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Evaluation of an automtic siphonic sampler for capillary zone electrophoresis
Journal of Chromatography, 404 (1987) 3 13-320
Elsevier Science Publishers B.V., Amsterdam -
Printed in The Netherlands
CHROM. 19 689
EVALUATION OF AN AUTOMATIC LARY ZONE ELECTROPHORESIS
SUSUMU HONDA*, SHIGEFUMI
SIPHONIC
SAMPLER
FOR CAPIL-
IWASE and SHIGERU FUJIWARA
Faculty of Pharmaceutical Sciences, Kinki University, 3-4-l Kowakae, Higashi-Osaka (Japan)
(First received February 23rd, 1987; revised manuscript received April 22nd, 1987)
SUMMARY
An automatic sampler based on siphoning was devised and its capability for capillary zone electrophoresis (CZE) was examined. The peak area of benzyl alcohol as a model compound introduced to a capillary tube was proportional to the sampling time, and the reproducibility of sample introduction was quite high. These results ensure the usefulness of this apparatus for quantitative as well as qualitative analyses by CZE.
INTRODUCTION
Capillary zone eletrophoresis (CZE) is a recently developed method for the separation of ions, based on a combination of the effects of electrophoresis and electroosmosis in a capillary tube. It was introduced by Mikkers et al.‘, and developed by Jorgenson and Lukacs2q3. Its applicability is very wide, ranging from small inorganic4 as well as organic ions 4-6 to biomacromolecules such as proteins7. The use of dynamic hydrophobic interaction by adding a detergent to the carrier has further extended its applicability to neutral molecules *a9.It exhibits high efficiency, the number of theoretical plates calculated in the same manner as in liquid chromatography often reaching several hundreds of thousands. The problems of quantification were studied by Fujiwara and HondalO, who also demonstrated the applicability of CZE to drug monitoring. However, all these studies on CZE were performed by manual sample introduction, because no automatic apparatus was commercially available. This has hampered the popularization of CZE. The most difficult problem in sample introduction in CZE is that an extremely small volume (less than several hundred nanolitres) of a sample solution has to be introduced into a capillary tube which is strictly insulated so as to withstand high voltages. Dipping one end of a capillary tube in a solution prepared by dissolving the sample in the carrier electrolyte solution and the other end in the same electrolyte solution, followed by application of an high voltage between the two ends for a short period l’, can result in sample introduction by the effect of electroosmosis. However, the quantity of the sample introduced is dependent on its concentration. In addition the sample is usually ionic and cannot easily be recovered from the electrolyte solution. Since the volume of sample introduced is extremely small, on-column detec0021-9673/87/$03.50
0
1987 Elsevier Science Publishers B.V
314
S. HONDA, S. IWASE, S. FUJIWARA
tion demands an high concentration of the sample solution, as compared to the detection in ordinary high-performance liquid chromatography. The recovery is critical, when only small amounts of samples are available, as is often encountered with biological and environmental samples. A small dual-barrelled pipette proposed by Wallingford and Ewing’ 2 is notable in that it minimizes the sample volume, but there is still the problem of the sample concentration and it is also difficult to automate. In an early study of CZE a microlitre syringe or a specially constructed four-way injection valve was used’, but the volumes of the sample solutions introduced were large. A few kinds of microinjectors designed for capillary liquid chromatography are commercially available, but they all use metallic parts which have high electric conductivity and cause a gradual change in the optical density of the carrier, resulting in baseline drift, presumably due to electrolysis in the flow path. We made some attempts to prepare an all-plastic valve to overcome this effect, but the results were not promising because no resins withstood repeated switching of such narrow-bore flow paths. The use of a ceramic valve might give better results. Deml et al.’ 3 devised an electric splitter based on sample division by differential electromigration in two electrical circuits. They demonstrated the capability of this apparatus by presenting a few improved separations of carboxylates in isotachophoresis, but did not give any data on reproducibility. It is a fundamental disadvantage that only a small part of the applied sample was analyzed, the greater part being discarded. As compared to the above techniques, the one based on siphoning is important. Since it can be performed manually, many studies on CZE5,* were done by this technique, though on a qualitative basis. We propose here an automatic sampler for both qualitative and quantitative studies, based on this principle. There have been no reports on the evaluation of such an apparatus from the viewpoint of quantification. EXPERIMENTAL
The siphonic sampler
