2


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PARAMETERS AFFECTING AMINO ACIDSEPARATION:3

1. Analytical column dimension The sensitivity increases while

column diameter decreases

resin bed length increases

2. Buffer composition

pH

Organic solvent content


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3. Timing of buffers

Adjusting the timing of the buffer isequivalent to adjusting the pH.

Timing of buffer adjustment : 1 to 2 min ata time

4. Buffer flow rate

5. Analytical column temperature

Temperature adjustment: 1 to 2O c at a time


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APPLICATION2 Primary tool in determination of amino

acid imbalance

Evaluation of functional vitamin andmineral deficiencies

For Diagnosis of various metabolicdisorders

AllergiesMechanism include disordered methionine

metabolism, taurine depletion and freeradical pathology

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Many people with food allergies report improve toleranceof food with amino acid supplements particularly whenplasma taurine and histidine levels are low

Cardiovascular disease:Taurine : Powerful antiplatelet aggregation property

(important in CVS diseases)

Depression and Behaviour disorders:Trptophan,tyrosine and phenylalanine:depressionTaurine:To control seizures

Others:In blood sugar disorder,immune

dysfunctions,trauma,post surgical recovery,sclerosis,

eating disorders

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LATEST APPLICATION

Determination of d- and l-amino acids by ionexchange chromatography as l-d and l-ldipeptides4

The free amino acids of human spinal fluiddetermined by ion exchange chromatography5

Quantitative amino acid analysis of food proteinsby means of a single ion-exchange column6

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Amino acid determination in biological fluidsby automated ion-exchange chromatography:performance of hitachi l-8500a7

Determination of the tryptophan content ofproteins by ion exchange chromatography ofalkaline hydrolysates8

Accelerated chromatographic analysis of aminoacids in physiological fluids containingglutamine and asparagine9

Ion exchange chromatography of the freeamino acids in the plasma of the newborninfant10 28


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REFERENCES

1) Peptide and Protein Drug Analysis by Reid, Marcel Dekker.

2) http://www.esu.edu/~jfreeman/317/chem317l/Lab%20folders/

317lamacion/317lamacionpro.

3) www.biochrom.co.uk

4) James M. Manning and Stanford Moore November 10,1968 The Journal of Biological Chemistry, 243, 5591-5597.

5) Dickinson, J. C. and Hamilton, P. B. (1966),Journal of

Neurochemistry, 13: 11791187. doi: 10.1111/j.1471-

4159.1966.tb04275.x

6) D.S. Bidmead and F.J. Ley Biochimica et Biophysica Acta

Volume 29, Issue 3, September 1958, Pages 562-567

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7) Jacques Le Boucher, Christelle Charret, Colette

Coudray-Lucas, Jacqueline Giboudeau and Luc Cynober

Clinical Chemistry 43: 1421-1428, 1997

8) Tony E. Hugli and Stanford Moore May 10, 1972 The

Journal of Biological Chemistry, 247, 2828-2834

9) James V. Benson, Jr. , Manuel J. Gordon and James A.

Analytical Biochemistry Volume 18, Issue 2, February

1967, Pages 228-240

10) Johanne C. Dickinson M.A., Herman Rosenblum M.D,

Paul B. Hamilton M.D., PEDIATRICS Vol. 36 No. 1

July 1965, pp. 2-1330


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