Anal. Chem. 1999, 71, 1119-1124
Capillary Electrochromatography. Abnormally High Efficiencies for Neutral-Anionic Compounds under Reversed-Phase Conditions F. Moffatt,* P. A. Cooper, and K. M. Jessop
Zeneca Agrochemicals, Jealotts Hill Research Station, Bracknell, RG42 6ET, U.K. Unusually high efficiencies (up to 2.5 million plates m-1) during the capillary electrochromatographic analysis of partially ionized anionic-neutral compounds have been observed under reversed-phase conditions using a standard C18 stationary phase. An explanation has been proposed in terms of nonequilibrium conditions caused by pulses of stronger or weaker solvent that arise from the sample. The increased efficiencies are observed when the migration time of the analyte is closely matched to the elution time of sample-induced discontinuities in the mobile phase. Repeatability and hence the feasibility to control the system have been demonstrated along with the effect that separation parameters such as mobile-phase organic solvent content, ionic strength, and separation voltage have on peak efficiencies, areas, heights, and asymmetry. Van Deemter plots show that the B term (axial molecular diffusion) is the only major contributor to peak dispersion. A reduced plate height of 0.1 was obtained. The implications of this phenomenon and its ability to cause concurrent electrophoretic effects during the analysis of charged species, thus leading to even greater efficiencies, are discussed. The attainment of high sensitivity and resolution in chromatographic and electrophoretic separations is facilitated when high efficiencies, characterized by minimal peak dispersion (i.e., minimum height equivalent to a theoretical plate, H), can be achieved during the separation process. The equations of van Deemter1 and Knox2 are among the most straightforward models that describe the contributors to peak dispersion in liquid chromatographic systems under which equilibrium exists between the stationary and mobile phases:
h ) Aυn + B/υ + Cυ
(1)
where n ) 0 in the van Deemter equation, n ) 0.33 in the Knox equation, h is the reduced plate height, υ is the reduced linear velocity, and A, B, and C are constants. The reduced plate height (h) and reduced velocity (υ) are related to their nonreduced forms (H and u) by eqs 2 and 3, where * Corresponding author: (tel) +44 1344 414689; (fax) +44 1344 413677; (e-mail)
[email protected]. (1) Giddings, J. C. Dynamics of Chromatography, Part 1; M. Dekker: New York, 1965. (2) Knox, J. H. J. Chromatogr. Sci. 1977, 15, 352-364. 10.1021/ac981117x CCC: $18.00 Published on Web 02/11/1999
© 1999 American Chemical Society
h ) H/dp
(2)
υ ) udp/Dm
(3)
dp is the particle diameter, u is the linear velocity of the mobile phase through the packed bed, and Dm is the diffusion coefficient of the solute in the eluent. The efficiency (N) and plate height (H) can be calculated from the peak width using eqs 4 and 5, where tr is the retention time
N ) 5.54(tr/w1/2)
(4)
H ) L/N
(5)
for an analyte, w1/2 is the peak width at half-height, and L is the column length. The A term in eq 1 is associated with eddy diffusion or nonlinear passage along the packed bed and is claimed to be reduced in a system that utilizes electrically driven flow.3 The B term comes from axial molecular diffusion (and hence increases with time), and the C term incorporates the effect of resistance to mass transfer. Knox2 described the situation in HPLC where typical values for A, B, and C are 1, 2, and 0.1, respectively. For a very uniformly packed column, the A term was expected to be 0.5 but could be as high as 2-3 for a poorly packed column. The theoretical minimum value of C for a fully porous particle is 0.01.4 The use of solvent gradients is common in HPLC in order to extend the range of substances that may be separated within a given period of time. The solvent gradient is, in effect, a continuous nonequilibrium situation which can result in reduced peak dispersion.5 Hence “apparent efficiencies” are generally higher than those achieved under isocratic elution conditions. However, under isocratic conditions, if the bulk liquid composition of the mobile phase and the sample are different, localized gradient waves will be produced that give rise to pulses of stronger or weaker solvent, thus generating discontinuous nonequilibrium conditions. This has been termed “vacancy chromatography” in gas-liquid, in gas-solid,6 and later in liquid-solid chromatog(3) Dittman, M. M.; Wienand, K.; Beck, F.; Rozing, G. P. LC-GC 1995, 13, 800-814. (4) Knox, J. H.; Pryde, A. J. Chromatogr. 1975, 112, 171-188. (5) Snyder, L. R.; Glajch, J. L.; Snyder, J. J. Practical HPLC Method Development; John Wiley and Sons Inc.: New York, 1988. (6) Zhukhovitski, A. A.; Turkel’taub, N. M. Dokl. Akad. Nauk USSR 1961, 143, 646.