Fig. 1 shows a frontal view of this apparatus. The whole apparatus was surrounded (not shown) by a rectangular, plastic cover to protect an operator from any hazard due to electrical leakage. This cover was designed so that no voltage could be applied while its front panel was open. Both ends of a capillary tube were placed in cylindrical glass vessels (a and c), into which electrodes (e) were inserted. Thin, spherical rubber plates were stuck on the underside of the vessels for insulation purposes. The cathode vessel (c) remains at the same position throughout an analysis, but the anode vessel (a) is placed on a turntable (t), whose level could be changed automatically by a microcomputer-controlled motor (m). The component ions separated can be monitored by an on-column detector (d) near the cathode. In analyses using capoillary tubes with inner diameters larger than 50 pm, commercial UV or multiwavelength detectors were usable with slight modification. A capillary tube (cap) was fixed, directly (in the case of a glass or FEP tube) or after removal of the coating resin (in the case of a fused-silica tube), at the centre of the monochromatic beam. Fig. 2 explains the.introduction of a sample solution into a capillary tube in one cycle of operation. The operation begins at stage I, where both electrode solutions
315
SAMPLER FOR CAPILLARY ZONE ELECTROPHORESIS
to a highvol taee supplier
-
t
m
I
Fig. 1. Frontal view of the automatic siphonic sampler. cap. = Capillary tube; a = anode vessel; b = ball-bearing; c = cathode vessel; d = detector; e = electrode; m = motor; n = nut; r = rubber stopper; s = switch: t = turntable.
316
S. HONDA,
S. IWASE,
S. FUJIWARA
Fig. 2. The sampling process. For each stag: are shown the frontal view of the whole sampler, and a circle representing the upper surface of the turntable. h = Hole; sv = sample vial; other parts as in Fig. 1. The arrows indicate the movement of the turntable from stage to stage.
SAMPLER FOR CAPILLARY ZONE ELECTROPHORESIS
317
are arranged at the same level. First the anode vessel is moved to a lower level, stage II, while the anodic end of the tube is held at the original level by means of a small rubber stopper (r). Then the turnable is rotated clockwise by 45”, and the anode vessel replaced by a round hole (h), indicated by a hatched circle, stage III. Subsequently the turntable is raised to an high level for sampling, stage IV. This level was set several centimetres higher than the starting level. The anodic electrode is undamaged by this movement, because it safely passes through the hole on the turntable. A sample solution in a small vial (sv) is introduced into the capillary tube by downhill flow due to the difference in levels between the sample and the cathode solutions. The sampling time can be specified on a programmer. After the sample solution is introduced, the turntable is lowered again, stage V, equivalent to stage III. The turntable is rotated counterclockwise, stage VI, which is equivalent to stage II. Finally it is raised to the initial level. All these movements of the turntable are indicated by arrows. Evaluation of the sampler
Two fused-silica capillary tubes (80 cm x 100 pm I.D.; 80 cm x 250 pm I.D.) were obtained from Scientific Glass Engineering (North Melbourne, Australia). The coating resin was removed by burning at a distance of 20 cm from one end of each tube. The tube was filled with 10 mM phosphate buffer (pH 7.0) by downhill flow, and set on the proposed apparatus. After 100 mM benzyl alcohol had been introduced for an appropriate period at a 5.0-cm head of water by use of the sampler, a voltage of 21 kV (for the 100~pm tube) or 11 kV (for the 250~pm tube) was applied between both ends of the tube by an high-voltage supply installed in an IP-1B isotachophoresis apparatus (Shimadzu, Kyoto, Japan). The peaks of benzyl alcohol were monitored by on-column detection at 254 nm using an UV detector contained in a Model 633 high-performance liquid chromatograph (Hitachi, Ibaragi, Japan). The volumes of the sample solution introduced to the lOO+m and 250~pm tubes by 5-s sampling were ca. 40 and 500 nl, respectively, estimated from the peak widths of benzyl alcohol. The separation of cinnamic acid (CA) and 3,4-dimethoxycinnamic acid (DMCA) was performed by the same system using the IOO-pm tube. RESULTS AND DISCUSSION
Fig. 3 gives examples of repeated sample introduction, as observed from the detector response of benzyl alcohol introduced as a model compound. The solid line in Fig. 4a indicates the relationship between the peak height of benzyl alcohol introduced to the 250-pm tube and the programmed sampling time. Even when the programmed sampling time was zero, a small volume of the sample solution was siphoned into the tube during the up-and-down movement of the anodic end of the tube, giving an intercept of 16 mm. Subtraction of this value from each plot gave the dotted line showing the relationship between the net peak height and the real sampling time. The convex nature of this curve indicates that the peak height is not proportional to the real sampling time, obviously due to peak broadening caused by overloading. On the other hand, a plot of peak area (as calculated from the weight of the cut-out peak) vs. sampling time gave a straight line (Y = 2.4 X + 3.2, r =
S. HONDA,
318
S. IWASE,
S. FUJIWARA
b a
Fig. 3. Repeated introduction of benzyl alcohol as a model compound by using the proposed sampler: (a) 5-s sampling to the IOO-nm tube; (b) 5-s sampling to the 250-pm tube. Capillaries: fused silica, (a) 80 cm x 100 nm I.D., (b) 80 cm x 250 grn I.D. Carrier: 10 mM phosphate buffer (pH 7.0). Concentration of the sample solution: 100 mM. Difference in levels between the sample and the cathodic solution: 5 cm. Current applied: 30 (a); 125 PA (b). Voltage: 21 (a); 11 kV (b). Detection: absorbance at 254 nm.
