Anal. Chem. 1999, 71, 995-1001
Large-Volume Sample Stacking in Acidic Buffer for Analysis of Small Organic and Inorganic Anions by Capillary Electrophoresis Yan He† and Hian Kee Lee*,‡
Departments of Chemical Engineering and Chemistry, National University of Singapore, Kent Ridge, Singapore 119260
This paper describes a straightforward approach for stacking extremely large volumes of sample solutions containing small organic and inorganic anions in capillary electrophoresis. The methodology involves the stacking of large sample volumes and the separation of the stacked anions in an acidic buffer (pH 1) and that of FUMA smaller (x < 1) than EOF in aqueous sample, the entire column can be filled with sample without loss of MALE but with loss of FUMA. The decrease in peak height of MALE after 53 cm is primarily due to the significant increase in peak width caused by band broadening whereas that of FUMA may mainly result from the loss of FUMA loaded onto the column. Hitherto, we assume that no loss of stacked anions occurs due to the removal of the aqueous sample plug. This assumption was verified by the following experiments: (1) A 1-mm plug of aqueous sample containing 10 ppm each of MALE and FUMA (defined as organion-2) is injected and then driven by pressure of 100 mbar (Figures 4a and 5a). (2) A 1-mm plug of neutral sample (10 ppm, 13-DNB) and 530-mm plug of aqueous sample containing 0.05 ppm organion-2 are injected consecutively. Negative voltage (-24 kV) was applied to remove the aqueous sample plug and switched off at 3.5 (Figures 4b and 5b), 3.7 (Figures 4c and 5c), and 4.0 min (Figures 4d and 5d). Then 100 mbar was applied to drive the neutral 13-DNB and stacked anions toward the detector. As can be seen, the retention times of stacked anions (Figure 4b-d) are shorter than that of anions attained under normal smallvolume injection (Figure 4a). This result indicates that the distance between the stacked anions and the detector window is shorter than the effective length of the capillary (the distance between the inlet tip and the detector window). Hence, the stacked anions do not reach the edge of the capillary inlet, and the loss of stacked anions is avoided. Additionally, Figure 4 reveals that the stacked anions begin to move away from the concentration boundary toward the detector before the removal of the sample plug is completed. This was verified by the gradual separation of stacked anions and neutral 13-DNB in Figure 4b-d and more clearly
illustrated in Figure 5b-d. As can be seen, stacked organion-2 first moves toward the inlet (Figures 4b and 5b) and elutes together with 13-DNB as one peak. When the time of water removal is over 3.7 min, stacked organion-2 begins to move away from the concentration boundary while 13-DNB keeps moving toward the inlet. This is indicated by the gradual decrease of the difference in retention times between the 13-DNB peak (Figure 4c and d) and the organion-2 peak (Figure 4a) and the increase in separation of 13-DNB and stacked organion-2 as shown in Figures 4c and 5c and Figures 4d and 5d. LVSEP of Inorganic Anions in Acidic and Basic Buffers. In Burgi’s work on the LVSEP of inorganic anions (e.g., nitrate and bromide ions), DETA was used to suppress the EOF.9 In this part of work, we want to demonstrate that no addition of DETA (and even no reduction of pH value) is required for the LVSEP of these fast-moving inorganic anions. It is well-known that nitric and bromic acids are strong electrolytes that can be completely dissociated even at very low pH values. Further, nitrate and bromide ions are much smaller than the organic anions studied above. Hence, nitrate and bromide anions have high electrophoretic mobility across the wide range of pH. Unlike those of MALE and FUMA, the electrophoretic mobilities of bromide and nitrate ions are greater not only in acidic buffer but also in basic buffer. This is illustrated in the separation of bromide and nitrate ions in acidic and basic buffers under the same negative voltage (Figure 6). The result means that the LVSEP of fast-moving inorganic anions can be performed in both acidic and basic buffers. Further, adjustment of pH in the acidic range need not be so delicate as that for organic anions. These results indicate that selection of pH is more flexible in stacking inorganic anions than in stacking organic anions. Table 1b displays the EOF and electrophoretic mobilities of bromide and nitrate ions in acidic and basic buffers. It is clear that EOF is reduced significantly with the decrease in pH from 9.26 to 2.68. On the other hand, the electrophoretic mobilities of Analytical Chemistry, Vol. 71, No. 5, March 1, 1999
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Figure 6. Electropherograms of bromide and nitrate ions attained in (a) basic borate buffer (pH 9.26, 20 mM tetraborate) and (b) acidic phosphate buffer (pH 2.68, 40 mM KH2PO4-10 mM H3PO4). Applied voltage, -24 kV. Peak identities: (1) bromide; (2) nitrate.
Figure 8. Influence of length of sample plug on the peak areas of bromide and nitrate ions.
