Isoelectric Focusing in Continuously Tapered Fused Silica Capillary

Technical Note pubs.acs.org/ac

Isoelectric Focusing in Continuously Tapered Fused Silica Capillary Prepared by Etching with Supercritical Water Karel Šlais,* Marie Horká, Pavel Karásek, Josef Planeta, and Michal Roth Institute of Analytical Chemistry of the ASCR, v. v. i., Veveří 97, 602 00 Brno, Czech Republic ABSTRACT: This communication indicates the potential of etching with sub- and/or supercritical water for reproducible preparation of fused-silica capillaries with tapered geometry suitable for capillary isoelectric focusing (CIEF) with electroosmotic displacement. The etching procedure provided a single-piece combination of the tapered separation space with a cylindrical connection of the detection window to the electrode vial. Selected proteins and colored pI markers were used as model analytes. A comparison with conventional cylindrical capillary under comparable applied voltage and analysis time was made, and the resultant peaks were compared in terms of peak resolution under optimized conditions. In CIEF carried out in a tapered capillary with the inlet cross-section three times larger than the cross-section at the detection window, three to four times higher resolutions of corresponding peak pairs were obtained. The method described opens the way to increase the number of separable compounds without resorting to excessively high voltage.

T

The description of migration of the focused zone in the nonuniform capillary of arbitrary shape is generally very difficult. Movement of the zone in nonuniform capillary along the length coordinate, y, occurs under velocity gradient, dv/dy. However, it was derived6,7 that the condition dv/dy = const (or v = const × y) required for optimum performance can be achieved for a narrow nonuniform capillary of special shape. Such a shape of the capillary can be specified by eq 1,

race analysis of ionized compounds often needs some means of sample concentration and the use of a large sample volume. The focusing electrophoretic methods including capillary isotachophoresis (ITP) and capillary isoelectric focusing (CIEF) with electroosmotic displacement conveniently combine the powerful focusing and separation features. To analyze the large amount of sample, a capillary with sufficient volume is needed. Since IEF with electroosmotic displacement and ITP enable also the transfer of analytes from the large capillary cross-section to a smaller one,1−3 the column coupling or the volume coupling is a possible approach to handle large sample volumes.1−3 Although the separation capacity and the detection sensitivity are better in this case, deterioration of the zones because of the transfer between different cross sections occurs, and the long detection capillary and thus high voltage has to be applied again to reestablish the steady state.4 The use of a shallow-cone capillary with continuous decrease of cross-section toward the detection point5 could meet both the suitable high separation capacity of the system and relatively lower voltage needed for the separation. A simple model of the electro focusing processes in the tapered capillary was described previously.6−8 © 2013 American Chemical Society

V A 1 1 = L Ad Ad ln(A 0 /Ad ) y

(1)

where A is the local cross-section and VL is the volume between the inlet (y0, A = A0) and the outlet (yd, A = Ad) of the capillary: VL =

∫y

yd

A dy (2)

0

The advantages predicted for the continuously tapered channel in comparison to the constant cross-section capillary include lower voltage and higher limiting peak capacity.6,7 Received: January 29, 2013 Accepted: March 26, 2013 Published: March 26, 2013 4296

