Flash Chromatography – Method Development

TLC method development

• Solvents classification: Snyder triangle

0.2 0.6

0.3

xd xe
proton donor proton acceptor
0.4

0.5 0.3

0.6 0.2
0.2 0.3 0.4
n x
dipole interaction
0.6 0.7
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 TLC method development

• Practical methodology for mobile phase determination

© Camag
1997

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 TLC method development

• Solution for plate conditioning with vapor phase: twin

through chambers ( + filter paper )

© Camag
1997

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 Transposition from TLC to flash
chromatography

• Transposition :
z sol u sol 1 1 Rf t r  t0
Rf    k with k
z front u 1 k Rf t0

• In practice :

1 1  dR f
dR f  k with d=1.5
1 k dR f

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 Transposition from TLC to flash
chromatography

CAMAG –Vario chamber

parallel test of 6 neat solvents
or solvent mixtures
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 Transposition, approach 1
(solvents screening J.Dingenen, Janssen Pharmaceutica)

1) 2 series of neat solvents covering a large domain of polarity and selectivity

Series 1 MTBE Toluene CH2Cl2 CH3CN EtAc THF

Series 2 MeOH EtOH IPA Acetic acid 2-propanone 2-butanone

2) Test series based on a methanol-dichloromethane mixture

N-Hexane 50%
Series 3 CH2Cl2 50 % MeOH 2% MeOH 5% MeOH 10% MeOH 20% MeOH 50%

Line 1 2 3 4 5 6

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 Transposition, approach 1
(solvents screening J.Dingenen, Janssen Pharmaceutica)

1) Good enough selectivity for one of the neat solvents or

one of the methanol-dichloromethane mixtures

Start tests on columns

2) Good enough selectivity for one of the neat solvents but too high Rf values
Addition of a non polar solvent to adjust the Rf values between 0.1 and 0.35
(corresponding to k values of 1.98 to 9)
3) Non satisfying results
Based on the observed spots shape during experiments with neat solvents,
combine the most appropriate solvents (binary or ternary mixtures) for some
more investigation

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 Transposition, approach 2
(ethyl acetate / hexane gradient ; HPLConsult, Dr. Thomas Maetzke)

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 onto column

regarding segment

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 Transposition, approach 2
(ethyl acetate / hexane gradient ; HPLConsult, Dr. Thomas Maetzke)

Transfer on LiChrospher Si 60, 12 µm (Selfpacker

column, length 125 mm, 25 or 50 mm I.D.)
(e ( at / T )  1)  (1  K )
Exponential gradient: c( EtAc)  K
e 1
a

T=gradient duration (min), K= c(EtAc) at t=0, exponential gradient parameter, t=time

Rf 0.05-0.25 0.25-0.60 0.60-0.90

a 7 6 8
K 0.15 0.09 0.05
T 8 6 6

Automated normal-phase preparative HPLC as a substitute for flash chromatography in the

synthetic research laboratory
P.Renold, E.Madero, Th.Maetzke, J.Chromatogr. 2001 (908), 143-148

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Transposition, approach 2
(ethyl acetate / hexane gradient ; HPLConsult, Dr. Thomas Maetzke)

organic modifier proportion

M.D. Palamareva and V.R. Meyer, J. Chromatogr. 641, 391, (1993)

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Transposition, approach 2
Reference system ; HPLConsult, Dr. Thomas Maetzke

ea / hex = 1:4
0.9
1.00
0.8

0.7 0.70 Bibenzyl 0.80

0.60 Ethylbenzoate
0.6

%B(C)
0.54 Dibutylphtalate 0.60

0.5 0.50 Acetophenone

G-1
0.43 Diethylphtalate 0.40
0.4
0.33 Dimethylphtalate G-2
G-3
0.3
0.20

0.2 0.22 Benzyl alcohol

0.00
0.1
0.1
0.7
1.3

1.9
2.5

3.1

4.3
4.9

5.5
6.1

6.7
7.3
7.9
8.5
9.1
9.7
3.7

10.3

11.5
12.1
12.7
10.9
t
0.05 Acetanilide

Ref1

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 Transposition, approach 3

• Determine the optimum TLC mobile phase in order to get

Rf values between 0.1 and 0.35 and Rf differences (Rf)
of at least 0.15 between 2 compounds.
• Determine the column dead volume. This volume
represents the necessary solvent volume to fill all the intra
and extra-particular spaces in a given column, it is usually
determined with injection of a non retained solute.
• The solute retention is measured in terms of Column
Volumes (CV) which represent the number of necessary
column volumes to elute the compound.

