Direct Imaging of the Stepwise Elution Process in High-Performance

In the stepwise elution mode, a rapid change of migration velocity of the solute band was observed when a second eluent just overtook the band. As a f...
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Anal. Chem. 1996, 68, 4000-4005

Direct Imaging of the Stepwise Elution Process in High-Performance Liquid Chromatography Atsushi Tamura, Keiko Tamura, Saeid Razee, and Tsutomu Masujima*

Institute of Pharmaceutical Sciences, Hiroshima University School of Medicine, Kasumi 1-2-3, Minami-ku, Hiroshima 734, Japan

The migrating bands of solutes in a liquid chromatography glass column were directly monitored by a video imaging system. In the stepwise elution mode, a rapid change of migration velocity of the solute band was observed when a second eluent just overtook the band. As a function of migration time and migration distance, the locus of each solute band showed two straight lines with a clear inflection point. It was shown that the solvent front of the second eluent was kept sharp on passing through the column. The inflection points of all bands showed linear correlation coefficients. This line of inflection points showed a locus of the average solvent front of the second eluent, and the slope of this line showed the linear flow velocity of the eluent. A momentary compression of the bandwidth was also observed when the solvent front of the second eluent just passed through the solute band. Reversed-phase (RP) high performance liquid chromatography (HPLC) has been widely used. It is easy to select a mobile phase which typically consists of acetonitrile or methanol and aqueous buffer solution.1 Owing to the difficulty in separating a mixture in a single operation that contains a wide range of polar solutes, the stepwise elution mode has been widely adopted.2-6 For setting a stepwise chromatographic condition, however, one relies heavily on the operator’s experience in choosing eluent and solvent changing time. Many theoretical approaches have been developed to clarify the band migration and the separation process.7-10 Gelderloos et al. showed the necessity and usefulness of whole-column detection with computer simulations.11 In an experimental ap(1) Krstulovic, A. M.; Brown, P. R. Reversed-Phase High-Performance Liquid Chromatography; John Wiley & Sons Inc.: New York, 1982. (2) Jandera, P.; Churacek J. J. Chromatogr. 1974, 91, 207-221; 1974, 91, 223235; 1979, 170, 1-10. (3) Schoenmakers, P. J.; Billiet, H. A. H.; Tijssen, R.; de Galan, L. J. Chromatogr. 1978, 149, 519-537. (4) Dolan, J. W.; Lommen, D. C.; Snyder, L. R. J. Chromatogr. 1989, 485, 91112. (5) Boro´wsko, M.; Jaroniec, M.; Narkiewicz, J.; Patrykiejew; A.; Rudzinski, W. J. Chromatogr. 1978, 153, 309-319. (6) Boro´wko, M.; Jaroniec, M.; Narkiewicz, J.; Patrykiejew, A. J. Chromatogr. 1978, 153, 321-328. (7) Giddings, J. C. Dynamics of Chromatography; Marcel Dekker: New York, 1965. (8) Kirkland, J. J. Modern Practice of Liquid Chromatography; J. Wiley & Sons Inc.: New York, 1971. (9) Snyder, L. R.; Kirkland, J. J. Introduction to Modern Liquid Chromatography; J. Wiley & Sons Inc.: New York, 1974. (10) Snyder, L. R. In Modern Practice of Liquid Chromatography; Kirkland, J. J., Ed.; Wiley: New York, 1971. (11) Gelderloos, D. G.; Rowlen, K. L.; Birks, J. W.; Avery, J. P.; Enke, C. G. Anal. Chem. 1986, 58, 900-903.

