Johannsen

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Fundamentals and applications

of chromatography

WS 2004/05

PD Dr. rer. nat. habil. Monika Johannsen

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Contents:

1 Introduction

2 Fundamentals of linear chromatography

3 Fundamentals of nonlinear chromatography

4 Adsorption isotherms

5 Process design

6 Equipment

7 Applications

Fundamentals and applications of chromatography

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Optimisation parameter

time

effort

amount

costs

purity

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Way to solve a chromatographic separation problem

1) in analytical scale

! method development (stationary and mobile phase combination, temperature, and in SFC: pressure)

! determination of thermodynamic data (solubility, adsorption isotherms)

2) selection of preparative mode and simulation of separation

3) in preparative scale

! separation of diluted feed

! increase productivity by increasing the injected amount of substances

! productive separation

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Selection of stationary phase for LC

Molecular weight of sample <1000, soluble in organic solvent

Normal phase

Reversed phase

Reversed phase

Reversed phase

Reversed phase with pH control

Reversed phase: Ion pair

chromatography

Reversed phase with

alkaline eluent

BasicIon

formingNon-ionic

Non-polar

Polar

[Chrombook, Merck]

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Selection of stationary phase for LC

Molecular weight of sample <1000, soluble in water

Reversed phase

Reversed phase

Reversed phase with pH control

Reversed phase:

Ion pairing

Mono-, Disaccharides

Ion forming

Non-ionic

Strong acids or bases

[Chrombook, Merck]

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Selection of stationary phase for LC

Molecular weight of sample >1000, soluble in water

Reversed phase

Hydrophobic interaction

(HIC)

Gel permeation (GPC, SEC)

Reversed phase

Saccharides Proteins, peptidesNon-ionic Nucleic

acids

Ion exchange

(Bio-)Affinity

Ion exchange

Normal phase

Gel permeation (GPC, SEC)

Ion exchange

[Chrombook, Merck]

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Unmodified NP material:

! Al2O3 from Bayerit (Al(OH)3) or Böhmit (AlO(OH))

! Florisil - natural Mg-Al-silicate

! SiO2 from silica sand (silica gel) after pulping with HF

! Polymers on polystyrene-di-vinylbenzene basis

Normal phase (NP-) chromatography

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Active sites in normal phase (NP-) chromatography

! covalent bonded hydroxyl groups (but: no RP-phases possible)

! combined retention by hydroxyl groups and Lewis centres

! pH stable from 2 -13

O O

AlO

Al

Lewis

basic

AlO

Al

OO

OHH2O

H

δ+

δ-

Alumina:

O O

AlO

Al

δ+

δ-

OH

chemisorbed wateracidic

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SiO

O O OO

Si

O

Si

O

O O

OSi

Fe

OO

OO

O

O

O Al

H

H

Si

H

strong medium

weakvery strong

O

H

OO

Active sites in normal phase (NP-) chromatography

Silica gel:

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Mobile phase: n-hexane or acetonitrile/MetOH:

Elution order: o, m, p-nitroaniline

H

O

H N2 NO

2

O

H H

O

H N2

NO2

O

H H

O

H N2

NO2 O

H

Retention mechanism in (NP-) chromatography

p-nitroaniline o-nitroanilinem-nitroaniline

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Eluotropic series of solvents

Eluotropic series:

! a relative ranking of LC solvents ranging from non-polar to very polar properties in order of their eluting power

! polarity effects are due, e.g., to dielectric constant, dipole moment and hydrophobic-hydrophilic properties

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Eluotropic series of solvents

n-hexanecyclohexene

toluenebenzene

diethyletherchloroform

dichloromethane1,2-dichloroethane

acetoneethylacetateacetonitrilepropanolethanol

methanolacetic acid

water

Incr

easi

ng p

olar

ity

On alumina:

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Eluotropic elution on alumina

Start with 100% benzene then add acetonitrile

gradientelution

Benzene: ε°=0.32

Acetonitrile: ε°=0.65

isocraticelution

12 3

4

12 3 4

12 3 4

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Common stationary phases (polar)

