CompoundPurificationFlashGuide SM
CompoundPurificationFlashGuide SM
CompoundPurificationFlashGuide SM
Fourth Edition
© 2003, 2005, 2008, 2010 Teledyne Isco, Inc. All rights reserved.
Printed in the United States of America
The material provided in this guide is from sources that are believed to be reliable.
Neither Teledyne Isco, Inc. nor any person acting on its behalf makes any warranty
with respect to accuracy, completeness, or usefulness of the material provided
herein. Teledyne Isco, Inc. shall not be liable for any damages that arise from the
use of the information provided herein.
Chapter 1
Introduction to Flash Chromatography
Chromatographic Purification in Organic Chemistry . . . . . . . 1
Chapter 2
Flash Chromatography Essentials
Compound Solubility. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Mobile Phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Mobile Phase Modifiers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Stationary Phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Using TLC to Predict Separation . . . . . . . . . . . . . . . . . . . . . . . . 8
Correlating TLC and Flash. . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Retention Factor and Column Volumes. . . . . . . . . . . . . . . . 8
Method Development Using TLC . . . . . . . . . . . . . . . . . . . . 10
TLC and Mobile Phase Techniques . . . . . . . . . . . . . . . . . . 13
Isocratic Elution. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Gradient Elution. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Stepped Gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Linear Gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Mixed Gradients. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Loading Capacity of Column . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Column Length Versus Resolution and Purity. . . . . . . . . . . . 26
Flash Column Packings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Particle Shape . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Particle Size. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Sample Loading Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Manual Glass Chromatography . . . . . . . . . . . . . . . . . . . . . 28
Automated Chromatography . . . . . . . . . . . . . . . . . . . . . . . 28
Chapter 3
From Traditional Glass Columns to
Automated Flash Chromatography
Manual Glass Column Chromatography . . . . . . . . . . . . . . . . . 35
Benefits of Automation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Column Packing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
Manually-packed Columns . . . . . . . . . . . . . . . . . . . . . . . . . . 38
Pre-packed Columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
TLC Plates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
High Performance Flash Chromatography . . . . . . . . . . . . . . . 44
Column Stacking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
Why spherical media?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
Higher Resolution with small spherical media . . . . . . . . . 48
Improved load capacity . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Faster purifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
Chapter 4
C18 Flash Chromatography
Overview of Reversed Phase Chromatography . . . . . . . . . . . 54
Normal Phase Silica. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
Reversed Phase Silica . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
C18 Method Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
Thin Layer Chromatography Plates . . . . . . . . . . . . . . . . . . 57
Using HPLC Systems to Generate Flash Methods . . . . . . . 58
Using the Flash Instrument for Method Development . . . 59
Loading Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
Column Care . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
Solvent Modifiers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
Low-solubility Polar Heterocycles . . . . . . . . . . . . . . . . . . . . . . 64
Primary Amines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Carbohydrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
Carboxylic Acids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
Ionic Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
RediSep Rf Gold High Performance C18 Columns . . . . . . . . . 70
RediSep Gold C18 Columns at High pH . . . . . . . . . . . . . . . . . . 73
Storage of the column after use in high pH . . . . . . . . . . . . 73
Chapter 5
Advanced Flash Chromatography
Alternative Chromatographic Media . . . . . . . . . . . . . . . . . . . . 75
Specialty Media. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
Amine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
Basic Alumina . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
Neutral Alumina . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
Cyano . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
Diol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
Ion Exchange Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
SCX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
SAX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
Natural Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
Cytotoxic Constituents from Butea superba . . . . . . . . . . 102
Alkaloids of Banisteria caapi . . . . . . . . . . . . . . . . . . . . . . . 103
Advanced Solvent Strategies . . . . . . . . . . . . . . . . . . . . . . . . . 105
Chapter 6
Detection Techniques
UV Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
Detection with UV-Vis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
All-Wavelength Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
Example with a compound mixture . . . . . . . . . . . . . . . . . 114
Example of unknown spectrum . . . . . . . . . . . . . . . . . . . . 114
Solvent spectrum overlaps compound . . . . . . . . . . . . . . 116
Sample overloads detector . . . . . . . . . . . . . . . . . . . . . . . . 117
Other Detectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
Appendix A
Column Media Selection
Media Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
Appendix B
Solvent and UV-Vis Wavelength
Selection Guide
Solvent Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
Wavelength Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
Appendix C
Theory & Application of Flash
Chromatography
Elementary theory. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
Appendix D
Troubleshooting LC Systems
Basic checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
List of Figures
1 Illustration of basic elements in a traditional Flash
column chromatography apparatus . . . . . . . . . . . . . . . . . . . . 2
2 Photo of TLC plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
3 Table of common solvents in liquid chromatography . . . . . 6
4 Illustration of mobile phase modifier . . . . . . . . . . . . . . . . . . . 7
5 Table of Rf to CV conversions . . . . . . . . . . . . . . . . . . . . . . . . . 9
6 Illustration of solvent strength optimization . . . . . . . . . . . . 10
7 Illustration of solvent system selectivity optimization. . . . 11
8 Illustration of a solvent system optimized for compound
selectivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
9 Table of suggested loading of RediSep Rf silica gel
columns based on Rf differences from TLC plates. . . . . . . . 12
10 Illustration of mobile phase techniques . . . . . . . . . . . . . . . . 13
11 Illustration of isocratic 20% EtOAc in hexane . . . . . . . . . . . 15
12 Illustration of isocratic 30% EtOAc in hexane . . . . . . . . . . . 15
13 Illustration of isocratic 40% EtOAc in hexane . . . . . . . . . . . 16
14 Illustration of isocratic 50% EtOAc in hexane . . . . . . . . . . . 16
15 Illustration of isocratic 70% EtOAc in hexane . . . . . . . . . . . 17
16 Illustration of a stepped gradient and chromatogram . . . . 19
17 Illustration of a linear gradient and chromatogram . . . . . . 20
18 Chromatograms resulting from various gradient slopes . . 21
19 Chromatograms of catechol and resorcinol separations . . 22
20 Illustration of CombiFlash Rf Gradient Optimizer and
resulting chromatogram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
21 Chromatogram indicating column loading capacity
near limit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
22 Chromatogram indicating column loading capacity
exceeded . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
23 Illustration of gradient mobile phase . . . . . . . . . . . . . . . . . . 25
24 Photos of particle shapes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
25 Illustration of sample injection on glass columns . . . . . . . . 29
26 Photo of syringe injection. . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
27 Photo of a solid sample load cartridge . . . . . . . . . . . . . . . . . 30
28 Photo of solid load cartridge connected to column . . . . . . 31
29 Chromatograms of syringe injection and dried solid
load cartridge techniques. . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
30 Photo of pre-packed cartridges . . . . . . . . . . . . . . . . . . . . . . . 33
31 Photo of dry loading sample onto the column. . . . . . . . . . . 33
32 Table of RediSep Rf solid load cartridges . . . . . . . . . . . . . . . 34
33 Photo of a manual Flash system in use . . . . . . . . . . . . . . . . . 35
34 Photo of Teledyne Isco’s CombiFlash Rf system . . . . . . . . . 37
35 Photo of glass column preparation . . . . . . . . . . . . . . . . . . . . 39
36 Chromatograms of compounds separated on a
hand-packed column . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Introduction to Flash
Chromatography
Compressed air
Solvent
(mobile phase)
Sand
Column media
Separated (stationary phase)
products
Frit
Tap
Empty Separated
collection fractions
tubes
Flash Chromatography
Essentials
Compound Solubility
The solubility of the crude products mixture to be separated is a
factor the organic chemist should consider when choosing the sol-
vent system mixture, or mobile phase.
A mobile phase with low polarity properties may precipitate oily
crude mixture products in the flask during dissolution prior to
loading the sample on the column, or after being loaded on top of
the column when the low polarity solvent mixture progression
starts.
To avoid having the sample precipitate unintentionally (or crash),
it is important to choose a solvent system polar enough to cover
both the solubility issue upon sample loading on column and the
maximized separation conditions obtained from thin-layer chroma-
tography (see Using TLC to Predict Separation, on page 8).
Should the sample precipitate in the flask prior to column loading
or be in an initial solid state, the solid loading technique is recom-
mended (see Solid sample loading, on page 28).
In the event the sample precipitates after being loaded onto the
column, increasing the polarity of the solvent system through gra-
dient solvent elution (see Gradient Elution, on page 17) would
eventually reach a solvent system mixture polar enough to solubi-
lize it. However, precipitated samples often raise the system
pressure thereby reducing the solvent flow. Higher pressure Flash
systems, such as the CombiFlash Rf with 200 psi capability, are
better able to push the solvent through making it easier to
increase the polarity. Once solubilized, the sample moves through
the stationary phase.
Mobile Phase
The solvent system or mobile phase choice for Flash chromatog-
raphy is dependent on the polarity of the product(s) to be isolated
and the type of stationary phase to be used.
Typically, the organic chemist will first proceed with a few TLC
analytical trials to determine which solvent system will provide
the optimal separation conditions with respect to the polarity of
the desired product(s) and the selected stationary phase.
The retention distance, Rf , on a TLC plate represents the distance a
given compound migrates from the origin with respect to the sol-
vent front on the plate. (See Method Development Using TLC, on
page 10.)
During the TLC analytical trials, the chemist will seek the solvent
system that moves the desired product to Rf =0.25±0.05 and keeps
other undesired products to a distance of at least ΔRf =0.2. These
TLC parameters constitute the ideal Flash chromatography condi-
tions because of high compound-stationary phase contact time
predisposing to high compound resolution during the column
separation.
Many organic solvents are available. Figure 3 lists commonly used
solvents. Figure 131 on page 135 lists additional solvents that may
be more suitable for specialized purifications.
The solvent system strength and selectivity refer respectively to
the solvent system’s ability to migrate all compounds simultane-
ously on the column (i.e. purification duration) and to migrate one
specific compound differently from the others (i.e. separation
resolution).
Typically, the solvent system is a binary mixture of a higher and a
lower strength (polarity) solvent. For instance, organic chemists
commonly initiate their solvent system evaluation and selection
Without With
Modifier Modifier
Solvent
Front
Base
Line
Stationary Phase
Stationary phase selection is driven by the nature of the products
to be separated. Factors such as compound polarity and function-
alities greatly influence the media selection.
The majority of reaction products organic chemists need to isolate
can be purified using a normal-phase or a reversed-phase silica gel
as the stationary phase.
For some specific types of compounds, however, it is difficult to
achieve an overall satisfactory degree of separation using these
common Flash chromatography stationary phases. The silica gel
suppliers have designed and marketed functionalized silica gel to
provide chemists additional purification media options. Thus,
organic chemists now have a wide range of purification tools avail-
able, which facilitates isolation of compounds with very different
physico-chemical properties.
Appendix A of this guide provides a stationary phase selection
guide and more information on media types.