Analytical Chemistry, Vol. 71, No. 6, March 15, 1999 1119
raphy by Scott et al.7 Theoretical descriptions of the perturbation caused by the injection of a mobile-phase component of lower or higher concentration than that of the bulk mobile phase have been reported, based upon plate theory7 and binomial expansion.8 When such waves can be made to coelute with an analyte, uncommonly high efficiencies may be achieved. This has been demonstrated in both ion-pair reversed-phase HPLC of substituted benzamides9 and supercritical fluid chromatography of clevidine.10 These mobile-phase discontinuities have also been termed ghost peaks,11 system peaks,12 vacant peaks,13 and induced peaks14 by other researchers. The discontinuities may be observed as positive or negative peaks or even go undetected. Strode et al.10 reported “system peaks” to originate when acetonitrile is displaced from a silica surface by an injection plug of water or alcohol. On silica, water-enriched negative peaks were observed to elute after solventenriched positive peaks. Optimization of peak compression in supercritical fluid chromatography by chemometrics was employed by Carlsson et al.15 Fornstedt and Westerlund16 observed peak compression in reversed-phase ion-pair chromatography. Coelution of imipramine (in cationic form) with a hydrophobic anion-containing system peak was responsible. The nature of the phenomenon is such that high efficiency is only associated with a narrow section of the elution window in which the migration rate of the analyte and system peak are sufficiently well matched. The limitation of using a single pulse of solvent introduced with the sample was overcome by Stranahan and Deming,17 who succeeded in improving the separation of a six-component mixture of anilines by introduction of octanesulfonate in regular pulses. The van Deemter plot reported by Dittman et al.3 for separations on a packed capillary column with electrically driven mobile phase gave values for A, B, and C (eq 1) of 1.35, 3.47, and 0.54, respectively (using anthracene as the analyte, k′ ) 1.4), which represents no marked improvement over HPLC. The authors attributed this to poor packing, but at face value the observations did not experimentally support the theory that peak dispersion had been reduced at all. Nevertheless, it is commonly observed in capillary electrochromatography (CEC) that most peaks are 2-3 times sharper than corresponding HPLC peaks.18 It is reasonable to suppose that many of these studies would have involved elution of analytes where the mobile phase and sample solvent were in equilibrium. This paper describes circumstances under which mobile phase discontinuities in capillary electrochromatography may be used to give unusually high efficiencies. (7) Scott, R. P. W.; Scott, C. G.; Kucera, P. Anal. Chem. 1972, 44, 100-104. (8) Reiley, C. N.; Hildebrand, G. P.; Ashley, J. W. Anal. Chem. 1962, 34, 1198. (9) Nilsson, L. B.; Westerlund, D. Anal. Chem. 1985, 57, 1835-1840. (10) Strode, J. T., II; Gyllenhaal, O.; Torstensson, A.; Karlsson, A.; Karlsson, L. J. Chromatogr. Sci. 1998, 36, 257-262. (11) Berek, D.; Bleha, T.; Pevena, Z. J. Chromatogr. Sci. 1976, 14, 560-563. (12) Denkert, M.; Hackzell, L.; Schill, G.; Sjogren, E. J. Chromatogr. 1981, 218, 31. (13) Slais, K.; Krejci, M. J. Chromatogr. Sci. 1974, 91, 161-166. (14) Stranahan, J. J.; Deming, S. N. Anal. Chem. 1982, 54, 1540-1546. (15) Carlsson, D.; Strode, J. T., II; Gyllenhaal, O.; Karlsson, A.; Karlsson, L. Chromatographia 1997, 44, 289-293. (16) Fornstedt, T.; Westerlund, D. J. Chromatogr. 1990, 506, 61-74. (17) Stranahan, J. J.; Deming, S. N.; Sachok, B. , J. Chromatogr. 1980, 202, 233237. (18) Gordon, D. B.; Lord, G. A.; Jones, D. S. Rapid Commun. Mass Spectrom. 1994, 8, 544-548.
