Capillary electrophoresis: the newest analytical separation technique.
Capillary electrophoresis is the newest technique to enter the realm of separation science. The technique enables separations to be carried out with an electrolyte (mobile phase) that can be modified as in HPLC, to move peaks relative to each other. The use of electrolyte with organic solvent also enables the technique to be readily used for organic or inorganic compounds. The other major advantage is high efficiencies are obtained, 100,000 plates is common, with some separations as high as 1,400,000 plates and higher. This brings the separation technique in line with capillary GC as far as efficiency is concerned.
Early publications in capillary electrophoresis have been most numerous in the analysis of biochemical compounds ie. proteins, DNA, etc. As capillary electrophoresis is developed it will become more widely used and will find its way into more labs for smaller molecules. One unique use that is presently available is the separation of anions. The technique uses a patent pending technology developed by Waters called Nice Chemistry enabling the separation, and detection of thirty (30) anions in 89 seconds (see Figure 1).
Capillary electrophoresis is performed using capillaries 50um - 100um in internal diameter and 30cm - 100cm in length. The fused silica capillaries are coated with polyamide making them easier to handle. To perform CE separations, both capillary ends are placed into separate vessels containing the same buffer solution. Into these buffered solutions are placed an anode and cathode connected to a power supply (see Figure 2). With the application of voltage, flow called electroosmotic flow begins from the anode toward the cathode as the hydrated positive counterions move toward the cathode. Electroosmostic flow is the bulk carrier that takes sample from the injection point past the detector. The other main forces are the electrical attraction toward either the anode or cathode. Compounds attracted to the anode will move more slowly than the bulk flow while compounds attracted to the cathode will move more quickly than the bulk flow. In this separation scheme called free zone capillary electrophoresis, neutral compounds will migrate with the bulk flow as the only active force on the molecule(s). Therefore, neutrals will migrate together in a single region. To detect compounds migrating through the capillary, a section of the polyamide coating is removed and the detector diode placed next to the transparent silica capillary. Detector sensitivity, the measure of light absorbed by the samples is dependent on the internal diameter of the capillary. The internal diameter of the capillary is the pathlength for the detector. It is, therefore, a major advantage to be able to, readily use varying diamter capillaries to increase sensitivity, because sample mass will limit sensitivity.
When voltage is applied across the capillary, current is generated. The amount of current generated will depend on the electrolyte characteristics and strength. The higher the current generated, the higher the heat produced. A second problem can arise from this current excess, bubble formation. If the current is too high, bubble formation occurs and this stops the electroosmotic flow and no separation occurs. To prevent bubble formation, the current is monitored by the instrument. Typically, if the current remains below 100uA, the electrolyte will not boil. The heat generated by the current must be dissipated from the capillary to ensure that the migration time is reproducible. The dissipation of heat is accomplished by different means from manufacturer to manufacturer. The simplest, requiring the least maintenance, is the use of fans which draw air across the capillary, thereby removing the generated heat. This also has the advantage that specialized cartridges are not necessary, thereby reducing the cost of capillaries and holders. This increases the flexibility of CE, allowing the use of any type or dimension of capillary.
Introduction of sample into the capillary is performed in one of two modes of injection. Sample can be introduced from a sample vial containing buffer by applying a low voltage for a specified period of time. This form of injection is called electromigration. Electromigration will selectively inject species that migrate fastest toward the cathode. A second means of injection is called hydrodynamic. This injection technique inserts a representative sample into the capillary. Typical injection volumes are 1-20nl, not the microlitre injections used in high-performance liquid chromatography (HPLC). Hydrodynamic injections are performed by an elaborate procedure drawing a vacuum on the capillary for a finite time, thereby drawing sample in to the capillary. Strict control of the time and vacuum must be maintained for reproducible results. A similar, more reliable and simpler method has the injection end of the capillary immersed in the sample vial. The sample vial and capillary are raised above the running electrolyte for a specified time at a fixed distance (10cm). The sample enters the capillary due to gravity force which applies a fixed pressure. This method, termed |hydrostatic' injection, has proven to be extremely reproducible and reliable. Figure 3 shows the data from an analysis of Diazide tablets. For six samples, the standard deviation of area for hydrochlorothiazide (HCT) and metahydroxy acetophenone (HAP) were 0.7% and 0.5%, respectively. The HAP and HCT show excellent reproducibility of injection.
