May 2017

Chromatography seminar

BÜCHI Labortechnik GmbH

May 2017

Agenda

Principles of chromatography · Retention time

· Resolution

· van Deemter equatation

· From TLC to column chromatography

· Optimization of TLC plates

· How to transfer results from TLC to a column

May 2017

Büchi: Solution for your synthesis

Design and

preparationSynthesis Evaporation Drying

Quality

control

Chromatography

May 2017

Büchi: Solution for extraction

May 2017

Chromatography

Chromatography is a method of separating and identifying the components of a

complex mixture by differential movement through a two-phase system, in which

the movement is effected by a flow of a liquid (mobile phase) through an adsorbent

(stationary phase).

Preparative Chromatography is the process of using liquid chromatography to

isolate a sufficient amount of material for other experimental or functional

purposes.

Preparative Chromatography can be separated into three major segments;

traditionally differentiated by the pressure range:

A low pressure Traditional Flash segment ( 0 -15 bar)

A medium pressure MPLC segment (20 – 50 bar)

A high pressure Prep HPLC segment (up to 200 – 300 bar)

Definitions:

May 2017

Chromatography – Goal

Source: www.kunstaufräumen.ch

May 2017

Source: www.kunstaufräumen.ch

Chromatography – Goal

May 2017

A separation technique, based on an equilibrium

between 2 phases

• Stationary phase

(column packing)

• Mobile phase

(solvent)

How does it work?Stationary and mobile phase

May 2017

Adsorption and Desorption

Adsorption: Binding of a molecule

Desorption: Detaching of a molecule

Adsorption

Desorption

May 2017

Mechanism of separation

Different

Adsorption/Desorption

charateristic

Adsorption

strength

Desorption

strength

May 2017

Stationary phases

- Normal phase:

polare silica gele

- Reversed phase:

modified silica gele

May 2017

Normal phase

Si

O

O

OHO

Si

Si

Si

O

O

OH

OH

Si O

Si O

Reversed Phase

Si

O

O

O

Si

Si

Si

O

O

OH

Si O

Si O

O

O

Si

CH3

(CH2)n CH

3

Stationary phases

May 2017

Stationary phases

May 2017

Mobile Phase: Eluotropic serie

Polarity Viscosity

Refractive Index

Dielectricity Constant

n-Pentane 0.00 0.24 1.358 1.84

n-Hexane 0.01 0.33 1.375 1.88

Isooctane 0.01 0.50 1.391 1.94

Tetracarbon 0.02 0.97 1.466 2.24

Di-isopropyl-ether 0.28 0.37 1.368 3.88

Toluene 0.29 0.59 1.496 2.38

n-Propylchloride 0.30 0.35 1.389 7.7/

Benzene 0.32 0.65 1.501 2.28

Ethylbromide 0.37 0.39 1.421 9.34

Di-Ethylether 0.38 0.23 1.353 4.33

Chloroform 0.40 0.57 1.443 4.80

The solvent strength is the property of displacing a substance from

the stationary phase.

May 2017

Mobile Phase: Parameter of solvents

relevant characteristic of the mobile phase

• Dipole-parameter

• Proton-aczeptor-parameter

• Proton-donor-parameter

Selectivity

May 2017

Mobile PhaseGroups of solvents with similar separation properties

IIsopropyl ether

Diethyl etherTriethyl amine

VIDioxane

Ethyl acetate

AcetoneAcetonitril

VIIToluene

Benzene

IVAcetic acidFormamide

VDichloromethane

1,2-Dichloroethane

IIPropanol

EthanolMethanol

VIIIChloroform

IIITetrahydrofuran

Pyridin

Dimethylforamide

May 2017

H-

Acceptor

DipoleH-

Donor

TolueneVII

V

Dichloro-methane

I

Diethyl-ether

II

Ethanol

IV

Aceticacid

III

Tetra-hydrofuran

VIII

Chloroform

VI

Ethylacetate

Selectivity triangle

May 2017

Influence of the selectivity in TLC

TLC: Silica Si60 Detection: UV lamp 254 nm

Tetrahydrofuran

Mix.

Dichloromethane

Mix.

Chloroform

Mix.

CH3

O

NH

May 2017

Influence of the solvent strength in TLC

THF/Hexan 1:1

Mix.

Si= 1.0

Tetrahydrofuran

Mix.

Si= 2.0

TLC: Silica Si60 Detection: UV 254 nm

CH3

O

NH

TLC 1 TLC 2

May 2017

Selectivity vs solvent strength

Selectivity interacts the distance of the components

Solvent strength interacts the moving velocity

For good results selectivity and solvent strength have

to be carefully adapted to the separation problem!

May 2017

Terms in thin layer chromatography (TLC)

Solvent front

Start position

D0

D1

D2

D3

Solute distanceMobile phase distance

D1

D0

Rf =

Rf =

Retention factor Rf

May 2017

Calculation of Rf-values (TLC)

D0 = 7,4 cm

D1 = 6,6 cm

D2 = 3,6 cm

D3 = 1,5 cm

89.04.7

6.61

Rf

49.04.7

6.32

Rf

20.04.7

5.13

Rf

May 2017

General terms in a chromatogram (1)

t0 = Dead time

tR = Retention time

H100% = Peak height

H50% = 50% of the total

peak height

H10% = 10 % of the total

peak height0 1 2 3 4 5 6

Time (min)

H100%

H50%

H10%

t0

tR

May 2017

0 1 2 3 4 5 6

Time (min)

H100%

H50%

H10%

t

0

t

R

General terms in a chromatogram (2)

b0,5 = Peak width at

half higth

a b

b0,

5

Base line

a = Peak width at 10%

of peak higth,

front part

b = Peak width at 10%

of peak higth,

back part

May 2017

General terms in a chromatogram (3)

Number of plates N: Number of theoretical equlibria of a substancebetween the stationary phase and the mobile phaseduring passage through the column

Chromatogram: Record of all detector signals during achromatographic separation

Eluent: Mobile phase, solvent

Flow rate u: Rate of flow of solvent through the column, in mm/s

Fronting: Modification of the shape of the front peak flank

Tailing: Modification of the shape of the rear peak flank

May 2017

The resolution

0 2 4 6 8 10 12 14

Time (min.)

