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ELUTION CHROMATOGRAPHY

DERYA KSE

MMN DURSUN


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OUTLINE

INTRODUCTION

BASIC PRINCIPLES

TYPESOFCHROMOTOGRAPHY YIELD AND PURITY

DISCRETE STAGE ANALYSIS

KINETIC ANALYSIS SCALINGUP CHROMOTOGRAPHY

CONCLUSION


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INTRODUCTION:

CHRO

MATOG

RAPHY Separation of substances with similar

chemical and physical properties can beachieved via chromatography

Most downstream processes include somechromatographic steps to obtain very pure

products

Different techniques and modes are available. Chromatography can be utilized for

separation or analysis of mixtures.

Reference:www.rpi.edu/dept/chem-eng/Biotech.../chromintro.html


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ELUTION CHROMATOGRAPHY

A procedure for chromatographic separation inwhich the mobile phase is passed through thechromatographic bed after the application of thesample.

Reference: IUPAC Compendium of Chemical Terminology 2nd Edition (1997)


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BASIC PRINCIPLES

There are two major components: stationaryphase and mobile phase

Reference: gcsescience.gcsescience.mobi


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Elution of a sample mixture

from a chromatographic

column a) A sample containing

3 different molecules is loaded

onto a column. As the mobile

phase carries the sample

components through the

column, their rate of movement

is affected by the relativetendency to adsorb to the

stationary phase, slowing their

elution. b) The three molecules

are graphed according to their

elution times.

Peak area or intensity = quantity

Resolution (retention time/volume)

= selectivity

Substances are separated based upon theirdifferential interaction with two phases, i.e.stationary and mobile phases.

Reference: http://serc.carleton.edu/microbelife/research_methods/biogeochemical/ic.html


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Stationary Phase: Geometry, Size,Porosity

In general, the solid matrix must be:

inert

porous chemically stable

contain low content of ionic groups

mechanically rigid

Reference:http://books.google.com.tr/books?id=v9NQ0GH18kC&pg=PA151&lpg=PA151&dq=chromatography+matrix+characteristic&s

ource=bl&ots=7EMup5X6e&sig=DX446WHUd54Xfd2u2y1C9EFRjFI&hl=tr&ei=olW4TeaMM8zTsgbejOHqAw&sa=X&oi=book_result&ct=r

esult&resnum=3&ved=0CCsQ6AEwAg#v=onepage&q=chromatography%20matrix%20characteristic&f=false


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Charge/FunctionalGroups Ion Exchange

Size/Molecullar Weight Size exclusion Solubility/Polarity Hydrophobic Interaction

Binding Specificity Bioafinity

TYPESOFELUTION

CHRO

MATOG

RAPHY


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Ion Exchange Chromatography

Separation is based on differences in net charge

Molecules with a net positive charge will beretained on negatively charged columns

Molecules with a net negative charge will beretained on positively charged columns

Reference: mach7.bluehill.com/proteinc/tutorial/iec.html


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Steps in Ion Exchange

Chromatography

Reference: pharamcytimes.wordpress.com


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Size Exclusion Chromotography

GelFiltration Chromatography (GFC)

Gel Permeation Chromatography (GPC)

Molecular Sieve Chromatography (MSC) Separation is based on hydrodynamic size

difference

Solutes larger than the pore size are excludedfrom the matrix and pass through the columnunimpeded.

Reference:chemphys.armstrong.edu/.../SizeExclusionChromatography.htm


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Major Applications of Size Exclusion

Chromotography Desalting or separation, in which the target

product (usually protein) and contaminatingsolutes differ substantially in molecular size.

Protein fractionation, where proteins with onlya small size difference must be separated.

A third application is the characterization of

molecular dimensions of proteins.


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Hydrophobic Interaction

Chromatography

Separation in HIC/RPC is based on differences in surfacehydrophobicity.

Molecules interact with a hydrophobic surface

Interaction between molecule and surface is reversed byreducing the ionic strength of the medium.


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Reverse Phase Chromatography

Interaction between molecule and surface is reversed byincreasing the organic content of mobile phase (=reducingpolarity of mobile phase)

The surface of an RPC medium is more hydrophobicthan that of a HIC medium. This leads to stronger interactions


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Separation is based on reversible biospecific interactions between abiomolecule (or group of biomolecules) and a specific ligandcoupled to a chromatography matrix.

High selectivity = high resolution , high purity, high capacity

Elution conditions and affinity matrix are the most criticalparts of affinity chromatography

Reference: http://www4.gelifesciences.com/aptrix/upp00919.nsf/Content/AD018C9E293F99BEC1256E92003E865A?OpenDocument

Bioaffinity Chromatography


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Methods for Elution

1. Change in buffercomposition

2. Extreme pH

3-4. Addition ofsubstances competing

for binding.