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proach, Bussolo and Gant discussed the visualization of protein retention and migration in RP liquid chromatography.12 Ilg et al. tried to observe the band profiles by magnetic resonance imaging and discussed the wall effect of a preparative column.13 To get a clearer visualization of the problem, we developed a video image analyzing system for liquid chromatography.14 Video detection methods have been previously used in separation sciences as an alternative to two-dimensional densitometers,15-17 as a detector for DNA molecular movement in pulse field gel electrophoresis,18 and in capillary electrophoresis.19-23 In the present work, we investigated the dynamic aspects of band migrating processes in HPLC and estimated the movement of the solvent front in a stepwise elution mode using video imaging techniques. EXPERIMENTAL SECTION Chemicals. [5-(Dimethylamino)-1-naphthalenesulfonyl]alanine (dansylalanine, DNS-Ala), dansylglycine (DNS-Gly), dansylleucine (DNS-Leu), and dansylvaline (DNS-Val) were purchased from Seikagaku Kogyo Co. (Tokyo, Japan). Acetonitrile was HPLC grade. Distilled water was purified by a Milli-Q Labo (Millipore Co., Bedford, MA) system. All other chemicals were of reagent grade and used without any further purification. Acetonitrile and 0.1 M acetic acid were used for the mobile phase. Apparatus. A schematic diagram of the video imaging system is shown in Figure 1. The HPLC apparatus consisted of two HPLC pumps (880-PU, JASCO, Hachioji, Japan) with a system controller (802-SC, JASCO), an injection valve (7125, Rheodyne, Cotati, CA), and a glass column (115 mm × 5 mm i.d.; Pharmacia Biotech, Uppsala, Sweden) which was packed with Chemcosorb ODS-H (9 µm, Chemco, Osaka, Japan) resin in our laboratory. A video camera with three charge-coupled devices (DXC-930, Sony, Tokyo, Japan) was used as a detector, and a triacetyl cellulose long-pass (12) Bussolo, J. M. D.; Gant, J. R. J. Chromatogr. 1985, 327, 67-76. (13) Ilg, M.; Maier-Rosenkranz, J.; Mu ¨ ller, W.; Bayer, E. J. Chromatogr. 1990, 517, 263-268. (14) Tamura, A.; Tamura, K.; Wada, K.; Masujima, T. Chem. Pharm. Bull. 1994, 42, 704-706. (15) Toda, T.; Fujita, T.; Ohashi, M. Electrophoresis 1984, 5, 42-47. (16) Sutherland, J. C.; Lin, B.; Monteleone, D. C.; Mugavero, J.; Sutherland, B. M.; Trunk, J. Anal. Biochem. 1987, 163, 446-457. (17) Koutny, L. B.; Yeung, E. S. Anal. Chem. 1993, 65, 183-187. (18) Schwartz, D. C.; Koval, M. Nature 1989, 338, 520-522. (19) Tsuda, T.; Ikedo, M.; Jones, G.; Dadoo, R.; Zare, R. N. J. Chromatogr. 1993, 632, 201-207. (20) Taylor, J. A.; Yeung, E. S. Anal. Chem. 1993, 65, 2928-2932. (21) Kuhr, W. G.; Licklider, L.; Amankwa, L. Anal. Chem. 1993, 65, 277-282. (22) Fishman, H. A.; Amudl, N. M.; Lee, T. T.; Schiller, R. H.; Zare, R. N. Anal. Chem. 1994, 66, 2318-2329. (23) Razee, S.; Tamura, A.; Khademizadeh, M.; Masujima, T. Chem. Lett. 1996, 1996, 93-94. S0003-2700(96)00274-0 CCC: $12.00

© 1996 American Chemical Society

Figure 1. Schematic diagram of chromatography video imaging system. (A) Pump, (B) injector, (C) glass column, (D) CCD video camera, (E) video printer, (F) flame memory unit, (G) videotape recorder, (H) image analyzer, (I) CRT display, (J) personal computer.