1120 Analytical Chemistry, Vol. 71, No. 6, March 15, 1999
Figure 1. Overlaid electrochromatograms of 1 at 40 (A), 30 (B), 20 (C), 10 (D), and 5% (E) acetonitrile.
EXPERIMENTAL SECTION 2-Amino-6-(hydroxymethyl)-5-methylpyrimidin-4-ol (1) and 6-(hydroxymethyl)-2-(methylamino)-5-methylpyrimidin-4-ol (2) were synthesized at Zeneca Agrochemicals (Bracknell, Berkshire U.K.). Trisma HCl, Trisma base, and thiourea were obtained from SigmaAldrich Co. Ltd. (Poole, Dorset, U.K.). All organic solvents and water were HPLC grade (Romil, Cambridge, U.K.). Buffers were prepared using HPLC-grade water and Trisma HCl and base, to give an overall pH of 8.6 at an ionic concentration of 5 mM. The pH of this solution was measured using a Whatman PHA 230 pH meter. The aqueous buffer was admixed with acetonitrile to give the required running buffer composition. Thiourea and 2 were dissolved in water to give concentrations of 2.1 mg mL-1 . 1 was dissolved in water-methanol (5:3, v/v) to give an overall concentration of 2.5 mg mL-1. Capillary electrochromatography was performed using a HewlettPackard HP 3DCE instrument with UV-visible absorbance detection at 214 nm. Nitrogen at between 10 and 12 bar was applied to both ends of the capillary during separations. Separation voltages were between 0.3 and 30 kV. The system temperature was set to 30 °C for all experiments. A 50-µm-i.d., 375-µm-o.d. polyimidecoated fused-silica CEC capillary (Hypersil, Runcorn, U.K.) was used for all the experiments. The capillary had a total length of 33 cm and was packed to the detector window (24.5 cm) with 3-µm-diameter C18 Hypersil CEC packing material (Hypersil, Thermo Instrument Systems Inc., Runcorn, U.K.). Thiourea coinjections (5 kV for 5 s) were made for every run using an injection program (thiourea followed by sample) to provide an estimate of the retention time for an unretained sustance (t0). RESULTS AND DISCUSSION As part of our ongoing investigation into the effect of electrically driven flow upon reversed-phase chromatographic behavior, a series of pyrimidines were analyzed using a range of five running buffers consisting of 5 mM aqueous TRIS with CH3CN added at 40, 30, 20, 10, and 5% v/v. The results showed that two of the pyrimidines (1 and 2) produced anomalously sharp peaks at certain running buffer compositions (Figures 1 and 2). Maximum efficiencies of up to 2 500 000 plates/m were observed when buffers containing 20 and 30% acetonitrile were used with 1 (Figure 3) and 2 (Figure 4), respectively. The resulting peak
Figure 2. Overlaid electrochromatograms of 2 at 40 (A), 30 (B), 20 (C), 10 (D), and 5% (E) acetonitrile.
Figure 5. Expanded electrochromatogram of 2. Table 1. Reproducibility of Peak Sharpening for Compounds 1 and 2a compound 1
RT (min) area (mAU) height (mAU) k′ effic (TPM) a
Figure 3. Electrochromatogram of 1 (A) coinjected with thiourea (B), 80:20 (v/v) 5 mM aqueous TRIS (pH 8.6)-acetonitrile.
Figure 4. Electrochromatogram of 2 (A) coinjected with thiourea (B), 70:30 (v/v) 5 mM aqueous TRIS (pH 8.6)-acetonitrile.
widths were as narrow as 1 s (Figure 5). The repeatability of the phenomenon was proven by making a series of 50 replicate injections. All associated peak parameters displayed reasonably low relative standard deviations (Table 1). A further set of experiments was performed using running buffer compositions which centered around the compositions of interest for 1 and 2. Six replicate injections were made for each
compound 2
mean
% RSD
mean
% RSD
2.50 806 1449 0.176 1644983
0.53 3.17 1.70 0.36 4.25
2.43 448 1039 0.173 2583877
1.11 8.47 9.28 1.2 8.44
n ) 50.