Reference:http://www.separations.eu.tosohbioscience.com/ServiceSupport/TechSupport/

ResourceCenter/PrinciplesofChromatography/Affinity/


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Preparation of Affinity Matrix


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Preparation of Affinity Matrix

Chemical activation of support surface for covalentattachment of bioaffinity ligand

Spacer Arm: Solid surface causes steric hindrancebetween ligand on the surface and its bioaffinity partner.Introducing a spacer arm on support surface beforecovalent attachment of bioaffinity ligand increasesaccessibility of ligand.


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Affinity Matrix

1. Single molecule specific

Too strong interaction: elution

difficult

2. Group specific

Group-specific ligands are used widely

in bioaffinitychromatography because of easy

elution


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Yield and Purity

y= solution concentration

H=solvent flow


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DISCRETE STAGE ANALYSIS


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DISCRETE STAGE ANALYSIS ( Contd)


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KINETIC ANALYSIS

Introduction- The column contains equilibrium stages

- The concentration profile is the result of diffusion and chemical

reaction

- The actual diffusion and chemical reaction between solute andpacking (5 step)

1. Starting Conditions

2. Adsorption of sample

substances

3. Start of desorption

4. End of desorption

5. Regeneration

+

++

+

++

++

+

++

+ +

++

1 2 3 4 5


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Bio process lab.

To see the effects of diffusion and reaction, a solute pulse fed into apacked column

Inlet

t or V

Outlet - Plug Flow without Dispersion

Outlet - Flow with Dispersion

No adsorption

Slow adsorptionfast adsorption

Slow adsorption fast adsorption

Figure 1. Modeling elution chromatography with rate processes


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Mass balance equation

A quantitative approximation

z

y

z

yE

t

q

t

y

x

x

x

x!

x

x

x

xYII

2

2

)1(

Bio process lab.

y, q = concentration of the solute re

inmobile and stationary phase

z = column length

t = timev = linear velocity of the mobile

phaseI = void fractionE = apparent axial dispersion

coefficient

- to solve these equation

if mass transfer is controlling

)()1(!

x

x

yykat

qI

K = mass transfer coefficient

a = packing area per bed volume

y* = concentration in solution at equilibrium

- initial condition

0!

!

q

zA

My Ht = 0, z = 0 ,

all z ,


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!!y

y yy

dy

kadzl

00 *

N Y

Bio process lab.

if reversible chemical reaction israte controlling

qkkytq ')1( !!x

x I

k, k = forward and reverse rate constants

of this reaction

Number of transfer units(NTU)

Height of a transfer unit(HTU)

NTUHTUl y!

!

!

2

00

2

2

00

/2

1/exp

2

1/exp

NTU

tty

ttyy

W

YW

kal

TU

lNTU !!!

2

1

)('

)1( !x

x yya

t

D

t

qI

D = effective diffusion coefficient

t = some characteristic time

if diffusion within pores is rate

controlling


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Bio process lab.

g

g! My dt

YIIYIIT lka

KAMkal

VKM

NTU

HtMyB ])1([

/])1([)(2

/ 00

!

!!

The concentration at the peak

The result

!

2

12

exp])1([

/ottkal

lka

KAMy

YYII

!

2

12

exp])1([

/

oV

Vkal

l

ka

K

AMy

YYII


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SSCALING UPCALING UP

Introduction

- At the larger scale, we want a bigger capacity but with the sameyield and purity

- To increase the capacity, we are able to increase the solute

concentration in the feed and the flow through the column

- parameter : y0, V/V0, W

Bio process lab.

!

2

2

00 21exp W

V

Vyy


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rate constant k :

-if the controlling step by diffusion

and fast reaction within the particles

- if the controlling step by mass transfer

between the bulk and the particle

Changes in the standard deviation

Bio process lab.

kal

YW !

2 - it is a function of the velocity, columnlength, rate constant

- d : spheres diameter

l

d

dk

2/32/12/1Y

WY

w

w

ld

l

E Y

YW w!

2

it may also change because of dispersion

l

d

dk

YW

221ww


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Bio process lab.

Table 1. Changes in the Standard Deviation

Controllingstep

The quantityW2 isProportional to Remarks

Internal diffusion andreaction

External masstransfer

External(Taylor)

dispersion

Axial diffusion

Column of actual

equilibrium stages

l

dY2 Probably the most commoncase

Supported by the most completeanalysis

Likely to become more important

at large scale

Rarely important

Assumes the number of stages Nis proportional to the length

ld 2/32/1Y

Dl

d Y2

Yl

D

l

1


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Scale up the separation

Bio process lab.

Keep the ratio of packing diameter to column diameter

preserve the character of the flow in the packed bed use larger, cheaper packing

Fix dand increase both Y and lat constant (Y/l)

at this case, the pressure drop can increase dramatically because of small constant d, the pressure drop isalready high

use short fat columns

Y

andl

used in the small scale separation increased capacity is due to their greater cross sectionalarea


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CONCLUSIONS

Elution chromatography is concerned with the product

purification.

Using packed beds of adsorbants and yielding a product pulse

diluted with solvent

The yield and purity are usually estimated by Gaussian shape.

Rationalizing in terms of either equilibrium stages or transport

processes.

Elution chromatography ;

* provides excellent purification at lab-scale,

* has also potential at larger scale.

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