0.997) over the whole range examined (O-l 5 s), as seen in Fig. 4b. Since the peak area is considered to be proportional to the amount of sample introduced, a plot of the sampling time vs. amount introduced, i.e., volume introduced should also be linear. These relationships are important for quantification. Similar results were obtained with the 100~pm tube. Table I gives the coefficient of variation (C.V., n = 12) of the peak height
Sampling
time,
8
Fig. 4. Relationships between the peak height (a) and peak area (weight of the cut-out alcohol and sampling time. Analytical conditions as in Fig. 3b.
peak) (b) of benzyl
SAMPLER
FOR CAPILLARY
ZONE ELECTROPHORESIS
319
obtained with the 250~,um tube. It was as low as 2.4 and 0.84% for l-s and 5-s samplings, respectively. The C.V. values for the peak area were much smaller, 1.3 and 0.55%, respectively. The narrower tube gave slightly higher C.V. values for both the peak height and peak area, but they were sufficiently low to allow practical analysis. For comparative purposes, the data obtained by manual introduction are also presented in Table I. Each value is much higher than the corresponding value obtained by automatic introduction. This neutral sample was migrated from the inlet of the capillary tube to the detector by electroosmotic flow under the conditions used, so the data described above include the variation due to dispersion during migration. Therefore, the actual C.V. values for sample introduction alone should be smaller than these values. In the most typical mode of determination in CZE, the concentration of the sample solution is estimated by using a calibration curve obtained from standard solutions at an appropriate sampling time. Since the precision of the sample introduction described above was as high as or higher than the reproducibilities (C.V. generally less than 3%) of other processes in CZE, including the preparation of sample and standard solutions, electrophoretic separation, optical detection and recorder or integrator response, the sample introduction by the proposed sampler will not be decisive in the reproducibility of a determination by CZE. The overall reproducibility will be at approximately the same level as those in high-performance liquid chromatography (HPLC). The use of an internal standard will give more reproducible results. Fig. 5 shows an example of the separation of CA and DMCA introduced into the 100~pm tube by the proposed sampler. The numbers of theoretical plates, calculated as 16 t2~-‘, where t is the retention time and w is the peak width, of CA and DMCA were 41000 and 45000, respectively, when they were introduced for 5 s by a 5-cm head of water. The column efficiency was quite high, compared to that in ordinary HPLC, and this result indicates the usefulness of this technique for the separation of ionic substances. Thus, the use of the proposed siphonal sampler allowed reproducible introduction of a sample to a capillary tube by the simple operation of setting a sample TABLE I REPRODUCIBILITY OF SAMPLE INTRODUCTION, AND PEAK AREA OF BENZYL ALCOHOL
ACCORDING
TO THE PEAK HEIGHT
The conditions used were as described in Fig. 3. Inner diameter (pm)
100 250
Programmed sampling time
Coejicienl
(.?I
Peak height
1 5 1 5
qf variation (o/o, n = 12) Peak area
Automatic sampling
Manual introduction
Automatic sampling
Manual introduction
3.2 I.1 2.4 0.84
8.1 5.6 6.5 4.4
2.3 0.92 1.3 0.55
7.8 5.2 5.9 4.0
S. HONDA, S. IWASE, S. FUJIWARA
320
Ll
1
-
Fig. 5. Separation by CZE of cinnamic acid (CA) and 3,4-dimethoxycinnamic by the proposed sampler. Conditions as in Fig. 3a. 1 = CA; 2 = DMCA.
