Thin-Layer-Chromatography

Rapid and reliable method transfer from TLC to Preparative Chromatography

Lab scale isolation of compounds by Flash Chromatography

Properties of Silica Gel Using Thin Layer Chromatography (TLC) for chromatographic method development

Transfer from TLC to prep. Chromatography

Applications

Rapid and reliable method transfer from TLC to Preparative Chromatography

Silica and Aluminium oxide production facilities, Gernsheim

Rapid and reliable method transfer from TLC to Preparative Chromatography

Production Plant
The biggest chromatographic silica gel plant in the world

Rapid and reliable method transfer from TLC to Preparative Chromatography

Production Plant
Four reactors for silica gel production, ... large scale production, batch size: several tons

Rapid and reliable method transfer from TLC to Preparative Chromatography

Current output
(with increasing volume)
Silica gel: > 1.500 tons (360 tons for internal use)

Aluminium oxide:
Products of spray drying: Miscellaneous products:

~ 150 tons (40 tons for internal use)

~ 120 tons ~ 120 tons

Rapid and reliable method transfer from TLC to Preparative Chromatography

Silica Production Process

Hydrosol
(oligosilicic acid, orthosilicic acid)

Na2SiO3
(water glass)

+ H2SO4

Hydrogel
temperature control
drying temperature control control of temperature and pH

Xerogel
(raw silica powder, particle size 0.5 - 6 mm)

Ground silica gel

(dp 5 - 500 m)

Classified silica gels for TLC

5-20m

washing pH-control, ion content

Rapid and reliable method transfer from TLC to Preparative Chromatography

Standardized Silica Gels

LiChrosorb Si60 LiChroprep Si 60 5, 10m 15-25 m

25-40 m
15-40m Silica Gel 60 40-63m [9385] > 63m + Silica Gel for TLC 63-100m 63-200m [7734] 200-500m All from one Production Process CONSTANT SELECTIVITY
8 Rapid and reliable method transfer from TLC to Preparative Chromatography

Which factors are influencing selectivity and which are usability

Form Surface irregular / spherical Modification Area / Pore size Purity pH Water content Efficiency, Pressure Drop Selectivity Loadability Selectivity, Resolution Selectivity Selectivity

Stability

mechanical
chemical (pH)

Life time
CIP - Conditions

Rapid and reliable method transfer from TLC to Preparative Chromatography

Separation Optimization
Resolution: the key parameter for preparative chromatography

1 k N RS ( )( ) 1 k 4
Maximize through sorbent screening Minimize: keep cycle time short maximize high efficiency sorbent

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Rapid and reliable method transfer from TLC to Preparative Chromatography

Method Development using Thin Layer Chromatography

You have to consider three phases in TLC : Gas phase

Sample

Stationary phase

Liquid phase

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Rapid and reliable method transfer from TLC to Preparative Chromatography

Method Development using Thin Layer Chromatography

Several parameters have to be considered when developing a TLC method: Activity of the stationary phase

Conditioning/preparation
methanol / isopropanol (plate developing or dipping) drying (20 min @ 120 C)

Support (plastic, aluminium, glas) Binder Fluorescent indicator Manufacturer/lot reproducibility Manufacturer/comparability with Flash / column chromatography

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Rapid and reliable method transfer from TLC to Preparative Chromatography

Method Development using Thin Layer Chromatography

The mobile phase should be: Dissolve the sample Desorb the sample from the stationary phase

Transport the sample over an acceptable distance

The mobile phase should in general be : Simple (maximum 4, better 2 components)

Non-toxic
of chromatographic quality Not cause any secondary reactions Not remixing

Of favorable low viscosity

13 Rapid and reliable method transfer from TLC to Preparative Chromatography

Solvents classification: Snyder triangle

Method Development using Thin Layer Chromatography

0.2

0.6

0.3

proton donor

xd

xe
0.4

proton acceptor

0.5

0.3

0.6 0.2
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0.3

0.4 n dipole interaction

0.6

0.2 0.7

Rapid and reliable method transfer from TLC to Preparative Chromatography

TLC/HPLC - Conversion

d oS Rf d of

ATTENTION: The migration distance is measured from the sample spot

15 Rapid and reliable method transfer from TLC to Preparative Chromatography

kDC

1 1 Rf

TLC - equipment

CAMAG vario chamber parallel use of 6 different solvents or solvent mixtures

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Rapid and reliable method transfer from TLC to Preparative Chromatography