Solvent
front
Rf 0.95
Rf 0.50
Rf 0.25
Base line
Optimal Rf
Solvent
front
ΔRf
Base line
Optimal
selectivity
ΔCV
Solvent
front 3
1 2
4
2 1
3
Base line 4
0 3 6 9 12
Column Volumes
Loading
Column size
(g silica) ΔRf < 0.2 0.2 – 0.4 0.4 – 0.6 > 0.6
4g 0.0004 – 0.004 0.004 – 0.16 0.16 – 0.28 0.28 – 0.4
(69-2203-304)
12 g 0.0012 – 0.012 0.012 – 0.48 0.48 – 0.84 0.84 – 1.2
(69-2203-312)
24 g 0.0024 – 0.024 0.024 – 0.96 0.96 – 1.68 1.68 – 2.4
(69-2203-324)
40 g 0.004 – 0.04 0.04 – 1.6 1.6 – 2.8 2.8 – 4
(69-2203-340)
80 g 0.008 – 0.08 0.08 – 3.2 3.2 – 5.6 5.6 – 8
(60-2203-380)
120 g 0.012 – 0.12 0.12 – 4.8 4.8 – 8.4 8.4 – 12
(69-2203-320)
125 g — — 5 – 8.75 8.75 – 12.5
(69-2203-314)
220 g 0.022 – 0.22 0.22 – 8.8 8.8 – 15.4 15.4 – 22
(69-2203-422)
330 g 0.033 – 0.33 0.33 – 13.2 13.2 – 23.1 23.1 – 33
(69-2203-330)
750 g 0.075 – 0.75 0.75 – 30 30 – 52.5 52.5 – 75
(69-2203-275)
1500 g 0.15 – 1.5 1.5 – 60 60 – 105 105 – 150
(69-2203-277)
Isocratic Stepped
Isocratic Elution
Most classical Flash chromatography uses an isocratic mobile
phase to separate compounds. In an isocratic separation, the
mobile phase may be a single solvent or a mixture, but the mobile
phase composition is the same throughout the separation.
TLC is an isocratic technique. Therefore, it can closely correlate to
isocratic separations scaled up to column chromatography.
An isocratic mobile phase can be optimized to purify nearly any
compound of interest. To ensure the separation is selective, the
chemist must control the isocratic conditions beyond just the
right solvent blend. Sample loading and column capacity also must
be closely controlled. But in the end, these efforts yield a special-
ized method that will not separate a wide variety of compounds.
Column capacity is typically limited when using isocratic mobile
phases. If the sample size is increased too much, the mixture’s
compounds will contaminate each other.
Figures 11 through 15 illustrate tests performed to optimize an iso-
cratic mobile phase. In this example, Sample A, a blend of
acetophenone (1), methyl paraben (2), and 4-aminobenzoic acid
(3) is separated using 20, 30, 40, 50, and 70% EtOAc and Hexane.
When reviewing the results of this TLC series, we learn that for the
purification of acetophenone 20% EtOAc is best. For methyl
paraben 20 to 30% EtOAc is best, and for the purification of 4-ami-
nobenzoic acid, 40 to 50% of EtOAc is best.
Solvent
front 1
Rf ~.7 1
2
2
3
Baseline
0 1 2 3 4 5 6 7 8 9 10
Column Volumes
Rf ~.9 1 1
Rf ~.1 3 3
0 1 2 3 4 5 6 7 8 9 10
Column Volumes
1
1
2
3
2
0 1 2 3 4 5 6 7 8 9 10
Column Volumes
1
1
2
3
2
0 1 2 3 4 5 6 7 8 9 10
Column Volumes
0 1 2 3 4 5 6 7 8 9 10
Column Volumes
Figure 15: Illustration of isocratic 70% EtOAc in hexane
Nothing is resolved at 70%.
Gradient Elution
Gradient elution describes techniques for decreasing overall purifi-
cation time, increasing resolution, and increasing column capacity
by varying the mobile phase composition during the chromato-
graphic separation. Gradient methods include both stepwise and
continuous changes in the solvent blend, with linear or
straight-line gradients being the most common form of continuous
gradients. A binary gradient is one in which the ratio of two sol-
vents (or solvent mixtures) is varied during the separation.
Ternary (3-solvent) and quaternary (4-solvent) gradients are also
used in some cases.
It is extremely powerful to have fully programmable control over
the mobile phase components during the course of a separation.
This capability allows you to tailor the resolving power for a par-
ticular set of species that need to be separated on a
chromatographic column.
Until the recent development of automated Flash chromatography
systems, the power of programmable gradients was not readily
available to organic chemists. Gradients are a means of controlling
resolution. By adroit use of gradients, closely eluting compounds
may be separated while compounds with long retention times
(they may be thought of having highly excess resolution) can be
run with reduced time and solvent.
Stepped Gradient
Stepped gradients are a classical technique used in Flash chroma-
tography. The solvent system is a blend of solvents. Several
different blends are prepared at increasingly polar solvent
strengths.
In the same way an optimal solvent is chosen for an isocratic sepa-
ration, optimal solvent blends for stepped gradients are identified
through TLC trials. The goal of the TLC trials is to determine a
blend that moves the compound of interest.
These blends are introduced onto the Flash column in turn. The
solvent strength is increased only after the previous compound
has separated, greatly improving selectivity. As a result, column
capacity can be increased.
Referring back to our example separating Sample A, Figure 16 illus-
trates a stepped gradient developed from the analytical TLC trials.
A stepped gradient starting at 20% EtOAc and moving to 40% after
4 column volumes will allow the separation of the three com-
pounds in a single run.
80% 1
60%
Gradient
40%
2
20% 3
0 1 2 3 4 5 6 7 8 9 10
Column Volumes
Linear Gradient
Linear gradients begin with a low-strength solvent blend. The gra-
dient is advanced in infinitesimal steps (limited by the resolution
of the solvent pumping system) until the separation ends at a
high-strength solvent blend.
With isocratic and stepped gradients, it is very important that a
sufficient number of TLCs be performed to determine just the right
solvent blend. In the case of linear gradients, this requirement is
reduced because the gradient that is best for separation of the
compounds is by default provided to the column. This is because
at one point along the gradient profile, the best solvent blend for
separation of the compounds is delivered to the column.
To continue the example, TLC determined that the ideal concentra-
tion of the solvents is between 20 and 40%. To determine this with
confidence it required that 20, 30, 40, 50, and possibly 70% blends
be prepared and evaluated for separation effectiveness. This is
because it is difficult to know at the outset what concentrations
will bracket ideal conditions.
However, when a linear gradient is used, since it starts at a concen-
tration that is lower than the optimal and increases to a
concentration that exceeds the optimal level, it is not necessary to
perform as many TLCs.
file
80% P ro
1
e nt
di
60% ra
G
Gradient
40%
2
20% 3
0 1 2 3 4 5 6 7 8 9 10
Column Volumes
1 CV
2.5 CV
5 CV
10 CV
20 CV
0 5 10 15
Minutes
Figure 18: Chromatograms resulting from various gradient
slopes The duration of the gradient can be manipulated to
optimize the purity required while minimizing the time needed to
complete the procedure.
Mixed Gradients
Mixed gradients are a combination of step and linear gradients.
These gradients are used to reduce run time while maintaining a
separation between closely eluting components. A linear gradient
is started and an isocratic hold is employed during the gradient to
maintain the resolution between closely eluting compounds.
Figure 19 shows catechol and resorcinol purified under isocratic
conditions and a linear gradient.
80%
B Solvent Strength
60% Isocratic
40%
20%
0 2 4 6 8 10 12 14 16 18 20
Column Volumes
80%
B Solvent Strength
60% Linear
40%
20%
0 5 10 15 20 25 30 35 40 45 50 55
Column Volumes
Figure 19: Chromatograms of catechol and resorcinol
separations using isocratic and linear mobile phases
Under isocratic conditions, the peaks are broad and run together.
The linear gradient, while sharpening the peaks, also causes
overlap. Reducing the slope of the gradient would separate the
peaks but they would also be broadened so there is still overlap
between the peaks. Combining a linear gradient with an isocratic
hold generates the chromatogram in Figure 20 where nearly com-
plete resolution is achieved between the two diols.
PeakTrak® software on CombiFlash systems makes it very easy to
create these gradients with just two TLC plates. The retention fac-
tors of the compound of interest and the closest impurity are
entered into the PeakTrak’s Gradient Optimizer window which
then calculates the optimal combination of linear gradient and iso-
cratic hold prior to elution of the compounds to give the best
separation.
80%
B Solvent Strength
60%
40%
20%
0 10 20 30 40 50 60 70
Minutes
0 1 2 3 4 5 6 7 8 9 10
Column Volumes
0 1 2 3 4 5 6 7 8 9 10
Column Volumes
80% 1
60%
Gradient
40%
2
20% 3
0 1 2 3 4 5 6 7 8 9 10
Column Volumes
Particle Shape
Silica is manufactured in either irregular or spherical particles
(Figure 24).
Irregular silica is produced as a large block of amorphous silica
which is ground and then sieved to produce different particle size
ranges.
Particle Size
In liquid chromatography, the smaller the particle size of the
column packing leads to greater plate count1. However, as particle
size decreases the back pressure increases. Typical Flash grade,
irregular silica is classified as 40–63 µm or 230 to 400 mesh which
refers to the sieves sizes used to produce that particle distribu-
tion. This particle size provided adequate resolution while creating
low back pressure so gravity and air pressure could produce a sep-
aration with glass columns.
Reducing the particle size generates greater back pressure due to
the viscosity of the solvent. Reversed phase solvents generally
have a higher viscosity, further increasing the back pressure. Irreg-
ular particles of the same specified size range typically have more
fine particles (>10 µm) in the mixture. Because of the manufac-
turing and handling processes, spherical media has fewer fine
particles than irregular of the same particle distribution, resulting
in lower back pressure than irregular particles. Typical Flash col-
umns of 40–63 µm will create back pressure of around 15–20 psi
with normal phase solvents and 40–60 psi with reverse phase sol-
vents (without consideration of sample interaction.)
Automated Chromatography
Syringe injection — Syringe injection (Figure 26) is a very
common technique as it is very simple and convenient. It also
allows equilibration of the column for improved separation.
Syringe injection requires that the compounds are soluble in
mobile phase at beginning of gradient.
Liquid Solid
sample sample
Sample coated
Sand on silica gel
Silica gel
Frit
O
Cl
O
HO N NH2
methyl-4-hydroxybenzoate (A) 2-amino-5-chloropyridine (B)
Syringe Injection
1.0 100%
B Solvent Strength
Absorbance
λ=280nm
0%
0 2 4 6 8 10
Minutes
Sample dissolved in 2mL of acetone loaded injected onto a
silica column and followed by a 1mL acetone chase. Acetone
used as a strong solvent to completely dissolve the sample.
Solvent system hexane/ethyl acetate.
0%
0 2 4 6 8 10
Minutes
Sample dissolved in 2mL of acetone and loaded in a 5g
pre-packed normal phase silica cartridge. Acetone removed
with a cartridge dryer.
Prepacked Cartridges
69-3873-238 Sample load prepacked silica gel Rf cartridges, 2.5 gram, pkg. of 20.
69-3873-236 Sample load prepacked silica gel Rf cartridges, 5 gram, pkg. of 20.
69-3873-243 Sample load prepacked silica gel Rf cartridges, 12 gram, pkg. of 15.
69-3873-241 Sample load prepacked silica gel Rf cartridges, 25 gram, pkg. of 15.
69-3873-310 Sample load prepacked silica gel Rf cartridges, 32 gram, pkg. of 12.