Figure 7. Comparison of stacking and separation of bromide and nitrate ions in (a) basic borate buffer (pH 9.26, 20 mM tetraborate) and (b) acidic phosphate buffer (pH 2.68, 40 mM KH2PO4-10 mM H3PO4). Injection, 600 mbar‚min. Other conditions as in Figure 6.
bromide and nitrate ions remain constant and become increasingly bigger than EOF when pH is decreased from 9.26 to 2.68. An acidic buffer has an advantage over a basic buffer in terms of separation speed and detection sensitivity as indicated by the shorter separation time and higher peak heights in Figure 6b. On the other hand, a basic buffer is more favorable from the point of view of enrichment speed as indicated by the shorter time (1.8 min in basic buffer vs 3 min in acidic buffer) required to remove the water plug (Figure 7). Nevertheless, an acidic buffer is more suitable for use in the enrichment and separation of small inorganic anions with respect to the total analysis time and detection sensitivity (Figure 7). The influence of the injection plug length on peak areas and heights of bromide and nitrate ions was also studied. Similar to those for small organic anions, the maximum peak height was observed when 87% (53 cm) of the capillary was filled with sample. Under this condition, enrichment of over 300-fold was attained 1000 Analytical Chemistry, Vol. 71, No. 5, March 1, 1999
Figure 9. Electropherogram of 0.8 M boric acid. Peak identity: (1) nitrate. Buffer, 40 mM KH2PO4-10 mM H3PO4 (pH 2.68); injection, 600 mbar‚min. Other conditions as in Figure 6.
with little loss in separation efficiency and resolution. However, the maximum peak areas were attained for both anions when the entire capillary was filled with sample (Figure 8), similar to the result for the fast-moving organic anion, MALE. This is obviously due to the fact that the electrophoretic mobilities of bromide and nitrate ions are higher than the EOF in aqueous sample. Determination of Nitrate Impurities in Boric Acid and Potassium Bromide by LVSEP in Acidic Buffers. The determination of trace inorganic anions in real samples containing a large excess of nonionic matrix components is a demanding task for CE because of the interference by the latter. This problem can be effectively resolved by removing the nonionic matrix components while concentrating the trace amount of small anions using sample stacking techniques. Boden et al.11 has investigated the addition of DETA in buffer for sample stacking without polarity switching in CE for analysis of nitrate ions in boric acid (pKa1 ) 9.27, boric acid present as neutral compounds when pH is ,9.27). (11) Boden, J.; Darius, M.; Bachmann, K. J. Chromatogr., A 1995, 716, 311317.
peak appearing at 1-3 min results from the boundary between boric acid and phosphate buffer that passes through the detector during the initial stage of sample stacking. LVSEP was also applied to the determination of nitrate ions in the ionic matrix, potassium bromide, as shown in Figure 10. Unlike the removal of nonionic boric acid, an excess amount of bromide ion was concentrated together with, and separated from, the trace amount of nitrate ion during the sample stacking and subsequent separation processes, respectively. Figure 10 clearly shows that 3 ppb nitrate ion can be detected in the presence of 50 ppm bromide ion with an ATMR of 1:2.5 × 104. CONCLUSIONS The reported data show that sample stacking using the EOF pump of extremely large sample volumes in acidic buffers provides a simple, rapid, and effective approach for on-line concentration of small organic and inorganic anions in CE. For some fast-moving inorganic anions (e.g., bromide), this method of sample stacking can be performed in both acidic and basic buffers. With the use of sample stacking, CE with UV detection can be successfully applied to the analysis of small organic and inorganic anions at low ppb levels. Figure 10. Electropherogram of 50 ppm potassium bromide. Peak identities: (1) bromide; (2) nitrate. Buffer, 40 mM KH2PO4-10 mM H3PO4 (pH 2.68); injection, 600 mbar‚min. Other conditions as in Figure 6.
To simplify this method, LVSEP was carried out in acidic buffer containing no DETA. Figure 9 shows the electropherogram attained by the stacking and separation of ∼1 µL 0.8 M (4.9 × 104 ppm) boric acid. The standard addition method was used for quantification. The concentration of nitrate ion is 19 ppb. Hence, nitrate ion can be determined in the presence of a boric acid matrix up to an analyte-to-matrix ratio (ATMR) of 1:2.6 × 106. It should be mentioned that complete removal of the boric acid matrix takes ∼8 min, which is much longer than that (3.0 min) for the removal of water. This is because the EOF in the sample region is partially suppressed by the high viscosity and low pH (3.5) of 0.8 M boric acid solutions. Also, note that the broadened
ACKNOWLEDGMENT The authors thank the National University of Singapore for financial support of this work. SUPPORTING INFORMATION AVAILABLE Plot of the influence of length of aqueous sample plug in the capillary on detection sensitivity (indicated by peak heights) and separation efficiency (indicated by peak widths at half-height) and plot of the influence of length of aqueous sample plug in the capillary on the amount of sample loaded onto the capillary (indicated by peak areas). This material is available free of charge via the Internet at http://pubs.acs.org.
Received for review October 6, 1998. Accepted December 12, 1998. AC981100E
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