compound at each composition. Table 2 shows the mean values of the separation parameters of interest. The electrochromatograms obtained for 1 are shown overlaid in Figure 6. The efficiency (and peak height) for each compound can clearly be seen to increase smoothly through a maximum before decreasing again (Figure 7). The van Deemter plot using reduced parameters is a useful measure of column performance.4 Using the running buffer compositions that led to the highly efficient peaks (20% acetonitrile for 1, 30% acetonitrile for 2), the effect of the linear velocity on efficiency was investigated by running the analyses at a range of separation voltages from 30 to 0.3 kV. The resulting van Deemter plot for 2 (Figure 8) gives a minimum reduced plate height value of 0.1. Using a value of 10-9 m2 s-1 for the diffusion coefficient,2 the A, B, and C values are calculated to be 0.00, 0.15, and 0.017, respectively, indicating that under these conditions the B term is by far the dominant factor and hence axial molecular diffusion is the main contributor to peak dispersion. The data set for 1 did not include a point of inflection, but the lowest calculated reduced plate height was 0.19 (Table 3). A plot of peak symmetry (as defined by Hewlett-Packard Chemstation data package version A.05.04) vs percent acetonitrile (Figure 9) indicates how the peaks show fronting characteristics at high acetonitrile content. As the acetonitrile content is decreased, the fronting is reduced until the peaks become highly symmetrical at the point where the high efficiencies are observed. As the acetonitrile content is increased further, the peaks then begin to show tailing characteristics. This effect can be readily observed in Figures 2 and 6. A graph of ln k′ against percent acetonitrile shows a zero slope around the set of conditions corresponding to the high efficiencies Analytical Chemistry, Vol. 71, No. 6, March 15, 1999
1121
Table 2. Effect of Running Buffer Composition for Compounds 1 and 2 compound 1
compound 2
% CH3CN
RT (min)
area (mAU)
height (mAU)
effic (TPM)
asymm
RT (min)
area (mAU)
height (mAU)
effic (TPM)
asymm
37.5 35 32.5 30 27.5 25 22.5 20 17.5 15 12.5
nda nd nd nd 2.45 2.48 2.5 2.51 2.53 2.54 2.56
nd nd nd nd 1239 1108 1035 1023 1080 1169 1319
n.d nd nd nd 980 1299 1422 1435 1355 1166 541
nd nd nd nd 306 724 688 630 960 006 1 011 826 819 835 521 576 89 977
nd nd nd nd 0.809 0.888 0.967 0.983 1.005 1.038 0.963
2.26 2.35 2.39 2.43 2.44 2.47 2.5 nd nd nd nd
492 655 575 560 513 572 690 nd nd nd nd
505 967 977 964 989 1005 764 nd nd nd nd
451 816 975 903 1 341 232 1 424 017 1 805 685 1 534 291 624 843 nd nd nd nd
0.744 0.961 0.99 0.997 0.945 1.01 1.164 nd nd nd nd
a
nd, not determined.
Figure 6. Effect of acetonitrile content on peak sharpness for 1. Run with buffers containing 27.5 (A), 25 (B), 22.5 (C), 20 (D), 17.5 (E), 15 (F), and 12.5% (G) acetonitrile.
(Figure 10), which would be characteristic if the compound were being “unretained” in a solvent-rich environment. The effect of ionic strength was also investigated and showed that increased ionic strengths led to lower k′, EOF, and peak areas, but increased retention times, as expected (Tables 4 and 5). Decreasing the injection times led to lower efficiencies (Table 6), which is probably more due to the decreased amount of solvent injected reducing the effect of the system peak rather than the lower amount of analyte injected. There are known focusing mechanisms which are applicable to ions in capillary electrophoresis. One of these (sample stacking or field amplification) arises when the ionic strength of the sample solution is lower than that of the running buffer.19 The rapid migration of sample ions up to the sample-buffer boundary occurs in order to conserve current along the capillary. A second mechanism, isotachophoresis,20 occurs when the sample is sandwiched between two electrolyte solutions, one that contains ions of greater mobility than the sample (leading electrolyte) and one that contains ions of lower mobility than the sample (terminating electrolyte). Upon application of the voltage, the sample ions (19) Wehr, T.; Zhu, M. Handbook of Capillary Electrophoresis Applications; Blackie Academic and Professional: London, 1997. (20) Burgi, D. S.; Chien, R. L. Handbook of Capillary Electrophoresis, 2nd ed.; CRC Press Inc.: New York, 1997.