69-3873-226 Sample load prepacked silica gel Rf cartridges, 65 gram, pkg. of 12.
69-3873-311 Sample load, prepacked silica gel cartridges 125 grams, pkg. of 4.
68-3873-202 Sample load, prepacked silica gel cartridges 260 grams, pkg. of 4.
69-3873-254 Sample load, prepacked silica gel cartridge. For use on the CombiFlash Tor-
rent and Companion XL systems only.
69-3873-255 Sample load, prepacked silica gel cartridge. For use on the CombiFlash Tor-
rent and Companion XL systems only.
69-3873-247 Sample load, prepacked, C18 Rf cartridges, 2.5 gram, pkg. of 5.
69-3873-237 Sample load, prepacked, C18 Rf cartridges, 5 gram, pkg. of 5.
69-3873-248 Sample load, prepacked, C18 Rf cartridges, 12 gram, pkg. of 4.
69-3873-242 Sample load, prepacked, C18 Rf cartridges, 25 gram, pkg. of 4.
69-3873-249 Sample load, prepacked, C18 Rf cartridges, 32 gram, pkg. of 3.
69-3873-250 Sample load, prepacked, C18 Rf cartridges, 65 gram, pkg. of 3.
69-3873-312 Sample load, prepacked, Celite® Rf cartridges, 2.5 gram, pkg. of 20.
69-3873-313 Sample load, prepacked, Celite® Rf cartridges, 5 gram, pkg. of 20.
69-3873-314 Sample load, prepacked, Celite® Rf cartridges, 12 gram, pkg. of 15.
69-3873-315 Sample load, prepacked, Celite® Rf cartridges, 25 gram, pkg. of 15.
69-3873-318 Sample load, prepacked, Celite® Rf cartridges, 32 gram, pkg. of 12.
69-3873-319 Sample load, prepacked, Celite® Rf cartridges, 65 gram, pkg. of 12.
Empty Cartridges
69-3873-235 Sample load, empty Rf cartridges (holds up to 5 gram), package of 30.
69-3873-240 Sample load, empty Rf cartridges (holds up to 25 gram) pkg. of 30.
69-3873-225 Sample load, empty Rf cartridges (holds up to 65 gram) pkg. of 12.
69-3873-201 Sample load, empty cartridges (holds up to 260 grams), pkg. of 6.
Benefits of Automation
For instance, the CombiFlash® Rf system offered by Teledyne Isco
provides automated Flash chromatography that is user-friendly
and reliable, without the downsides of manual glass Flash
chromatography.
Column Packing
Manually-packed Columns
Manual column packing can be used to load specialized column
media. However, commonly-used media (listed in Appendix A) are
readily available as pre-packed columns in a variety of sizes.
Pre-packed Columns
Pre-packed columns improve the efficiency of compound purifica-
tion, offering greater productivity and reproducibility.
By using pre-packed columns, scientists can purify compounds
more quickly because they save the time required to pack the
column. Pre-packed columns also show higher purification effi-
ciency since the silica is more tightly packed allowing the
compounds more interaction with the stationary phase (see
Figure 36)
1.0
0.8 Hand-packed
Absorbance
0.6
0.4
0.2
0.0
0 2 4 6 8 10 12 14 16
Minutes
2.0
1.6 Pre-packed
Absorbance
1.2
0.8
0.4
0.0
0 2 4 6 8 10 12 14 16
Minutes
Figure 36: Chromatograms of compounds separated on a
hand-packed column (top) and pre-packed RediSep Rf
column (bottom). The prepacked column shows near baseline
separation and would result in pure fractions. The hand-packed
column gives mixed fractions that would need to be run again at
additional time to improve product yield.
TLC Plates
RediSep TLC plates are useful to run compounds to determine
optimal purification conditions or to confirm purity at the end of a
run. The plates are small enough to run quickly yet long enough to
allow accurate measurement of Rf differences for programming the
Gradient Optimizer. RediSep TLC plates are available in a variety of
materials to match your purification needs.
Figure 38: Photo of matching TLC media TLC media can be prepared
to match the performance characteristics of a column. Matched
media ensures that favorable analytical TLC results can be
developed into effective column purification methods.
Solvent Migration
Isopropanol Full, 45 min
Water Plate degrades
Methanol Full, 15 min
Water/Methanol 1:1 Full, 45 min
Acetonitrile (ACN) Full, 15 min
Water/ACN 1:1 Full, 45 min
Water/ACN 6:1 75%, 45 min
Water/ACN 7:1 60%, 45 min
Water/ACN 8:1 20%, 45 min
Water/ACN 9:1 10%, 45 min
Solvent Migration
Hexane Full, 15 min
Ethyl acetate Full, 15 min
Isopropanol Full, 30 min
Hexane/Ethyl acetate 1:1 Full, 15 min
Hexane/Isopropanol 5:1 Full, 30 min
Dichloromethane Full, 15 min
Methanol Full, 15 min
Dichloromethane/Methanol 9:1 Full, 15 min
0.6 min
1 column
1.2 min
2 columns
1.8 min
3 columns
2.3 min
4 columns
3.0 min
5 columns
3.5 min
6 columns
4.1 min
7 columns
4.9 min
8 columns
5.7 min
9 columns
0 2 4 6 8 10 12 14 16
Run Time (min)
2 4 6 8 10 12 14 16 2 4 6 8 10 12 14 16
Run Time (min) Run Time (min)
0.4
Absorbance
0.3
0.2
0.1
0.0
0 5 10 15 20 25 30
Minutes
0.4
Absorbance
0.3
0.2
0.1
0.0
0 5 10 15
Minutes
Figure 44: Chromatogram of two 24 g stacked columns
compared to a chromatogram of a single 40 g RediSep Rf
Gold column
Absorbance
%B Solvent
R CH3
Peak 2
R
CH2Br
Peak 3 0.0 0
Run Conditions:
Column size: 40 g RediSep Rf Gold silica
Load: 500 mg (on 3.0 100
5 g cartridge)
%B Solvent
Solvents: Hexane and
Ethyl Acetate
Gradient: 0–20%
Flow rate: 40 mL/min
Run time: 15 min.
0.0 0
Wavelength: 254 nm
0 5 10 15
Minutes
Figure 45: Chromatogram of bromotoluenes purification A 1%
sample load of a bromotoluene mixture on an irregular shaped,
larger particle media (RediSep column, top) and a spherical
shaped, smaller particle media (RediSep Rf Gold, bottom)
RediSep Rf silica
2.5 100
Absorbance
%B Solvent
0.0 0
Run Conditions:
Column size: 40 g RediSep Rf Gold silica
Load: 500 mg 2.5 100
(1.1% load)
%B Solvent
Solvents: Hexane and
Ethyl Acetate
Gradient: 0–20%
Flow rate: 40 mL/min
Run time: 19 min.
0.0 0
Wavelength: 254 nm
0 5 10 15 18
Minutes
Figure 46: Chromatogram of minor compound separation
of a proprietary sample using an irregular shaped, larger particle
media (RediSep column, top) and a spherical shaped, smaller
particle media (RediSep Rf Gold, bottom).
50
Irregular silica, peak 1
20
10
0
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
Peak Width (min)
50
Run Conditions:
Column size: 12 g Irregular silica, peak 2
Sample Load (mL)
40
Solvent: Isocratic Spherical silica, peak 2
EtOC:hexane 30
(10:90)
Flow rate: 30 mL/min 20
0
0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4
Peak Width (min)
NO 2 NO 2
+ CN
CN
NH 2 N
H
Standard Flash Column
2.0 100
19 minutes
Run Conditions: 1.1 L solvent used
UV Absorbance
Solvent %B
Column size: 40 g
Load: 400 mg
Solvent: Hexane:ACN
Flow rate: 40 mL/min
Run time: 19 min.
0 0
Wavelength: 229 nm
0 5 10 15
Run Time (min)
0.4
B
A
AU
Analytical HPLC
A: Acetone
B: peak 2 compound 0
40
Solvent %B
Load: 400 mg
Solvent: Hexane:ACN
Flow rate: 80 mL/min
Run time: 5 min.
Wavelength: 229 nm
0 0
0 1 2 3 4 5
Run Time (min)
0.4
A
B
Analytical HPLC
A: Acetone
B: peak 2 compound 0
40
Figure 48: Chromatograms of 3-(2-nitrophenyl amino)
propionitrile purifications compared to standard Flash
grade silica, the RediSep Rf Gold column demonstrated 60% time
savings, 30% solvent savings, with no loss in purity.
Faster purifications
The higher resolution of RediSep Rf gold columns can be used to
more quickly purify compounds saving time and solvent. This is
accomplished by increasing the gradient; in some cases the flow
rate can be increased as well. Using CombiFlash Rf systems, the
fast parameters are optimized as a “Gold Speed” method.
To use Gold Speed, the retention factor difference between the two
compounds of interest should be greater than 0.1.
The resolution of the spherical column under Gold Speed condi-
tions is equivalent to that of a standard Flash column run under
standard conditions, but less solvent was used (Figure 48). Since
the peaks are much narrower, there is less solvent collected with
the peaks reducing the time required to dry the fractions. Gold
Speed also allows compounds sensitive to silica gel to be purified
since the compound has less contact time on the silica.
Figure 49: Table of RediSep Rf Gold Silica Gel Disposable Flash Col-
umns, spherical, 20–40 microns
Column Teledyne
Sample Size Isco Part
Size (g) Number Description
20 mg - 0.4 g 4 69-2203-344 4 gram RediSep Rf Gold Silica Gel Disposable
columns, pkg. of 14.
60 mg - 1.2 g 12 69-2203-345 12 gram RediSep Rf Gold Silica Gel Disposable
columns, pkg. of 14.
120 mg - 2.4 g 24 69-2203-346 24 gram RediSep Rf Gold Silica Gel Disposable
columns, pkg. of 10.
200 mg - 4 g 40 69-2203-347 40 gram RediSep Rf Gold Silica Gel Disposable
columns, pkg. of 10.
400 mg - 8 g 80 69-2203-348 80 gram, RediSep Rf Gold Silica Gel Dispos-
able columns, pkg. of 6.
600 mg - 12 g 120 69-2203-349 120 gram, RediSep Rf Gold Silica Gel Dispos-
able columns, pkg. of 6.
1.1 - 22 g 220 69-2203-359 220 gram, RediSep Rf Gold Silica Gel Dispos-
able columns, pkg. of 4.
1.65 - 33 g 330 69-2203-369 330 gram, RediSep Rf Gold Silica Gel Dispos-
able columns, pkg. of 3.
3.8 - 75 g 750 69-2203-427 750 gram, RediSep Rf Gold Silica Gel Dispos-
able columns, pkg. of 3.
7.5 - 150 g 1500 69-2203-428 1500 gram, RediSep Rf Gold Silica Gel Dispos-
able columns, pkg. of 2.
15 - 300g 3000 69-2203-529 3000 gram, RediSep Rf Gold Silica Gel Dispos-
able column, pkg. of 1.
C18 Flash
Chromatography
O O
Si O Si OH
O O
Si O Si OH
O O
Si O Si OH
O O
Figure 50: Diagram of normal phase silica Bare silica allows silanol
groups to form on the exposed surfaces.
