Pseudo‐Isotachophoresis: II. Visualization of the Stacking

Abstract

In order to investigate the stacking step by pseudo‐isotachophoresis (p‐ITP) completely apart from the separation step, wide capillaries 10–20 cm×0.5–0.8 cm (I.D.) were completely filled with coloured analytes, such as Br‐cresol green and ponceau S dissolved in both acetonitrile and salts and imaged at different times. The compounds concentrated visibly with efficiency of ∼10–12. Chrmogenic salts, such as chromate (ions), migrated to the anode ahead of the Br‐cresol green. It was apparent in this work that acetonitrile and salts are necessary for the stacking. Unexpectedly, the speed of the stacking and its association with some boundary separation were evident in this set‐up. As the salt concentration in the sample is decreased, the stacking speed increased greatly. Also, as the concentration of the analytes in the sample decreased the speed of the stacking increased. Indirectly, EOF affected the concentration by moving the anodic band slowly to the cathode and prevented reaching equilibrium. In most cases, when two anionic compounds are present in the sample, there was concentration but without separation. However, as the length of the tubes (sample) is increased some boundary separation is present. As the sample concentration decreased the separation between the two compounds improved. Thus, the stacking step under proper conditions of analysis such as sample plug dimensions, buffers, and analyte concentration, can be accompanied by some separation. This simple approach should be helpful to investigate other types of stacking.