What impact can choice of solvent have on the separations obtained in CE?

Sample diluent ionic strength, pH, organic solvent composition and viscosity can all have a significant impact on the performance of CE separation. It is, therefore, essential that the choice of solvent is optimized during method development. The optimized diluent should be fully specified in the method and used consistently in method validation and all subsequent applications of the method.

What is the effect of sample diluent ionic strength?

In CE the typical length of an injection inside the capillary is a few mm. This is a significant length given that the total capillary length may be 30 cm and the detection window may be 0.1 mm. The peak width is directly related to the injection zone length. Ideally the injection zone length would be very small but this would result in sensitivity issues.

There are many different in-capillary concentration approaches in CE that can be used to improve method sensitivity and separation efficiency by reducing peak width after injection. These have been summarized and interested readers should read these more in-depth publications.^1,2

The most common approach to reducing the zone length of the sample injection is by using a process termed "stacking". Stacking reduces the width of the sample zone at the start of the separation when the voltage is initially applied and results in an improved sensitivity (because the sample becomes more concentrated inside the capillary) and increased peak efficiency.

"Failure to use the exact sample diluent can lead to irreproducible separations and/or poor quantitative performance."

Figure 1

Stacking occurs when the sample is dissolved in a lower ionic strength solution than the separation electrolyte. Under these circumstances the field strength is higher in the sample zone than in the rest of the capillary, which is filled with electrolyte (Figure 1). The sample ions move forwards rapidly in the sample zone until they encounter the electrolyte boundary where they experience a lower applied field and their migration rate slows down. In this way the sample zone is focused/condensed/concentrated and stacking can lead to as much as a 10-fold reduction in the starting peak width. This process is optimized if the sample is dissolved in a 1:10 dilution of the run electrolyte.

Figure 2(a)

Figure 2 illustrates the stacking principle in action. Figure 2(a) is the separation achieved for a 0.5 mg/mL solution of a drug dissolved in 50 mM phosphate buffer. Separation was performed using the same 50 mM phosphate buffer. The peaks in the separation are relatively wide, which gives poor sensitivity and limited resolution. Figure 2(b) shows the stacking effect — in this instance a 0.5 mg/mL solution of the same drug batch was dissolved in a 5 mM phosphate buffer and separated with a 50 mM buffer.

Figure 2(b)

The sample is concentrated within the capillary to give both smaller peak widths and higher sensitivity. This concentration results in better sensitivity as a result of taller peaks and better resolution because peaks are more efficient/thinner.

Stacking can also be deliberately performed if the solute is zwitterionic (i.e., it has a positive charge at low pH and a negative one at high pH).¹ For example, samples can be deliberately prepared in a high pH diluent and separated using a low pH buffer. In this instance the samples would be negatively charged and would move to the back of the sample zone when the voltage is initially applied. When the sample ions reach the back of the zone they protonate and move forward and the sample zone length is greatly reduced.