filter (cutoff wavelength, 450 nm; SC-42, Fuji Photo Film Co., Tokyo, Japan) was placed before the camera in order to cut off the excitation light. The movement of dansyl amino acid bands along a column length was monitored by their fluorescence emission (365 nm excitation, 520 nm emission) under a longwave UV lamp (with broad emission band at 315-400 nm, peaks at 365 nm) (UVP Inc., Upland, CA), and the video image was recorded using a standard video recorder (AG-7355, Panasonic, Osaka, Japan) or a time-lapse video recorder (AG-6720A, Panasonic). The image data were analyzed by an 8-bit image processor (PIP-4000, ADS Co., Osaka, Japan), and the video image was printed out by a video printer (CP-11, Mitsubishi Co., Tokyo, Japan). Data Analyzing Procedures. Figure 2 shows densitograms (with peaks) of separating bands along the center line of the HPLC column (dotted straight lines) which were obtained by 8-bit digitizing of the brightness (fluorescence intensity) of each separating band of dansyl amino acid as seen along the axis of the HPLC column. Migration distances and bandwidths of solutes were obtained by analyzing these densitograms. RESULTS AND DISCUSSION Migration Profiles. Figure 2 shows typical consecutive video densitograms in the stepwise elution mode. By accumulating these densitograms, a contour map of fluorescence intensity with axes of migration time and distance along the column was obtained, which is shown in Figure 3A. Four solute bands are clearly shown, and each straight line experiences a distinct inflection point. A round shape at each bending point was not found, even for slowly migrating solutes like DNS-Leu and DNSVal. This indicates that the migration velocity changed not gradually but sharply for all solute bands through the whole column length. In isocratic elution, the migration distance had a good linear relationship with the migration time, and the slope is the apparent migration velocity.14 In the case of stepwise elution, the migration velocities of solute bands were changed to another constant velocity after the inflection points. Table 1 shows the migration velocities calculated from in Figure 3 and the isocratic elution by the second eluent. The migration velocities after the inflection point correspond nicely with isocratic elution. This finding shows that the mobile phase was rapidly replaced from the first eluent to the second one. Figure 3 shows that the inflection points follow a straight line (r > 0.999), as shown by the solid line. This line shows the movement of the solvent front of the second eluent. The intercept of this line to the time axis is 538 s, which is close to the experimental time for solvent change from the first eluent to the

Figure 2. Time course of typical densitograms with stepwise elution mode. Each densitogram shows fluorescence intensity at (a) 3, (b) 5, (c) 7, (d) 9, (e) 11, and (f) 13 min. Mobile phase was changed from acetonitrile-0.1 M acetic acid (20:80) to acetonitrile-0.1 M acetic acid (40:60) at 9 min after injection. Samples are DNS-Leu, DNSVal, DNS-Ala, and DNS-Gly.

second one (540 s). These results showed that the solvent front of the second eluent kept fairly sharp on moving through the column, and the change in migration velocity of a solute band is instantaneous as soon as the second eluent overtakes this band. Chromatographic Parameters. The velocity of the mobile phase was preferably expressed as the linear flow velocity u (cm/ min) rather than the volume flow rate F (mL/min), because u corresponds to the average velocity of the solvent molecules.24 Linear flow velocity is a useful parameter. However, it has not been used since the actual flow velocity of the mobile phase in liquid chromatography was not directly observed. In the present method, evaluation of the linear flow velocity is feasible by analyzing the solvent changing process. Using the data presented in Figure 3B, one can directly calculate the linear flow velocity of the mobile phase by measuring the slope of the solid line. These were found to be 6.51 and 8.05 cm/min at flow rates of 0.8 and 1.0 mL/min, respectively. (24) Engelhardt, H. High Performance Liquid Chromatography: Chemical Laboratory Practice; Springer-Verlag: Berlin-Heidelberg-New York, 1979.

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V0 ) (L/u)F

(1)

phase was calculated by the above-mentioned method. By this approach, the V0 was calculated to be 1.77 and 1.79 mL at flow rates of 0.8 and 1.0 mL/min, respectively. This present method of video imaging LC system might be useful for investigating other chromatographic parameters; for instance, we could look into column free cross section, void volume of the column, and solvent flow in the column. The migration velocity changed abruptly in the stepwise elution mode, and an apparent migration velocity could be easily estimated by video image analysis. Then, the linear flow velocity of the mobile phase could also be evaluated by this method. Thus, the migration profiles of the solute band could be simulated. Furthermore, the elution (retention) time (tR) could be estimated by eq 2 (see Appendix below). Accordingly, the retention time of

tR )