Thin Layer Chromatography (TLC) as pilot technique

Some general rules: Adjust polarity to lower retention values on TLC plates

Try to maintain the same relative activity on the plate and on the column
Do not use volatile mobile phase components

Literature: TLC as a pilot technique for transferring retention data to HPLC W.Jost, H.-E.Hauck, F.Eisenbeiss, Kontakte 1984 (3), p.45-52
17 Rapid and reliable method transfer from TLC to Preparative Chromatography

TLC/HPLC - Conversion
Place a spot of the sample on a TLC-plate Develop chromatogram with mobile phases of different elution strength

0 (elution strength)

far too low

too low

perfect

too high

Literature: E.Stahl, Chemiker-Ztg. 82 (1958) 323

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Rapid and reliable method transfer from TLC to Preparative Chromatography

TLC/HPLC - Conversion
Non-polar compound n-Heptane Cyclohexane 0.01 0.04 Polar compound 0.38 0.56 0.57 0.58 0.82 0.88 0.95 0 MTBE Dioxane THF Etat IPA EtOH MeOH ACN

MTBE Dichloromethane

0.38 0.42

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Rapid and reliable method transfer from TLC to Preparative Chromatography

Solvent Screening model J.Dingenen, Janssen Pharmaceutica

TLC-Approach 1
1.) Two series of neat solvents covering a broad polarity and selectivity range
MeOH MTBE EtOH Toluene IPA CH2Cl2 Acetic acid CH3CN 2-propanone EtAc 2-butanone THF

Run 1 Run 2

2.) Solvent series based on dichloromethane methanol mixture Run 3 Lane

N-Hexane 50% CH2Cl2 50 % MeOH 2% MeOH 5% MeOH 10% MeOH 20% MeOH 50%

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Rapid and reliable method transfer from TLC to Preparative Chromatography

Solvent Screening model J.Dingenen, Janssen Pharmaceutica

TLC-Approach 1
2.) Solvent series based on dichloromethane methanol mixtures

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Rapid and reliable method transfer from TLC to Preparative Chromatography

TLC-Approach 1
1) Good selectivity for one of the tested neat solvents or for one of the dichloromethane methanol mixtures

Start experiments on the preparative chromatography column

2) Good selectivity for one of the tested neat solvents but Rf values to high Addition of an apolar solvent to adjust the Rf values between 0.10 and 0.35 (corresponding to a k` value between 1.85 and 9)

3) Unsatisfactory results
On the basis of the observed spot shapes in the experiments with neat solvents the most suitable solvent combinations (binary ternary) are chosen and further inverstigated
Solvent Screening model J.Dingenen, Janssen Pharmaceutica
22 Rapid and reliable method transfer from TLC to Preparative Chromatography

Solvent Screening model J.Dingenen, Janssen Pharmaceutica

TLC-Approach 1
Binary solvent mixtures: THF/acetone or Ethanol/Acetone

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Rapid and reliable method transfer from TLC to Preparative Chromatography

Solvent Screening model J.Dingenen, Janssen Pharmaceutica

TLC-Approach 1
Ternary solvent mixtures

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Rapid and reliable method transfer from TLC to Preparative Chromatography

EtAc/Heptane-Gradient, Solvias

TLC-Approach 2
TLC-Screening with Ethyl acetate / n-hexane (1:4) Segment 3
(Rf=0.60-0.90)

Segment 2
(Rf=0.25-0.60)

Segment 1
(Rf=0.05-0.25)

Transfer to preparative normal phase silica, according to segment

25 Rapid and reliable method transfer from TLC to Preparative Chromatography

Diastereomers + impurities

Example for TLC method development

100 % n-Heptan

95 % n-Heptan 5 % Ethylacetat

90 % n-Heptan 10 % Ethylacetat

85 % n-Heptan 15 % Ethylacetat
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80 % n-Heptan 20 % Ethylacetat

