3 Chromatography

3.1 Basic principles

3.2 Gel filtration

3.3 Ion exchange

3.4 Affinity chromatography

3.5 HPLC FPLC

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Historical review

Round filter paper

Long rectangle paper

Thin layer(TLC)

Column

Samplecapacity increased

Largercapacity

Development

Adapted from Scope RK (1987) Protein Purification Principles and Practice p.9

Paper partition chromatography (PPC)

Martin, Synge (1952)

Essential mechanism

Polar PolarNon-polar Non-polar

LikeDissolves

Like

LikeDissolves

Like

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LL

Two-phase separation system

Mobilephase

Stationaryphase

ADSORPTION PARTITIONLiquid - Solid Liquid - Liquid

Two-phaseA B C

One Plate ofSeparationTheoretical

platenumber

SPolar

L

Non-Polar

PolarNon-polar

Sample Sample

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One plate

One separation, one plate

B

B BB B

B B BB

BBA

A

AAA

A

A

A

AA

AA

AA

AA

AA

AA

B

B BB BB B

B

B

B

80

20

10

90

6416

19

164

981

5113

01

31

873Polar

Non-polar

100A+

100B

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Common chromatographic methods

Gel filtrationReverse phase

chromatography

Ion exchangeAffinity

chromatographyHydrophobic

interactionHydroxyapatite

AdsorptionPartition

PPC, TLC, GC TLC, GCSmall

molecules

Largemolecules

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Thin-layer chromatography

Thin-layer plate

Sample spotting Add developer Developing

ActivatedActivated

NotNot--activatedactivated

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3.2 Gel filtration

3.2.1 Basic principles partition

3.2.2 Gel materials

3.2.3 Gel and column

3.2.4 Column operation

3.2.5 Problem and solution

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Smaller molecules are retarded

Largermolecules

Small molecules

Partition

Sample

Stationary phase

Mobilephase

Stokes radiusMolecular sizeand shape

Elute out fasterGel filtration is a partition type chromatography Juang RH (2005) EPA

Pharmacia

Bio-Rad

SephadexSepharoseSephacrylSephacel

BioGel PBioGel A

glucose (dextrose)agaroseglucose + acrylamidecellulose

acrylamideagarose

FPLC Superose, SuperdexMono Q, Mono S

Gel material is a polymer of carbohydrate or acrylamide Juang RH (2005) EPA

Pha

rmac

ia (1

991)

Gil

Filtr

atio

n

Prin

cipl

es a

nd M

etho

ds p

.35

Sephadex

glucose unit

Space for filtration

Sephadex is the polymer of glucose with chemical cross-linking

Sephadex G

MW ranged from ten to several hundred thousands

Pharmacia (1991) Gil Filtration Principles and Methods p.37

G-200 could be smashed flat easily

From powder to gel form1 g 20 mL gel1 g 7.5 mL

(its backbone support is too weak)

Small proteins

Pha

rmac

ia (1

991)

Gil

Filtr

atio

n

Prin

cipl

es a

nd M

etho

ds p

.22

Sephacryl

AcrylamideCross-linking

Bigger spaceStronger support for backbone

Agar gel formation

Pharmacia (1991) Gil Filtration Principles and Methods p.38, 39

Even stronger backboneMuch bigger space

Sepharose

MW ranged from ten thousands to several millions

Pharmacia (1991) Gil Filtration Principles and Methods p.40

2% gel6% gel

Large proteins

Sepharose is rigid

Sephadex

Sephacry

Cross-linking

Gel filtration beads

Spaces in a column

Adapted from Pharmacia (1991) Gil Filtration Principles and Methods p.11

Vt Vo Vt - Vo

Mobile phase Stationary phase

A typical chromatogram

XY

ZEnzymeactivity NaCl

Vo

Vt

(E) (Y)

Elution Volume (mL)

A

VoVe

Vt0

E

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Poor separationPeak flattened

Best separation

0

50

100

0.5 1.0 1.5

Superfine

Fine

Corse

Ve/Vt

0

50

100

0

50

100Rel

ativ

e am

ount

Ada

pted

from

Pha

rmac

ia: G

il Fi

ltrat

ion

P

rinci

ples

and

Met

hods

Bead size is critical to the resolution of gel chromatography

Dispersion

Diffuse in and out bead

Sample or buffer is diffusing in and then out of the gel particle.

DiffusionJuang RH (2005) EPA

Two types of gel

Dispersion ()

Small particle size reduces the diffusion time and increases the resolution of the gel

Dispersion type gels let the sample molecules flow directly through the gel body, and have better resolution

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Bead size vs flow rate

Adapted from Scope RK (1987) Protein Purification Principles and Practice p.192

Smaller particles also reduce the space between the beads, and prevent the turbulence as the buffer flows, but the flow rate might be decreased

Sephacryl S-200

Elution volume

Sephacryl S-300

Ads

orba

nce

Ada

pted

from

Pha

rmac

ia: G

il Fi

ltrat

ion

P

rinci

ples

and

Met

hods

0

50

100

0

50

100

Choose the gel which brings your target protein out of the column earlier

Sample volume at 1% of Vt

Sample volume at 1~2% of total gel volume

Adapted from Pharmacia (1991) Gil Filtration Principles and Methods p.46

Superdex 200Transferrin (81 kD)& IgG (160 kD)

0 1 2 3 4 5

0.5

1.0

1.5

Sample volume (% of Vt)

Resolution

a

cb

R = a(b+c)/2

Ada

pted

from

Pha

rmac

ia (1

991)

Gil

Filtr

atio

n

Prin

cipl

es a

nd M

etho

ds p

.59

Elution time (h)

Con

cent

ratio

n

Bed height (cm)

Flow rate (mL/cm2/h)

85

85

20

2

25

25

0 1 2 3

0 10 20 30Increaseflow rate

Reducecolumn height

A B C

A B C

AB C

10 cm2

30 cm

10 cm

30 cm2

300 cm3

Short and fat

Long and slim

Fat column cannot tolerate poor separation

But it has better flow rate and higher capacity

Adapted from Scope RK (1987) Protein Purification Principles and Practice p.195

A

Buffer

Gradient mixer

Pump

Recorder

Monitor

Column

Gel

Fraction collector

(1)

(2)

(3) (4)

(5)

adapter

reservoir

Connector

The whole family of liquid chromatography apparatus Juang RH (2005) EPA

Packing column step by step

Equilibratedin buffer

Temperatureequilibrated

Gel stands o/n

Wash gel well

Estimate

gel volume Pouring gelsmoothly

Stop elution X

Keep eluting

Put on reservoir

Sediment

In progressing

Supernatant

Elute under

High pressure

Put on adaptor

12 Gel

GelCheck flow rate of empty column

Gel should be packed tightlyJuang RH (2005) EPA

Packing column

Pour gel slurry smoothly (non-stop), avoid trapping any bubble Ph

arm

acia

(199

1) G

il Fi

ltrat

ion

P

rinci

ples

and

Met

hods

General principle for column chromatography

Gel selection Make target protein elute out column earlier

Bead size Finer bead has better resolution, slower flow rate

Column size Use larger column size but consider practical need

Flow rate Fast flow reduces resolution, slow

Sample volume Apply 1% of total gel volume for sample

Column shape Slim column for gel filtration, fat column for others

Pack tightly Pack the gel tightly for better resolution

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