High Pressure Liquid Chromatography (HPLC), Capillary Electrophoresis (CE), and Isoelectric Focusing (IE) are newer methods for detecting hemoglobin variants.
The principle of HPLC is that mobile buffers at different concentrations pass through a stationary ion exchange column. As the programmed buffer gradient of increasing ionic strengh and pH is delivered by pumps, the hemoglobins are separated according to their ionic interactions. As each separated hemoglobin passes through the flow cell photometer, each hemoglobin is characterized by a specific retention time, which is the elapsed time from the sample injection to the peak of the hemoglobin. An example of some common elution times is seen below.
In CE, an electro-osmotic flow is established in a capillary, and differently charged hemoglobins are thus separated. Although it is an electrophoretic method, it has been shown to be superior to conventional methods, especially if the CE utilizes an internal standard such as HbA2, and is said to be more user-friendly.
IE focusing is an electrophoretic method using polyacrylamide gels. In this method, the hemoglobin will move through a pH gradient to the point where its net charge is zero. This technique does require high voltages, but gives good separation with higher resolution compared to other methods.