acid (DMCA) introduced
solution, followed by pushing a starter button. The results ensured quantification by the absolute amount and internal standard methods. It is also a great advantage that the sample amount could be minimized. The application of this sampler to quantitative studies of CZE will be described elsewhere. ACKNOWLEDGEMENT
We thank Mr. K. Hishikawa of Ekikuro Science Company (Tenri, Nara, Japan) for his skilful assistance in preparing the automatic sampler. REFERENCES I 2 3 4 5 6 7
8 9 10 11 12
13
F. E. P. Mikkers, F. M. Everaerts and Th. P. E. M. Verheggen, J. Chromatogr., 169 (1979) 11-20. J. W. Jorgenson and K. D. Lukacs, Anal. Chem., 53 (1981) 1298-1302. J. W. Jorgenson and K. D. Lukacs, J. Chromatogr., 218 (1981) 209-216. T. Tsuda, K. Nomura and G. Nakagawa, J. Chromatogr., 264 (1983) 385-392. T. Tsuda, K. Nomura and G. Nakagawa, J. Chromatogr., 248 (1982) 241-247. T. Tsuda, G. Nakagawa, M. Sato and K. Yagi, J. Appl. Biochem., 5 (1983) 330-336. H. H. Latter and D. McManigill, Anal. Chem., 58 (1986) 166170. S. Terabe, K. Otsuka, K. Ichikawa, A. Tsuchiya and T. Ando, Anal. Chem., 56 (1984) 113-116. S. Terabe, K. Otsuka and T. Ando, Anal. Chem., 57 (1985) 834841. S. Fujiwara and S. Honda, Anal. Chem., 58 (1986) 1811-1814. W. Jorgenson and K. D. Lukacs, Science (Washington, D.C.), 222 (1983) 266-272. R. A. Wallingford and A. G. Ewing, Anal. Chem., 59 (1987) 681-684. M. Deml, F. Foret and P. BoEek, J. Chromurogr., 320 (1985) 159-165.
Elsevier Science Publishers B.V., Amsterdam -
Printed in The Netherlands
CHROM. 19 689
EVALUATION OF AN AUTOMATIC LARY ZONE ELECTROPHORESIS
SUSUMU HONDA*, SHIGEFUMI
SIPHONIC
SAMPLER
FOR CAPIL-
IWASE and SHIGERU FUJIWARA
Faculty of Pharmaceutical Sciences, Kinki University, 3-4-l Kowakae, Higashi-Osaka (Japan)
(First received February 23rd, 1987; revised manuscript received April 22nd, 1987)
SUMMARY
An automatic sampler based on siphoning was devised and its capability for capillary zone electrophoresis (CZE) was examined. The peak area of benzyl alcohol as a model compound introduced to a capillary tube was proportional to the sampling time, and the reproducibility of sample introduction was quite high. These results ensure the usefulness of this apparatus for quantitative as well as qualitative analyses by CZE.