1. Diol [R = (CH2)2OCH2CH(OH)CH2OH ]

2. Cyano/Nitrile [R = (CH2)3CN ]

3. Amino [R = (CH2)3NH2 ]

! similar separations as with SiO2

! shorter equilibration times

! suitable for gradient elution

! usable for NP- and RP-chromatography

NP-chromatography with chemically bonded phases

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SiO

O O OO

Si

O

Si

O O

OSi

O O

OO

OO

H

H

Si

HO

H

Si Si

Si

(CH )2 n

Cl

CH 3

CH 3

CH 3 Si

(CH )2 3

R

CH 3

CH 3

Cl

R= CN, Phenyl, NO , NH

2 2

SiO

O O OO

Si

O

Si

O O

OSi

O O

OO

OO

H

H

Si

O

Si Si

Si

(CH )2 n

CH 3

CH 3

CH 3

Si

(CH )2 3

R

CH 3

CH 3

Cl

R= CN, Phenyl, NO , NH

2 2

-H Cl-H

Chemically bonded silicas chromatography

silica backbone

spacer, e.g., propyl

R: end group

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! stationary phase less polar than mobile phase

! mobile phase water or solvent mixtures with water

! the less polar the sample is the higher is retention

! C18 (octadecylsilan – “ODS“, RP18) phases most frequently used

Common stationary phases (non-polar)

1. Octadecyl, C18 [R = (CH2)17CH3 ]

2. Octyl, C8 [R = (CH2)7CH3 ]

3. Phenyl [R = (CH2)3C6H5 ]

Reversed Phase (RP-) chromatography

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O OH

OH

O

Si

CH 3

CH 3

O

HO

Si

CH 3

H3C

O

HCH

3Si

CH 3

CH 3

H3C

OH

H3C H

3C

Mobile phase: H2O or H2O / ACN

Stat. phase: C-18

Reversed Phase (RP-) chromatography

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! for separation of mixtures of acids, bases and neutral compounds

! RP phases used as stationary phases

! counterion influences separation factor

! separations mostly in buffered systems

! alternative to ion exchange chromatography

for acids ⇒ quaternary amines as counterion

for bases ⇒ alcyl and aryl sulfonate as counterion

Ion pair chromatography

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O OH

O HH

O

Si

CH 3

CH 3

O

HO

Si

CH 3

H3 C

O

HCH 3Si

CH 3

CH 3H3 C H3 C

counterion+

Sample ion -

+sample

-

[

[paircounterion+

Stat. Phase: C18Mobile Phase:H2O oder H2O / AcN

Ion pair chromatography

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! for separation of ionic organic and inorganic compounds

! analyses of chloride, nitrate, nitrite und sulfate

! separation of amino acids

! active sites fixed on organic resin (e.g. polystyrene-di-vinylbenzene)

! anion exchange: quaternary ammonium groups ( NR3+)

! cation exchange: sulfonic acid or carbonic acid groups ( SO3-)

Ion exchange chromatography

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Stationary phase – cross linked polymer

CH CH2

vinyl benzene

CH CH2

CH CH2

di-vinyl benzene

! vinyl benzene ⇒ polystyrene

! 1 - 16% divinyl benzene as cross linker links polystyrene chains together (amount changes pore size and rigidity)

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Stationary phase – cross linked polymer

CH CH2 CHCH CH2

CH CH2 CHCH CH2

CH CH2

R

Polystyrene-di-vinylbenzene:

cation exchange R = SO3-

anion exchange R = NR3+

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! competition between sample and ions from the mobile phase

! separation influenced by type of ion exchanger, pH value, ionic strength and counterion of the mobile phase

NR3

+

Cl -

sample -

sample -

Cl -

NR3

+

SO3

-

sample + Na+

sample + SO3

-Na+

Ion exchange chromatography

Anion exchange Cation exchange

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! Gel permeation chromatography - GPC (organic mob. phase)

! Gel filtration chromatography – GFC / Size exclusion chromatography - SEC (aqueous mob. phase)

! classification after molecular size (no adsorptive interactions with stat. Phase)

! elution volume only dependent on molecular size

! the smaller the molecule the higher the usable pore volume

! for separation between to molecules at least 10% difference in molecular mass required

