High-Performance Membrane Chromatography of Small Molecules

However, no study about the separation of small molecules has been performed until now. In this work, we investigated the possibility of gradient and ...
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Anal. Chem. 1999, 71, 2986-2991

High-Performance Membrane Chromatography of Small Molecules Alesˇ Podgornik, Milosˇ Barut, Janez Jancˇar, and Alesˇ S ˇ trancar

BIA Separations d.o.o., Teslova 30, SI-1000 Ljubljana, Slovenia Tatiana Tennikova*

Institute of Macromolecular Compounds, Russian Academy of Sciences, 199 004 St. Petersburg, Russia

High-performance membrane chromatography (HPMC) proved to be a very efficient method for fast protein separations. Recently, it was shown to be applicable also for the isocratic chromatography of plasmid DNA conformations. However, no study about the separation of small molecules has been performed until now. In this work, we investigated the possibility of gradient and isocratic HPMC of small molecules with Convective Interaction Media disks of different chemistries and tried to explain the mechanism that enables their separation. We demonstrated that it is possible to achieve efficient separations of oligonucleotides and peptides in the ion-exchange mode as well as the separation of small hydrophobic molecules in the reversed-phase mode. It was shown that similar peak resolution can be provided in both gradient and isocratic modes. High-performance membrane chromatography (HPMC) is one of the significant chromatographic inventions of the past decade.1,2 Here, the modern highly efficient process of chromatographic separation was proposed to realize with the use of thin layers of finely organized and well-controlled macroporous polymeric stationary phase designed in the mode of rigid monolithic disks of 2-3 mm thickness.3,4 The term HPMC was suggested to take into account the flat geometry of the stationary phase. An analogy of the proposed method, membrane adsorption (MA), was developed at about the same time and successfully applied for the separations of biological molecules.5-10 (1) Tennikova, T. B.; Belenkii, B. G.; Svec, F. J. Liq. Chromatogr. 1990, 13, 63. (2) Tennikova, T. B.; Bleha, M.; Svec, F.; Almazova, T. V.; Belenkii, B. G. J. Chromatogr. 1991, 555, 90. (3) Svec, F.; Tennikova, T. B. J. Biocompat. Polym. 1991, 6, 393. (4) Svec, F.; Jelinkova, M.; Votavova, E. Angew. Makromol. Chem. 1991, 188, 167. (5) Unarska, M.; Davies, P. A.; Esnouf, M. P.; Bellhouse, B. J. J. Chromatogr. 1991, 519, 53. (6) Upshall, A.; Kumar, A. A.; Bailey, M. C.; Parker, M. D.; Favreau, M. A.; Lewson, K. P.; Joseph, M. L.; Maraganore, J. M.; McKnight, G. L. Bio/ Technology 1987, 5, 1301. (7) Briefs, K.-G.; Kula, M.-R. Chem. Eng. Sci. 1992, 47, 141. (8) Langlotz, P.; Kroner, K. H. J. Chromatogr. 1992, 591, 107. (9) Klein, E. Affinity Membranes; Wiley: New York, 1991. (10) Roper, D. K.; Lightfoot, E. N. J. Chromatogr., A 1995, 702, 3. (11) Snyder, L. R.; Stadalius, M. A. In High Performance Liquid Chromatography: Advances and Perspectives; Horvath, Cz., Ed.; Academic Press: New York, 1980; Vol. 1, p 207.

