Capillary electrophoresis (CE) is an electrophoresis technique utilizing small bore capillaries. CE provides methods for the separation of ionic species including macromolecules. The efficiency of CE can be relatively high, i.e. in excess of 400,000 theoretical plates, and thus is being explored for a number of different applications.
A typical CE system includes a 50-100 micrometer internal diameter silica capillary tube filled with a suitable electrically conducting buffer. The outlet end of the capillary is immersed in a reservoir containing the buffer and an electrode. A sample containing ions of interest is introduced into the inlet end of the capillary and then the inlet is placed in another reservoir containing the buffer and another electrode. Since a small diameter capillary is used in CE, a relatively high applied voltage can be used without the generation of thermal gradients in the capillary; thus the electrodes are connected to a power supply capable of delivering .sup..about. 30 kV per 100 cm of capillary. A detector is placed between the two electrodes to permit detection of various ionic species migrating in the capillary. A detector so positioned is often referred to as an on-column detector. Typically, an integrator is attached to the detector, such that the peak areas may be measured.
The movement of the sample ions of interest is controlled by two factors: the electrophoretic velocity and the electroosmotic flow velocity. The total migration velocity is the vector sum of these two terms.
Electrophoretic migration is the migration of the sample ion towards the oppositely charged electrode under the influence of the electric field. The electrophoretic mobility of any particular ion is the electrophoretic velocity per unit field strength.
Electroosmotic flow (EOF) is the bulk flow of the buffer in the capillary. EOF is due to the charge of the inside surface of the capillary which is in contact with buffer containing mobile counterions. For example, an unmodified silica capillary surface comprises silanol (Si--OH) groups that are negatively charged (Si--O.sup.-) when the pH of the buffer is greater than about 2, and positively charged (Si--OH.sub.2 +) when the pH is less than about 2. Alternatively, hydrophobic cations may be adsorbed onto the inside surface of the capillary to obtain a positively charged surface at higher pHs.
When the surface is negatively charged, then the mobile counterions, for example, sodium ions (Na.sup.+), migrate under the influence of the electric field and in the process drag the bulk solvent with them. Thus the direction of the electroosmotic flow is from the positive to the negative electrode when the surface is negatively charged.
When the surface is positively charged, then the mobile counterions of the positively charged surface, e.g. biphosphate ions (HPO.sub.4.sup.2-), migrate under the influence of the electric field and in the process drag the bulk solvent with them. Thus the direction of the electroosmotic flow is from the negative to the positive electrode when the surface is positively charged.
When the surface is not charged, then there is no electroosmotic flow, and any movement of analyte ions is due solely to electrophoretic mobility.
Thus, depending on the charge of the ions of interest, the nature and the extent of capillary surface charging and the polarity of the applied voltage, electroosmosis can augment, counteract or even override the electrophoretic movement. Since sample components to be determined must travel from the inlet end of the capillary to the detector which is located near the outlet end of the capillary, it is essential that they move in the desired direction. However, since the total migration velocity of the sample is the vector sum of the electrophoretic velocity and the electroosmotic flow velocity, it is possible that the charge of the sample is such that it would move away from the outlet electrode in the absence of electroosmotic flow; under these conditions the electroosmotic flow velocity of the bulk solution must be greater than the electrophoretic mobility of the analyte.
The detector used in the CE system is very important, and the type of detector used will usually depend on the properties of the compounds under analysis. Currently, there are a number of different detector schemes utilized in CE. These include direct and indirect photometric detection, direct and indirect fluorescence detection, as well as suppressed and non-suppressed conductometric detection. Other types of detection which may be utilized are mass spectrometry, radiometry and other electrochemical methods such as amperometry. These methods may be used either on-column or end-column, i.e. at some point after the outlet electrode.
While most organic molecules of interest display significant ultraviolet absorption such that direct photometric detection is practical, this is not the case for many inorganic ions or aliphatic carboxylic acids that display very low optical absorption. Thus indirect photometric or fluorescence detection may be utilized in these cases. For example, indirect fluorometric detection is described in Gross et al., Anal. Chem. 62:427-431 (1990); Bachmann et al., Journal of Chrom. 626:259-265 (1992); and Gross et al., Journal of Chrom. 480:169-178 (1989). Indirect photometric detection is described in Foret et al., Journal of Chrom. 470:299-308 (1989); Foret et al., Electrophoresis 11:780-783 (1990); and Henshall et al., Journal of Chrom. 608:413-419 (1992). Additionally, since electrical mobility is an intrinsic property of all ions, detection based on conductivity can be a desirable method for many uses of CE.
However, conductivity detectors are nonselective bulk property detectors. The signal arises from the difference in equivalent conductance or mobility of the charge carrier electrolyte ion and the analyte ion. In CE, a large difference in mobility of the carrier electrolyte ion and the analyte ion leads to excessive peak tailing/fronting, which means that there are practical restrictions on the choice of the eluent ion. This conflict between optimum sensitivity and separation efficiency represents the ultimate limitation of nonsuppressed conductivity detection in CE.
Electrolyte suppression, or the post run alteration of the electrolyte buffer such that the background "noise" of the buffer is decreased, has been explored for ion chromatography (see for example U.S. Pat. Nos. 3,897,213; 3,920,397; 3,925,019; 3,956,559; 4,474,664; 4,751,004; 4,459,357 and 4,999,098), and recently for CE (U.S. Ser. Nos. 07/771,336 and 07/771,597, filed Oct. 4, 1991, herein incorporated by reference). These systems are referred to as "suppressed" systems.
When conductometric detection is utilized, aspects of the electropherogram from a CE run are different from an optically detected CE run. These phenomena are the subject of the present invention.
One problem with CE is that the sample volume introduced into the capillary may vary; samples are not easily injected onto the column by a fixed volume valve. This can be a problem in other chromatography systems as well, although many systems do employ a fixed volume valve. Instead, in CE, the samples are introduced into the capillary in several ways. Typical injection modes for CE are pressurization of the sample- or standard-containing vial for a fixed length of time (pressure injection), or the application of an electrical field for a fixed length of time (electrostatic injection). Thus CE sample injection methods are based on time of injection. This means that small variabilities in time, sample viscosity, pressure or hydrostatic height may result in variabilities of the sample volume injected. This may have a profound impact on the migration time and the size of the peaks.
Furthermore, these potential variabilities may make quantification of sample peaks difficult. Quantification requires that the injected volume of the sample and the injected volume of the standard be essentially exactly the same, or that the volume of each be precisely known to compensate for injected volume differences.
Another problem with CE and other systems relying on electroosmotic flow is that it is difficult to determine the electroosmotic flow velocity, and to detect any variations in this rate. For example, in CE, the electroosmotic flow velocity in a bare silica capillary can be affected by material adsorbed on the wall from the previous injection, which may alter the flow rate. Similarly, the flow rate of different capillaries may be different due to a variety of factors. The electroosmotic flow velocity will also change as a result of a change in applied voltage. Since the flow velocity will affect the migration times of the analyte peaks, i.e. total migration velocity of the analyte, this may be a serious problem. This takes on an increased significance if an integrator is used, since the integration is dependent on time; thus a decrease in the flow rate for a sample run may result in an increase in the peak area, and vice versa.
These two limitations are addressed by the present invention.