Figure 3. Relationship between migration time and migration distance. (A) Plots of fluorescence intensity as a function of migration time and migration distance. (B) Relationship between migration time and migration distance. Dotted lines were obtained by linear leastsquares method, and a bold line shows the correlation line of inflection points. Samples are DNS-Leu (b), DNS-Val (O), DNS-Ala (9), and DNS-Gly (0). Other chromatographic conditions are the same as described in the legend of Figure 2. Table 1. Apparent Migration Velocities Obtained by Stepwise Elution and Isocratic Elution

DNS-Leu DNS-Val DNS-Ala DNS-Gly

first eluent by stepwise,a cm/min

second eluent by stepwise,b cm/min

isocratic,b cm/min

0.07 0.18 0.60 0.87

1.30 1.91 3.12 3.59

1.28 1.89 3.44 4.06

a Mobile phase was acetonitrile-0.1 M acetic acid (20:80). b Mobile phase was acetonitrile-0.1 M acetic acid (40:60).

Determination of the column void volume (V0) has been the subject of a number of studies.25-28 This is an important parameter which should be calculated for kinetics and dynamics. Many socalled “nonretained” solutes (sodium nitrate,26 acetone,29 uracil,30 etc.) were used to estimate the V0; however, these materials have a weak interaction with stationary and/or mobile phases, and the real value for V0 could not be obtained. On the other hand, we propose a new method to calculate V0 by simply using eq 1, where L is the column length (cm). Linear flow velocity of the mobile (25) Wainwright, M. S.; Haken, J. K. J. Chromatogr. 1980, 184, 1-20. (26) Krstulovic, A. M.; Colin, H.; Guiochon, G. Anal. Chem. 1982, 54, 24382443. (27) Alhedai, A.; Martire, D. E.; Scott, R. P. W. Analyst 1989, 114, 869-875. (28) Wells, M. J. M.; Clark, C. R. Anal. Chem. 1981, 53, 1341-1345. (29) Johnson, H. J., Jr.; Cernosek, S. F., Jr.; Gutierrez-Cernosek, R. M. J. Chromatogr. 1979, 177, 297-311. (30) Karger, B. L.; Gant, J. R.; Hartkopf, A.; Weiner, P. H. J. Chromatogr. 1976, 128, 65-78.

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u(K2 - K1) L + t - V0 K2 uK2 - K1 c

(2)

some solute or the elution time from the column could be easily estimated using the data of column length, L, apparent migration velocities of the first solute, K1, and second eluents, K2, as well as linear flow velocity of the mobile phase, u, and the time of solvent change, tc. Change of Bandwidth. Bandwidths of migrating solutes were estimated from the densitograms of Figure 2. In Figure 4, the bandwidths are also affected by the solvent change, and two regions with different slopes are shown. In isocratic elution, the square of the bandwidth is proportional to the migration distance in the column.14 According to the data shown in Figure 5, a nearly linear dependence of the square of the bandwidth on the migration distance also applies in the stepwise elution mode. These results show that the band broadening depends on migration distance rather than migration time. Furthermore, the extent of band broadening of DNS-Leu does not depend on the acetonitrile content of the mobile phase, as shown in Figure 6. This also supports the conclusion that the bandwidth of the solute in the column is independent of the migration velocity. This means that the bandwidth of a fast-eluting solute does not stay sharp during migration, and band broadening depends mainly on the migration distance in these typical RP partition chromatography experiments. The main sources of band broadening have been discussed as multiple-path diffusion, molecular diffusion, and resistance to mass transfer.7,31 When the flow of the mobile phase, which was 40% acetonitrile in 0.1 M acetic acid, was stopped in the course of migration, the bandwidths of DNS-Gly did not change for a period of 30 min. This showed that molecular diffusion has a minor effect on band broadening under these conditions. In general, molecular diffusion may be ignored since the linear flow velocity is quite fast in HPLC.1 Giddings showed that multiple-path diffusion and resistance to mass transfer have not independent but rather a combined effect.7 Our results also showed that the bandwidth in the column depended on migration distances, and the band broadening was not influenced by the linear flow velocity. (31) Nambara, T.; Ikekawa, N. Modern High-Performance Liquid Chromatography; Hirokawa: Tokyo, 1988.