INTRODUCTION
Capillary zone eletrophoresis (CZE) is a recently developed method for the separation of ions, based on a combination of the effects of electrophoresis and electroosmosis in a capillary tube. It was introduced by Mikkers et al.‘, and developed by Jorgenson and Lukacs2q3. Its applicability is very wide, ranging from small inorganic4 as well as organic ions 4-6 to biomacromolecules such as proteins7. The use of dynamic hydrophobic interaction by adding a detergent to the carrier has further extended its applicability to neutral molecules *a9.It exhibits high efficiency, the number of theoretical plates calculated in the same manner as in liquid chromatography often reaching several hundreds of thousands. The problems of quantification were studied by Fujiwara and HondalO, who also demonstrated the applicability of CZE to drug monitoring. However, all these studies on CZE were performed by manual sample introduction, because no automatic apparatus was commercially available. This has hampered the popularization of CZE. The most difficult problem in sample introduction in CZE is that an extremely small volume (less than several hundred nanolitres) of a sample solution has to be introduced into a capillary tube which is strictly insulated so as to withstand high voltages. Dipping one end of a capillary tube in a solution prepared by dissolving the sample in the carrier electrolyte solution and the other end in the same electrolyte solution, followed by application of an high voltage between the two ends for a short period l’, can result in sample introduction by the effect of electroosmosis. However, the quantity of the sample introduced is dependent on its concentration. In addition the sample is usually ionic and cannot easily be recovered from the electrolyte solution. Since the volume of sample introduced is extremely small, on-column detec0021-9673/87/$03.50
0
1987 Elsevier Science Publishers B.V
314
S. HONDA, S. IWASE, S. FUJIWARA
tion demands an high concentration of the sample solution, as compared to the detection in ordinary high-performance liquid chromatography. The recovery is critical, when only small amounts of samples are available, as is often encountered with biological and environmental samples. A small dual-barrelled pipette proposed by Wallingford and Ewing’ 2 is notable in that it minimizes the sample volume, but there is still the problem of the sample concentration and it is also difficult to automate. In an early study of CZE a microlitre syringe or a specially constructed four-way injection valve was used’, but the volumes of the sample solutions introduced were large. A few kinds of microinjectors designed for capillary liquid chromatography are commercially available, but they all use metallic parts which have high electric conductivity and cause a gradual change in the optical density of the carrier, resulting in baseline drift, presumably due to electrolysis in the flow path. We made some attempts to prepare an all-plastic valve to overcome this effect, but the results were not promising because no resins withstood repeated switching of such narrow-bore flow paths. The use of a ceramic valve might give better results. Deml et al.’ 3 devised an electric splitter based on sample division by differential electromigration in two electrical circuits. They demonstrated the capability of this apparatus by presenting a few improved separations of carboxylates in isotachophoresis, but did not give any data on reproducibility. It is a fundamental disadvantage that only a small part of the applied sample was analyzed, the greater part being discarded. As compared to the above techniques, the one based on siphoning is important. Since it can be performed manually, many studies on CZE5,* were done by this technique, though on a qualitative basis. We propose here an automatic sampler for both qualitative and quantitative studies, based on this principle. There have been no reports on the evaluation of such an apparatus from the viewpoint of quantification. EXPERIMENTAL
The siphonic sampler
Fig. 1 shows a frontal view of this apparatus. The whole apparatus was surrounded (not shown) by a rectangular, plastic cover to protect an operator from any hazard due to electrical leakage. This cover was designed so that no voltage could be applied while its front panel was open. Both ends of a capillary tube were placed in cylindrical glass vessels (a and c), into which electrodes (e) were inserted. Thin, spherical rubber plates were stuck on the underside of the vessels for insulation purposes. The cathode vessel (c) remains at the same position throughout an analysis, but the anode vessel (a) is placed on a turntable (t), whose level could be changed automatically by a microcomputer-controlled motor (m). The component ions separated can be monitored by an on-column detector (d) near the cathode. In analyses using capoillary tubes with inner diameters larger than 50 pm, commercial UV or multiwavelength detectors were usable with slight modification. A capillary tube (cap) was fixed, directly (in the case of a glass or FEP tube) or after removal of the coating resin (in the case of a fused-silica tube), at the centre of the monochromatic beam. Fig. 2 explains the.introduction of a sample solution into a capillary tube in one cycle of operation. The operation begins at stage I, where both electrode solutions
315
SAMPLER FOR CAPILLARY ZONE ELECTROPHORESIS
to a highvol taee supplier
-
t
m
I
Fig. 1. Frontal view of the automatic siphonic sampler. cap. = Capillary tube; a = anode vessel; b = ball-bearing; c = cathode vessel; d = detector; e = electrode; m = motor; n = nut; r = rubber stopper; s = switch: t = turntable.
316
S. HONDA,
S. IWASE,
S. FUJIWARA
Fig. 2. The sampling process. For each stag: are shown the frontal view of the whole sampler, and a circle representing the upper surface of the turntable. h = Hole; sv = sample vial; other parts as in Fig. 1. The arrows indicate the movement of the turntable from stage to stage.
