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pH Gradient Elution Conditions

There is an increasing interest in pH gradient cation exchange chromatography for the characterization of mAbs.  In pH gradient elution the pH varies with time, while the ionic strength remains constant.  Commonly, mixtures of amine buffering species are used at high pH range, while a mixture of weak acids is employed for low pH range separations. 

However, maintaining linearity of the pH gradient slope can often prove challenging.  A mixture of Tris base, piperazine, and imidazole generates a linear pH range from 6 to 9.5 (9.6 mM Tris base, 6.0 mM piperazine, 11.0 mM imidazole at pH 6 or 9.5).4  Linear pH gradients in the range 7.5-10.0 can be generated using a triethylamine and diethylamine based buffer system (12.5 mM of each compound).5

Elution in pH gradient mode is achieved by modifying the charge state of the protein, and hence, the interaction with the stationary phase surface.  In cation exchange chromatography the protein is expected to elute at or close to its pI.  Therefore, the pH range used dictates which proteins will be eluted successfully.

The ionic strength (salt content) of the mobile phase should remain low for analysis and elution of proteins as this causes analytes to be focused on the column resulting in narrower bands and better resolution.

Similarly to salt gradient analysis, temperature does not have a profound impact on the selectivity of the separation; however, it does affect peak capacity as it influences peak width.  The significant impact of temperature on peak width will have an overall effect on resolution, therefore, temperature is important in the optimization of pH gradient cation IEX analysis.

Fast Optimization of pH Gradient Analysis

The key parameters for optimization of pH gradient cation IEX are pH and temperature.6   Gradient scouting runs with two gradient times and two temperatures can be used to optimize analyses (Table 1).  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 25 and 55 °C
Detector Fluorescence 280-360 nm
Gradient time tg1 = 10 min.
tg2 = 30 min.
Flow rate 0.6 mL/min

Table 1: Method conditions for pH gradient optimization.

Generic pH gradient method for the characterization of 10 intact mAbs

Figure 1: Generic pH 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: CX-1 buffer A pH 5.6
B: CX-1 buffer B pH 10.2 0-100%B, 20 min. 
Flow rate: 0.6 mL/min.6

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