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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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DISCRETE STAGE ANALYSIS ( Contd)
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DISCRETE STAGE ANALYSIS ( Contd)
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DISCRETE STAGE ANALYSIS ( Contd)
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DISCRETE STAGE ANALYSIS ( Contd)
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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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