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Analytical Chemistry

Technical Note

niger (Mr 97 000, pI 3.6)25 were obtained from Fluka Chemie GmbH (Buchs, Switzerland). The solution of synthetic carrier ampholytes (Biolyte, pH 3−10) was obtained from Bio-Rad Laboratories (Hercules, CA). N-(2-Acetamido)-2-aminoethanesulphonic acid (ACES) and 2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulphonic acid (HEPES) were obtained from Merck (Darmstadt, Germany). L-Aspartic acid (Asp) was obtained from LOBA Chemie (Vienna, Austria), other spacers including tartaric (Tart), malic (Mal), formic (Form), succinic (Suc), acetic (Acet), pivalic (Piv), glutamic (Glu), and nicotinic (Nic) acids, as well as the nonionogenic detergent Brij 35 were purchased from Sigma (St. Louis, MO). Polyethylene glycol (Mr 10 000, PEG 10 000), bovine serum albumin (Mr 67 000, pI 4.9),25 β-lactoglobulin (Mr 35 000, pI 5.1),25 and pepsin (Mr 33 000, pI 2.86−2.94)25 were obtained from Sigma-Aldrich (Milwaukee, WI). Specifications26,27 of the simple ampholytic electrolytes used as spacers were described elsewhere.28 The low-molecular pI markers, pI = 2.0,12 4.0,13 and 5.3,12 and 4morpholinyl acetic acid (MAA)28 were synthesized at the Institute of Analytical Chemistry of the ASCR, v. v. i. All chemicals were of electrophoresis, analytical grade. CIEF Equipment and Procedures. CIEF was carried out using a laboratory-made apparatus28 at a constant voltage (−20 kV on the detector side) supplied by high-voltage unit Spellman CZE 1000 R (Plainview, NY). The lengths of the cylindrical fused silica (FS) capillaries, 100 μm I.D. and 360 μm O.D. (Agilent Technologies, Santa Clara, CA), were 67.6 cm + 20 cm to the detector. The length of the tapered FS capillary, I.D. from 170 μm at the inlet of the capillary tapered to 100 μm at the detection window, was 45 cm +20 cm to the cathode vial. The ends of either capillary were dipped in 3 mL-glass vials with the electrodes and with the anolyte or the catholyte solutions in CIEF or with the background electrolyte (BGE) in capillary zone electrophoresis (CZE). An on-column UV−vis detector LCD 2082 (Ecom, Prague, Czech Republic) connected to the detection cell by optical fibers (Polymicro Technologies, Phoenix, AZ) was operated at 235 nm (CZE) and 280 nm (CIEF). Sample injection was performed by siphoning action achieved by temporary elevation of the inlet reservoir on the side of anode relative to the outlet reservoir on the side of cathode. Height difference of the reservoirs for the sample injection, Δh, was 10 cm (tapered FS) and 20 cm (cylindrical FS) whereas the injection time, tinj, ranged from 5 to 35 s. The detector signals were acquired and processed with a Clarity chromatography data station (DataApex, Prague, Czech Republic). Aqueous sodium hydroxide, 4 × 10−2 mol L−1, and 0.1 mol L−1 ortho-phosphoric acid were used as the catholyte and the anolyte solutions, respectively, with the addition of 1−3% (v/v) ethanol (EtOH) and 0.5% (w/v) PEG 10 000. In CZE, 20 mM phosphate buffers pH 2−10 (Figure 2, curves 1 and 2) were used as the BGEs in the EOF measurements. Thiourea was used here as the neutral marker of the EOF. The CIEF separation was carried out using the segmental injection of the sample pulse into the capillary.28,29 The sample was injected in three parts. The first segment contained the spacers, i.e., a solution of selected simple ampholytic electrolytes, 15 × 10−5 mol L−1, dissolved in 2 × 10−2 mol L−1 NaOH. The second segment was composed of the sample of proteins, albumin, amyloglucosidase, β-lactoglobulin, and pepsin (50 μg mL−1 each), dissolved in distilled water. The third segment contained a 5% (w/v) aqueous solution of commercial carrier ampholytes, Biolyte, pH 3−10, and ampholytes pH 3.0−4.5