O O
Si O Si ( )n CH3
O O
Si O Si ( )n CH3
O O
Si O Si ( )n CH3
O O
Column Teledyne
Sample Size Isco Part Reverse Phase (C18) RediSep Rf
Size (g) Number Column Description
4.3 - 86 mg 4.3 69-2203-410 4.3 gram columns, pkg. of 2.
13 - 260 mg 13 69-2203-411 13 gram column, pkg. of 1.
26 - 520 mg 26 69-2203-412 26 gram column, pkg. of 1.
43 - 860 mg 43 69-2203-413 43 gram column, pkg. of 1.
86 mg - 1.72 g 86 69-2203-416 86 gram column, pkg. of 1.
130 mg - 2.6 g 130 69-2203-414 130 gram column, pkg. of 1.
240 mg - 4.8 g 240 69-2203-418 240 gram column, pkg. of 1.
360 mg - 7.2 g 360 69-2203-415 360 gram column, pkg. of 1.
Solvent Migration
Isopropanol Full, 45 min
Water Plate degrades
Methanol Full, 15 min
Water/Methanol 1:1 Full, 45 min
Acetonitrile (ACN) Full, 15 min
Water/ACN 1:1 Full, 45 min
Water/ACN 6:1 75%, 45 min
Water/ACN 7:1 60%, 45 min
Water/ACN 8:1 20%, 45 min
Water/ACN 9:1 10%, 45 min
CO2H
Gallic Acid Pyrogallol
(A) (B)
HO OH HO OH
OH OH
0.30 60
Absorbance
0.25 50
(B)
0.20 40
0.15 30
0.10
(A) 20
0.05 10
0.00 0
0 2 4 6 8 10 12 14 16
Minutes
The first step is to run a gradient and determine where the com-
pound of interest elutes. Adjust the gradient so the desired
compound is resolved from contaminants. The next step is to
transfer the method to the Flash system. Figure 54 shows gallic
acid and pyrogallol developed on an HPLC system and transferred
to Flash. The only change was to lengthen the run time, which “flat-
tened” the gradient to allow better resolution.
The compounds eluted at three to four minutes in the analytical
method. When this method was transferred to the Flash system,
the compounds eluted at the same time as the HPLC. Starting the
gradient at 0% acetonitrile gave baseline separation while allowing
a higher loading of 10 mg. Using an analytical HPLC allows
methods to be easily developed with minimal compound use.
OH OH
0.40 80
0.35 70
(B)
Gradient % Solvent B
0.30 60
Absorbance
0.25 50
0.20 40
0.15 30
0.10 20
0.00 0
0.45 90
0 2 4 6 8 10 12 14 16
Minutes 80
0.40
0.35 70
Gradient % Solvent B
0.30 60
(A)
Absorbance
0.25 (B) 50
0.20 40
0.15 30
0.10 20
0.05 10
0.00 0
0 2 4 6 8 10 12 14 16
Minutes
Loading Compounds
Loading techniques for C18 are similar to those for silica. The com-
pound can be liquid-loaded onto the column. Alternatively,
compounds may be adsorbed onto an inert material such as celite.
The compound may also be loaded onto C18 solid load cartridges.
If C18 cartridges are used they should be conditioned by washing
them with the “B” solvent to be used in the separation (typically
methanol or acetonitrile) followed by the “A” solvent. This
double-wash step “raises” the C18 chain from the silica bed so it
can interact with the sample. The compound can be loaded in the
same fashion as done with a Solid Phase Extraction (SPE)
cartridge.
If loose C18 is used, the compound should be dissolved in an
organic solvent prior to adding the C18 and drying. This will acti-
vate the C18. The compound adsorbed on the C18 can be added to
an empty RediSep Rf solid load cartridge.
Using C18 as an adsorbent (either loose or in a pre-packed car-
tridge) also acts as a guard column that protects the main column.
Column Care
With proper care, RediSep Rf C18 columns may be used for over 20
purifications. RediSep Rf C18 columns are shipped dry-packed;
before its first run, the C18 column must be washed with at least 6
column volumes of water:organic solvent prior to use. The organic
solvent is typically the “B” solvent that will be used for the separa-
tion. The minimum concentration of organic solvent is 1:1; higher
concentrations of organic solvent are generally better. Once
wetted, a C18 column should never be allowed to dry out or chan-
nels will form that will adversely affect future separations.
After initial conditioning, use three column volumes of the initial
solvent conditions prior to the run to equilibrate the column. After
the run is complete, make sure the system does not purge the sol-
vent from the column (change the method to turn off “Air Purge”
prior to the run if needed). If using a CombiFlash Rf system with
RediSep Rf columns, the column’s RFID tag will tell the system not
to purge the solvent.
Solvent Modifiers
The most common solvent modifier used for reversed phase is TFA
(TriFluoro acetic Acid). TFA is compatible with the stainless steel
fittings used in HPLC and CombiFlash systems. Use of a pH above
7.5 to 8 causes the silica gel supporting the C18 to dissolve; TFA
keeps the solvent system at a low pH. TFA can also be easily
removed by lyophilization. RediSep Rf Gold columns have demon-
strated the ability to perform multiple runs at pH 10 (page 73).
At neutral pH, acids and bases may form their conjugates and
appear as two peaks or as a broad peak. Figures 56 and 57 illus-
trate this effect and how the use of TFA forces these compounds
into a single peak.
When diphenyl acetic acid is purified from esculin (Figure 57)
without TFA, the diphenyl acetic acid shows as two peaks at 3.5
and 9 column volumes. The chromatogram appears as if it has con-
tains three compounds with the first tailing into the esculin peak.
One hint that the first peak is diphenyl acetic acid is that it tails
back while the peak at 6 column volumes tails forward due to the
conversion between diphenyl acetic acid and its conjugate base. In
more extreme examples, the two peaks will appear to be joined
together by the tailing (“bridging”).
Solvent modifiers are not required when purifying neutral
compounds.
O O
OH O
Without TFA
2.00 100
1.80 90
1.60 80
1.40 70
Gradient % Solvent B
1.20 60
Absorbance
1.00 50
0.80 40
0.60 30
0.40 20
0.20 10
0.00 0
0 2 4 6 8 10 12 14 16
Run Length 16.0 CV (28.3 min)
With TFA
2.00 100
1.80 90
1.60 80
1.40 70
Gradient % Solvent B
1.20 60
Absorbance
1.00 50
0.80 40
0.60 30
0.40 20
0.20 10
0.00 0
0 2 4 6 8 10 12 14 16
Run Length 16.0 CV (28.3 min)
O
CN
N
NH2
N N
(C)
H2N
0.50 100
0.45 90
0.40 80
0.35 70
Gradient % Solvent B
0.30 60
Absorbance
0.25 50
0.20 (C) 40
0.15 30
0.10 (B) 20
(A)
0.05 10
0.00 0
0 10 20 30 40 50
Column Volumes
Primary Amines
The separation of a mixture of primary amines was investigated:
(A) (B) (C) NH2
N
N
N
N NH2 N NH2
(D)
N NH2
N
1.00 100
0.90 90
0.80
(C) 80
0.70 70
Gradient % Solvent B
0.60 60
Absorbance
0.50 50
(B)
0.40 40
(A) 30
0.30
0.20 (D) 20
0.10 10
0.00 0
0 20 40 60 80 100
Column Volumes
Carbohydrates
The separation of a mixture of carbohydrates was investigated:
OH
(A)
O
HO
HO
OH
(B) O
OH
HO O O
O
HO
HO
OH
O
NO2
0.4 (A)
0.3
Absorbance (214 nm)
(B)
0.2
0.1
0.00
0 10 20 30
Run Time (min)
Peptides
The separation of a mixture of peptides was investigated:
Gly-Pro-Ala (A) Val-Tyr-Val (B)
1.00 100
0.90 90
0.80 80
0.70 (A) 70
Gradient % Solvent B
0.60 60
Absorbance
0.50 50
0.40 40
0.30 (B) 30
0.20 20
0.10 10
0.00 0
0 5 10 15 20 25
Column Volumes
Carboxylic Acids
The separation of a mixture of shikimic acid (A) and maleic acid
(B) was investigated:
COOH
H2OC CO2H
(B)
HO OH
OH
(A)
1.00 100
0.90 90
0.80 (B) 80
0.70 70
Gradient % Solvent B
0.60 60
Absorbance
0.50 50
0.40
(A) 40
0.30 30
0.20 20
0.10 10
0.00 0
0 5 10 15 20 25 30 35 40 45 50
Column Volumes
Ionic Compounds
The separation of a mixture of pyrrolidinium (A) and piperidinium
chlorides (B) was investigated:
Cl Cl
N N
N
Cl
NO2 NO2
NO2
(A) (B)
0.50 100
0.45 90
(A)
0.40 80
0.35 70
Gradient % Solvent B
0.30 60
Absorbance
0.25 50
0.20
(B) 40
0.15 30
0.10 20
0.05 10
0.00 0
0 5 10 15 20 25 30
Column Volumes
O O
H
N
OH
O
N
N
O
100
0.80
Absorbance
%B Solvent
0 0
0 10 20 30 40 50 CV
Figure 64: Chromatogram of 10 mg compound A purification on
a 13g RediSep Rf C18 column
1.50 100
Absorbance
%B Solvent
0.75
0 0
0 10 20 30 40 50 CV
Figure 65: Chromatogram of 46 mg compound A purification on
a 15.5g RediSep Rf Gold C18 column
100
Absorbance
%B Solvent
0
Time
Figure 66: Chromatogram of 10 mg compound A purification on a
Waters DeltaPrep 4000 system using a Vydac 10x250 mm
column, 5µ particle size
23.186
STOP
23.005
STOP
Advanced Flash
Chromatography
Specialty Media
Some specialty media have the carbon tether chain “end-capped”
with a functional group conferring the stationary phase specific
properties useful for some delicate separations.
O O
Si O Si ( )n FG
O O
Si O Si ( )n FG
O O
Si O Si ( )n FG FG = NH2
= CN
O O =...
Amine
Compounds that have an acidic or basic moiety may streak or tail
with normal or reversed phase silica. Streaking or tailing will ulti-
mately cause overlapping fractions.
Typically, when using silica, chemists spike their solvents with
either triethylamine (TEA) if they have a basic component, or
acetic acid (AcOH) if they have an acidic component in their target
compound. The problem here is that solvents have to be swapped
and primed before compounds can be separated, then purged from
the system after the run.
With an acid or base moiety covalently bound to the stationary
phase, the need to switch solvents is eliminated. Additionally, TEA
or AcOH are not used so they don’t need to be removed after the
purification is complete.
Si NH2
(A) N
N
N (B)
2 3 45
0.50 100
0.45 90
0.40 80
Gradient % Solvent B
0.30 60
0.25 50
0.20 40
0.15 (A+B) 30
0.10 20
0.05 10
0.00 0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Column Volumes
0.50 6 7 8 9 10 11 100
0.45 90
0.40 80
0.35
(A) 70
Absorbance (AU)
Gradient % Solvent B
0.30 60
0.25 50
0.20 40
(B)
0.15 30
0.10 20
0.05 10
0.00 0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Column Volumes
Column Teledyne
Sample Size Isco Part
Size (g) Number Description
23 - 235 mg 4.7 69-2203-350 4.7 gram Amine RediSep Rf columns, pkg. of 2.