Molecular exclusion chromatography

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tR e t e n t i o n

Sig

nal

Molecular exclusion chromatography

! stationary phase contains small pores that analytes can diffuse into (depending upon size)

! larger molecules cannot fit into pores so they elute faster than smaller molecules

! pore size determines range of MW which can be separated

! exclusion limit: smallest molecule which can not fit into the pores; any larger molecule will have same VR

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! stationary phase typically a cross-linked polymer, e.g. :

– Sephadex (glucose/glycerol polymer)– Bio-Gel (polyacrylamide gel)

! separation of complex mixtures (low molecular compounds from biological matrices)

! determination of distribution of molecular mass in quality control

! characterization of origin biological samples

! preparative isolation of single proteins

Molecular exclusion chromatography

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! For analytical or preparative separation of complex biological molecules

! Very powerful method of purification in biology

! Selective separation of compounds or classes of compounds (nucleotides, proteins, enzymes, hormones, glycopeptides)

! makes use of tight, specific complex (highly selective):– antigen/antibody – enzyme/substrate

! Support from silica gel, glass, polystyrene or cellulose

(Bio-)Affinity chromatography

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Ligand

sample

δ+δ−

δ+δ+δ−

δ− δ+ δ−

+δ+−

δ−

δ− δ+δ+ δ−

δ− δ+

δ+−

δ−+

δ+ δ−

δ+δ−

Stronger affinity pH change excessive ions

Elution by

(Bio-)Affinity chromatography

Support

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Method development

Optimisation of:

! stationary phase

! mobile phase (liquid or supercritical)

! mobile phase modifier (e.g.alcanol)

! temperature

! pressure/density (in SFC)

Optimisation criteria:

separation factor / peak resolution, solubility """" productivity

cycc

Feedspecific tV

mPR⋅

=

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! Reduction of organic solvents (→ no/less solvent recovery)

! complete separation of product from solvent by decrease in density(→ no further concentration step, high product purity)

! low operating temperature (suitable for separation of temperaturesensitive substances)

! high diffusion coefficient → high number of theor. plates/low viscosity→ high flow rates → high productivity

! selectivity variation by change in P/ρ and T

! increase of limited solubilities by modifier adding

Preparative SFC - Advantages

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Polar substances in SFC

Methods for increasing migrations rate of medium polar substances:

1) Increase polarity of mobile phase (different fluid or modifier)

2) Increase density

3) Increase temperature

4) Decrease polarity of stationary phase

SFC with CO2 is normal phase chromatography !!!

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Criteria- separation factor- peak resolution- retention factor

Optimization- stationary phase- mobile phase- modifier- mod. concentration- temperature- pressure

Method development in SFC

2,0 2,5 3,0 3,5 4,0 4,5 5,0

1,14

1,16

1,18

1,20

1,22

1,24

1,26

1,28

1,30 30°C 40°C 50°C 60°C 70°C 80°C

(α−/α3)-tocopherol; 22 MPa

Sep

arat

ion

fact

or [-

]

Modifier concentration [vol.-%]

Separation of tocochromanols by SFC on Kromasil

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Criteria- separation factor- peak resolution- retention factor

Optimization- stationary phase- mobile phase- modifier- mod. concentration- temperature- pressure

Method development in SFC

30 40 50 60 70 805

6

7

8

9

10

11

12

13α-tocopherol, 2 % 2-propanol

15 MPa 19 MPa 22 MPa

Ret

entio

n fa

ctor

[-]

Temperature [°C]

Separation of tocochromanols by SFC on Kromasil

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Design of SMB processes

Bel

adun

g q

Konzentration c

0 2 4 6 8-400

-200

0

200

400

600

800

1000

Retentionszeit [min]

Det

ekto

rsig

nal [

mAU

]Measurements Simulation I

4 6 80,0

0,2

0,4

0,6

0,8

1,0

Retentionszeit [min]

Det

ekto

rsig

nal [

mAU

]

m2

m3

Simulation II

Operating point

Production!