Recent development of etched flexible fused silica capillaries with variable cross-section9 allows us to create tapered capillaries that could be potentially applicable in focusing modes of electrophoresis. To verify the benefits of tapered capillaries, this work extends the previous one9 with the technique for production of tapered capillaries useful for CIEF. The CIEF experiments with both cylindrical and etched tapered fused silica capillaries were carried out, with the low-molecularweight colored pI markers prepared and described previously10−13 and selected proteins serving as the model substances for the proof-of-feasibility studies. Proteins tend to adhere strongly to the FS capillary inner surface.14,15 Therefore, the possible differences in the inner surface properties between the tapered capillary etched with supercritical water (SCW) and the original, nonetched, cylindrical FS capillary must be taken into account, and the separation conditions must be optimized.16−19 Simultaneously, it is necessary to minimize the strong electroosmotic flow (EOF) on both uncoated FS capillaries. The simplest suggested solutions include a dynamic modification of the inner capillary surface20,21 by soluble polymers.20,22,23 At the same time, the adsorbed proteins must be washed out from the capillary wall prior to the subsequent run. Certainly, the rinsing procedure between the individual runs was shown to have a strong effect on the reproducibility.24

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EXPERIMENTAL SECTION Capillary Preparation. The tapered capillary has been prepared by etching with SCW using the apparatus designed and built in our laboratory.9 Undeactivated, polyimide-coated capillary (100 μm I.D., 360 μm O.D.) was delivered by Agilent Technologies, Waldbronn, Germany (Part No. 160-2634-10). Water treatment prior to use included double distillation, purification with reverse osmosis system (Ultra Clear UV, Barsbüttel, Germany), and stripping with helium (Linde, Brno, purity 4.5) to remove oxygen and CO2. Several tapered capillaries with length 90 cm, inlet I.D. 170 μm, and outlet I.D. 100 μm were prepared by a dynamic flow-mode method with water flow rate of 0.272 g min−1, temperature of 400 °C, pressure of 300 bar, and treatment duration of 40 min. To minimize the pressure drop along the treated capillary, a fused silica restrictor (50 μm I.D., 130 cm) was located at the system outlet downstream of the cooling unit (Grant LTD6G, Grant Instruments, United Kingdom). The diameter changes were checked with an optical microscope (Olympus BX-51, Prague, Czech Republic) equipped for operation in both reflected and transmitted light and featuring an image processing software (Deep Focus ver. 3.1.). To examine the possible usability of the particular segment of the capillary (cut before the segmental measurements, input to detection window = 45 cm), the segment was fitted into the CIEF instrument, the capillary was filled with the 3 mM solution of KCl, and the current under 20 kV voltage was measured equal to 24 microamperes. In parallel, the cylindrical capillary with length 67.5 cm and the same volume (5.31 μL) as the tapered one was also examined under the same voltage, and the current found was 12 microamperes while the expected value was 10 microamperes. Chemicals. High resolution ampholyte, pH 2−4, ampholyte pH 3−4.5, 2-morpholino-ethanesulphonic acid monohydrate (MES), 3-morpholino-propanesulphonic acid (MOPS), N-[tris(hydroxymethyl)-methyl]-3-amino-2-hydroxy-propanesulphonic acid (TAPSO), and amyloglucosidase from Aspergillus 4297

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Analytical Chemistry

Technical Note

and pH 2.0−4.0 in the ratio 1:2:5, and low-molecular pI markers (25 μg mL−1 each), pI 2.0, 4.0, and 5.3, for tracing of the pH gradient employed, pH range 2.0−5.3. Injection time of the spacers segment was 25 s, 10 s for the sample segment, and 35 s for the segment of carrier ampholytes and pI markers. Before each injection, the capillaries were hydrodynamically rinsed with EtOH for 5 min and then back-flushed with catholyte or BGE for 5 min. Safety Considerations. CIEF employs high voltage requiring due care and caution from the operator. Because of its high temperature, high pressure, and high compressibility, SCW is a difficult-to-handle and potentially dangerous medium. The device for etching the capillaries should therefore be covered with shatterproof panels to protect the operator against accidental release of overheated steam. Eye protection and protective clothing are absolutely necessary.