SAMPLER FOR CAPILLARY ZONE ELECTROPHORESIS
317
are arranged at the same level. First the anode vessel is moved to a lower level, stage II, while the anodic end of the tube is held at the original level by means of a small rubber stopper (r). Then the turnable is rotated clockwise by 45”, and the anode vessel replaced by a round hole (h), indicated by a hatched circle, stage III. Subsequently the turntable is raised to an high level for sampling, stage IV. This level was set several centimetres higher than the starting level. The anodic electrode is undamaged by this movement, because it safely passes through the hole on the turntable. A sample solution in a small vial (sv) is introduced into the capillary tube by downhill flow due to the difference in levels between the sample and the cathode solutions. The sampling time can be specified on a programmer. After the sample solution is introduced, the turntable is lowered again, stage V, equivalent to stage III. The turntable is rotated counterclockwise, stage VI, which is equivalent to stage II. Finally it is raised to the initial level. All these movements of the turntable are indicated by arrows. Evaluation of the sampler
Two fused-silica capillary tubes (80 cm x 100 pm I.D.; 80 cm x 250 pm I.D.) were obtained from Scientific Glass Engineering (North Melbourne, Australia). The coating resin was removed by burning at a distance of 20 cm from one end of each tube. The tube was filled with 10 mM phosphate buffer (pH 7.0) by downhill flow, and set on the proposed apparatus. After 100 mM benzyl alcohol had been introduced for an appropriate period at a 5.0-cm head of water by use of the sampler, a voltage of 21 kV (for the 100~pm tube) or 11 kV (for the 250~pm tube) was applied between both ends of the tube by an high-voltage supply installed in an IP-1B isotachophoresis apparatus (Shimadzu, Kyoto, Japan). The peaks of benzyl alcohol were monitored by on-column detection at 254 nm using an UV detector contained in a Model 633 high-performance liquid chromatograph (Hitachi, Ibaragi, Japan). The volumes of the sample solution introduced to the lOO+m and 250~pm tubes by 5-s sampling were ca. 40 and 500 nl, respectively, estimated from the peak widths of benzyl alcohol. The separation of cinnamic acid (CA) and 3,4-dimethoxycinnamic acid (DMCA) was performed by the same system using the IOO-pm tube. RESULTS AND DISCUSSION
Fig. 3 gives examples of repeated sample introduction, as observed from the detector response of benzyl alcohol introduced as a model compound. The solid line in Fig. 4a indicates the relationship between the peak height of benzyl alcohol introduced to the 250-pm tube and the programmed sampling time. Even when the programmed sampling time was zero, a small volume of the sample solution was siphoned into the tube during the up-and-down movement of the anodic end of the tube, giving an intercept of 16 mm. Subtraction of this value from each plot gave the dotted line showing the relationship between the net peak height and the real sampling time. The convex nature of this curve indicates that the peak height is not proportional to the real sampling time, obviously due to peak broadening caused by overloading. On the other hand, a plot of peak area (as calculated from the weight of the cut-out peak) vs. sampling time gave a straight line (Y = 2.4 X + 3.2, r =
S. HONDA,
318
S. IWASE,
S. FUJIWARA
b a
Fig. 3. Repeated introduction of benzyl alcohol as a model compound by using the proposed sampler: (a) 5-s sampling to the IOO-nm tube; (b) 5-s sampling to the 250-pm tube. Capillaries: fused silica, (a) 80 cm x 100 nm I.D., (b) 80 cm x 250 grn I.D. Carrier: 10 mM phosphate buffer (pH 7.0). Concentration of the sample solution: 100 mM. Difference in levels between the sample and the cathodic solution: 5 cm. Current applied: 30 (a); 125 PA (b). Voltage: 21 (a); 11 kV (b). Detection: absorbance at 254 nm.
0.997) over the whole range examined (O-l 5 s), as seen in Fig. 4b. Since the peak area is considered to be proportional to the amount of sample introduced, a plot of the sampling time vs. amount introduced, i.e., volume introduced should also be linear. These relationships are important for quantification. Similar results were obtained with the 100~pm tube. Table I gives the coefficient of variation (C.V., n = 12) of the peak height
Sampling
time,
8
Fig. 4. Relationships between the peak height (a) and peak area (weight of the cut-out alcohol and sampling time. Analytical conditions as in Fig. 3b.