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The model of gradient HPLC separations of proteins was applied to develop the theoretical and practical views of HPMC.11-17 The physical and chemical properties of protein macromolecules, which explain their behavior in dynamic separation, gave the possibility of transferring rather easily the separation process onto thin throughput layers with an adsorption functionality identical to that of sorbents used in conventional HPLC.18-21 The experimental comparison of protein behavior under HPLC and HPMC conditions showed that the general principles of gradient chromatography can be applied in both cases.22-25 The advantages of the later are separation of proteins (including affinity HPMC) in the seconds time range, low-pressure drop, and high-flow unaffected dynamic binding capacity.26-31 Experience in this area (12) Snyder, L. R.; Stadalius, M. A.; Quarry, M. A. Anal. Chem. 1983, 55, 1412A. (13) Kopaciewicz, W.; Rounds, M. A.; Regnier, F. E. J. Chromatogr. 1983, 266, 3. (14) Geng, X.; Regnier, F. E. J. Chromatogr. 1984, 296, 15. (15) Yamamoto, S.; Naganishi, K.; Matsuno, R.; Ion-Exchange Chromatography of Proteins; Marcel Dekker: New York, Basel, 1988, p 94. (16) Regnier, F. E.; Chicz, R. M. In HPLC of Biological Molecules, Methods and Applications; Gooding, K. M., Regnier, F. E., Eds.; Marcel Dekker: New York, 1990; p 89. (17) Snyder, L. R. In HPLC of Biological Molecules, Methods and Applications; Gooding, K. M., Regnier, F. E., Eds.; Marcel Dekker: New York, 1990; p 231. (18) Abou-Rebyeh, H.; Korber, F.; Schubert-Rehberg, K.; Reusch, J.; Josic´, Dj. J. Chromatogr. 1991, 566, 341. (19) Josic´, Dj.; Reusch, J.; Loster, K.; Baum, O.; Reutter, W. J. Chromatogr. 1992, 590, 59. (20) Luks´a, J.; Menart, V.; Milic´ic´, S.; Kus, B.; Gaberc-Porekar, V.; Josic´, Dj. J. Chromatogr., A 1994, 661, 161. (21) Josic´, Dj.; Lim, Y.-P.; Sˇ trancar, A.; Reutter, W. J. Chromatogr., B 1994, 662, 217. (22) Belenkii, B. G.; Podkladenko, A. M.; Kurenbin, O. I.; Maltsev, V. G.; Nasledov, D. G.; Trushin, S. A. J. Chromatogr. 1993, 646, 1. (23) Tennikova, T. B.; Svec, F. J. Chromatogr. 1993, 646, 279. (24) Dubinina, N. I.; Kurenbin, O. I.; Tennikova, T. B. J. Chromatogr., A 1996, 753, 217. (25) Tennikov, M. B.; Gazdina, N. V.; Tennikova, T. B.; Svec, F. J. Chromatogr., A 1998, 798, 55. (26) Sˇtrancar, A.; Koselj, P.; Schwinn, H.; Josic´, Dj. Anal. Chem. 1996, 68, 3483. (27) Josic´, Dj.; Schulz, P.; Biesert, L.; Hoffer, L.; Schwinn, H.; Kordisˇ-Krapezˇ, M.; Sˇ trancar, A. J. Chromatogr., B 1997, 768, 24. (28) Kasper, C.; Meringova, L.; Freitag, R.; Tennikova, T. J. Chromatogr., A 1998, 798, 65. (29) Josic´, Dj.; Schwinn, H.; Sˇ trancar, A.; Podgornik, A.; Barut, M.; Lim, Y.-P.; Vodopivec, M. J. Chromatogr., A 1998, 803, 61. (30) Sˇ trancar, A.; Barut, M.; Podgornik, A.; Koselj, P.; Josic´, Dj.; Buchacher, A. LC-GC Int. 1998, 11, 660. (31) Josic´, Dj.; Sˇ trancar, A. Ind. Eng. Chem. Res. 1999, 38, 333. 10.1021/ac981350v CCC: $18.00