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RESULTS AND DISCUSSION Capillaries. This study builds up on the etching experiments described in our previous paper.9 A set of six etched capillaries was prepared under the same conditions and five of them were cut into 1 cm segments and measured microscopically to obtain the actual local cross-section and, consequently, the variability of etching process. The result can be seen in panel A of Figure 1. It can be seen that the etching variability does not exceed about 10% expressed in terms of relative deviation of local internal diameter. Therefore, one can reasonably anticipate that the sixth capillary’s internal diameter can be approximated by the average of the measured five capillaries. Next, the segment of 45 cm separation length to detector window together with an additional 20 cm to cathode was cut out (Figure 1A) to obtain the tapered capillary useful for CIEF experiments. The positions of the cut and the detection window are marked in Figure 1A. The volume of segment was estimated to be a sum of 5.34 μL separation space and 1.57 μL volume from the detection window to the electrode vial, i.e., 6.91 μL. The estimated course of internal diameter with the length of separation space is shown by the points in panel B of Figure 1. Now, the important question arises whether the obtained diameter vs the capillary position dependence has the suitable shape. It was derived previously6,7 that the optimal capillary taper should obey the reciprocal decrease in the local cross-section with the length coordinate (eq 1). Thus, the course of actual cross-section with capillary length is shown by the average line in Figure 1B together with the theoretical curve predicting the optimal shape determined by the particular inlet and outlet cross sections of the tapered separation space, A /Ad = 69/(24 + y),

Figure 1. (A) Dependence the local internal diameter of etched fused silica capillary on the capillary length. The cutout of the segment used as the tapered capillary in cIEF and the detection window are indicated. (B) Relative mean value of the inner cross-section of the tapered capillary (SDs are also shown) versus the profile predicted by the theory.7

the tapered separation space with the capillary connection to electrode vial. Effect of Etching on EOF. The electroosmotic flow is strongly controlled by the numerous silanol groups on the inner surface of the FS capillary. SCW used for etching may alter the form of silanol groups. Therefore, modification of capillary wall during preparation of the tapered FS capillary can modulate the EOF, and it can also act differently on the possible adsorption of proteins onto the inner surfaces of both examined capillaries, etched and nonetched. At first, therefore, it is important to compare the effect of etching of the inner capillary surface by SCW on the EOF in the pH range of 2.0− 10.0. The results are illustrated for both the cylindrical (L = 67.6 + 20 = 87.6 cm) and the tapered (L = 45 + 20 cm) capillaries by curves 1 or 2, respectively, in Figure 2. The electroosmotic mobility, μEOF, is somewhat lower on the tapered capillary compared to the cylindrical capillary, notably in the pH range from 5.0 to 10.0; see curves 1 vs 2. The reduction in EOF appears to be useful for the CIEF separation of proteins on tapered FS capillary. CIEF of Selected Proteins and pI Markers on Cylindrical and Tapered FS Capillaries. The CIEF experiment was designed to compare separation performances of the tapered and the cylindrical capillaries of equal volumes

y = [0; 45] cm

This curve almost coincides with the function resulting from eq 1 where the real values were inserted (VL is in cm3, A0 and Ad are in cm2, and y is in cm), A /Ad = 0.00534/0.0000785 × 1/1.054 × 1/y = 65.46 × 1/y

Taking the variability of local capillary cross-section into account, it can be concluded that the shape of prepared capillary is not too far from the optimal one and the prepared segment could have beneficial properties when used in CIEF. The prepared capillary (see Figure 1A) conveniently combines 4298

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Analytical Chemistry

Technical Note

Figure 2. Effect of pH on the electroosmotic flow in a cylindrical (1) and a tapered (2) FS capillary. Conditions: FS capillaries: (1) 100 μm ID, 360 μm OD, 87.6 cm length (67.6 cm length to the detection window +20 cm toward the electrode vial); (2) capillary ID from 170 μm at the inlet of the capillary, tapered to 100 μm at the detection window, taper length 45 cm +20 cm of 100 μm ID toward the electrode vial, BGE, 2 × 10−2 mol L−1 phosphate buffer from pH 2 to pH 10; siphoning injection (Δh = 10 cm, 5 s); applied voltage (−)20 kV; neutral marker of EOF, thiourea; UV detection, 235 nm; μEOF, electroosmotic mobility [cm2V−1s−1].

