TECHNICAL INFORMATION

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1 T-1860A TECHNICAL INFORMATION Capillary Electrophoresis PRECISION IN CAPILLARY ELECTROPHORESIS Harry Whatley and Jeff Chapman Beckman Coulter, Inc. Introduction Instrumentation for routine chemical analysis must have an acceptable degree of precision. The instrument must give the same result for a given sample from run to run and from day to day. A technology must be shown to be sufficiently reproducible for the task at hand before it can be accepted for routine use. Many advances in instrument system design have been made since capillary electrophoresis was commercialized in the late 1980s. As this note will demonstrate, P/ACE System MDQ is capable of achieving the degree of migration time and peak area precision necessary for routine chemical analysis. Reproducibility in CE Many factors are involved in reproducibility. Some of these, such as temperature control, voltage control, and sample injection precision, are inherent in the design of the instrument. Other factors, such as the quality of the reagents used and the manner in which the instrument is programmed and operated, are completely in the hands of the user. These two factors, the quality of the instrument and the quality of the operation, are both required in order to achieve reproducible results. To determine the typical reproducibility that can be achieved on the P/ACE System MDQ, we evaluated the separation of two very similar analytes in a commercially available test mixture (System Performance Test Mix B, Beckman Coulter, Inc.). This test mix contains two organic acids: p-hydroxybenzoic acid (0.72 mmol/l) and p-hydroxyphenylacetic acid (0.66 mmol/l). Figure 1 illustrates a typical separation of these two compounds. Figure 2 shows the peak area plotted against run number from 100 successive runs of a single sample of the test mixture. The peak area %RSD for the entire set of 100 runs is 1.52% for Peak A and 1.67% for Peak B. It is not usual for analyses to require this many replicates: six is a typical number. Figure 3 shows the moving %RSD of the area of Peak A for any six consecutive runs (1-6, 2-7, etc., giving 95 sets of data points). The average %RSD for the sets of six runs is 0.89% with a range from 0.42% to 1.48%. The above data represent peak areas which have not been corrected for velocity. In CE, peak area is not only a function of concentration but also of an analyte s velocity through the detection window. Any irreproducibility in migration time will compound the error in peak area determination. In capillary electrophoresis, it is usual to normalize peak areas for migration velocity. The resulting parameter is referred to as corrected peak area. The corrected area %RSD for the entire set of 100 runs is 1.43% for Peak A and 1.56% for Peak B. The average %RSD for the sets of six runs is 0.90% with a range from 0.44% to 1.46%. The fact that the %RSD values from both methods of area calculation are essentially the same indicates that the migration velocities were nearly constant throughout this experiment. Peak migration time is another factor for which precision is important. Very often the migration time of a given analyte is used to identify the species present in a mixture. Peaks that drift from run to run make identification difficult. In the present experiment the migration times of the two components in

2 Run # 20 of 100 Peak B %RSD, A Absorbance A = p-hydroxyphenylacetic acid B = p-hydroxybenzoic acid Peak A %RSD Minutes Figure 1. Typical separation from this experiment. Peak Area Area A Area B Run Number Figure 2. Peak area for 100 runs Set Number (n=6) Figure 3. %RSD values for sets of 6 consecutive runs, Peak A (Peak B is similar). mt, Minutes mt A mt B Run Number Figure 4. Migration time of Peaks A and B. the mixture were quite consistent (as shown in Figure 4). The migration time %RSD for the entire set of 100 runs is 0.19% for both Peaks A and B. Using the six-run-moving-average method for %RSD gives values ranging from 0.00% to 0.34% for Peak A and 0.00% to 0.38% for Peak B. Reproducibility of Mobility Separation of analytes in capillary zone electrophoresis is a function of charge and mass. The calculated mobility (µ, cm 2 /Volt-sec) of the analytes is an important factor to be considered. Mobility is useful in analyte identification strategies as it accounts for normal variations in such run factors as electroosmotic flow and applied voltage. Automatic mobility determination is one of the CE-specific functions of the P/ACE System MDQ software. Accurate determination of the voltage profile up to the point where the peak emerges is critical to the correct calculation of mobility. P/ACE System MDQ integrates the voltage profile on a point-by-point basis to account for variations such as voltage ramps. In the determination of mobility, a neutral reference marker such as acetone or benzyl alcohol is used to take into consideration the impact of electroosmotic flow (EOF). This is often inconvenient in routine analyses. Mobility values suitable for peak identification can be obtained by using a reference marker that is similar in chemical structure to the analytes of interest in place of the neutral marker. By way of example, we can consider Peak A as a reference marker and assign it a value of µ = cm 2 /Volt-sec (this value having been previously obtained using a neutral marker). The P/ACE System MDQ software can use Peak A as a mobility standard against which other peaks can be compared. Using this technique to calculate the mean mobility of Peak B for the entire set of 100 runs gives a value of µ = cm 2 /Volt-sec with a %RSD of 0.07%. Using the six-run-movingaverage method for %RSD gives values ranging from 0.01% to 0.10%. 2