© 1999 American Chemical Society Published on Web 06/22/1999

recently allowed for development of an original theoretical model of protein separation by the HPMC method called the “one-step desorption process”.32 Despite important results obtained in protein separations, other possibilities for its application were little studied until the present time. The first paper dealing with the analysis of molecules quite different from proteins, i.e., DNA plasmids, opened some unexpected aspects of this method.33 An especially interesting result emerging from that study was the possibility of using fast isocratic elution. These results generated additional research where the oligonucleotides were presented as the object of an isocratic HPMC separation study.34 The goal of the present paper was to investigate the separation of substances of low molecular masses, in both gradient and isocratic modes, with the purpose of explaining the mechanism that enables the separation of this type of molecules on thin monoliths. Anion and cation exchange as well as reversed-phase modes of HPMC were applied. The quantitative comparison of isocratic and shallow gradient separations was carried out in order to explain the results from the point of view of existing chromatographic models and by taking into account the particularities of the behavior of molecules to be separated in monolithic macroporous media. THEORETICAL SECTION One can suggest a priori several differences in protein and small-molecule behavior. These differences, which are common to HPLC and HPMC methods, will undoubtedly influence the process of chromatographic separation.32 First, the coefficients of free diffusion of proteins and low molecular mass substances differ by 3-4 orders of magnitude, providing huge differences in the diffusional mobility of the molecules to be separated. Second, the heterogeneity of surface interaction energy is much lower in the case of low molecular mass homologues than that observed in the case of different proteins. HPMC also demonstrates significant differences in comparison with conventional HPLC. The main difference is that this process is not limited by pore diffusion described thoroughly in the case of HPLC columns filled with porous particles. It is sensible to discuss only the diffusive movement of a molecule from the mobile phase flowing through the pore to the pore walls. In fact, the absence of pore diffusion gives the possibility of performing the process of separation within very short times. This characteristic should be beneficial also in the case of HPMC of small molecules. According to the classic dynamics of chromatography,35 the theoretical number of possible contacts of molecules with the pore wall can be calculated using the well-known equations

d2 ) 2DtD

(1)

where d is the distance that a molecule in a liquid travels to reach the pore wall, D is the free diffusion coefficient of a substance to be separated, and tD is the time necessary to travel the distance (32) Tennikova, T. B.; Freitag, R. In Analytical and Preparative Separation Methods of Biomolecules; Aboul-Ehnen, H. Y., Ed.; Marcel Dekker: New York, Basel, in press. (33) Giovannini, R.; Freitag, R.; Tennikova, T. Anal. Chem. 1998, 70, 3348. (34) Podgornik, A.; Barut, M.; Jancˇar, J.; Sˇ trancar, A. J. Chromatogr., in press. (35) Giddings, J. C. Unified Separation Science; Wiley: New York, 1991.

from some point in the liquid phase to a pore wall by free diffusion.

tres ) L/U

(2)

where tres is the residence time of molecules within the separation layer, L is the length of the separation layer, and U is the linear flow velocity. Combining these two equations, it is possible to express the theoretical number of molecule contacts with the pore surface within a defined time range:

N ) tres/tD ) 2DL/Ud2

(3)

Recently it was suggested that the same approximation can be used to calculate the pore size optimum of the membrane adsorbers.36 The calculated value of N (eq 3) for a low molecular mass substance with a diffusion coefficient of 10-3 cm2/s at its pass through a layer of 3 mm thickness with average pore size 0.5 µm at 8 mL/min rate is equal to 105. This value greatly exceeds the theoretical value of N found for the globular proteins, due to their much lower diffusivity. It is thus possible to conclude that, because of the high number of interactions, even in the case of isocratic adsorptive separation of small molecules the use of thin layers of stationary phase seems possible. Furthermore, since the binding characteristics among small molecules are more similar than in the case of proteins, shallow gradients have to be applied to obtain improved separation. This is especially important when the peak broadening of later eluting components in isocratic separation is too pronounced. Recently it was shown that to obtain maximum resolution for protein separation, the gradient can be defined in a way that the working part X0 of the stationary phase is similar to its physical length.24 X0 was defined as the distance in which the quasi steady state is attained. It can be calculated according

X0 ) λUCc/(ZB)