Figure 4. Relationship between migration time and bandwidth. Samples are DNS-Leu (b), DNS-Val (O), DNS-Ala (9), and DNSGly (0). Other chromatographic conditions are the same as described in the legend of Figure 2.

Figure 5. Relationship between migration distance and the square of the bandwidth. Samples are DNS-Leu (b), DNS-Val (O), DNSAla (9), and DNS-Gly (0). Other chromatographic conditions are the same as described in the legend of Figure 2.

Figure 6. Effect of acetonitrile content in the mobile phase on bandwidth. Contents of acetonitrile in the mobile phase are 20% (b), 30% (O), 40% (9), 50% (0), and 60% (2). Sample is DNS-Leu.

We know the highly retained solutes show broad bands at longer retention time zones in conventional chromatograms. Since all bands show similar bandwidths at the bottom of the column, it was shown that the broad and low band shapes in the chromatograms are due to the increased volume of eluent, which is necessary to elute the highly retained solute from the column. Band Shape while the Front of the Second Solvent Just Passes Through. Densitograms were analyzed in shorter intervals in order to observe the change of band shape of the migrating solute while the solvent front of the second eluent was just passing through the band. However, no band contraction, which is caused by differences within migration velocities of individual solute molecules in a single band, was observed. This means that the second solvent front was not strictly flat in detail,

Figure 7. Densitograms after solvent change. Each densitogram shows fluorescence intensity at (a) 0, (b) 20, (c) 30, (d) 40, (e) 50, (f) 60, (g) 70, (h) 80, and (i) 90 s after solvent change. Arrows in each densitogram show estimated solvent front of the second eluent.

to show differences in migration velocities of solute molecules dispersed in a single band. Therefore, in order to show the change of band shape, a trial to broaden the initial bandwidth was performed by injecting ∼350 µL of a second eluent just after loading of sample onto the column. The successive densitograms obtained after this treatment are shown in Figure 7. We observed a band contraction in the case of DNS-Ala from positions d to f. However, in position i, the bandwidth broadens again. The Analytical Chemistry, Vol. 68, No. 22, November 15, 1996

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Figure 8. Changes of bandwidth upon solvent passing. Samples are DNS-Leu (b), DNS-Val (O), DNS-Ala (9), and DNS-Gly (0).

estimated solvent fronts, which were previously discussed, are shown as arrows in Figure 7. This figure shows the inflection point through the peak of DNS-Val from positions b to c, DNSAla from positions e to f, and DNS-Gly from positions g to h. As shown in Figure 8, bandwidths of DNS-Val, DNS-Ala, and DNSGly became narrow, due to the passage of the solvent front of the second eluent. The momentary contraction of bandwidth was observed while the solvent front was just passing through the band. However, after complete passage of the band, again the band broadening was only a function of the migration distance. Reversed Stepwise Elution Mode. In RP-LC, when we practice a separation with a stepwise elution mode, the content of organic solvent in the mobile phase is usually changed from lower concentrations to higher ones in order for a loosely retained solute to be eluted faster and a strongly retained solute to be eluted later. Although a reversed pattern in which the content of organic solvent in the mobile phase was changed from a higher concentration to a lower one is not common, such a pattern is informative for analyzing the stepwise elution mechanism.32 Three straight lines with sharp inflection points were observed in Figure 9, when the content of acetonitrile in the mobile phase was dropped from 40% to 20% and again raised to 40%. It was found that the change of migration velocity was very rapid, even though the organic content of the mobile phase was decreased. Two parallel straight lines were obtained connecting inflection points. However, the intercept of the line obtained by extrapolating the first inflection points to the time axis (45 s) showed a delay from the actual changing time (20 s). A similar delay was also observed when the mobile phase was changed from acetonitrile-0.1 M acetic acid (40:60) to acetonitrile-0.1 M acetic acid (20:80). It seemed that these delays were not caused by differences in the pumping flow rate, because the two fitting lines were almost parallel. Therefore, this delay occurred when organic solvent in the mobile phase was decreased in a stepwise manner. We presume that the lipophilic layer formed over the ODS packing was not easily exchanged when the concentration of the acetonitrile in the mobile phase was decreased, whereas in the ordinary case, the layer which has more lipophilic character was smoothly exchanged. (32) Tamura, A.; Tamura, K.; Enoki, C.; Masujima, T. Chem. Pharm. Bull. 1994, 42, 2379-2381.

