chromatography

tip sheet

Guidelines for Using UNOsphere™ S Cation Exchange Support
UNOsphere S support exhibits a pH shift during conductivity
fluctuations. These pH fluctuations can be modulated
through careful selection of buffering and sample conditions.
This phenomenon is common in strong cation exchangers
containing carboxyl groups in their support structures, as wellas in weak carboxymethyl (CM)-containing cation exchange
resins. The following recommendations (Table 1) will help you
choose the appropriate buffers and experimental conditions
when using UNOsphere S support.

Elution Buffer Selection and ConditionsSample
Preparation

Rapid Column
Equilibration

BufferPreparation

Buffer Selection

Table 1. Recommendations for experimental conditions.
Guideline

Rationale

Use an anionic or zwitterionic buffer.

Standard cation exchange chromatography buffers (anionic (–) and zwitterionic(+/–)) will not associate with the negatively charged groups on the cationexchanger and will be more available to provide pH control in the mobile phase.

Use a buffer that has a pKa within 0.5 pH units of the desired pH of theexperiment (Table 2).

Buffering capacity and pH control are greatest near the pKa of the buffer. Any
typical cation exchange buffer may be used; however, Table 2 lists the best
buffers, organized by target pH.

Do not back-titrate buffer pH.

Use particular care when using buffer salts to ensure that back-titration does
not occur. If the pH is overshot during preparation, discard the buffer and begin
again. Back-titration will produce inconsistent buffers due to the formation ofdiffering amounts of salt depending on the severity of the back-titration.

Preequilibrate the column (Figure 1).

A 1 column volume (CV) preequilibration with the appropriate buffer at aconcentration of at least 0.5 M will bring the column into operating conditions
more efficiently than using equilibration buffer alone. Experiments may be
conducted to further optimize equilibration, possibly by lengthening thepreequilibration step (Figure 1).

Use an equilibration buffer with a concentration of at least 0.05 M(Figure 2).

Equilibration with 0.05 M buffers reduces the equilibration time and provides
better pH modulation than 0.02 M buffers (Figure 2).

The salt concentration and conductivity of the sample should be as
close as possible to that of the equilibration buffer.

pH fluctuation can occur if the sample conductivity and salt concentration differ
from that of the equilibration buffer. This can create changes in protein andcontaminant binding. If the pH is reequilibrated during sample loading, theseeffects should be minimized.

Avoid the use of NaCl* to produce conductivity gradients; use higher
buffer concentrations to formulate elution buffer.

Maximum pH control upon elution occurs when elution is with a concentrated
buffer instead of a salt gradient. Using buffers to increase conductivity
increases buffering capacity and dramatically reduces the amplitude andduration of pH transients in comparison to NaCl.*

If NaCl* must be used to create conductivity gradients, make the steps
as shallow as possible.

The amplitude and duration of the pH transient is proportional to the change
in NaCl* concentration. Minimizing the interval of the step will minimize theseverity of the pH fluctuation.

When making multiple gradient steps, allow the pH to return to its
target value during one step before starting the next step.

Allow the operating pH to be restored before the next gradient step. If the pH
is not allowed to return to its target value prior to the next gradient step, thepH fluctuation may result in significant changes in selectivity that may or may
not be reproducible. This can be avoided by extending each gradient step until
reequilibration to the target pH is achieved.

Conduct method scouting and development with step gradients rather
than linear gradients.

The pattern of pH transients with linear gradients is different from the patternwith step gradients. Steps produce a transient pH value before reachieving
pH equilibrium. Linear gradients suffer from a lag in reequilibration. The
combination of pH fluctuation and changing salt concentration could make it
difficult to predict the correct step based on a linear gradient.

* NaCI is used as an example in this paper, but other salts will have similar effects.

Table 2. Buffers that provide the lowest total equilibration volume(<10 CV) and most effective pH control for UNOsphere S support.

12.0

120.01 N
NaOH

11.011pH

Recommended Buffer

Alternative Buffers

4.0Succinate

Citrate4.5Acetate

Succinate, citrate

5.0Citrate

Succinate

5.5Citrate

MES

6.0MESCitrate

5.0

6.5MES—

4.0

7.0Phosphate

—

10.0

109.0

9pH

8.0

87.0

7PE

6.0

6~4 CV

5~9 CV

40510		15CV
11 0.01 N

Fig. 2. Effect of buffer concentration on the number of CV required toachieve equilibration following a 1 CV preequilibration (PE) with 0.5 M buffer.
A 10 ml UNOsphere S column (10.4 cm x 1.1 cm) was run at 250 cm/hr. A 1 CV
preequilibration with 0.5 M citrate, pH 5.5 was followed by equilibration with either
0.02 M citrate, pH 5.5 (—) or 0.05 M citrate, pH 5.5 (—). The total numberof CV for the column to equilibrate was determined by calculating the volume
from the beginning of preequilibration until the pH stabilized at pH 5.5. The trend
is consistent for other buffer systems, but the degree of improvement varies frombuffer to buffer.

NaOH

109pH

876PE

5~5 CV43~14 CV
0510CV

15Fig. 1. Importance of preequilibration (PE) with concentrated buffer. A 10 ml
UNOsphere S column (10.4 cm x 1.1 cm) was run at 250 cm/hr. Equilibration
with 0.05 M acetate, pH 5.0 was preceded by a 1 CV preequilibration with thesame buffer (—) or with 0.5 M acetate, pH 5.0 (—). The total number of CV
required for the column to equilibrate was determined by calculating the volume
from the beginning of preequilibration until the pH stabilized at pH 5.0. The trend
is consistent for other buffer systems, but the degree of improvement varies
from buffer to buffer.

20Ordering Information
Catalog #Description

156-0111
156-0113
156-0115
156-0117

UNOsphere S Support, 25 ml
UNOsphere S Support, 100 ml
UNOsphere S Support, 500 ml
UNOsphere S Support, 10 L

For more information about UNOsphere S support, refer to the
UNOsphere™ Q and S Ion Exchange Media instruction manual,contact your local Bio-Rad sales representative, or visit Bio-Rad
Technical Support on the Web at consult.bio-rad.com

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