(4)

where U represents linear velocity of mobile phase through the stationary phase (cm/s), Cc is the critical concentration of displacer (M) at which the solute molecule is desorbed, Z is the effective charge on the solute ion divided by the charge on the mobile-phase ion, B is the steepness of linear gradient, and λ is an auxiliary parameter estimated to be 0.5. Although this approach was derived for proteins, it might also be applicable for smaller molecules. EXPERIMENTAL SECTION Mobile Phase. High-purity water and chemicals of analytical grade quality were used throughout the experimental work. Tris(hydroxymethyl)methylamine (Tris) was purchased from Aldrich (Steinheim, Germany), sodium chloride from Fluka (Buchs, Switzerland), sodium phosphate from Kemika (Zagreb, Croatia), and HPLC grade acetonitrile from Rathburn (Walkerburn, Scotland). Samples. Oligonucleotides. The oligonucleotides, synthesized and purified at the National Institute of Chemistry, Ljubljana, (36) Safert, F. T.; Etzel, M. R. J. Chromatogr., A 1997, 764, 3.

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Slovenia, were of the following lengths and structure: oligodeoxynucleotide 8 (oligo 8), C CAT GTC T3′; oligodeoxynucleotide 10 (oligo 10), GTC CAT GTC T3′; oligodeoxynucleotide 12 (oligo 12), AG GTC CAT GTC T3′; oligodeoxynucleotide 13 (oligo 13), GAG GTC CAT GTC T3′; oligodeoxynucleotide 14 (oligo 14), C GAG GTC CAT GTC T3′; oligodeoxynucleotide 15 (oligo 15), CC GAG GTC CAT GTC T3′; oligodeoxynucleotide 16 (oligo 16), GCC GAG GTC CAT GTC T3′. Peptides. Three peptides, a kind gift from Institute of Applied Microbiology (IAM), Vienna, Austria, with the following amino acid sequences were used: Pep-9, EYIKWEEFK; Pep-11, KSGDWKSKCFY; Pep-15, QISTKSGDWKSKCFY. Aromatic Compounds. Benzene and toluene were purchased from Merck (Darmstadt, Germany). Homologues of paraben (4hydroxybenzoate) were from U. S. P. C. Inc.: methylparabene, ethylparabene, propylparabene, and butylparabene. Steroids. The following steroids from Sigma (St. Louis, MO) were used: progesterone (4-pregnene-3,20-dione), 11-ketoprogesterone (4-pregnene-3,11,20-trione), and 11(R)-hydroxyprogesterone (4-pregnene-11(R)-ol-3,20-dione). Stationary Phase. Monolithic Convective Interaction Media (CIM) disk stationary phases (BIA Separations d.o.o., Ljubljana, Slovenia) used for gradient and isocratic HPMC had a diameter of 12 mm and a thickness of 3 mm. The 0.3-mm disks were additionally prepared by the same technology used in preparing standard disks. The base material was a macroporous glycidyl methacrylate-co-ethylene dimethacrylate (GMA-EDMA) polymer matrix. The anion-exchange disks bearing diethylaminoethyl groups (CIM DEAE-anion-exchange type), strong cation-exchanger disks with introduced sulfo charged groups (CIM SO3cation-exchange type), and strong hydrophobic disks with inserted C18 groups (CIM C18-reversed phase type) were used in chromatographic experiments. Instrumentation. A gradient HPLC system built with two pumps 64, an injection valve with a 20-µL SS sample loop, a variable-wavelength monitor with a 10-mm optical path set to 215, 254, or 260 nm (depending on samples) and with a 10-µL flow cell, connected by means of 0.25-mm-i.d. PEEK capillary tubes, and HPLC hardware/software (data acquisition and control station), all from Knauer (Berlin, Germany), was used in all fast analytical separations. A Knauer mixing chamber with its relatively large dead volume was replaced by the PEEK mixing tee with an extralow dead volume (Jour Research, Uppsala, Sweden). RESULTS AND DISCUSSION Anion-Exchange HPMC Separation of Oligonucleotides. The applied set of oligonucleotides differ by the number of nucleotide bases. Since the molecules to be studied are strongly charged poly(anions), the anion-exchange mode of HPMC was used for these experiments. The same as at the HPMC separation of plasmid DNA molecules,33 the high effective charge of the oligonucleotides molecules (oligo n) was taken into account to form the gradient conditions. Figure 1 demonstrates gradient HPMC separations of four oligonucleotides (oligo 8, 10, 12, and 14) on a CIM DEAE disk. Taking into account the particularities of adsorption of small molecules (the small differences in the interaction energy of homologues), very shallow gradients of salt concentration were used to provide a significant difference in retention of each 2988 Analytical Chemistry, Vol. 71, No. 15, August 1, 1999