70 mg - 0.7 g 14 69-2203-351 14 gram Amine RediSep Rf column, pkg. of 1.
140 mg - 1.4 g 28 69-2203-352 28 gram Amine RediSep Rf column, pkg. of 1.
235 mg - 2.35 g 47 69-2203-353 47 gram Amine RediSep Rf column, pkg. of 1.
470 mg - 4.7 g 94 69-2203-356 94 gram Amine RediSep Rf column, pkg. of 1.
0.7 - 7 g 140 69-2203-354 140 gram Amine RediSep Rf column, pkg of 1.
1.3 - 12.8 g 260 69-2203-358 260 gram Amine RediSep Rf column, pkg. of 1.
1.92 - 19.25 g 385 69-2203-355 385 gram Amine RediSep Rf column, pkg. of 1.
Column Teledyne
Sample Size Isco Part
Size (g) Number Description
5.5 - 110 mg 5.5 69-2203-504 5.5 gram Amine RediSep Rf Gold columns,
pkg. of 2.
15.5 - 310 mg 15.5 69-2203-505 15.5 gram Amine RediSep Rf Gold column,
pkg. of 1.
30 - 600 mg 30 69-2203-506 30 gram Amine RediSep Rf Gold column,
pkg. of 1.
50 mg - 1.0 g 50 69-2203-507 50 gram Amine RediSep Rf Gold column,
pkg. of 1.
100 mg - 2 g 100 69-2203-508 100 gram Amine RediSep Rf Gold column,
pkg. of 1.
150 mg - 3 g 150 69-2203-509 150 gram Amine RediSep Rf Gold column,
pkg. of 1.
275 mg - 5.5 g 275 69-2203-510 275 gram Amine RediSep Rf Gold column,
pkg. of 1.
415 mg - 8.3 g 415 69-2203-511 415 gram Amine RediSep Rf Gold column,
pkg. of 1.
0.9 - 19 g 950 69-2203-512 950 gram Amine RediSep Rf Gold column,
pkg. of 1.
1.9 - 38 g 1900 69-2203-513 1.9 kg Amine RediSep Rf Gold column,
pkg. of 1.
3.8 - 76 g 3800 69-2203-534 3.8 kg Amine RediSep Rf Gold column,
pkg. of 1.
Basic Alumina
Basic alumina is a mixture of different aluminum oxides that are
partially dehydrated. The intrinsic basicity of this media gives it
similar applications to the amine media.
To illustrate how a basic alumina media can be of assistance, the
separation of a mixture of quinazolinone and benzimidazole deriv-
atives was investigated.
(A) 3-(1-piperdinylmethyl)-4(3H)-quinazolinone
O
N N
(B) 5,6-dimethyl-1-(piperdinomethyl)benzimidazole
N
0.50 100
0.45
0.40
75
0.35
Absorbance (AU)
Gradient % Solvent B
0.30
0.25 50
0.20
(A+B)
0.15
25
0.10
0.05
0.00 0
2 4 6 8 10 12 14 16 18 20
Time (minutes)
0.50 100
0.45
0.40
75
0.35
Gradient % Solvent B
Absorbance (AU)
0.30
0.25 50
0.20
(A) (B)
0.15
25
0.10
0.05
0.00 0
2 4 6 8 10 12 14
Time (minutes)
Solvent Migration
Hexane Full, 15 min
Ethyl acetate Full, 15 min
Isopropanol Full, 30 min
Hexane/Ethyl acetate 1:1 Full, 15 min
Hexane/Isopropanol 5:1 Full, 30 min
Dichloromethane Full, 15 min
Methanol Full, 15 min
Dichloromethane/Methanol 9:1 Full, 15 min
Neutral Alumina
Neutral alumina is a useful media in situations when acid-sensitive
products partially or fully degrade during purification due to the
intrinsic slight acidity of normal phase silica gel. The neutral prop-
erties of this media also allows the purification of substances
holding basic properties.
To illustrate this latter possibility, the separation of a mixture of
two pyridine derivatives was investigated.
(A) 2-(2-piperdinoethyl)pyridine
N N NH
0.50 100
0.45
0.40
75
0.35
Gradient % Solvent B
Absorbance (AU)
0.30
0.25 50
0.20
0.15
25
0.10
0.05
0.00 0
0 5 10 15 20 25 30 35 40 45 50
Column Volumes
1.00 100
0.90
(B)
0.80
75
0.70
Gradient % Solvent B
Absorbance (AU)
0.60
0.50 (A) 50
0.40
0.30
25
0.20
0.10
0.00 0
0 10 20 30 40 50
Column Volumes
Cyano
Cyano functionalized silica acts very similar to normal phase silica
when using similar solvents.
In reversed phase conditions, it is similar to C4 reversed phase col-
umns, although the elution order and selectivity of compounds
may be different. This allows chemists to purify compounds that
may not be well resolved on C18.
C N
Si
Cyano columns are reusable. After the first use, do not allow the
column to dry out since drying the column will adversely affect
future purifications. Turn off the air purge on your Flash system’s
method. The CombiFlash Rf system will turn off the air purge by
reading the column RFID tag. Remove all organic modifiers by
flushing with three column volumes of 80% acetonitrile in water or
100% isopropanol and store the column in the wash solvent. If the
storage solvents are immiscible with the solvents used for the sep-
aration, you may need to wash the column with an intermediate
solvent prior to storage.
Column Teledyne
Sample Size Isco Part
Size (g) Number Description
5.5 - 110 mg 5.5 69-2203-494 5.5 gram Cyano RediSep Rf Gold columns,
pkg. of 2.
15.5 - 310 mg 15.5 69-2203-495 15.5 gram Cyano RediSep Rf Gold column,
pkg. of 1.
30 - 600 mg 30 69-2203-496 30 gram Cyano RediSep Rf Gold column, pkg.
of 1.
50 mg - 1.0 g 50 69-2203-497 50 gram Cyano RediSep Rf Gold column, pkg.
of 1.
100 mg - 2 g 100 69-2203-498 100 gram Cyano RediSep Rf Gold column, pkg.
of 1.
150 mg - 3 g 150 69-2203-499 150 gram Cyano RediSep Rf Gold column, pkg.
of 1.
275 mg - 5.5 g 275 69-2203-500 275 gram Cyano RediSep Rf Gold column, pkg.
of 1.
415 mg - 8.3 g 415 69-2203-501 415 gram Cyano RediSep Rf Gold column, pkg.
of 1.
0.9 - 19 g 950 69-2203-502 950 gram Cyano RediSep Rf Gold column, pkg.
of 1.
1.9 - 38 g 1900 69-2203-503 1.9 kg Cyano RediSep Rf Gold column,
pkg. of 1.
3.8 - 76 g 3800 69-2203-533 3.8 kg Cyano RediSep Rf Gold column,
pkg. of 1.
Diol
Diol functionalized silica is less polar and has higher retention time
than normal phase bare silica. Diol functionalized silica offers an
interesting alternative to normal phase bare silica for difficult sep-
arations. The OH moieties on diol are less active than those on
silica making this media useful for compounds that decompose or
are difficult to elute with silica. Being normal phase, diol uses sol-
vents that are easy-to-evaporate compared to solvents used in
reverse phase.
Si O OH
OH
Diol columns are reusable. After the first use, do not allow the
column to dry out since drying the column will adversely affect
future purifications. Turn off the air purge on your Flash system’s
method. The CombiFlash Rf system will turn off the air purge by
reading the column RFID tag. Remove all organic modifiers by
flushing with three column volumes of 80% acetonitrile in water or
100% isopropanol and store the column in the wash solvent. If the
storage solvents are immiscible with the solvents used for the sep-
aration, you may need to wash the column with an intermediate
solvent prior to storage.
Diol columns work well with long-chain compounds. Diol columns
are useful for compounds that are unstable on silica or irreversibly
bind to silica.
Method development with diol columns is similar to that of silica
gel. TLC plates can be used for non-aqueous solvent systems. If
aqueous systems are required, it is faster to develop the method
with a small diol column using a small amount of sample. Although
diol columns are compatible with reverse phase solvents, it is eas-
iest to think of the column as always working in normal phase with
the ability to run aqueous solvent systems. The combination of a
less active surface and the ability to elute with a strong water sol-
vent allows diol to be used with a wide range of compounds.
HO O
HO
0.12
0.10
Absorbance (AU)
0.08
0.06
0.04
0.02
0.00
0 5 10 15 20 25 30
Run Time (min)
3. Fong, C.; Wells, D.; Krodkiewska, I.; Booth, J.; Hartley, P.G.
Synthesis and Mesophases of Glycerate Surfactants J. Phys
Chem B 2007, 111, 1384
HO
100
1.50
Absorbance (AU)
% Solvent B
0.00 0
0 3 6 9 12 15 18
Column Volumes
100
2.50
Gradient– Gradient–
hexane/isopropanol isopropanol/water
Absorbance (AU)
(C)
% Solvent B
(B)
(A)
0.00 0
0 10 20 30 40 50
Column Volumes
Column Teledyne
Sample Size Isco Part
Size (g) Number Description
5.5 - 110 mg 5.5 69-2203-514 5.5 gram Diol RediSep Rf Gold columns, pkg.
of 2.
15.5 - 310 mg 15.5 69-2203-515 15.5 gram Diol RediSep Rf Gold column, pkg.
of 1.
30 - 600 mg 30 69-2203-516 30 gram Diol RediSep Rf Gold column, pkg. of
1.
50 mg - 1.0 g 50 69-2203-517 50 gram Diol RediSep Rf Gold column, pkg. of
1.
100 mg - 2 g 100 69-2203-518 100 gram Diol RediSep Rf Gold column, pkg.
of 1.
150 mg - 3 g 150 69-2203-519 150 gram Diol RediSep Rf Gold column, pkg.
of 1.
275 mg - 5.5 g 275 69-2203-520 275 gram Diol RediSep Rf Gold column, pkg.
of 1.
415 mg - 8.3 g 415 69-2203-521 415 gram Diol RediSep Rf Gold column, pkg.
of 1.
0.9 - 19 g 950 69-2203-522 950 gram Diol RediSep Rf Gold column, pkg.
of 1.
1.9 - 38 g 1900 69-2203-523 1900 gram Diol RediSep Rf Gold column, pkg.
of 1.
3.8 - 76 g 3800 69-2203-535 3.8 kg Diol RediSep Rf Gold column, pkg. of 1.
SCX
The SCX (Strong Cation Exchange) media is a silica-bound tosic
acid.
Si O
S OH
The strong acidity of this media induces the full retention of any
compounds with basic properties subjected through it. This
intrinsic media property can be exploited several ways.
SCX media can be used as a practical and efficient tool for the
selective isolation of either basic or non-basic compounds from a
crude reaction mixture containing both.
To illustrate how SCX media can be of assistance, the separation of
a mixture of chromone and a benzimidazole derivative was
investigated.