under the same applied voltage. Mixtures of proteins and pI markers were separated by CIEF in the pH gradient 2.0−5.3; see Figure 3, on a cylindrical (A,C) and tapered (B,D) FS capillary. In the preliminary experiments, the compositions of the catholyte and the anolyte were 4 × 10−2 mol L−1 NaOH and 0.1 mol L−1 H3PO4, respectively. The resultant separations of two pI markers and albumin are shown in Figure 3A,B. The separation on tapered capillary is apparently better than that on the cylindrical one; however, it is problematic to express the improvement quantitatively. Therefore, the solutions were further optimized with respect to the differences in the surface properties of the cylindrical and the tapered capillaries. As a result of this optimization, 0.5% (w/v) PEG 10 000 was dissolved in the catholyte and the anolyte for CIEF on the cylindrical capillary and 0.3% (w/v) PEG 10 000 and 5% (v/v) EtOH for CIEF on the tapered capillary; see Figure 3C,D. Other compositions and concentrations of the additives in the catholyte and the anolyte resulted in deterioration of the resolution on the examined capillaries, both etched and nonetched. The pH gradient was traced by the pI markers with pI 4.0 and 5.3 (Figure 3C). The isoelectric point for the albumin zone was calculated from the isoelectric points and the migration times of the pI markers used, and it was determined as 4.9. That is in agreement with the tabulated value of pI.25 The relative standard deviations (RSDs) of the migration times were always arround 1.7%. In order to give a better illustration of the separation potential of IEF on the tapered capillary, more analytes were separated under the optimized conditions. The pH gradient in the electropherogram depicted in Figure 3D was traced by the pI markers with pI 2.0, 4.0, and 5.3. The observed isoelectric points of proteins, β-lactoglobulin, albumin, amyloglucosidase, and pepsin, are in agreement with the respective tabulated25 values of pI. The positions of protein peaks in the record follow from protein successive addition in the sampling solution. The relative standard deviations of the migration times, RSDs, were always arround 1.8%.

Figure 3. Influence of the capillary geometry, cylindrical (A, C) and tapered (B, D), on CIEF separation of pI markers and proteins in the pH gradient 2.0−5.3. Conditions and designations, see Figure 2; the catholyte, 4 × 10−2 mol L−1 NaOH, the anolyte, 0.1 mol L−1 H3PO4; sample composition, segment of spacers28 dissolved in the catholyte, tinj, 25 s, segment of carrier ampholytes, 5%(w/v) of synthetic carrier ampholytes, Biolyte, pH 3−10, ampholyte pH 3−4.5 and pH 2−4 (1:2:5), tinj, 35 s; UV detection, 280 nm; A: sample: pI markers pI 5.3 and 4.0 (25 μg mL−1 of each), albumin, 50 μg mL−1, dissolved in water; tinj, 10 s (Δh, 20 cm); B: see A, tinj, spacers, 10 s, sample 10 s and carriers 17 s; C: see A, 0.5%(w/v) PEG 10 000 was dissolved in the catholyte and the anolyte; D: see A, 0.3% (w/v) PEG 10 000 and 5% (v/v) EtOH were dissolved in the catholyte and the anolyte; sample: pI markers pI 5.3, 4.0, and 2.0 and proteins, β-lactoglobulin, albumin, amyloglucosidase, and pepsin, 50 μg mL−1 of each, tinj, 10 s; rinsing procedure, EtOH for 5 min, and then back-flushed with the catholyte for 1 min; t, migration time [s].