The detection schemes available in capillary electrophoresis are numerous because equipment suppliers are able to draw on experience from HPLC and apply this to capillary electrophoresis. The most widely used detectors to date are UV/VIS adsorption detectors. These detectors are used due to the ease of incorporating capillaries into the path of the photodiode and the sensitivity available. The small amount of sample mass injected make it necessary, that the most sensitive UV/VIS detectors be used. The most sensitive and linear UV/VIS detectors are able to quantitate trace levels of compounds in the presence of larger quantities of the major compound. The sensitivity of the Waters Quanta 4000 detector make it possible to see impurities below 0.1% (see Figure 4).
Uses for Capillary Electrophoresis
The technique of capillary electrophoresis lends itself well to charged molecules when performed in its simplest form, free-zone capillary electrophoresis. To increase CE usefulness as an analytical technique there is a need to separate uncharged molecules and to perform analyses not presently available with other techniques, or at least to make such separations simpler to obtain. The technique used to separate neutral molecules is termed micellar electrokinetic capillary chromatography (MECC). MECC is performed when a detergent is added to the electrolyte above the concentration necessary to form micelles. The most widely-used detergent is sodium dodecyl sulfate (SDS). The micelle will act as a |psuedo-stationary phase'. In the case of SDS, it will have a net negative charge and will be attracted to the anode, thereby slowing its movement, relative to the electroosmotic flow set up by the application of voltage across the capillary. The SDS slower rate of movement will allow other molecules usually swept toward the cathode with the bulk flow, to interact with the SDS micelle due to their degree of hydrophobicity. The differences in interaction with the micelle allow these molecules to be separated from each other even if the molecules do not have a charge.
An area where capillary electrophoresis has shown excellent potential, as a separations technique, is in the analysis of isomers. The high efficiencies generated enable isomers to be readily resolved. One particular type of analysis performed more easily and quickly by capillary electrophoresis is the separation of optical isomers. Using a micelle that will physically discriminate by preferential inclusion of one form relative to the other enables enantiomers to be separated. Cyclodextrins are one of the most common chiral micelle discriminators. Cyclodextran micelles are cone shaped and more readily include the linear form of an enantiomer. The form not readily included will migrate at a faster rate relative to the included form and this will readily resolve the enantiomers (see Figure 5). Other materials are also used to perform enantiomer separations such as bile salts, crown ethers and metal ions.
The separation of anions by capillary electrophoresis was mentioned earlier in this article. The separation is done with simple and easy modifications to the basic CE system. The power supply which applies voltage across the capillary is normally a positive power supply for positively changed molecules. By a simple exchange of the power supply for a negative power supply (see Figure 6), the anions migrate toward the anode, separate and are detected. This movement eliminates the interferences found in the separation of anions by ion chromatography (IC). The term coined for this type of separation is inorganic capillary electrophoresis (ICE). ICE uses chemistry called Nice-Pak from Waters. Nice-Pak Chemistry enables the migration to be controlled to allow rapid, high resolution separations to occur, as well as making it possible to visualize the anions using a photometric detector. The detection limits for this technique in the low PPM range and lack of interfering compounds make it an excellent screening technique due to the rapid separations that occur (see Figure 7). This separation also lends itself to the analysis of organic acids, a more difficult separation to perform in the food industry (see Figure 8).
The technique of capillary electrophoresis is in its infancy, but it can be expected to move at a more rapid pace than either capillary GC or HPLC, because of the previous developments in these separation techniques. Development of capillaries with specific internal coatings, the development of modifiers, both designed to control the flow through the capillary and to control migration of sample molecules should be a major area of change. Improvement in the detection of samples. The advantages of the technique come in the areas of, rapid separations, high efficiency, good sensitivity and the ease with which changes to separations can be made. It is an analytical technique we will all be reading much about in the near future.
Thanks to Peter Rahn, Mike Swartz and Bill Jones from Waters, Milford, MA.
PHOTO : Figure 1 30 Anions in 89 Seconds (Analysis time 3.1 min)
PHOTO : Figure 2 Basic CE Instrumentation
PHOTO : Figure 3 Analytical Results for Dyazide
PHOTO : Figure 4 Capillary Electrophoresis Impurity Profile
PHOTO : Figure 5 Capillary Electrophoresis Curve for Salicylamide
PHOTO : Figure 6 Capillary Electrophoresis Setup for ICE* with Negative Power Supply
PHOTO : Figure 7 Analysis of Anions Fast Anion Separation Using Inorganic Capillary Electrophoresis
PHOTO : Figure 8 Analysis of wine organic acids provides producers with authentically, stability and flavour information. Separation is done using Waters Nice-Pak Chemistry. Sample concentration range from 3,500 to 70 ug/ml.
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|Publication:||Canadian Chemical News|
|Date:||Feb 1, 1991|
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