(A) Chromone
O
(B) 1-(1-piperidinylmethyl)-1H-benzimidazole
N
N
1.75
1.50 75
Gradient % Solvent B
Absorbance (AU)
1.25
1.00 50
0.75
0.50 25
(B)
0.25
0.00 0
2 4 6 8 10 12 14 16 18 20 22 24 26 28
Time (minutes)
1.75
1.50 75
Gradient % Solvent B
Absorbance (AU)
1.25
1.00 50
0.75
0.50 25
0.25
0.00 0
2 4 6 8 10 12 14 16 18 20 22 24 26 28
Time (minutes)
Column Teledyne
Sample Size Isco Part
Loada (g) Number Description
≤3.5 mMol 5 69-2203-390 5 gram Strong Cation Exchange RediSep Rf
columns, pkg. of 2.
≤10.5 mMol 15 69-2203-391 15 gram Strong Cation Exchange RediSep Rf
column, pkg. of 1.
≤21 mMol 30 69-2203-392 30 gram Strong Cation Exchange RediSep Rf
column, pkg. of 1.
≤35 mMol 50 69-2203-393 50 gram Strong Cation Exchange RediSep Rf
column, pkg. of 1.
≤70 mMol 100 69-2203-396 100 gram Strong Cation Exchange RediSep Rf
column, pkg. of 1.
≤105 mMol 150 69-2203-394 150 gram Strong Cation Exchange RediSep Rf
column, pkg. of 1.
≤192 mMol 275 69-2203-398 275 gram Strong Cation Exchange RediSep Rf
column, pkg. of 1.
≤287 mMol 410 69-2203-395 410 gram Strong Cation Exchange RediSep Rf
column, pkg. of 1.
SAX
The SAX (Strong Anion Exchange) media is a silica-bound quater-
nary amine.
CH3
Cl -
Si N+
CH3
HC3
OH
(A)
Chromone OH
(B)
2,4-Dihydroxybenzoic acid
(B)
1.50 100
1.25
75
(A)
Gradient % Solvent B
1.00
Absorbance (AU)
0.75 50
0.50
25
0.25
0.00 0
0 2 4 6 8 10 12 14
Column Volumes
1.00 100
(A only)
0.90
0.80
75
0.70
Gradient % Solvent B
Absorbance (AU)
0.60
0.50 50
0.40
0.30
25
0.20
0.10
0.00 0
0 2 4 6 8 10 12 14
Column Volumes
Column Teledyne
Sample Size Isco Part
Sizea (g) Number Description
≤6.27 mMol 5.7 69-2203-381 5.7 gram Strong Anion Exchange RediSep Rf
columns, pkg. of 2.
≤18.7 mMol 17 69-2203-382 17 gram Strong Anion Exchange RediSep Rf
column, pkg. of 1.
≤37.4 mMol 34 69-2203-383 34 gram Strong Anion Exchange RediSep Rf
column, pkg. of 1.
≤62.7 mMol 57 69-2203-384 57 gram Strong Anion Exchange RediSep Rf
column, pkg. of 1.
≤125.4 mMol 114 69-2203-387 114 gram Strong Anion Exchange RediSep Rf
column, pkg. of 1.
≤187 mMol 170 69-2203-385 170 gram Strong Anion Exchange RediSep Rf
column, pkg. of 1.
≤344.3 mMol 313 69-2203-389 313 gram Strong Anion Exchange RediSep Rf
column, pkg. of 1.
≤517 mMol 470 69-2203-386 470 gram Strong Anion Exchange RediSep Rf
column, pkg. of 1.
Natural Products
Natural products chemistry is unique because the chemist gener-
ally doesn’t know the structure of the compound until after the
material is purified. Natural products must be removed from their
matrix (plant, fermentation, or marine organism) and the desired
compound isolated from a host of other materials without a priori
knowledge of which compound is active.
Automated Flash chromatography helps the natural products
chemist with well-packed columns that improve resolution, pre-
cise gradient control, large sample capacity, and systems such as
the CombiFlash Rf that allow easy scale-up. UV detection also
helps to find compounds easily, although this is of greater use later
in the purification. Automated Flash chromatography systems can
be used with a wide variety of pre-packed columns that work well
on a wide range of compounds. Columns that are usually
hand-packed due to variable swelling of the stationary phase in dif-
ferent solvents, such as LH-20 or CHP-20, are easily adapted to
CombiFlash systems to provide the benefits of automation to these
purifications as well. The parameters for a particular sized column
can be entered manually into the system and saved for future use.
Automated gradients avoid the need for pre-mixing solvents.
Purification of natural products starts with a chemical screen.
Compounds that extract into ethyl acetate are good candidates for
silica gel using hexane/ethyl acetate or hexane/acetone gradients.
Compounds that adsorb onto XAD-16 can be purified with silica gel
using more polar solvents systems including methylene chlo-
ride/methanol. The use of RediSep TLC plates also aids in
determining which solvent system provides the best resolution of
components in the mixture. CombiFlash Rf systems allow easy
changing of solvent systems during the run with their four-solvent
inlet lines which allows single-step purification of a wider range of
compounds in a single run.
Plant alkaloids can be easily purified on RediSep C18 columns after
extraction. The mixture is first dissolved in methylene chloride
and extracted into acidic water. The water is adjusted to pH 10 and
back-extracted into methylene chloride to yield only the free
bases. The resulting mixture is often clean enough for C18 purifica-
tion that yields clean compounds.
H O OCH3
HO
3-hydroxy-9-methoxypterocarpan (A)
O H
R’
R’’
N N
H3CO O
N N
H H3C H
CH3 CH3
Harmine Harmaline
(A) (B)
1.00 100
90
80
0.75
70
Gradient % Solvent B
Absorbance (AU)
60
0.50 50
40
30
0.25
20
10
0.00 0
0.50 100
0.45 90
0.40 80
0.35 70
Gradient % Solvent B
Absorbance (AU)
0.30 60
0.25 50
0.20 40
0.15 30
0.10 20
0.05 10
0.00 0
H Acceptors
8 6
5
H 7 Large
Donors Dipole
For our example: 0.16 = 0.2 × 3.4 ⁄ 4.4, so the new solvent system is
0.84 : 0.16 hexane : chloroform.
If this system failed to work well, a solvent from group 2 such as
n-propanol could be used.
Choosing a solvent from the same group will make little difference
to the selectivity. Changing from ethyl acetate to acetone, both in
group 6, makes little difference in the selectivity. This knowledge
can be used to choose a solvent in the same group that may have
physical properties, such as absorbance, more appropriate for the
purification.
For reverse phase, changing the less polar solvent (solvent B) is
less effective in changing selectivity. Changing from methanol to
acetonitrile generally results in little change to the chromatogram.
A change to tetrahydrofuran sometimes produces good results.
In addition to determining the best solvent system for purifying
compounds, the concept of solvent groups can also be used to find
solvents that allow easier detection of compounds. When using
0.75
0.65
Ethyl Acetate
0.55
Absorbance (AU)
0.45
0.35
0.25
Acetone
0.15
0.05
-0.05
200 225 250 275 300 325
Wavelength (nm)
NO 2 NO 2
+ CN
CN
NH 2 N
H
3-(2-nitrophenylamino) propionitrile absorbs most strongly at
230 nm, lower than the UV cutoff for ethyl acetate. Setting the
detection wavelength to 230 nm will cause the detector to see the
absorbance of both the ethyl acetate and the desired compound.
0.75
0.65
0.55
Absorbance (AU)
0.45
0.35
0.25
0.15
0.05
-0.05
200 250 300 350 400 450
Wavelength (nm)
2.00 100
90
80
1.50
70
Gradient % Solvent B
Absorbance (AU)
60
1.00 50
40
30
0.50
20
10
0.00 0
2.00 100
90
80
1.50
70
Gradient % Solvent B
Absorbance (AU)
60
1.00 50
40
30
0.50
20
10
0.00 0
0.45
Absorbance (AU)
0.35
0.25
0.15
0.05
0.00
-0.05
200 210 220 230 240 250
Wavelength (nm)
1.50 100
90
80
70
Gradient % Solvent B
Absorbance (AU)
60
0.75 50
40
30
20
10
0.00 0
Detection Techniques
UV Detection
UV detection is the most common detection technique for Flash
chromatography. Most compounds can be detected within the
range of 200–360 nm used by UV detectors.
The default wavelength commonly used is 254 nm because many
compounds absorb at this wavelength. Since some compounds
exhibit weak absorbance at this wavelength (Figure 109), they will
only show a small peak on the detector.
For this reason, the absorbance spectrum of the compound should
be known before starting the purification.
RediSep columns can be loaded with enough sample that the
detector becomes saturated as the peak elutes. The detection
wavelength can be moved to a different value during the elution on
CombiFlash systems such as a minor absorbance (280 nm using
the spectrum in Figure 109), or a shoulder of the major absorbance
if saturation of the detector prevents fractionation of closely
eluting peaks.
0.5
0.4
Absorbance (AU)
0.3
0.2
0.1
0.0
480 nm
630 nm
700 nm
660 nm
360 nm
3.00 100
90
80
70
Gradient % Solvent B
Absorbance (AU)
60
1.50 50
40
30
20
10
0.00 0
0 2 4 6 8 10 12
Run Length 12.8 minutes
All-Wavelength Detection
All-Wavelength Collection in the CombiFlash Rf 200 and Torrent
systems measures the average absorbance on all wavelengths
detected on a photodiode array. The signal is processed to remove
baseline drift caused by solvent absorbance. This creates a single
voltage that allows the fraction collection program in the MPLC or
Flash chromatography system to properly cut the peak. All-wave-
length detection is useful when:
• The UV-vis spectrum is unknown, such as compounds puri-
fied from natural products.
• There is a mixture of compounds with various absorbances
such that a single wavelength can’t “see” all the com-
pounds in the mixture.
• The elution solvent spectrum overlaps that of the desired
compound.
• Compounds with similar spectra overload the detector,
making it difficult to properly fractionate compounds.
• Only compounds within a specified range of absorbance
are desired. This method would exclude some starting
materials, of the products have a different absorbance
spectrum.
All-wavelength detection enhances the ability for a CombiFlash
system to purify compounds in an automated fashion.
(C)
2.50 100
Solvent Gradient: Solvent Gradient: 90
A1: hexane A2: isopropanol
B1: isopropanol B2: water 80
70
Gradient % Solvent B
Absorbance (AU)
60
50
40
(B) 254 nm
30
All Wavelength
20
(A) 10
0.00 0
0 10 20 30 40 50
Column Volumes
0.25 100
(B) (C) 90
80
70
Gradient % Solvent B
Absorbance (AU)
60
50
40
30
(A) 254 nm
All Wavelength 20
10
0.00 0
0 20 40 60 80
Column Volumes
OH
0.5 OH
(A) Catechin
Not observed with OH
AU
single wavelength HO O
detection
OH
0.0
200 254 300 400
O CH3
1.0 H3C N
N
N
AU
(B) Caffeine O N
CH3
0.0
200 300 400
1.0
AU
0.08 0.08
AcO
All-Wavelength Absorbance
210 nm Absorbance (AU)
O
AcO
AcO
OAc
OAc
0.00 0.00
-0.08
0 5 10 15
Run Time (min)
2.0 0.7
207 nm Absorbance (AU)
All-Wavelength Absorbance
207 nm
All Wavelength
0.0 0.0
0 20 40 60
Column Volumes
Other Detectors
Occasionally, other detectors are used to purify compounds such
as refractive index (RI), fluorescence, or evaporative light scat-
tering (ELSD) detectors. External detectors may be connected to
CombiFlash Rf and Torrent systems. These systems will then cut
peaks using an input from the external detector.