75 % n-Heptan 25 % Ethylacetat

Rapid and reliable method transfer from TLC to Preparative Chromatography

Diastereomers + impurities

Example for TLC method development

100 % n-Heptan

95 % n-Heptan 5 % Ethylacetat

90 % n-Heptan 10 % Ethylacetat

85 % n-Heptan 15 % Ethylacetat
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80 % n-Heptan 20 % Ethylacetat

75 % n-Heptan 25 % Ethylacetat

Rapid and reliable method transfer from TLC to Preparative Chromatography

Diastereomers + impurities

Example for TLC method development

100 150 50

Retention

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Rapid and reliable method transfer from TLC to Preparative Chromatography

200

250

300

2
1,55 1,68 1,89 2,62 3,08

4
5,04

8 10 12 14 16 18 Retention Time (min)

7,85

HPLC chromatogram

Intensity (mV)

TLC chromatogram

Comparison of TLC and HPLC

Used stationary phases: TLC HPTLC Silica gel 60 HPTLC RP-8 HPLC LiChrospher Si 60 LiChrospher 100 RP-8, LiChrospher 60 RP select B HPTLC RP-18 LiChrospher 100 RP-18

HPTLC CN
HPTLC NH2 HPTLC Diol

LiChrospher 100 CN
LiChrospher 100 NH2 LiChrospher 100 Diol

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Rapid and reliable method transfer from TLC to Preparative Chromatography

Comparison of TLC and HPLC

Example: Triazines TLC: HPLC: HPTLC RP-8 F254s plate LiChroCART LiChrospher 100 RP-8 or LiChroCART LiChrospher 60 RP select B Mobile phase: Acetonitril/Water 60/40
Pesticide Terbutryn Prometryn Terbuthylazin Propazin Sebuthylazin Atrazin Metribuzin hRf 19,3 21,4 27,1 30,0 30,0 35,7 45,7 KP 4,18 3,67 2,69 2,33 2,33 1,80 1,19 KS (RP-8) 15,70 14,18 2,10 1,89 1,85 1,46 1,06 KS (select B) 3,20 2,90 1,91 1,71 1,68 1,31 0,94

Simazin
Cyanazin Hexazinon Desethylatrazin Metamitron Desisopropylatrazin

45,7
47,1 52,1 55,0 60,7 61,4

1,19
1,12 0,92 0,82 0,65 0,63

1,10
0,95 0,81 0,66 0,61 0,52

1,01
0,85 0,79 0,62 0,54 0,49

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Rapid and reliable method transfer from TLC to Preparative Chromatography

Example: Triazines

Comparison of TLC and HPLC

a1) RP-8 (without Terbutryn and Prometryn): r = 0,996; KS = 0,11 + 0,75 KP

31 Rapid and reliable method transfer from TLC to Preparative Chromatography

Example: Triazines

Comparison of TLC and HPLC

a2) RP-select B: r = 0.996; KS = 0,03 + 0,75 KP

32 Rapid and reliable method transfer from TLC to Preparative Chromatography

Transfer of a real sample (salad)

Comparison of TLC and HPLC

HPTLC-Chromatogramm ( = 200 nm)

33 Rapid and reliable method transfer from TLC to Preparative Chromatography

HPLC-Chromatogramm ( = 200 nm)

Transposition, example
In ideal conditions, transposition from TLC to Flash Chromatography should give such results

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Rapid and reliable method transfer from TLC to Preparative Chromatography

Particle size comparisons at column Chromatography

Test silice Si60 40-63 m Test silice Si60 15-40 m
300000 250000 200000 150000 100000 50000

Test silice Si60 63-200 m

0
0 1 2 3 4 5 6 7 8 9 10 11 12 13

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Rapid and reliable method transfer from TLC to Preparative Chromatography

Conclusion
Method transfer from Thin-Layer-Chromatography to Preparative Chromatography

Easy transposition TLC / Flash/prep. HPLC.

TLC has very good purification efficiency. TLC show quicker purifications than Glass columns. Smaller fractions to evaporate.

Secure working area (no glass breakage risk).

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Rapid and reliable method transfer from TLC to Preparative Chromatography