peak) (b) of benzyl
SAMPLER
FOR CAPILLARY
ZONE ELECTROPHORESIS
319
obtained with the 250~,um tube. It was as low as 2.4 and 0.84% for l-s and 5-s samplings, respectively. The C.V. values for the peak area were much smaller, 1.3 and 0.55%, respectively. The narrower tube gave slightly higher C.V. values for both the peak height and peak area, but they were sufficiently low to allow practical analysis. For comparative purposes, the data obtained by manual introduction are also presented in Table I. Each value is much higher than the corresponding value obtained by automatic introduction. This neutral sample was migrated from the inlet of the capillary tube to the detector by electroosmotic flow under the conditions used, so the data described above include the variation due to dispersion during migration. Therefore, the actual C.V. values for sample introduction alone should be smaller than these values. In the most typical mode of determination in CZE, the concentration of the sample solution is estimated by using a calibration curve obtained from standard solutions at an appropriate sampling time. Since the precision of the sample introduction described above was as high as or higher than the reproducibilities (C.V. generally less than 3%) of other processes in CZE, including the preparation of sample and standard solutions, electrophoretic separation, optical detection and recorder or integrator response, the sample introduction by the proposed sampler will not be decisive in the reproducibility of a determination by CZE. The overall reproducibility will be at approximately the same level as those in high-performance liquid chromatography (HPLC). The use of an internal standard will give more reproducible results. Fig. 5 shows an example of the separation of CA and DMCA introduced into the 100~pm tube by the proposed sampler. The numbers of theoretical plates, calculated as 16 t2~-‘, where t is the retention time and w is the peak width, of CA and DMCA were 41000 and 45000, respectively, when they were introduced for 5 s by a 5-cm head of water. The column efficiency was quite high, compared to that in ordinary HPLC, and this result indicates the usefulness of this technique for the separation of ionic substances. Thus, the use of the proposed siphonal sampler allowed reproducible introduction of a sample to a capillary tube by the simple operation of setting a sample TABLE I REPRODUCIBILITY OF SAMPLE INTRODUCTION, AND PEAK AREA OF BENZYL ALCOHOL
ACCORDING
TO THE PEAK HEIGHT
The conditions used were as described in Fig. 3. Inner diameter (pm)
100 250
Programmed sampling time
Coejicienl
(.?I
Peak height
1 5 1 5
qf variation (o/o, n = 12) Peak area
Automatic sampling
Manual introduction
Automatic sampling
Manual introduction
3.2 I.1 2.4 0.84
8.1 5.6 6.5 4.4
2.3 0.92 1.3 0.55
7.8 5.2 5.9 4.0
S. HONDA, S. IWASE, S. FUJIWARA
320
Ll
1
-
Fig. 5. Separation by CZE of cinnamic acid (CA) and 3,4-dimethoxycinnamic by the proposed sampler. Conditions as in Fig. 3a. 1 = CA; 2 = DMCA.
acid (DMCA) introduced
solution, followed by pushing a starter button. The results ensured quantification by the absolute amount and internal standard methods. It is also a great advantage that the sample amount could be minimized. The application of this sampler to quantitative studies of CZE will be described elsewhere. ACKNOWLEDGEMENT
We thank Mr. K. Hishikawa of Ekikuro Science Company (Tenri, Nara, Japan) for his skilful assistance in preparing the automatic sampler. REFERENCES I 2 3 4 5 6 7
8 9 10 11 12
13
F. E. P. Mikkers, F. M. Everaerts and Th. P. E. M. Verheggen, J. Chromatogr., 169 (1979) 11-20. J. W. Jorgenson and K. D. Lukacs, Anal. Chem., 53 (1981) 1298-1302. J. W. Jorgenson and K. D. Lukacs, J. Chromatogr., 218 (1981) 209-216. T. Tsuda, K. Nomura and G. Nakagawa, J. Chromatogr., 264 (1983) 385-392. T. Tsuda, K. Nomura and G. Nakagawa, J. Chromatogr., 248 (1982) 241-247. T. Tsuda, G. Nakagawa, M. Sato and K. Yagi, J. Appl. Biochem., 5 (1983) 330-336. H. H. Latter and D. McManigill, Anal. Chem., 58 (1986) 166170. S. Terabe, K. Otsuka, K. Ichikawa, A. Tsuchiya and T. Ando, Anal. Chem., 56 (1984) 113-116. S. Terabe, K. Otsuka and T. Ando, Anal. Chem., 57 (1985) 834841. S. Fujiwara and S. Honda, Anal. Chem., 58 (1986) 1811-1814. W. Jorgenson and K. D. Lukacs, Science (Washington, D.C.), 222 (1983) 266-272. R. A. Wallingford and A. G. Ewing, Anal. Chem., 59 (1987) 681-684. M. Deml, F. Foret and P. BoEek, J. Chromurogr., 320 (1985) 159-165.