)2(5,0)1(5,0

12177.1

bb

ttR RR

O

O

O

O

CH3

CH3

O

O

O

O

C

H2

CH

2

CH

2

C

H2

C

H2

CH

2

CH3

CH3

O

O

O

OC

H2

CH

2

CH3

CH3

CH3

The resolution is a measure

for the separating power of

a columnR

May 2017

Theoretical plates

Theoretical distance for

1 adsorption/desorption

step = 1 theoretical plate

Adsorption Desorption

2

5,0

54.5

bR

tN

(N = Number of theoretical plates)

May 2017

Plate height

2

5,0

54.5

bR

tN

Number of theoretical plates N:C

olu

mn

len

gth

L

Plate height H (or HETP):

N

LH

Height equivalent of a theoretical plate

May 2017

Retention factor

0

1

D

DRf

Theoretical plates

2

5,0

55.5

b

tN R

Resolution

)2(5,0)1(5,0

12177,1

bb

ttR RR

Peak symmetry

a

bIS ..

Linear flow rate

26

4

d

Fu m

Summary

May 2017

Effects on column efficiency

Particle

size

Flow rate

Loading

May 2017

Flow rate (u)

Pla

te h

eig

ht

(H

)Effect of the particle size (1)

udp

udpH

16

63

2

The plate height H and

hense the number of

theoretical plates N of

a column are strongly

determined by the

particle size dp

H0

Van Deemter curve1)

1) Simplified by Halasz et al., Z Anal.Chem. 277 (1975)257

May 2017

0

100

200

300

400

500

600

700

800

0 0,5 1 1,5 2 2,5 3

Linear flow rate (mm/s)

Pla

te h

eig

ht

H (

mic

ron

)

Effect of the flow rateDependence of the plate height H on the linear flow rate for

adsorbents of different particle size

The column efficiency

shows greater dependence

on the flow rate in the case

of coarse adsorbents than

in case of fine particle sizes

dp = 100

dp = 60

dp = 50

dp = 30

May 2017

Loading (B)

Pla

te h

eig

ht

(H

)Effect of the loading (1)Dependence of the plate height H on the loading B

High loading increases

the plate height and is

therefore lowering the

column efficiency

B0

Some facts:

Loading B0 200 g / g SiO2

Analyt. loadings: B < B0

Prep. loadings: B > B0

Good prep. separations

up to 30 mg / g silica gele

May 2017

How to optimize?

Rf1= 0.49

Rf2= 0.30

Rf3= 0.14

Rf1= 0.97

Rf2= 0.84

Rf3= 0.73

May 2017

What can we optimize?

Solvent 1 Solvent 2 Solvent 3

Optimize the selectivity

May 2017

H-

Acceptor

DipoleH-

Donor

TolueneVII

V

Dichloro-methane

I

Diethyl-ether

II

Ethanol

IV

Aceticacid

III

Tetra-hydrofuran

VIII

Chloroform

VI

Ethylacetate

Selectivity triangle

May 2017

Link between TLC and column? (1)

TLC Column

0

1

D

DRf

1'

'

141

2

2

k

kNR

Calculate the required theoretical plates of the column

2

2

20,1

'1

1'4

k

kNR

May 2017

Link between TLC and column? (2)

TLC Column

0

1

D

DRf

Calculate the required theoretical plates of the column?

2

2

20,1

'1

1'4

k

kNR

11

' fR

k

1

2

'

'

k

k

May 2017

Terms in the formulae

1'

'

141

2

2

k

kNR

R = Resolution, measure for

the separation of

2 components (distance

between 2 peaks)

N = Number of theor. plates

NR1,0 = Number of plates for

a resolution of 1.0

= Separation factor

(selectivity)

k = Capacity factor

(solvent strength)

2

2

20,1

'1

1'4

k

kNR

May 2017

Rule for optimization

2

2

20,1

'1

1'4

k

kNR

Rf1= 0.49

Rf2= 0.30

k1 = 1.04

k2 = 2.33

= 2.24

N = 106

Rf1= 0.97

Rf2= 0.84

k1 = 0.03

k2 = 0.19

= 6.33

N = 885

The capacity factor k

affects the required

column efficiency to a

greater extend than the

separation factor .

Optimum k values are 1...5(Rf values between 0.2 and 0.5)

Optimum values are 1.2

(Rf differences 0.05)

May 2017

Evaluation of the mobile phaseSample: Crude reaction mixture

Tetrahydrofuran

Ethanol

Ethylacetate

Chloroform

Dichloromethane

Diethylether

Step 1

Different solvents,

diluted 1:1 with

hexane

Step 2

solvent , &

diluted 1:3 with

hexane

May 2017

Appoint the parameters for preparative

separation on the column

Selectivity:

Diethylether

()

Adsorbent:

Silica Gel Si 60(same as TLC)

Solvent strength:

15% Diethylether

in Hexane( 1/3 of TLC, k)

Column:

15 x 230 mm

( 20 g silica gel

for 600 mg sample,

loading

30mg/g)Delivery 10 ml/min

(linear flow rate

1,25 mm/s)

May 2017

BÜCHI Labortechnik GmbH

Thank you for your attention!