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.

CONTENTS
Introduction

Principle

Stationary

& mobile phase Derivatization Amino Acid Analyser Parameters affecting Amino acid separation Application References
2

ION EXCHANGE CHROMATOGRAPHY

Each

protein has overall net charge at a particular pH are negatively positively charged charged and some

Some

This

property of protein is the basis for ion exchange chromatography

3

 Fine

cellulose resins are used that are either negatively(cation exchanger) or positively charged (anion exchanger). of opposite charge to the resin are retained, as a solution of proteins passed through the coloumn. bound protein are then eluted by passing a solution of ions bearing a charge opposite to that of the column.
4

Protein

The

E.g. Dowex-50 is a cation-exchange resin It has covalently attached sulfonic acid groups which, at pH 3, are deprotonated and charged-balanced by associated sodium ions. Proteins with significant regions of opposing charge will bind to this column material by ionic attraction, displacing the sodium ions.

The charges of the aspartic acid at different pH: oAt pH=1 the molecule has one positive charge, but if the pH value is increasing, larger number of molecules situated in the -carboxil group will have a negative charge up to the limit of pH=2.8 owhen all of them disposes it. This is the isoelectric point of the aspartic acid. o The carboxylic group in the side chains less acid than the -carboxilic acid, and the concentration of the hydrogen ions is suffcient enough to prevent its ionization. oIf the pH value rises to 6.6, the carboxylic group of the side chain will be ionised, and the molecule will get two negative and one positive charge o if the pH rise to 11.0, the molecule will dispose only two negative charges.
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PRINCIPLE OF SEPARATION
The

positively charged amino acids are bound to the resin which is negatively charged. conditions are then altered to increase the pH, temperature and the concentration of the buffer counter ion. the isoionic point of an amino acid is being reached, the ionic attraction to the resin is lost and the amino acid elutes from the column. 7

The

When

STATIONARY PHASES OF IEC

1. FUNCTIONAL CATION EXCHANGER

GROUP

ANION

EXCHANGER

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+

Quaternary amine N(CH3)3+ OHQuaternary amine N(CH3)2(EtOH)+OHTertiary amine NH(CH3)2+ OHSecondary amine NH2(CH3)2+ OHPrimary amine -NH3+ OH-

2. MATRIXES

1. Silica-based

Better chromatographic efficiency, stability and durability in high pressure limited pH range : 2< pH <7

2. Polymer-based

chemically derivatization of synthetic organic polymers most widely used types of ion-exchangers tolerance towards eluents and samples with extreme pH, between 0-14.

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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 sample components organic modifiers

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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 PO4313

ELUENTS FOR CATIONS

Inorganic

acids such as nitric acid Organic bases

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DERIVATIZATION1
Amino

acids are colourless and most of them have very little absorption in the UV region. in detecting amino acid

Problem To

overcome the difficulty, amino acids are converted into its derivative by using ninhydrin
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O OH 2 OH O
+

O H O H3N C CO R(any) O O N O

Ninhydrin (2 mol) reacts with one mol of ANY amino acid to give the SAME blue colored product.
This reaction is performed post-column, after Ion Exchange Chromatography separation of a mixture of amino acids. The area of each peak in the chromatogram is proportional to the relative molar amount of the amino acid of that retention time.

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EXAMPLE

A simple mixture of three amino acids having very different isoelectric points
H O H3N C CO Mixture of: buffered at pH 6.0 CH2CO2
+

H O H3N C CO CH3 A (pI=6.0)

Alanine
+

H O H3 N C CO CH2CH2CH2CH2NH3 K (pI= 9.7)

Lysine

D (pI=2.8)
Aspartic acid

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SO3 K sulfonated polystyrene

(strongly retained) A (slightly retained, &

SO3

D (unretained) SO3

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

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In the simple mixture D- elutes first, followed by A; K elutes last, and only after the pH of buffer is increased and K+ is deprotonated

injection Increase pH of buffer

Retention time

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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 of free amino acids.

PRINCIPLE The system utilises ion-exchange chromatography incorporating post column reaction with ninhydrin and subsequent detection in the visible region spectrum.
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 WORKING PROCEDURE STEPS

1. Mixture Purification: Colum Preparation Sample loading Elution 2. Establishing Standard Rfs: 3. Identification:

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PARAMETERS AFFECTING AMINO ACID SEPARATION: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 is equivalent to adjusting the pH. Timing of buffer adjustment : 1 to 2 min at a 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 mineral deficiencies For Diagnosis disorders of vitamin and

various

metabolic

Allergies Mechanism include disordered methionine metabolism, taurine depletion and free radical pathology

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Many people with food allergies report improve tolerance of food with amino acid supplements particularly when plasma 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:depression Taurine: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 ion exchange chromatography as l-d and l-l dipeptides4 The free amino acids of human spinal fluid determined by ion exchange chromatography5 Quantitative amino acid analysis of food proteins by means of a single ion-exchange column6
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Amino acid determination in biological fluids by automated ion-exchange chromatography: performance of hitachi l-8500a7 Determination of the tryptophan content of proteins by ion exchange chromatography of alkaline hydrolysates8 Accelerated chromatographic analysis of amino acids in physiological fluids containing glutamine and asparagine9 Ion exchange chromatography of the free amino acids in the plasma of the newborn infant10

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REFERENCES
1) 2)

Peptide and Protein Drug Analysis by Reid, Marcel Dekker. http://www.esu.edu/~jfreeman/317/chem317l/Lab%20folders/ 317lamacion/317lamacionpro.

3) 4)

www.biochrom.co.uk James M. Manning and Stanford Moore November 10, 1968 The Journal of Biological Chemistry, 243, 55915597. Dickinson, J. C. and Hamilton, P. B. (1966),Journal of Neurochemistry, 13: 11791187. doi: 10.1111/j.14714159.1966.tb04275.x 29 D.S. Bidmead and F.J. Ley Biochimica et Biophysica Acta Volume 29, Issue 3, September 1958, Pages 562-567

5)

6)

7)

Jacques Le Boucher, Christelle Charret, Colette Coudray-Lucas, Jacqueline Giboudeau and Luc Cynober Clinical Chemistry 43: 1421-1428, 1997 Tony E. Hugli and Stanford Moore May 10, 1972 The Journal of Biological Chemistry, 247, 2828-2834
James V. Benson, Jr. , Manuel J. Gordon and James A. Analytical Biochemistry Volume 18, Issue 2, February 1967, Pages 228-240

8)

9)

10)

Johanne C. Dickinson M.A., Herman Rosenblum M.D, Paul B. Hamilton M.D., PEDIATRICS Vol. 36 No. 1 July 1965, pp. 2-13

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