1122 Analytical Chemistry, Vol. 71, No. 6, March 15, 1999
Figure 7. Changes in peak efficiency of 1 and 2 with increasing acetonitrile content in the running buffer.
Figure 8. Van Deemter plot for 2 (the line was fitted by Microsoft Excel using a nonlinear least-squares method).
separate into discrete bands (according to their electrophoretic mobility), all of which have the same velocity and concentration as the leading electrolyte. Both of these mechanisms rely on the sample to be present in ionic form. The state of ionization of 2 was determined in order to assess the likelihood that ionic mechanisms were contributing to peak
Table 3. Reduced Plate Height for Compounds 1 and 2a effic theor plates
reduced plate ht (h)
t0 (min)
tr (min)
W1/2 (min)
2.111 3.416 7.106 14.218 75.789 273.809
2.471 4.022 8.399 16.834 88.98 319.172
Compound 2 0.011 279 557 0.011 740 642 0.024 678 488 0.054 538 390 0.607 119 047 6.182 14 767
0.29 0.11 0.12 0.15 0.69 5.53
2.112 2.655 3.399 4.653 7.16 14.554
2.485 3.129 4.013 5.503 8.473 17.222
Compound 1 0.009 422 355 0.012 376 668 0.015 396 520 0.021 380 426 0.034 344 054 0.073 308 341
0.19 0.22 0.21 0.21 0.24 0.26
a From eqs 2, 4, and 5; h ) L/(Nd ) where L ) 24.5 cm and d ) p p 3 µm.
Figure 10. Changes in Ln k′ of 1 and 2 with increasing acetonitrile content in the running buffer. Table 4. Variations in Ionic Strength for Compound 1 mM
k′
RT (min)
area (mAU)
height (mAU)
tailing
effic (TPM)
0.5 1 5 10 20 30 40 50
0.17 0.17 0.17 0.17 0.17 0.14 0.15 0.15
2.40 2.36 2.44 2.49 2.55 2.56 2.85 3.23
912 956 1,026 1,022 1,045 902 801 593
1351 1284 1344 1379 1497 570 856 495
1.101 1.028 0.998 1.008 1.247 1.292 1.023 1.319
1 023 123 819 031 831 254 920 192 1 090 042 221 711 758 931 605 898
Table 5. Variations in Ionic Strength for Compound 2
Figure 9. Changes in peak symmetry of 1 and 2 with increasing acetonitrile content in the running buffer.
sharpness. The effects of an organic modifier upon the pH of buffers and the pKa of analytes have been described by Kenndler.21 The relationship between pH and the degree of ionization should be established by measurement of both pH and pK in the solvent system used for separation. In general, increasing the proportion of acetonitrile can elevate the pKa of neutral acids by up to several pH units. The effect upon pKa for cationic acids, such as TRIS, is much less but can be positive or negative relative to water. At an acetonitrile content of 30%, the pKa of 2 was found to be 9.08 and the apparent pH of the buffer was 8.35. The degree of dissociation (R) for a neutral acid, calculated from eq 6, is 0.16. Therefore 2
mM
k′
RT (min)
area (mAU)
height (mAU)
tailing
effic (TPM)
0.5 1 5 10 20 30 40 50
0.18 0.17 0.16 0.17 0.16 0.16 0.13 0.14
2.55 2.42 2.49 2.47 2.45 2.54 2.76 3
855 706 545 633 934 806 338 439
1327 1046 876 1017 1342 1367 382 656
1.196 1.022 1.001 0.995 1.143 1.034 0.966 1.180
1 278 401 1 051 677 1 308 438 1 282 435 1 009 724 1 512 657 779 790 1 479 667
(6)
against electrophoretic contributions to peak sharpening. The data presented are fully consistent with the system peak effect. This point is established further by the loss of efficiency observed when the sample was dissolved in mobile phase instead of water and run under the same conditions (Figure 11). Evidence that capillary electrochromatography can produce localized high efficiencies has been observed previously in a small isolated number of studies. Frame et al.22 found that one component of a steroid test mixture had an efficiency of 1 million plates/m by capillary electrochromatography using a Hypersil
is partially anionic. This would rule out ion exchange with the silica support. The relatively low level of ionization would argue
(21) Kenndler, E. Capillary Electrokinetic Technology; Marcel Dekker: New York, 1993. (22) Frame, L. A.; Robinson, M. L.; Lough, W. J. J. Chromatogr., A 1998, 798, 234-249.