3 How Are Results Like This Obtained? Table 1 describes the separation conditions employed in this study. The results of these experiments are summarized in Appendix A. The complete set of MDQ data files is available for review contact a Beckman Coulter, Inc., representative for details on how to obtain a copy. The key factors in obtaining the reproducible results shown in this report are: Control of buffer ion depletion Proper injection conditions Adequate capillary conditioning Buffer Ion Depletion: During any electrophoretic separation, buffer ions migrate from one buffer reservoir to another along with the analytes. Over large numbers of injections, the buffer composition in the reservoirs will change sufficiently that the migration of the analytes will be affected. This can be controlled by limiting the number of samples run with any one pair of buffer vials or by using larger buffer reservoirs. The separations shown here were performed with the large buffer reservoir accessory on the P/ACE System MDQ (Figure 5). Each buffer tray has two 25 ml reservoirs. This allows extended analyses without the effects of ion depletion. Injection Conditions: P/ACE capillary electrophoresis instruments utilize a pressure-time integral calculation to correct for variances during injection. With positive pressure injection, this is made necessary by the time required for pressure to ramp up and ramp down and by the response time of pressure-sensing devices. These variances are monitored and the injection time adjusted so that the product (pressure time) is constant from run to run. In theory, a 5-psi, 1-second injection and the 0.5-psi, 10- second injection used in this study should give the same injected volume. In practice, a longer, lower pressure injection gives the instrumentation more time to respond to variances and will give a better result. Another useful technique that was employed here is to dip the capillary into water between injection and separation. The parallel electrode-capillary design of P/ACE System MDQ makes a simple water dip effective for reducing sample transfer from vial to vial. This step is accomplished through the P/ACE System MDQ software by programming a wait function while sending the capillary to a water vial. Using a wait time of zero will result in a brief dip followed immediately by the next method step. Table 1. Separation Conditions Instrument: P/ACE System MDQ Capillary: 75 µm internal diameter 50 cm to the detector window 60 cm total length Run Buffer: P/ACE Capillary Performance Test Buffer A (P/N ) in Large Reservoir Tray (P/N ) Sample: P/ACE Capillary Test Mixture B (P/N ) (A mixture of p-hydroxybenzoic and p-hydroxyphenylacetic acids) Detection: UV absorbance at 214 nm Method: Inject Positive pressure, 0.5 psi for 10 seconds Wait Water vial dip, time = 0.0 Separate 30 kv constant voltage, 5 minutes, normal polarity, 25 C Rinse Run buffer, 20 psi for 1 minute Figure 5. Buffer Reservoir Tray as used in this study (P/N ). Injection precision is a prerequisite for peak area reproducibility. If injection volumes are not controlled from run to run, it will be impossible to construct a calibration curve that will be suitable for quantitative analysis. When using external standards to construct a calibration curve, the standards and the samples should be matched as closely as possible. If the viscosity of the sample and the standards is not matched (such as sampling blood serum against standards dissolved in water), the actual volume of sample injected may be different from the volume of standard injected. Similarly, if the conductivity of the standards and the samples is not matched, the migration times or peak widths of the analytes may differ between the standards and the 3

4 samples. If it is not possible to match the standards with the samples, spiking or co-injection of standards into samples will provide a better way to identify and quantitate unknowns than will using an external calibration curve. At very high analyte concentrations, it may be observed that peak area increases without a corresponding increase in peak height (i.e., the peaks become wider). With oncapillary detection, this is seldom a problem except with very closely spaced peaks. With extended-path detection schemes, this peak broadening may increase the probability of the co-detection of multiple components. Capillary Conditioning: Capillary conditioning is often underestimated as a source of reproducibility problems. A surface should be chosen that is compatible with the types of analytes that are being separated. When using a non-coated, fused-silica capillary, it is important to understand the nature of the charge at the walls. At ph values above 3.0, the capillary wall begins to be charged and electroosmotic flow becomes a significant factor in the separation. A used capillary surface will also exhibit a memory effect it remembers the last buffer system to which it was exposed. Time is required for equilibration with a new buffer system. A new, bare fused-silica capillary should be cleaned and regenerated prior to use. A suggested protocol for starting up is given in Table 2. After cleaning (and again after storage), the capillary must be equilibrated with the buffer system that will be used for analysis. A simple way to do this is to run five to ten blank runs (no sample injection) prior to running sample analyses. The need for this can be seen in Figure 4. For the first few runs, the peak migration times drift slightly, even though this capillary had been used a few days earlier for this same analysis. It is a good practice to dedicate a piece of capillary to one type of buffer and preferably to one analytical task. Alternating a single piece of capillary between different buffer systems (such as borate and phosphate) may necessitate very long equilibration times. Table 2. Regeneration Procedure for Uncoated Silica Capillary For Maximized EOF Methanol (HPLC grade or better) 10 minutes* HCl, 1 N, 10 minutes NaOH, 0.1 N, 10 minutes Run buffer, 10 minutes Use of 0.1 N NaOH between sample runs may be necessary to maintain consistent EOF flow. For Analyses with Low-pH Phosphate Buffer (EOF Minimized) Methanol (HPLC grade or better) 10 minutes HCl, 1 N, 10 minutes Phosphoric acid, 1 N, 10 minutes Run buffer, 10 minutes When using low-ph buffers, do not rinse with NaOH between runs. If clean-up is necessary, use 1 N acid. * All times are for a 75 µm capillary, 60 cm total length, 20 to 30 C, 20 psi. For other capillary dimensions, adjust time and/or pressure so that the wash solution is replaced at least 20 times. CE Expert (available from Beckman Coulter, Inc.) is a software package that can be used to calculate fluid delivery under various conditions. Conclusion Capillary electrophoresis can be a reproducible technique. The basic requirements are a quality instrument system that is properly operated, calibrated, and equilibrated. In addition to the factors covered here, reproducible results require the use of clean, fresh chemistries and good day-to-day system cleaning and maintenance. By taking care to recreate the run conditions, capillary electrophoresis can be a valuable tool for routine analytical tasks. 4

5 Appendix A. Results from 100 Runs Migration (min) Mobility Raw Peak Area Corrected Area Rep mt A mt B Peak B Area A Area B c Area A c Area B

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