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After considering the results of the brilliant cresyl blue stain, the clinical laboratory scientist decided to repeat the hemoglobin electrophoresis on this patient. This time, she shortened the electrophoresis time by fifteen minutes.The results of the electrophoresis, represented in the image below, show a band in the area of Hb H. Hemoglobin H travels quickly during alkaline electrophoresis, and a shorter electrophoresis time was needed to ensure that HbH remained on the acetate paper. HbF is still present as it was on the original electrophoresis, but it is blended into the Hb A band.

Hemoglobin electrophoresis is the movement of hemoglobin proteins in an electric field at a fixed pH.Because the various hemoglobins are comprised of different combinations of globin chains (normal or abnormal), they will demonstrate different degrees of mobility. Typically, when a thalassemia or hemoglobinopathy is suspected, an alkaline electrophoresis is performed which may be confirmed with acid electrophoresis.For an alkaline hemoglobin electrophoresis, a hemolysate is applied to cellulose acetate which is electrophoresed in a buffer at pH 8.4-8.6. At this pH hemoglobin proteins move from cathode to anode. The proteins are visualized by the application of a dye which also makes them measurable by densitometry.

For electrophoretic separation of solutes, the sample of solutes is placed on a gel or membrane in contact with buffer for separation. Common gels are cellulose acetate, agarose, and polyacrylamide gels. These gels are formed into sheets, slabs, or inserted into columns or tubes. The gel can be positioned horizontally or vertically.Cellulose is chemically reacted with acetic anyhdride to form a cellulose acetate gel. Because cellulose requires soaking before sample application and a clearing step for detection of separated solutes or bands, agarose gel is more often used than cellulose acetate gel for clinical electrophoresis.

Isoelectric focusing is a type of separation where the solutes migrate based upon a different principle. The separation takes place on a gel where a pH gradient has been created using ampholytes. Ampholytes are a mixtures of amphoteric polyaminocarboxylic acids. This mixture possesses a range of pIs, a high buffering capacity at each pH, and is used to create pH gradients.When ampholytes undergo electropohoresis, each individual ampholyte migrates to its own region, an area that matches its pI. After migration of ampholytes, the gel has stable pH zones of increasing pH or a pH gradient. The solutes in the specimen do not migrate to the electrode of opposite charge but to the zone or area that matches their pI. IEF is performed on a gel in a capillary tube, strip, or plate. Gels used are most commonly polyacrylamide gels but agarose and cellulose acetate can also be used.