Figure 1. Effect of the working part length X0 on the resolution of the gradient separation of four oligonucleotides. Conditions: stationary phase, CIM DEAE disk; mobile phase, (buffer A) 20 mM Tris-HCl, pH 8.5 (buffer B) 1 M sodium chloride in 20 mM Tris-HCl, pH 8.5; gradient, different X0; flow rate, 8 mL/min; detection, UV at 260 nm; sample (1) oligo 8, (2) oligo 10, (3) oligo 12, and (4) oligo 14; sample concentration, 50 µg/mL of each; injection volume, 20 µL.

Figure 2. Comparison of gradient and isocratic separations of oligonucleotides. Conditions: stationary phase, CIM DEAE disk; mobile phase, (buffer A) 20 mM Tris-HCl, pH 8.5, (buffer B) 1 M sodium chloride in 20 mM Tris-HCl, pH 8.5; flow rate, 5 mL/min; detection, UV at 260 nm; sample (1) oligo 8, (2) oligo 10, (3) oligo 12, and (4) oligo 14; sample concentration, 50 µg/mL of each; injection volume, 20 µL; (a) gradient mode, gradient X0 ) 3 mm; (b) isocratic mode, mobile phase a mixture of 43% buffer A and 57% buffer B.

component. The resulting separations at different values of gradient parameters, defined as working part X0 of the length of the separation layer,22,24,25 are presented in this figure. Although this term was proposed for the gradient chromatography of proteins, this approach seem to be valid, also for the comparable experiments in gradient HPMC of small molecules. As in the case of proteins,24 the resolution improves when X0 approaches the physical length of the monolith. Figure 2 demonstrates the comparable separations of the same oligonucleotide mixture by gradient (a) and isocratic (b) modes. The separations were carried out at the same flow rate, 5 mL/ min. The composition of eluent for isocratic chromatography approximately corresponded to the salt concentration providing

Figure 3. Isocratic oligonucleotide separations on a 0.3-mm disk at two different flow rates. Conditions: stationary phase, CIM DEAE disk (0.3 mm thickness); mobile phase, 0.48 M sodium chloride in 20 mM Tris-HCl, pH 8.5; flow rate, 4 and 8 mL/min; detection, UV at 260 nm; sample (1) oligo 8, (2) oligo 10, (3) oligo 12, and (4) oligo 14; sample concentration, 50 µg/mL of each; injection volume, 20 µL.

the desorption of oligo 14 in gradient HPMC, resulting in a longer analysis time of the later. The parameter X0 in the gradient mode was close to the real thickness of disks used in both types of HPMC (3 mm). The values of RS of two last pairs presented (oligos 10 and 12, oligos 12 and 14) seem to be close. Actually, gradient chromatography at very shallow gradient shapes approaches the isocratic separation when the gradient slope tends to zero. However, the increase of the resolution factor of the last pair in gradient elution can be explained by the effect of gradient compression of a zone of strongly adsorbed component. In contrast to that, in isocratic elution, the same zone has to be mostly spread since the mobile-phase composition is constant. As was discussed in the theoretical section, the quality of the chromatographic separation depends on column efficiency or on a number of theoretical adsorption-desorption acts along the column. The question still is, what is the minimum number of these contacts to provide the chromatographic separation? Recently it was shown34 that the increase of the layer thickness increases the peak separation of isocratic HPMC separation of oligonucleotides but that even a thickness of only 0.75 mm is enough to carry out isocratic separation. In this work, we further decreased the monolith thickness to 0.3 mm, approaching the thickness of the membranes. In this way, the number of theoretical contacts was more than halved; however, the separation still took place. Furthermore, it remained almost unaffected when the flow rate was doubled (Figure 3), indicating that even thinner monoliths could provide separation in the isocratic mode. The HETP value practically defined from the isocratic HPMC data for oligo 8 34 gave the possibility of making a conclusion about the multiple acts of adsorption-desorption in high-performance membrane chromatography separations. In contrast to that, an attempt at isocratic separation of a protein mixture did not give positive results.33 It is necessary to emphasize, however, that the isocratic HPLC of proteins is rarely practically applied. The positive results for isocratic HPMC of plasmid DNA33 can be explained