R ) 1/(1 + 10pKa-pH)
Analytical Chemistry, Vol. 71, No. 6, March 15, 1999
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Table 6. Variations in Efficiency with Injection Time injection time (s)
compound 1
compound 2
1 2 3 4 5 6
1 031 760 1 483 068 1 574 867 1 627 295 1 348 023 1 158 745
976 822 1 214 949 1 028 867 1 706 021 2 786 841 2 785 854
Figure 11. Electrochromatograms of 2 injected in (A) water and (B) buffer solution (70% 5 mM aqueous TRIS, pH 8.6-30% acetonitrile).
ODS stationary phase. Efficiencies in the tens of million of theoretical plates were discovered when a series of strongly basic antidepressants were separated on a strong cation-exchange phase at low pH.23 Another group24 have subsequently claimed that a different compound had given an efficiency of 16 million theoretical plates/m on the same Spherisorb-based cation exchanger. The paper by Smith and Evans is particularly noteworthy because high resolution was demonstrated in the separation of four “focused” components over a 2-min time span. However, in the studies cited above, no explanation was put forward that could account for these observations. The production of narrow peaks seen on the cation exchangers has been described as a “focusing effect”,23 but this (23) Smith, N. W.; Evans, M. B. Chromatographia 1995, 41, 197-202. (24) Euerby, M. R.; Gilligan, D.; Johnson, C. M.; Roulin, S. C. P.; Myers, P.; Bartle, K. D. J. Microcolumn Sep. 1997, 9, 373-388. (25) Moffatt, F.; Chamberlain, P.; Cooper, P. A.; Jessop, K. M. Chromatographia 1998, 48, 481-490.
1124 Analytical Chemistry, Vol. 71, No. 6, March 15, 1999
term perhaps requires some qualification. Separations performed under continuous or discontinuous gradient conditions can lead to high “apparent efficiencies”. When charged solutes are being analyzed, it is possible to envisage a situation where the sample ions would be sandwiched between the electrolyte and the discontinuous solvent system peak. If the electrolyte ion was of higher mobility than the sample ions, then the conditions would be in place for isotachophoretic focusing to occur. In this situation there may be twin sharpening mechanisms in action, with contributions from the system peak effect and an isotachophoretic effect, which would lead to even greater efficiencies than are reported here. CONCLUSIONS High apparent efficiencies may be achieved in CEC under reversed-phase conditions by the use of pulsed gradients generated by differences in composition of the mobile phase and the sample. The reproducibilities of retention times, peak heights, and areas are close to values found previously in capillary electrochromatography.25 This indicates that the “focusing” effects are sufficiently reproducible to be considered for analytical applications where sensitivity is of major importance. There is scope to extend the effect to other modes of separation, for instance, those involving ionic species that could well exhibit properties that are not shared by either liquid chromatography or electrophoresis alone, as well as to realize greater flexibility through use of pulsed gradients. Ultimate sensitivity for samples of very low analyte concentration remain to be demonstrated but would appear to be attainable in principle. Claims of particularly high efficiencies under isocratic conditions should be supported by evidence that the analyte was separated under equilibrium conditions between the stationary and mobile phases. For a “focusing effect” to be anomalous, it would have to be an effect that could not be explained by the generation of pulsed solvent or ionic gradients. Injection of a sample in mobile phase provides unequivocal evidence of whether any focusing effects are due to localized waves caused by the sample solvent or components therein. ACKNOWLEDGMENT The authors thank Dr. T. Fraser and Mr. M. Waller (Zeneca Agrochemicals) for their technical advice and help in the determination of pKa values and statistical analysis. Received for review October 12, 1998. Accepted January 3, 1999. AC981117X