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Figure 9. Relationship between migration time and migration distance at stepwise elution. Chromatographic conditions: mobile phase was changed from acetonitrile-0.1 M acetic acid (40:60) to acetonitrile-0.1 M acetic acid (20:80) at 20 s, and again to acetonitrile-0.1 M acetic acid (40:60) at 200 s. Sample are DNS-Leu (b), DNS-Val (O), DNS-Ala (9), and DNS-Gly (0). Bold lines show the correlation lines of inflection points.

CONCLUSION An imaging system based on a charge-coupled device video camera was used to form direct images of migrating solute bands in a liquid chromatography column. The present method showed the ability of video image analysis to seize many phenomena taking place in a column by pursuit of change of movement which had not been previously observed. This video imaging LC system might be useful for investigating some other chromatographic parameters; for instance, we could now look into column free cross section, void volume of the column, and solvent flow in the column. ACKNOWLEDGMENT This study was partly supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan. APPENDIX: ESTIMATION OF ELUTION (RETENTION) TIMES OF SOLUTES Under ideal conditions, the relationship between migration time (t) and migration distance (d) can be expressed as

d ) K1t (0 < t < ti)

(3)

where K1 is the migration velocity in the first eluent and ti is the inflection time. After the inflection time, the migration distance could be correlated to the migration time by

d ) K1ti + K2(t - ti)

(t > ti)

(4)

where K2 is the solute migration velocity of the second eluent. The migration distance of the second eluent front (dsf) could be calculated as follows:

dsf ) u(t - tc)

(t > tc)

(5)

where tc is the time of solvent change. The migration distance, di, at inflection time, ti, can be obtained by substituting ti, di in eqs 3 and 5, respectively:

di ) K1ti

(6)

di ) u(ti - tc)

(7)

Upon substituting eq 11 into eq 2 or 10, we have

tR )

Solving eqs 6 and 7 for inflection time, we obtain

ti ) utc/(u - K1)

(8) Further rearrangement could be done by substituting u in eq 1 into eq 12:

Substituting eq 8 in eq 4 and rearranging it for d,

d ) K2t -

u(K2 - K1) t u - K1 c

(9)

Using this equation, the elution time (tR) could be obtained by substituting L for d, and tR for t:

tR )

u(K2 - K1) L + t - V0 K2 uK2 - K1 c

(10)

This is eq 2. The migration velocity, K, relates to the conventional capacity factor, k′, as follows:14

k′ ) (u/K) - 1

u(k2′ - k1′) (k2′ + 1)L + t + V0 (12) u u(k1′ + 1) - (k2′ + 1) c

(11)

tR )

LF(k2′ - k1′) (k2′ + 1)V0 + t + V0 (13) F LF(k1′ + 1) - (k2′ + 1)V0 c

This shows that tR in the stepwise elution mode could be calculated using ordinary chromatographic parameters with one uncertain parameter, V0. Equations 12 and 13 also show that V0 and u can be estimated from the values of tR, k1′, and k2′ which were obtained in a conventional HPLC condition. Received for review March 20, 1996. Accepted August 15, 1996.X AC960274Y X

Abstract published in Advance ACS Abstracts, October 1, 1996.

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