Chromatography_theory -Pak Sulis

7/28/2019 Chromatography_theory -Pak Sulis

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Chromatography

Separates

components in

mixture:Based on

- polarity

- boiling point- ionic strength

- size


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Chromatography

Mobile phase: phase which sample

is dissolved in may be gas, liquid, or

supercritical fluid Stationary phase: phase which

mobile phase is forced through

Mobile and stationary phases are

chosen so the analyte will distribute

itself between the two phases


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Partition

Chromatography Movie Used in GC & LC

Molecules will partitioninto the stationaryphase based uponaffinity for stationaryphase & eventuallypartition into mobilephase again

Thin layer is coated

onto inside of GCcolumn or on smallparticles on LC column
http://localhost/var/www/apps/conversion/movies/bla.movhttp://localhost/var/www/apps/conversion/movies/bla.mov


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Adsorption

Chromatography Very similar to

partitionchromatography

Adsorption just onsurface, partitioninto thin layer

Not used as widelyas partition usedmainly in TLC &very small particlesin LC

Movie
http://localhost/var/www/apps/conversion/movies/adsorp.movhttp://localhost/var/www/apps/conversion/movies/adsorp.mov


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Ion Exchange

Chromatography Movie Separation of

either cations oranions

Separtion based onrelative strength ofionic bond

Anion exchangehas cations onsurface

Used in LCexclusively
http://localhost/var/www/apps/conversion/movies/iex.movhttp://localhost/var/www/apps/conversion/movies/iex.mov


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Molecular Exclusion

Chromatography Separation

based on size

Small moleculesget trapped in

pores & take

longer to get out

Movie
http://localhost/var/www/apps/conversion/movies/mex.movhttp://localhost/var/www/apps/conversion/movies/mex.mov


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Gel Electrophoresis

Separation

based on size

and charge Smaller

molecules will

migrate further,

less tangled

Movie
http://www.dnalc.org/ddnalc/resources/electrophoresis.htmlhttp://www.dnalc.org/ddnalc/resources/electrophoresis.html


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Affinity Chromatography

Very selective

Specific binding

site is used toconcentrate

analyte on

column

Used a lot in

biological

applications

Movie
http://localhost/var/www/apps/conversion/movies/exc.movhttp://localhost/var/www/apps/conversion/movies/exc.mov


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Typical Gas Chromatogram


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Typical Liquid

Chromatogram


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Introduction to

Chromatography - Theory

General Relationships

1. Distribution constant

a. Craig counter currentexperiment

2. Retention time

3. Relationship between distribution

constant and retention time4. Capacity factork

5. Selectivity factora besaan yg menunjukkan kemampuanselktivitas


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Introduction to

Chromatography - Theory

Peak Broadening

1. Shapes

2. Column efficiency

a. plate height

b. number of plates

3. Kinetic factorsVan Deemter

equation


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Craig counter current

movie
http://localhost/var/www/apps/conversion/movies/counterc.movhttp://localhost/var/www/apps/conversion/movies/counterc.mov


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2. Retention time tr

Time it takes for

analyte to reach

detector aftersample injection

Tm = retention

time for material

to come throughcolumn which is

not retained also

called dead time

or void volume

tm rate of migration is the

same as the average rate of

motion of the mobile phase

molecules u = L/tm


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3. Distribution constant

& retention timev = u x moles of analyte in mobile phase

total moles of analyte

v = u x CmVm = 1

CmVm + CsVs 1 + CsVs/CmVm

v = u x 1

1 + KVs/Vm


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4. Capacity factor k Describes migration rates of analytes in columnFor a species A

k = KAVs v= u x 1/(1 + k)

kA = (tr- tm)/tm

For separations involving few components idealcapacity factors are between 1 - 5

What is k

for this

peak?


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5. Selectivity factora

Ability to

distinguish

between 2species, A & B


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Purpose of

Chromatography Achieve separation

Elution movie
http://localhost/var/www/apps/conversion/movies/elution.movhttp://localhost/var/www/apps/conversion/movies/elution.mov


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Peak Broadening


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Peak Broadening


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Is peak broadening a good or

bad thing?

BAD Why?


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Column Efficiency

Plate height (H)

# theoretical

plates (N)

N = L/H

Efficiency of a

column goes up

as N increasesand H decreases

Typical 250

10,000 plates


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Plate Height


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3. Kinetic Factors: The

Van Deemter Equation

Reality: column efficiency is

affected by kinetic factors


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What variable do you think are

important in determining the

efficiency of a separation?

In o r notebook predict hat the


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In your notebook predict what the

effect of increasing linear

velocity (flow rate) will have on

column efficiency (H)


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H = A + B/u + CuA = Eddy diffusion: Due to

different pathsmolecules can take as

they go throughparticles

B/u = longitudnal diffusion Band diffuses in

and against direction of mobile phase

movement

Cu often broken into 2 terms Csu + Cmu

Mass transfer coefficient: Time it takes for

analyte to diffuse into stationary phase


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How can band broadening be

reduced? (and thus column

efficiency be enhanced)1. Decrease particle diameter

2. Decrease column width

3. Lowering temperature in GC

(reduces diffusion coefficient)

4. Minimize thickness of liquid

stationary phase


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Resolution

Rs = 2((tr)B(tr)A)

wA + wB

This is called General

Elution Problem

Chromatography_theory -Pak Sulis

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