Figure 4. Two-step isocratic separation of a seven-oligonucleotides mixture. Conditions: stationary phase, CIM DEAE disk; mobile phase, (buffer A) 20 mM Tris-HCl, pH 8.5, (buffer B) 1 M sodium chloride in 20 mM Tris-HCl, pH 8.5; isocratic elution at 53% buffer B for 250 s and then at 68% buffer B for 120 s; flow rate, 3 mL/min; detection, UV at 260 nm; sample, mixture of seven oligonucleotides; sample concentration, 20 µg/mL of oligo 8, 100 µg/mL of oligo 14, and 50 µg/mL of others; injection volume, 20 µL.

by the much less rigid conformation of this very large molecule and a rather regular distribution of a charge along its chain. The existing peak widths in HPMC of oligonucleotides does not allow us to expect high peak capacity of the separation method under isocratic conditions. However, by increasing the adsorption ability through changes in mobile-phase composition, it is possible to improve significantly this very important chromatographic parameter. Moreover, the high speed and, as a consequence, the short time required for method optimization are the powerful advantages of the method discussed. Figure 4 demonstrates fast separation of a seven-oligonucleotide mixture by applying “twostep” isocratic elution. The thinness of the stationary phase absolutely excludes any alterations of gradient of displacer concentration along the separative matrix. Understanding the physical sense of the process and using the possibility of very fast optimization, the separation efficiency can be significantly improved. From this point of view, and taking into account the pressure drop lower than 1 MPa in all described analyses, the monolithic disks seem to be very competitive in comparison with their closest analogues, namely, “continuous separation media” or “monolithic rods”.37-41 Cation-Exchange HPMC Separation of Synthetic Peptides. Another very important class of small molecules is peptides. For our investigations, we used synthetic peptides of different molecular masses (different number of amino acid residues). Similarly to the proteins, these molecules do not have any regular distribution of adsorption sites. In contrast to the oligonucleotides, where the variation of bound units is equal to 4, the set of possible amino acids is spread up to 20. (37) Hjerten, S. Nature 1992, 356, 810. (38) Hjerten, S.; Liao, J. L.; Zhang, R. J. Chromatogr. 1989, 473, 273. (39) Liao, J. R.; Chang, N.; Ericson, C.; Hjerten, S. Anal. Chem. 1996, 68, 3468. (40) Svec, F.; Frechet, J. M. J. Anal. Chem. 1992, 64, 820. (41) Svec, F.; Frechet, J. M. J. Science 1996, 273, 205.

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Figure 5. Separation of a peptide mixture in gradient and isocratic modes. Conditions: stationary phase, CIM SO3 disk; mobile phase, (buffer A) 20 mM sodium phosphate buffer, pH 7, (buffer B) 1 M sodium chloride in 20 mM sodium phosphate buffer, pH 7; sample (1) Pep-9, (2) Pep-15, and (3) Pep-11; flow rate, 4 mL/min; detection, UV at 215 nm; injection volume, 20 µL; (a) gradient mode, 10085% of buffer A in 60 s; sample concentration, 90 µg/mL of Pep-9, 180 µg/mL of Pep-11, and Pep-15; (b) isocratic mode, mobile phase a mixture of 92% buffer A and 8% buffer B; sample concentration, 90 µg/mL each.

