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Salt Gradient Elution Conditions

During linear salt gradient elution the salt concentration (ionic strength) is varied with time during the gradient.  Samples are loaded in a low salt environment to promote interaction with the stationary phase.  Commonly used salts are sodium and potassium chloride and acetate.  An adequate salt concentration is required to disrupt the stationary phase/analyte interaction in order to elute the analytes.  Normal elution concentrations are in the range 200-500 mM.

pH is one of the key variables for altering retention in IEX as retention is strongly affected by the ionization state of the analyte.  The mobile phase pH must be set at least 1 pH unit (ideally not less than 2) below the protein pI for cation IEX. The charged functional group on the stationary phase must also be considered as this must also be in its ionized form in order to interact with the analyte.

Therefore the mobile phase buffer pH should be between the protein pI and the pKa of the surface functional group (for both cation and anion IEX).  In cation IEX a lower pH value produces a protein with more positive charge, resulting in greater retention.  IEX is normally carried out with pH values in the range 5.5 and 7.0, however, some applications may require a lower pH (3.5) to optimize retention and selectivity.  To provide suitable buffering capacity the buffer concentration is usually between 10-50 mM.  The most common buffers used for IEX are shown in Table 1.

 
Buffer pKa pH range UV cut off (nm)
Formate 3.8 2.8-4.8 210 (10 mM)
Acetate 4.8 3.8-5.8 210 (10 mM)
MES 6.10 5.5-6.7 215
Phosphate 2.1
7.3
12.3
1.1-3.1
6.2-8.2
11.3-13.3
<200
HEPES 7.48 6.8-8.2 225
Citrate 3.13
4.76
6.40
2.2-6.5
3.0-6.2
5.5-7.2
230
Acetic acid   4.8-5.2  
Citric acid   4.2-5.2  
Lactic acid   3.6-4.3  
MOPS   6.5-7.9  
PIPES   6.1-7.5  
TES   7.2-7.8  
Tricine   7.8-8.9  
Buffer pKa pH range UV cut off (nm)
Bis-tris 6.46 5.8-7.2 215 (10 mM)
Tris 8.06 7.5-9.0 205 (10 mM)
Piperazine 5.33
9.73
5.0-6.0
9.5-9.8
<200
Bicine   7.6-9.0  
Diethanolamine   8.4-8.8  
Diethylamine   9.5-11.5  
Triethanolamine   7.3-8.3  
Tricine   7.4-8.8  
Pyridine   4.9-5.6  
Imidazole   6.6-7.1  
L-histidine   5.5-6.0  

Table 1: Properties of common buffers used for ion exchange chromatography.

It has been noted that lower flow rates provide higher efficiencies for monoclonal antibodies in cation IEX, therefore, reducing the flow rate can help to improve resolution of charge variants.2


Fast Optimization of Salt Gradient Analysis

The important parameters for optimization of selectivity and resolution in salt gradient elution are salt gradient steepness (gradient time) and mobile phase pH.  Temperature does not have a pronounced impact on selectivity and resolution, however, working at low temperatures guarantees the highest peak capacity, therefore, a temperature of 30 °C is appropriate.   

Fekete et al. have shown that a salt gradient method can be optimized using only 6 scouting runs, comprised of two gradient times and three pH values (Table 2).3  The results from these experiments and modelling software can then be utilized to optimize separation conditions.

Column 4.6 x 100 mm, 5.0 μm non-porous SCX
Temperature 30 °C
Detector Fluorescence 280-360 nm
Gradient time tg1 = 10 min.
tg2 = 30 min.
pH 5.6, 6.0, and 6.4
Flow rate 0.6 mL/min

Table 2: Method conditions for salt gradient optimization.

The methodology of these six scouting runs (two gradient times at three pH values) was applied to 10 mAbs (pI 6.7-9.1) and their charge variants (Figure 1).  Acidic and basic mAb variants are eluted before and after the main peak respectively.  Optimal conditions on a strong cation exchange column were developed from the six scouting runs to give a generic salt gradient method that could be applied to several different types of mAbs and adjusted as needed. 

Generic salt gradient method for the characterization of 10 intact mAbs

Figure 1: Generic salt gradient method for the characterization of 10 intact mAbs.

Column: 4.6 x 100 mm, 5.0 μm non-porous SCX.
Temperature: 30 °C.
Detector: Fluorescence 280-360 nm.
Mobile phase:
A: 10 mM MES pH 5.7
B: 10 mM MES pH 5.7 + 1 M NaCl 0-20%B, 20 min.
Flow rate: 0.6 mL/min.3

 
 
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