22
Biotage
®
Flash 75/150 User Manual | © Biotage 2018
Optimizing Flash Separations
The following section is a summary of work completed by
Biotage application chemists in an effort to reduce the amount
of trial and error that is typically associated with optimizing
flash separations. The four guidelines to be addressed will
focus on the following issues:
1. Predicting column resolution using TLC.
2. Determining the best solvent strength for flash separations.
3. Determining the best solvent selectivity for flash separations.
4. Determining the optimum column size and sample load.
Column Separations are
Governed by ΔCV, Not ΔR
f
When transferring a separation from TLC to column
chromatography, the use of ΔR
f
as the primary factor to predict
the degree of resolution is not necessarily valid.
As shown in Figure 27, a good separation on a TLC plate does
not always translate into a good column separation. This is
due to the fact that R
f
values are inversely proportional to the
elution time of a component from a chromatography column
(see Table 5). Therefore, a better predictor of column behavior
is not ΔR
f
, but ΔCV
*
.
Generally, TLC conditions that will provide
R
f
values in the range of 0.15 to 0.40 should be chosen
, which
for column chromatography, would result in CVs of 2.5 to 6.7
†
.
Components having R
f
values above 0.40 will elute too early
(i.e. less than 2.5 CVs) whereas those with values much below
0.15 will consume excessive quantities of solvent.
The following sections address the two parameters used to
optimize ΔCV; solvent strength and solvent selectivity.
R
f
CV
R
f
CV
0.90
1.10
0.45
2.20
0.85
1.17
0.40
2.50
0.80
1.25
0.35
2.80
0.75
1.33
0.30
3.33
0.70
1.40
0.25
4.00
0.65
1.54
0.20
5.00
0.60
1.65
0.15
6.67
0.55
1.81
0.10
10.0
0.50
2.00
Table 5. Predicting elution behavior.
*
The difference in the number of column volumes in which the two
components elute.
†
Due to normal surface area and pore volume variability, CV values may
be different than the values predicted.
Vary Solvent Strength to Maintain
R
f
Values Between 0.15 and 0.40
Using the previous separation as a guide (see Figure 27), the
separation in Figure 28 shows that a significant improvement
was obtained by operating with a “slower” solvent system.
In this case, the separation was optimized by simply changing
the relative proportions of the strong and weak solvents.
Optimizing Flash Separations
So
lvent Fro
nt
Origin
Inject
Inject
So
lvent Fro
nt
Origin
CV
CV
Figure 27. Good separation on a TLC plate does not always translate into a good column separation.
So
lvent Fro
nt
Origin
Inject
Inject
So
lvent Fro
nt
Origin
CV
CV
Figure 28. Using a “slower” solvent system (B) can greatly improve the degree of separation.
R
f
A = 0.51
R
f
B = 0.39
ΔR
f
= 0.12
ΔCV = 0.6
Solvent system: 95 % toluene,
5% hexane
R
f
A = 0.32
R
f
B = 0.21
ΔR
f
= 0.11
ΔCV = 1.6
Solvent system: 70 % toluene,
30% hexane
Содержание Flash 150
Страница 1: ...Biotage Flash 75 150 User Manual ...
Страница 34: ...32 Biotage Flash 75 150 User Manual Biotage 2018 Notes Notes ...
Страница 35: ......