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Optimization SMB-SFCInfluence of the feed concentration on productivity and solvent consumption

System:α-/δ-tocopherol on Kromasil 60-10; T = 40 °C; p = 20 MPa; 5 wt.-% 2-propanol, Lcolumn = 14.4 cm; 2/2/2/2 configuration

0 2 4 6 8 10 12 14 16 18 20 220

5

10

15

20

25

30

35

40

45

feed

flow

rate

[g/m

in]

feed concentration [mg/ml]

0 2 4 6 8 10 12 14 16 18 20 220

5

10

15

20

25

prod

uctiv

ity [g

Toco

/(l*h

)]

concentration [mg/ml]0 2 4 6 8 10 12 14 16 18 20 22

0

5

10

15

20

25

400

600

800

1000

1200

1400

1600

400

600

800

1000

1200

1400

1600

solvent consumption

productivity

solv

ent c

onsu

mpt

ion

[gC

O2 /

gTo

co]

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4 6 8 10 12 14 165

10

15

20

25

30

35

40

45

50

55

solv

ent c

onsu

mpt

ion

[gC

O2/g

Toco

]

Prod

uctiv

ity [g

Toco

/(l*h

)]

length of column [cm]

550

600

650

700

750

solvent consumption

productivity

Optimization SMB-SFCInfluence of column lengthSystem: α-/δ-tocopherol on Kromasil 60-10; T = 40 °C; p = 20 MPa;

5 wt.-% 2-propanol, 2/2/2/2 configuration; cfeed = 6 mg/ml

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0 10 20 30 40 50 60

0,0

0,5

1,0

1,5

2,0

feed concentration cfeed= 6 mg/ml

Exp α-Tocopherol Exp δ-Tocopherol Sim α-Tocopherol Sim δ-Tocopherol

conc

entra

tion

[mg/

ml]

column length [cm]

Experimental results SMB-SFC

Comparison simulation – experimental results

System: α-/δ-tocopherol on Kromasil 60-10; T = 40 °C; p = 20 MPa; 5 wt.-% 2-propanol, Lc = 7.5 cm; 2/2/2/2 configuration

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Optimization Batch-SFC Influence of column length

System: α- and δ-tocopherol on Kromasil 60-10; T = 40 °C; p = 20 MPa; 5 wt.-% 2-propanol; umax = 0.55 cm/sec

10 15 20 25 30 35 40 45 50 55

0

20

40

60

80

100

column length [cm]

max

imum

of V

inj [m

l]

10 15 20 25 30 35 40 45 50 55 6070

80

90

100

110

120

130

prod

uctiv

ity [g

/(l*h

)]

column length [cm]

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Comparison SMB - SFC and Batch-SFC

# System: α- and δ-tocopherol on Kromasil 60-10; T = 40 °C; p = 20 MPa; 5 wt.-% 2-propanol; umax = 0.55 cm/sec

# Purity: Both components > 99 %

120.8620400.07220Elution

58.3495150.1306(sum: 36)

SMB(6 columns)

productivity[gToco/lSP⋅h]

CO2 consumption[gco2/ gToco]

cfeed

[mg/ml]mstat.phase

[kg] Lcolumn

[cm]

# higher specific productivity of the Batch SFC (factor 2)

# higher solvent consumption of the Batch SFC (factor 1,3)

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Maximal loading in chromatography

Surface area of silica gel at 300 m2/g can adsorb 3 x 1019 molecules of

a size of 1 nm2 and a molecular mass of 150-250 g/mol.

These have a total mass of around 10 mg.

Loading: max. 3%!

Productivity for example with 60 injections per hour: 1800g/kgSGh

Rule of thumb: 10 – 30 g Feed per kg silica gel

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Production costs

Influence of production capacity on relative production costs of EPA from fish oil

[Lembke, 1998]

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Production costsBreakdown of EPA cost price (100 €/kg) generated by 30 tons per year

[Lembke, 1998]Depreciation

18%

Stationary phase17%

Continguency9%

Energy10%

Ethyl Ester Formation

2%

Rent2%

Carbon dioxide

1%

Staff & Maintenance

incl. Overhead

34%

Consumables2%

Fish oil5%