Teledyne ISCO C18Aq User Manual

Desalting Samples with
RediSep Rf Gold

®

C18Aq

Columns

Abstract

Compounds are often purified with solvents that
contain salts or buffers that need to be removed
prior to subsequent studies with the compound. The
RediSep Rf Gold® C18Aq columns have the ability to
adsorb compounds under highly aqueous conditions
and are ideal for removing the salt from a sample.
The method described also removes a large portion
of water from samples dissolved in aqueous solvents
facilitating evaporation. The procedure is similar to
solid phase extraction, but on a larger prep scale.

Background

Compounds run on ion exchange media are often
eluted with buffers that are difficult to remove
from the sample after purification. Buffers, such as
phosphate salts, are often used to improve resolution
during purification on C18 by maintaining the pH of
the solvent at a value such that the eluted compounds
remain in a consistent ionization state.

The RediSep Rf Gold C18Aq columns are well suited
to desalting applications because they resist phase
collapse. Samples requiring desalting are generally
dilute and the large volume of water eventually causes
phase collapse with other types of C18 (Figure 1).

Figure 1: C18 chains are fully extended when organic solvent
is present but undergo “phase collapse” (left) under highly
aqueous conditions. Hydrophilic groups (right) reduce phase
collapse (right).

Chromatography Application Note

AN95, August 2020

The general procedure for desalting samples is as follows:

1. Remove any organic solvents by rotary evaporation.
Organic solvents tend to reduce the binding of
compounds to C18. Stop evaporation if the sample
appears to be precipitating.

2. Condition the RediSep Rf Gold C18Aq column by
washing with an organic solvent such as methanol
or acetonitrile for 5 column volumes (CV) followed
by water for 5 CV.

3. Load the sample. For desalting purposes, load up
to 5% by weight on the column because the desired
compound is captured from the salt solution and
released. For example, a 5.5 g RediSep Rf Gold
C18Aq column (PN 69-2203-558) can be used
to capture up to ~250 mg compound. If further
purification is performed after washing off the salt,
use a 1% load. Larger volumes can be loaded with a
CombiFlash® sample load pump

(PN 60-5247-007). Since the sample is being loaded
in a weak, polar solvent the total volume injected
may exceed the column volume without sample loss.

a. Place the CombiFlash in manual control, and

select Liquid Injection (Pumps Disabled) from
the Valve Position control. Place the column on
the instrument and load the sample.

b. For an Rf-150 or NextGen 100, place the

column on the system and load the sample.

c. Verify the column has been conditioned

according to the manufacturer’s instructions.

4. Wash the salt from the column using manual
control. On the CombiFlash, select Through column
and flow cell from the Valve Position control. On an
Rf-150 or NextGen 100, connect the solvent line to
the column shuttle. Set the flow rate to the value
listed on the column label and press the Prime
A button. Wash the column for at least one, and
preferably five, column volumes.

5. Elute the compound. Change the method to a step
gradient after 1 CV. The step should go to 100% B.
If the compound requires further purification, use
a gradient suitable for the purification. From the
Run Requirement screen, select None-On Column
from the Sample Loading Technique control
because the column was equilibrated and the
sample is on the column.

Chromatography Technical Note TN95

2.0

Absorbance (214 nm)

100

Methanol (%)

–

O

H

Always run a small sample to verify the compound will
adsorb on the column before running the entire sample
mixture.

Experiment and Results

The example in Figure 2 is brilliant blue purified from
a weak anion exchange (WAX) column eluted with 1M
NaH2PO4 in water. As there was no organic solvent
involved, the mixture was not evaporated prior to
loading on the column.

The procedure listed above was altered slightly for
this run; the column was not washed prior to the run
as described in step 4, but was washed with water
for 10 CV during the run to demonstrate that the
compound remained on the column during the wash.
This compound elutes with 30% methanol in water
on a RediSep Rf Gold C18Aq column1 under gradient
conditions and demonstrates that compounds that
normally elute with high water concentrations can be
desalted with this method. The solvent used to elute

1.5

1.0

0.5

0.0

0510 15 20

80

60

40

20

0

NH

O

the sample from the C18Aq column is methanol.

The step gradient provides the following features:

•Run time and solvent usage is reduced.

•The compound elutes in a sharper band reducing

the volume of solvent to evaporate, saving time.

•There is relatively little water in the sample, so
drying time is reduced because methanol is easier
to evaporate.

Conclusion

RediSep Rf Gold C18Aq columns provide a convenient
way to desalt samples. Other benefits include removing
most of the water and reducing the volume of the
fractions to facilitate evaporation. If desired, a regular
gradient can also be run if the compound requires
further purification instead of the step gradient.

O

S

O

+

N

O

O

N

S

O

Figure 2: Desalting of brilliant blue with a RediSep Rf Gold C18Aq column.

1. http://www.isco.com/WebProductFiles/Applications/101/Application_Notes/AN76_RediSep_Rf_Gold_C18A_for_Highly_Aqueous_Mobile_Phases.pdf
retrieved 15 Aug 2012

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