This application relates in general to separation techniques such as capillary electrophoresis and in particular, to apparatus and method for on-column derivatization in capillary electrophoresis.
Capillary electrophoresis (CE) is rapidly emerging as one of the separation methods of choice in resolving a complex mixture into its constituents (see Jorgenson et al., Science 1983, 222, 266; Gordon et al., Science 1988, 242, 224; Ewing et al., Anal. Chem., 1989, 61, 292A). In this procedure an electric field is applied across a capillary structure with typical dimensions of 2-200 .mu.m inside diameter and 10-100 cm length. The medium is an electrolyte or a gel. Because the volumes are small, typically nanoliters of injected sample, a major challenge is to find suitable detection schemes. In past work, many detection schemes have been used, such as optical absorption (see Lauer et al., Anal. Chem., 1986, 58, 166), optical fluorescence (see Gassman et al., Science, 1985, 230, 813), electrochemical (see Wallingford et al., Anal. Chem., 1987, 59, 1762), conductimetric detection (see Huang et al., Anal. Chem.. 1987, 59, 2747), radioactivity (see Pentoney et al., Chromatog., 1989, 4809, 259, refractive index change (see Bruno et al., Appl. Spec., 1991, 45, 462, and mass spectrometry (see Olivares et al., Anal. Chem., 1987, 60, 1230). In each of these procedures, it may be advantageous to attach to the molecular constituent to be detected a label or tag that aids/enables its detection. Examples are labels for fluorescence, absorption, electrochemical detection and radioactivity.
Frequently, the sample is derivatized off-column prior to being injected. In such pre-separation derivatization schemes, usually the sample to be separated is mixed with a labeling agent or compound in a vial where the compound or agent would react chemically with the sample to label or tag the sample. Such method of labeling or tagging is disadvantageous. Small samples will become diluted by the procedure since, for convenient handling, the vials used cannot be too small. After the labeling or tagging process has been completed, a portion of the labeled sample is then injected into a capillary column for separation. Thus, pre-column labeling requires two or more steps before the labeled sample is ready for separation. Post-column separation techniques have also been used, such as in U.S. Pat. No. 4,729,947 to Middendorf et al. Post-column labeling techniques share the same disadvantages as pre-column labeling techniques.
In view of the above-described drawbacks of off-column type derivatization techniques, on-column derivatization for fluorescence detection using a specially built T-shaped or "cross"-shaped structure in the capillary is proposed in U.S. patent application Ser. No. 07/235,953, filed Aug. 24, 1988 by Richard N. Zare et al., which is incorporated herein in its entirety by reference. See also the article entitled "On-Line Connector for Microcolumns: Application to the On-Column o-Phthaldialdehyde Derivatization of Amino Acids Separated by Capillary Zone Electrophoresis," by Pentoney et al., Anal. Chem., 1988, 60:2625-2629.
Another type of on-column derivatization technique is disclosed in a poster presentation by U. R. Tjaden et al., entitled "On-Line Derivatization and High Performance Capillary Electrophoresis," in the Thirteenth Symposium on Column Liquid Chromatography, Stockholm, Sweden, Jun. 25-30, 1989. In the technique proposed by Tjaden et al., a labeling reagent is added to the electrophoresis buffer in the capillary before the sample to be derivatized is introduced into the capillary column. Such technique also has a number of disadvantages. First, if fluorescent detection is used, the reagent present throughout the buffer in the capillary will cause background fluorescence which degrades the signal-to-noise ratio of the fluorescence detector. Furthermore, the reagent used must be carefully chosen so that the background fluorescence caused by the reagent will not be so high as to render fluorescence detection impossible. This severely limits the type of reagent that can be used and is undesirable.
None of the above-described off-column or on-column techniques is entirely satisfactory. It is therefore desirable to provide an improved on-column derivatization scheme in which the above-described difficulties are alleviated.
In particular, it is desirable to provide an improved on-column derivatization scheme where the unreacted reagent, dye or any other labeling compound would not reach the detector.