Figure 5 presents a comparison between gradient and isocratic separation of a mixture of three synthetic peptides differing by their molecular masses (Peps 9, 11, and 15). Taking into account the chemical properties of the amino acid set of each peptide, the cation-exchange type of HPMC was chosen for the analysis of a mixture, and accordingly, the CIM disk with bound sulfo groups was used as a stationary phase. Different gradient shapes, just similar to the separation of oligonucleotides, allowed regulation of the resolution of chromatographic zones. The direct dependence of a retention on chain length was not observed in this case, since the difference in the composition of amino acids strongly defines the electrostatic interaction of peptide with the charged surface. Reversed-Phase HPMC Separations of Small Hydrophobic Compounds. The next type of high-performance membrane chromatography, i.e., the reversed-phase mode, was chosen to improve our knowledge of the processes discussed. Figure 6 shows how it is possible to explore correctly the method offered, taking into account all fundamental realities. The RP chromatogram represents separation of a mixture of six substances including benzene, toluene, and four homologues of 4-hydroxybenzoate differing only by one CH2 group. The separation demonstrates the high peak capacity of the proposed method combined with short analysis time and low back pressures. It is important to add here that according to the peaks’ shape of small molecular mass compounds, which is very close to Gaussian, this indicates a high number of interactions with the active surface, which is assumed to be necessary for the standard chromatographic process. Again, all dependencies observed and discussed above also took place in this type of HPMC. Finally, one more example of RP HPMC of small molecules is presented. This is the separation of a mixture of three steroids 2990 Analytical Chemistry, Vol. 71, No. 15, August 1, 1999

Figure 6. Isocratic reversed-phase separation of a mixture of benzene and toluene combined with the mixture of four homologues of 4-hydroxybenzoate. Conditions: stationary phase, CIM C18 disk; mobile phase, 30% acetonitrile in distilled water; flow rate, 3 mL/min; detection, UV at 254 nm; sample (1) methylparabene, (2) ethylparabene, (3) propylparabene, (4) benzene, (5) butylparabene, and (6) toluene; sample concentration, 10 µg/mL each; injection volume, 20 µL.

Figure 7. Isocratic reversed-phase separation of a mixture of steroids. Conditions: stationary phase, CIM C18 disk; mobile phase, 30% acetonitrile in distilled water; flow rate, 4 mL/min; detection, UV at 254 nm; sample (1) 11(R)-hydroxyprogesterone, (2) 11-ketoprogesterone, and (3) progesterone; sample concentration, 50 µg/mL each; injection volume, 20 µL.

with very similar chemical structure (in fact, these are the close homologues of progesterone) in isocratic mode (Figure 7). CONCLUSIONS Investigations carried out in this work open new areas of application for thin monolithic separation units. In contrast to the gradient protein separation based on selective desorption, where the column length does not play an important role, for isocratic separations long columns were assumed to be necessary. However, in the case of rigid monolithic supports, it seems that such a process can also be performed using a very thin separation layer.

This is especially true for small molecules, where the diffusion coefficients are a couple of orders of magnitude lower than that of the protein molecules. Besides isocratic separations, shallow gradients can also be successfully applied. Short separation times, combined with a low back pressure drop, could make these units very competitive with other conventional units in the area of the separation of small molecules. ACKNOWLEDGMENT The authors are grateful to Dr. M. Prhavc and S. Jaksˇa, National Institute of Chemistry, Ljubljana, Slovenia, for providing

samples of oligonucleotides and R. Hahn, Institute of Applied Microbiology, Vienna, Austria, for providing samples of peptides, MSc P. Zˇnidarsˇicˇ, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Slovenia, for providing steroids samples, and MSc V. Pavli, Krka d.d., Novo mesto, Slovenia, for providing samples of parabens.

Received for review December 4, 1998. Accepted April 27, 1999. AC981350V

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