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 Transposition, approach 3

• The “ideal” Flash Chromatography system is the

one which elutes the compound of interest within
CV values of 3 to 6 and separates this compound
from the others with a CV difference (CV)
greater than 1.
• The relationship between CV and Rf values is
given by:
CV = 1 / Rf
CV = 1 / Rf1 - 1 / Rf2
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 Transposition, approach 3

• Due to the differences between TLC and Flash

Chromatography working conditions (mobile phase
velocity variation, binders, relative humidity,...), the
optimum TLC separation, conditions may not be directly
transposable to Flash Chromatography.
• The chromatographers should take care of some
parameters during transposition from TLC to Flash
Chromatography.

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 Transposition, approach 3

1. Use of identical stationnary phases chemistry (same manufacturer).

2. Optimize the TLC solvent mixtures in order to obtain Rf values in between
0.1 to 0.35 and Rf values greater than 0.20.
3. Do not forget that the lesser the compounds are retained in TLC the lower
the CV differences and charge capacity values shall be obtained in Flash
Chromatography.
Rf A Rf B Rf CV
0.51 0.39 0.12 0.60
0.32 0.21 0.11 1.60
0.22 0.14 0.08 2.60
4. Adjust the Flash Chromatography mobile phase selectivity and use a feed
solvent having less eluting power than the mobile phase itself.
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 Transposition, approach 4
• Control of TLC parameters:
– Mobile phase measured in volume/volume in order to be
consistent with the Flash Chromatography system mixer.
– TLC plate and chamber equilibrated with the mobile phase vapors
prior to separation (twin through chamber).
– Solvent front position at 1-4 cm of the plate edge.
– Sample application and solvent front positions shall be marked
with a black pencil.
• Recommendations :
– Use the same type of stationary phases (TLC and Flash
Chromatography)
– Use the same solvent for the mobile phase (TLC and Flash
Chromatography)

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 Transposition, approach 4

• If the compound Rf value is between 0.1 to 0.5, the

following formulas may be used to determine the number
of column volumes (CV) corresponding to the first and last
collected fractions:
First tube = (Rf-1 . 2 . Vc) / Vf
Last tube = (Rf-1 . 3 . Vc) / Vf
With :
Vc : column dead volume (volume for CV = 1)
Vf : collected volume in each tube
C.F. Poole, S.K. Poole, « Chromatography today », Elsevier Science Publishers B.V.,
649-667, (1991)
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 Transposition, approach 5
• Experimental approach, based on the distance covered by
the compound on the TLC plate.
– If Rf = 0.25, this means that the compound covered ¼ of the
distance covered by the mobile phase (from sample application
position).
– In a cartridge having the same length than the TLC plate, the
compound shall, under same elution conditions, cover ¼ of the
stationary phase bed length while a solvent volume of L..r² is
used, i.e. an elution volume of 4.L..r² for the compound.
– Taking into account the differences between TLC and Flash
Chromatography:

Ve = Vc / d.Rf
With Ve the elution volume, Vc the cartridge stationary phase
volume, and d the correction coefficient (d = 1.3 - 1.5).
© T. Poncet, emeritus chemist, Merck Chimie SAS
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 Transposition, load effects

• The TLC step

shall not only
serve for ideal
Rf values
determination,
it shall also
allow sample
feed
concentration
and volume
determination

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 Transposition, load effects

• The TLC step

shall not only
serve for ideal
Feed concentration : 385 mg/ml
Rf values Dilutions : 1/20 1/10 2/10 4/10 1/1
determination,
it shall also
allow sample
feed
concentration
and volume
determination

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 Transposition, load effects

Feed concentration 385 mg/ml Feed concentration 385 mg/ml

2 µl applied 3 µl applied

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 Transposition, load effects

• The TLC step shall not only serve for ideal Rf values
determination, it shall also allow sample feed
concentration and volume determination:
– Perform TLC feed tests:
• Take note of the ideal feed concentration solution dilution,
• Take note of the ideal feed concentration solution applied volume.
– Determine the ratio between the applied volume and the
stationary phase volume used for separation on the TLC plate.
– From this, deduct the ratio between the column feed volume and
the amount of silica contained in the column.

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 Transposition, load effects

• Example :
– 2 µl sample application on a TLC plate (silica bed thickness 0.25 mm)
– Maximum spot width, after elution: 5 mm
– Solvent front migration distance: 70 mm
• A volume of 0.025 x 0.5 x 7 = 0.0875 cm3 of the silica bed was used, the
applied volume represents 100 x 0.002 / 0.0875 = 2.3 % of the stationary
phase used volume (2.3 % v/v)
• Considering that the stationary phase mean specific weight is around 0.5,
the possible feed, in ml, represents in value 4.6 % (5 %) of the silica
amount in the cartridge. :
– In our example, the column feed volume is 1.5 ml for a 30 g column (for an
ideal TLC sample application volume of 2 µl)

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