Chromatofocusing

 Biological macromolecules differ in their compositions of buffering groups, such as amino acids and purine or pyrimidine bases, thus giving each biomolecule its own unique charge properties. Generally, at low or acidic pH values, most biomolecules have a net positive charge, whereas at a higher or basic pH they carry a net negative charge. Of course, at a certain pH, the isoelectric pH or isoelectric point (pI), these biomolecules do not possess a net electric charge. This unique isoelectric pH provides the basis for the selectivity of chromatofocusing in chromatographically separating biomolecules.

 Chromatofocusing is a special kind of technique classified within the ion-exchange chromatography of biomolecules. A weak ion-exchange matrix and a pH gradient are used in chromatofocusing, instead of a strong ion exchanger and a salt gradient as in normal ion-exchange chromatography. This technique was first described theoretically and experimentally demonstrated by Sluyterman and co-workers (1, 2). They proposed that a pH gradient could be produced in an ion-exchange column packed with an appropriate ion-exchange resin with good buffering capacity. Although a pH gradient in a column can be produced in a manner similar to that of a salt gradient by using two different pH buffers in a mixing chamber of a gradient maker, a pH gradient can be created internally in the column by taking advantage of the buffering capacity of the weak ion-exchange resin. In practice, a certain pH buffer is used to equilibrate a column packed with a weak ion exchanger. Then another buffer with a different pH is passed through the column, which generates a pH gradient in the column. If such a pH gradient is used to elute biomolecules bound to the ion-exchange resin, the biomolecules elute in order of their isoelectric pH.

 The mechanism of chromatofocusing is based on the buffering action of the charged groups on the ion-exchange resin and on the fact that a biomolecule has a net negative charge at a pH above its isoelectric point. In a descending pH gradient, a single molecular species exists in three charged states—negative, neutral, and positive. When a positively charged column (ie, packed with an anion exchange resin) is equilibrated with a starting buffer of high pH, biomolecules that become negatively charged are initially retained on the column. When an elution buffer of lower pH is passed through the column, a pH gradient develops and the individual molecules continuously change their charged states. The molecules at the rear of the sample zone are the first to be titrated by the low-pH buffer. These molecules become positively charged when the pH is less than their pI, so that they are repelled from the column matrix and are carried rapidly to the front of the sample zone, because of the high velocity of the moving buffer. In traveling to the front of the sample zone, the molecules encounter an increase in pH and are titrated from their positive form to neutrality and back to their negative form. Once the molecules become negatively charged again, they readsorb on the matrix and again fall back to the rear of the sample zone. The cycle between the front and rear of the sample zone results in “focusing” (ie, a continuous narrowing of this zone) until the molecules elute from the column. At this point, the pH of the column effluent is approximately the same as pI of the components eluting.

 Chromatofocusing is an analytical or preparative technique for separating biomolecules according to their pI. The details of this technique relating to column packing, sample preparation, and sample application used in this technique are similar to those for affinity chromatography. A detailed operational protocol is beyond the scope of this article, so the interested reader is directed to other reviews (3, 4).

 There are a number of advantages in using chromatofocusing. In this technique, a biomolecule is not subjected to a pH greater than its pI, and the resulting focusing effects concentrate the sample into a sharp, highly resolved band. One of the great benefits of chromatofocusing is its ease of operation. No gradient-forming devices or mixers are required. The pH gradient is formed with a single isocratic eluent. Chromatofocusing is used widely in research as the method of choice for resolving isozymes and molecular species with very similar charge characteristics, such as transferrin, ferritin, and hemoglobins.

References

1. L. A. A. Sluyterman and O. Elgersma (1978) J. Chromatogr. 150, 17–30. 

2. L. A. A. Sluyterman and J. Wijdenes (1978) J. Chromatogr. 150, 31–44. 

3. L. Giri (1990) in Guide to Protein Purification (M.P. Deutscher, ed.), Methods in Enzymology 182, Academic Press, New York, pp. 380–392. 

4. T. W. Hutchens (1989) in Protein Purification: Principles, High Resolution Methods, and Applications (J.-C. Janson, and L. Rydén, eds.), VCH, New York, pp. 149–174. 

5. Pharmacia (1985) FPLC Ion Exchange and Chromatofocusing-Principles and Methods, Offsetcenter AB, Uppsala, Sweden.