The resolution of peaks in Figure 3C,D was calculated according to eq 3, t 2 − t1 1.18Δt = Rs = 2(σ1 + σ2) w0.5(1) + w0.5(2) (3) where w0.5 is the peak width at half height. The results are summarized in Table 1. It can be seen that the resolutions, Rs, of corresponding peaks focused in the tapered capillary are substantially larger than those in the cylindrical capillary. They are even larger than what could be estimated from the simple theory derived before.6−8 The reason can be seen not only in the numerous simplifications of the theory but also in the fact 4299

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Table 1. Parameters for the Calculation of the Resolution, Rs, on the Cylindrical and Tapered FS Capillary capillary cylindrical analyte

tapered

marker pI 5.3

albumin

marker pI 4.0

marker pI 5.3

albumin

marker pI 4.0

36 0.22 13.45

36 0.31 14.21

36 0.23 15.55

40 0.19 11.41

40 0.15 13.12

40 0.18 16.95

Vinj [nL] w0.5 [min] t [min] Δt [min] Rs

0.76 1.7

1.34 2.9

1.71 5.9

(12) Št’astná, M.; Trávníček, M.; Šlais, K. Electrophoresis 2005, 26, 53−59. (13) Št’astná, M.; Šlais, K. J. Chromatogr., A 2003, 1008, 193−203. (14) Roosjen, A.; Kaper, H. J.; van der Mei, H. C.; Norde, W.; Busscher, J. Microbiology-SGM 2003, 149, 3239−3246. (15) Rodriguez-Diaz, R.; Wehr, T.; Zhu, M. Electrophoresis 1997, 18, 2134−2144. (16) Armstrong, D. W.; Schulte, G.; Schneiderheinze, J. M.; Westenberg, D. J. Anal. Chem. 1999, 71, 5465−5469. (17) Hjertén, S. J. Chromatogr. 1985, 347, 191−198. (18) Oscarsson, S. J. Chromatogr., B 1997, 699, 117−131. (19) Issaq, H. J. Electrophoresis 2000, 21, 1921−1939. (20) Righetti, P. G. J. Chromatogr., A 2004, 1037, 491−499. (21) Kilár, F. Electrophoresis 2003, 24, 3908−3916. (22) Girod, M.; Armstrong, D. W. Electrophoresis 2002, 23, 2048− 2056. (23) Shimura, K. Electrophoresis 2002, 23, 3847−3857. (24) Molteni, S.; Thormann, W. J. Chromatogr. 1993, 638, 187−193. (25) Righetti, P. T.; Caravaggio, T. J. Chromatogr. 1976, 127, 1−28. (26) Hirokawa, T.; Nishino, M.; Aoki, N.; Sawamoto, Y. K. T. Y.; Akiyama, J. I. J. Chromatogr., A 1983, 271, D1−D106. (27) Acevedo, F. J. Chromatogr., A 1991, 545, 391−396. (28) Horká, M.; Růzǐ čka, F.; Holá, V.; Šlais, K. Anal. Bioanal. Chem. 2006, 385, 840−845. (29) Horká, M.; Růzǐ čka, F.; Horký, J.; Holá, V.; Šlais, K. J. Chromatogr., B 2006, 841, 152−159.

that it does not take account of the transition sample stacking. Further, the positive effect of higher focusing rate due to the higher current caused by lower electrical resistance of the tapered capillary of the same volume can be further supported by lower electroosmosis on the etched capillary and/or different composition of the catholyte and the anolyte at the optimized separation conditions of both the cylindrical and the tapered capillaries.

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CONCLUSION This work indicates the potential of supercritical water to reproducibly prepare fused-silica capillaries with tapered geometry suitable for CIEF experiments. The method described opens the way to increase the number of separable compounds without the limitation by the excessive high voltage needed. In the future, the experimental conditions including the temperature and pressure of the capillary etching as well as the influence of the surface structures and the prospects of this effect will further be studied. Despite the complicated reasoning of improved CIEF performance, further research appears promising, namely, toward obtaining the capillaries with a larger ratio of the inlet to the outlet cross-section.

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3.83 13.6

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel. +420 532 290 211. Fax: +420 541 212 113. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Ministry of the Interior of the Czech Republic (Grant VG20112015023), by the Czech Science Foundation (Grant P106/12/0522), and by the Academy of Sciences of the Czech Republic (Institutional Support RVO:68081715).

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REFERENCES

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