1.0
UV Absorbance (AU)
0.0
0 6 12 18
Run Time (min)
Media Selection
The following charts and figures can assist with the selection of
stationary phase media based on sample properties and size.
• Figure 117: Chart for column media selection
• Figure 118: Table of RediSep Rf Gold Silica Gel Disposable
Flash Columns, 20–40 microns
• Figure 119: Table of RediSep Rf Silica Gel Disposable Flash
Columns, 40–60 microns
• Figure 120: Table of Reusable RediSep Rf Gold C18 Reversed
Phase columns, 20–40 microns
• Figure 121: Table of Reusable RediSep Rf C18 Reversed
Phase columns, 40–60 microns
• Figure 122: Table of Reusable RediSep Rf Gold Amine Col-
umns, 20–40 microns
• Figure 123: Table of Reusable RediSep Rf Amine Columns,
40–60 microns
• Figure 124: Table of Reusable RediSep Rf Gold Cyano Col-
umns, 20–40 microns
• Figure 125: Table of Reusable RediSep Rf SAX Columns
• Figure 126: Table of Reusable RediSep Rf SCX Columns
• Figure 127: Table of Reusable RediSep Rf Gold Diol Columns,
20–40 microns
• Figure 128: Table of RediSep Rf Alumina Acidic Columns
• Figure 129: Table of RediSep Rf Alumina Neutral Columns
• Figure 130: Table of RediSep Rf Alumina Basic Columns
High C18
Polarity Cyano
C18
Normal Phase
Amine
Basic
Properties Basic Alumina
SCX
Cyano
Neutral Alumina
Sample
Normal Phase
C18
Diol
Acid
Neutral Alumina
Sensitive
Cyano
C18
Charged
Cyano
Column Teledyne
Sample Size Isco Part
Size (g) Number Description
20 mg - 0.4 g 4 69-2203-344 4 gram RediSep Rf Gold Silica Gel Disposable
columns, pkg. of 14.
60 mg - 1.2 g 12 69-2203-345 12 gram RediSep Rf Gold Silica Gel Disposable
columns, pkg. of 14.
120 mg - 2.4 g 24 69-2203-346 24 gram RediSep Rf Gold Silica Gel Disposable
columns, pkg. of 10.
200 mg - 4 g 40 69-2203-347 40 gram RediSep Rf Gold Silica Gel Disposable
columns, pkg. of 10.
400 mg - 8 g 80 69-2203-348 80 gram RediSep Rf Gold Silica Gel Disposable
columns, pkg. of 6.
600 mg - 12 g 120 69-2203-349 120 gram RediSep Rf Gold Silica Gel Dispos-
able columns, pkg. of 6.
1.1 - 22 g 220 69-2203-359 220 gram RediSep Rf Gold Silica Gel Dispos-
able columns, pkg. of 4.
1.65 - 33 g 330 69-2203-369 330 gram RediSep Rf Gold Silica Gel Dispos-
able columns, pkg. of 3.
3.8 - 75 g 750 69-2203-427 750 gram RediSep Rf Gold Silica Gel Dispos-
able columns, pkg. of 3.
7.5 - 150 g 1500 69-2203-428 1.5 kg RediSep Rf Gold Silica Gel Disposable
columns, pkg. of 2.
15 - 300 g 3000 69-2203-529 3 kg RediSep Rf Gold Silica Gel Disposable
columns, pkg. of 1.
Column Teledyne
Sample Size Isco Part
Size (g) Number Description
20 mg - 0.4 g 4 69-2203-304 4 gram RediSep Rf Disposable Flash columns,
pkg. of 20.
60 mg - 1.2 g 12 69-2203-312 12 gram RediSep Rf Disposable Flash columns,
pkg. of 20.
120 mg - 2.4 g 24 69-2203-324 24 gram RediSep Rf Disposable Flash columns,
pkg. of 15.
200 mg - 4 g 40 69-2203-340 40 gram RediSep Rf Disposable Flash columns,
pkg of 15.
400 mg - 8 g 80 69-2203-380 80 gram RediSep Rf Disposable Flash columns,
pkg. of 12.
600 mg - 1 2g 120 69-2203-320 120 gram RediSep Rf Disposable Flash col-
umns, pkg. of 10.
N/A 125 69-2203-314 125 gram RediSep Rf Disposable Filter column,
pkg. of 6.
1.1 - 22 g 220 69-2203-422 220 gram RediSep Rf Disposable Flash column,
pkg. of 6.
1.65 - 33 g 330 69-2203-330 330 gram RediSep Rf Disposable Flash col-
umns, pkg. of 4.
3.8 - 75 g 750 69-2203-275 750 gram RediSep Disposable Flash columns,
pkg. of 4.
7.5 - 150 g 1500 69-2203-277 1.5 kg RediSep Disposable Flash columns, pkg.
of 3.
15 - 300 g 3000 69-2203-527 3 kg RediSep Silica Gel Disposable columns,
pkg. of 1.
Column Teledyne
Sample Size Isco Part
Size (g) Number Description
5.5 - 110 mg 5.5 69-2203-328 5.5 gram RediSep Rf Gold C18 columns, pkg.
of 2.
15.5 - 310 mg 15.5 69-2203-334 15.5 gram RediSep Rf Gold C18 column, pkg.
of 1.
30 - 600 mg 30 69-2203-335 30 gram RediSep Rf Gold C18 column, pkg.
of 1.
50 mg - 1.0 g 50 69-2203-336 50 gram RediSep Rf Gold C18 column, pkg.
of 1.
100 mg - 2 g 100 69-2203-337 100 gram RediSep Rf Gold C18 column, pkg.
of 1.
150 mg - 3 g 150 69-2203-338 150 gram RediSep Rf Gold C18 column, pkg.
of 1.
275 mg - 5.5 g 275 69-2203-339 275 gram RediSep Rf Gold C18 column, pkg.
of 1.
415 mg - 8.3 g 415 69-2203-341 415 gram RediSep Rf Gold C18 column, pkg.
of 1.
0.95 - 19 g 950 69-2203-492 950 gram RediSep Rf Gold C18 column, pkg.
of 1.
1.9 - 38 g 1900 69-2203-493 1.9 kg RediSep Rf Gold C18 column, pkg. of 1.
3.8 - 76 g 3800 69-2203-528 3.8 kg RediSep Rf Gold C18 column, pkg. of 1.
Column Teledyne
Sample Size Isco Part
Size (g) Number Description
4.3 - 86 mg 4.3 69-2203-410 4.3 gram Reverse Phase (C18) RediSep Rf col-
umns, pkg. of 2.
13 - 260 mg 13 69-2203-411 13 gram Reverse Phase (C18) RediSep Rf col-
umn, pkg. of 1.
26 - 520 mg 26 69-2203-412 26 gram Reverse Phase (C18) RediSep Rf col-
umn, pkg. of 1.
43 - 860 mg 43 69-2203-413 43 gram Reverse Phase (C18) RediSep Rf col-
umn, pkg. of 1.
86 mg - 1.72 g 86 69-2203-416 86 gram Reverse Phase (C18) RediSep Rf col-
umn, pkg. of 1.
130 mg - 2.6 g 130 69-2203-414 130 gram Reverse Phase (C18) RediSep Rf col-
umn, pkg. of 1.
240 mg - 4.8 g 240 69-2203-418 240 gram Reverse Phase (C18) RediSep Rf col-
umn, pkg. of 1.
360 mg - 7.2 g 360 69-2203-415 360 gram Reverse Phase (C18) RediSep Rf col-
umn, pkg. of 1.
Column Teledyne
Sample Size Isco Part
Size (g) Number Description
5.5 - 110 mg 5.5 69-2203-504 5.5 gram Amine RediSep Rf Gold columns,
pkg. of 2.
15.5 - 310 mg 15.5 69-2203-505 15.5 gram Amine RediSep Rf Gold column,
pkg. of 1.
30 - 600 mg 30 69-2203-506 30 gram Amine RediSep Rf Gold column, pkg.
of 1.
50 mg - 1.0 g 50 69-2203-507 50 gram Amine RediSep Rf Gold column, pkg.
of 1.
100 mg - 2 g 100 69-2203-508 100 gram Amine RediSep Rf Gold column, pkg.
of 1.
150 mg - 3 g 150 69-2203-509 150 gram Amine RediSep Rf Gold column, pkg.
of 1.
275 mg - 5.5 g 275 69-2203-510 275 gram Amine RediSep Rf Gold column, pkg.
of 1.
415 mg - 8.3 g 415 69-2203-511 415 gram Amine RediSep Rf Gold column, pkg.
of 1.
0.9 - 19 g 950 69-2203-512 950 gram Amine RediSep Rf Gold column, pkg.
of 1.
1.9 - 38 g 1900 69-2203-513 1.9 kg Amine RediSep Rf Gold column, pkg.
of 1.
3.8 - 76 g 3800 69-2203-534 3.8 kg Amine RediSep Rf Gold column, pkg.
of 1.
1.3 - 12.8 g 260 69-2203-358 260 gram Amine RediSep Rf column, pkg. of 1.
1.92 - 19.25 g 385 69-2203-355 385 gram Amine RediSep Rf column, pkg. of 1.
Column Teledyne
Sample Size Isco Part
Size (g) Number Description
5.5 - 110 mg 5.5 69-2203-494 5.5 gram Cyano RediSep Rf Gold columns,
pkg. of 2.
15.5 - 310 mg 15.5 69-2203-495 15.5 gram Cyano RediSep Rf Gold column,
pkg. of 1.
30 - 600 mg 30 69-2203-496 30 gram Cyano RediSep Rf Gold column, pkg.
of 1.
50 mg - 1.0 g 50 69-2203-497 50 gram Cyano RediSep Rf Gold column, pkg.
of 1.
100 mg - 2 g 100 69-2203-498 100 gram Cyano RediSep Rf Gold column, pkg.
of 1.
150 mg - 3 g 150 69-2203-499 150 gram Cyano RediSep Rf Gold column, pkg.
of 1.
275 mg - 5.5 g 275 69-2203-500 275 gram Cyano RediSep Rf Gold column, pkg.
of 1.
415 mg - 8.3 g 415 69-2203-501 415 gram Cyano RediSep Rf Gold column, pkg.
of 1.
0.9 - 19 g 950 69-2203-502 950 gram Cyano RediSep Rf Gold column, pkg.
of 1.
1.9 - 38 g 1900 69-2203-503 1.9 kg Cyano RediSep Rf Gold column, pkg.
of 1.
3.8 - 76 g 3800 69-2203-533 3.8 kg Cyano RediSep Rf Gold column, pkg.
of 1.
Column Teledyne
Sample Size Isco Part
Sizea (g) Number Description
≤6.27 mMol 5.7 69-2203-381 5.7 gram Strong Anion Exchange RediSep Rf
columns, pkg. of 2.
≤18.7 mMol 17 69-2203-382 17 gram Strong Anion Exchange RediSep Rf
column, pkg. of 1.
≤37.4 mMol 34 69-2203-383 34 gram Strong Anion Exchange RediSep Rf
column, pkg. of 1.
≤62.7 mMol 57 69-2203-384 57 gram Strong Anion Exchange RediSep Rf
column, pkg. of 1.
≤125.4 mMol 114 69-2203-387 114 gram Strong Anion Exchange RediSep Rf
column, pkg. of 1.
≤187 mMol 170 69-2203-385 170 gram Strong Anion Exchange RediSep Rf
column, pkg. of 1.
≤344.3 mMol 313 69-2203-389 313 gram Strong Anion Exchange RediSep Rf
column, pkg. of 1.
≤517 mMol 470 69-2203-386 470 gram Strong Anion Exchange RediSep Rf
column, pkg. of 1.
Column Teledyne
Sample Size Isco Part
Sizea (g) Number Description
≤3.5 mMol 5 69-2203-390 5 gram Strong Cation Exchange RediSep Rf
columns, pkg. of 2.
≤10.5 mMol 15 69-2203-391 15 gram Strong Cation Exchange RediSep Rf
column, pkg. of 1.
≤21 mMol 30 69-2203-392 30 gram Strong Cation Exchange RediSep Rf
column, pkg. of 1.
≤35 mMol 50 69-2203-393 50 gram Strong Cation Exchange RediSep Rf
column, pkg. of 1.
≤70 mMol 100 69-2203-396 100 gram Strong Cation Exchange RediSep Rf
column, pkg. of 1.
≤105 mMol 150 69-2203-394 150 gram Strong Cation Exchange RediSep Rf
column, pkg. of 1.
≤192 mMol 275 69-2203-398 275 gram Strong Cation Exchange RediSep Rf
column, pkg. of 1.
≤287 mMol 410 69-2203-395 410 gram Strong Cation Exchange RediSep Rf
column, pkg. of 1.
Column Teledyne
Sample Size Isco Part
Size (g) Number Description
5.5 - 110 mg 8 69-2203-430 8 gram Alumina Acidic RediSep Rf columns,
pkg of 20.
15.5 - 310 mg 24 69-2203-431 24 gram Alumina Acidic RediSep Rf columns,
pkg. of 20.
30 - 600 mg 48 69-2203-432 48 gram Alumina Acidic RediSep Rf columns,
pkg. of 15.
50 mg - 1.0g 80 69-2203-433 80 gram Alumina Acidic RediSep Rf columns,
pkg. of 15.
100 mg - 2 g 160 69-2203-436 160 gram Alumina Acidic RediSep Rf columns,
pkg. of 12.
150 mg - 3 g 240 69-2203-434 240 gram Alumina Acidic RediSep Rf columns,
pkg. of 10.
415 mg - 8.3 g 440 69-2203-438 440 gram Alumina Acidic RediSep Rf columns,
pkg. of 6.
0.9 - 19 g 660 69-2203-435 660 gram Alumina Acidic RediSep Rf columns,
pkg. of 4.
Column Teledyne
Sample Size Isco Part
Size (g) Number Description
40 - 320 mg 8 69-2203-440 8 gram Alumina Neutral RediSep Rf columns,
pkg. of 20.
120 - 960 mg 24 69-2203-441 24 gram Alumina Neutral RediSep Rf columns,
pkg. of 20.
240 mg - 1.92 g 48 69-2203-442 48 gram Alumina Neutral RediSep Rf columns,
pkg. of 15.
400 mg - 3.2 g 80 69-2203-443 80 gram Alumina Neutral RediSep Rf columns,
pkg. of 15.
800 mg - 6.4 g 160 69-2203-446 160 gram Alumina Neutral RediSep Rf col-
umns, pkg. of 12.
1.2 - 9.6 g 240 69-2203-444 240 gram Alumina Neutral RediSep Rf col-
umns, pkg. of 10.
2.2 - 17.6 g 440 69-2203-448 440 gram Alumina Neutral RediSep Rf col-
umns, pkg. of 6.
3.3 - 26.4 g 660 69-2203-445 660 gram Alumina Neutral RediSep Rf col-
umns, pkg. of 4.
Column Teledyne
Sample Size Isco Part
Size (g) Number Description
40 - 320 mg 8 69-2203-450 8 gram Alumina Basic RediSep Rf columns,
pkg. of 20.
120 - 960 mg 24 69-2203-451 24 gram Alumina Basic RediSep Rf columns,
pkg. of 20.
240 mg - 1.92 g 48 69-2203-452 48 gram Alumina Basic RediSep Rf columns,
pkg. of 15.
400 mg - 3.2 g 80 69-2203-453 80 gram Alumina Basic RediSep Rf columns,
pkg. of 15.
0.8 - 6.4 g 160 69-2203-456 160 gram Alumina Basic RediSep Rf columns,
pkg. of 12.
1.2 - 9.6 g 240 69-2203-454 240 gram Alumina Basic RediSep Rf columns,
pkg. of 10.
2.2 - 17.6 g 440 69-2203-458 440 gram Alumina Basic RediSep Rf columns,
pkg. of 6.
3.3 - 26.4 g 660 69-2203-455 660 gram Alumina Basic RediSep Rf columns,
pkg. of 4.
Solvent Selection
Figure 131 lists typical chromatography solvents and their proper-
ties by increasing polarity. Figure 132 may be used to select
miscible solvents.
Wavelength Selection
Figure 133 lists substances and the wavelength at which they typi-
cally may be detected. Figure 134 lists wavelengths that are
available with Teledyne Isco optical detection units and sub-
stances that have good absorbance at these wavelengths.
Selectivity Group
UV Cutoff (nm)
(Fig. 102)
Polarity
SOLVENT
Pentane 0.00 0.23 36 210 —
Petroleum ether 0.01 0.30 30—60 210 —
Hexane 0.06 0.33 69 210 —
Cyclohexane 0.10 1.00 81 210 —
Isooctane 0.10 0.53 99 210 —
Trifluoroacetic acid 0.10 — 72 — —
Trimethylpentane 0.10 0.47 99 215 —
Cyclopentane 0.20 0.47 49 210 —
n-Heptane 0.20 0.41 98 200 —
Trichloroethylene 1.00 0.57 87 273 —
Carbon tetrachloride 1.60 0.97 77 265 —
i-Propyl ether 2.40 0.37 68 220 1
Toluene 2.40 0.59 111 285 7
Chlorobenzene 2.70 0.80 132 — 7
o-Dichlorobenzene 2.70 1.33 180 295 —
Ethyl ether 2.90 0.23 35 220 1
Benzene 3.00 0.65 80 280 7
Isobutyl alcohol 3.00 4.70 108 220 2
Selectivity Group
UV Cutoff (nm)
(Fig. 102)
Polarity
SOLVENT
Methylene chloride 3.40 0.44 40 245 5
Ethylene dichloride 3.50 0.79 84 228 —
n-Butanol 3.90 2.95 117 210 2
n-Butyl acetate 4.00 — 126 254 —
n-Propanol 4.00 2.27 98 210 2
Tetrahydrofuran 4.20 0.55 66 220 3
Ethanol 4.30 1.20 79 210 2
Ethyl acetate 4.30 0.45 77 260 6
i-Propanol 4.30 2.37 82 210 2
Chloroform 4.40 0.57 61 245 8
Dioxane 4.80 1.54 102 220 6
Acetone 5.40 0.32 57 205, 6
225–300
Acetic acid 6.20 1.28 118 230 4
Acetonitrile 6.20 0.37 82 210 6
Dimethyl formamide 6.40 0.92 153 270 3
Methanol 6.60 0.60 65 210 2
Ethylene glycol 6.90 19.90 197 210 4
Dimethyl sulfoxide 7.20 2.24 189 268 —
Water 10.20 1.00 100 — 8
yl A
6.89 Di
-et ce
tat
0.0003 He hy
lE e
pta the
ne r
Miscible
0.001 He
xa
Immiscible
100 Me ne
tha
4.8 M eth n ol
Pe yl-
0.004
Values provided are solubility in water, %w/w
nta t-B
ne uty
100 n-P
ro lE
100 I s o-p p an
the
r
r ol
1.71 Di op
-is an
Te o-P ol
100
Solvent and UV-Vis Wavelength Selection Guide
Effective Organic Compound Purification
tra ro
T hy py
l
0.051 olu dro Eth
en er
W e fur
an
ate
r
137
Effective Organic Compound Purification
Appendix B
a. Amino acids which have greater absorbance at 280 nm are too dependent on pH for adequate
accuracy at 254 nm.
Wavelength Compound
(nm)
214 non-aromatic peptides, amino acids, nucleotides, and lipids, steroids.
254 aromatics, proteins, nucleic acids, nucleoproteins, aromatic amino acids, pterins, and vita-
mins.
280 proteins and amino acidsa.
310 natural tropolene, ferretin, vitamins, antibiotics, and substituted benzophenones.
326 oxidized rubredoxin.
340 enzymes; NADH, NADPH.
365 acridine, tropolene derivatives, steroids, porphyrin derivatives, o-napthaquinone, naptha-
lenes, ferricyanide, ferroproteins, ninhydrin-primary and secondary amino acids.
435 porphyrins, chlorophyll a, carotenoids, ninhydrin, proline, and hydroxyproline.
470 flavoproteins.
546 porphyrins, chlorophylls and derivatives, and carotenoids.
580 porphyrins, chlorophylls, and ninhydrin-amino acid reaction product (DYDA).
620 lactase and pyr-heme a2 hemachrome.
636 heme proteins, oxidized.
a. Amino acids which have greater absorbance at 280 nm are too dependent on pH for adequate
accuracy at 254 nm.
Elementary theory
Given a compound X in a Flash column:
( X ) s = concentration of X in stationary phase (g/mL or g/g)
t r = V r ⁄ F, tw = Vw ⁄ F
where: V r = retention volume of band
V w = baseline bandwidth (in mL)
Therefore:
( tr – to )
t r = t o ( 1 + k′ ) and k′ = -----------------
-
to
and V r = V m ( 1 + k′ ) = V m + V s K
where:
t0.5w = bandwidth at one-half the peak height.
Application
The basic function of LC is to separate a mixture of two or more
substances. Given two compounds, X and Y, in a column, their rela-
tive separation or resolution is defined as:
t r, y – t r, x
R s = ----------------------------------------
-
1 ⁄ 2 ( t w, y + t w, x )
where:
k′ is the capacity factor of either compound
84
88
92
98 99.4
95
90 93
95 98 99.5
96
97 99 99.6
Charts adapted by permission of L.R. Snyder and Preston Technical Abstracts Co., from
Journal of Chromatographic Science 10, 364 (1972)
Troubleshooting
LC Systems
Basic checklist
The list below summarizes common problems that can be quickly
checked and remedied.
• Instrument(s) not plugged in
• Instrument(s) not turned on
• Fuse(s) blown
• No mobile phase
• Air lock in pump lines
• Leaks
• No sample being introduced
• Temperature gradients across system
• Contaminated or plugged column
• Wrong column type or size
• Flow through column reversed
• Wrong detector setting
• Dirty detector cell
• Indicator light or gauge malfunction
• Sample chemistry misinterpreted
Troubleshooting
The following figures list common LC problems and solutions.
• Figure 136: Table for troubleshooting peak problems
• Figure 137: Table for troubleshooting baseline problems
Fourth Edition