Effects of Inner Diameter of Monolithic Column on Separation of Proteins In

中国科技论文在线 http://www.paper.edu.cn Available online at www.sciencedirect.com

Journal of Chromatography A, 1170 (2007) 15–22

Effects of inner diameter of monolithic column on separation of proteins in capillary-liquid chromatography Congying Gu a,LiLinb, Xiaodong Chen c, Jinping Jia a, Jicun Ren b, Nenghu Fang b,∗ a Department of Environmental Science & Engineering, Shanghai Jiaotong University, 800 Dongchuan Road, Shanghai 200240, China b Department of Chemistry & Chemical Engineering, Shanghai Jiaotong University, 800 Dongchuan Road, Shanghai 200240, China c Department of Chemical Engineering, Liaodong University, Dandong 118002, China Received 28 July 2007; received in revised form 28 August 2007; accepted 3 September 2007 Available online 12 September 2007

Abstract Polymer monolithic columns with I.D. between 100 and 320 ␮m were prepared by in-situ polymerization of styrene and divinylbenzene in fused silica capillaries. The effects of monolithic column I.D. on the separation of proteins in reversed-phase capillary-liquid chromatography under gradient elution were systemically studied. The loading capacity was positively proportional to the volume of the stationary phase. It was found that the smaller diameter columns showed better performance for protein separation. The minimum plate height decreases from 34.99 ␮m (320 ␮m I.D. column) to 5.39 ␮m (100 ␮m I.D. column) for a retained protein. After studying the three parameters of the Van Deemter equation, it was interpreted that the smaller diameter can provide less ﬂow resistance and the better performance may also be improved by the increasing of the effective diffusion. This conclusion was also supported by the data of separation permeability and breakthrough curves. © 2007 Elsevier B.V. All rights reserved.

Keywords: Capillary HPLC; Inner diameter; Monolithic columns; Protein

1. Introduction put forward a completely new solution to those problems. Cap- illary monolithic columns get many improvements including There is an increasing need to separate and determine small the simplicity of preparation, avoidance of frits and column amounts of the compounds of interest in minute samples, and packing procedures, robustness, high porosity and permeabil- there is a simultaneous requirement that the speed and efficiency ity, high chemical stability over a wide pH range. Thus the of separations should be maximized. This is especially true monolithic columns have the merits of fast mass transfer and in fields of proteomics, genomics and forensic sciences [1–6]. low-pressure resistance. Additionally, chemical functionaliza- Miniaturization of HPLC provides an available option for these tion of the monolith can be easily realized for special separation. requirements and has attracted strong interest [7,8]. Microflow All of these make this separation media an excellent alterna- or nanoflow chromatographic methods offer the advantages of tive for packed capillary columns and a number of research fast separation, good selectivity, low sample and mobile phase groups are studying a variety of issues related to their prepa- consumption. Also, the low volumetric flow rates make easy ration, properties and applications [11–16]. And several good their couplings to mass-spectrometry [8–10]. reviews are available [3,12,17–24]. Polymer-based monoliths For traditional packed columns used in capillary-HPLC, tech- are commonly for the separation of macromolecules such as nological barriers are often conquered, such as packing particle proteins [11,16,25], nucleotides, oligonucleotides and synthetic in a narrow column, ultra high-pressure resistance that exceeds polymers [26]. the pressure limit of the pump and tedious procedure for frits Gradient elution is often needed for protein separation in RP- preparation. The appearance of capillary monolithic columns HPLC, because they vary widely in retention factors [27–29]. Modeling of gradient elution for protein separation on monolithic capillary column is more complex than that of isocratic ∗ Corresponding author. Tel.: +86 21 54742802/54742803; elution. On the one hand, some important physical parameters fax: +86 21 54741297. of proteins, such as the diffusivity, the thermodynamically based E-mail address: [email protected] (N. Fang). steric factor and the size of the molecule, are different from

16 C. Gu et al. / J. Chromatogr. A 1170 (2007) 15–22 the low-molecular-weight compounds. With respect to the chro- 2.2. Instrumentation matography, they affect the kinetics of the adsorption process, thermodynamics and the character of the interaction between The experiments were performed on a set of TriSep-2100GV the protein molecules and the surface, which make the sep- from Unimicro Technology (Pleasanton, CA, USA) equipped aration of proteins more complicated than the separation of with a Data Module UV-visible detector (wavelength contin- low-molecular-weight compounds in gradient or isocratic elu- uously adjustable) and the injection was performed through a tion [3]. On the other hand, although the study of monolithic six-port injector. The morphology examination of the monolith columns has exhibited an increasing trend, some mechanisms cross-section was taken by scanning electron micrograph (SEM, of this new stationary phase are still unclear up to now. As a 7401F, JEOL, Japan). result, there is little available theoretical guide for the optimiza- tion of the experimental conditions of proteins separation based 2.3. Column preparation on gradient elution. Equations and concepts for evaluating the properties of the monoliths are mostly adopted from isocratic Pretreatment and vinylization of the fused-silica capillary chromatography of the traditional packed capillary columns. was based on the proto reported elsewhere [32]. In brief, fused- Therefore, extensive investigations are meaningful and neces- silica capillary tubings with different I.D. (100–320 ␮m) were sary to obtain better understanding of the separation process and rinsed with acetone for 30 min, activated with 0.1 mol/L sodium develop broader applications of this new promising stationary hydroxide for 2 h at 100 ◦C, washed with 0.1 mol/L hydrochlo- phase. ric acid for 30 min, then with water and acetone, and dried Up to now, there are several key factors used to evaluate by passing through nitrogen. The capillaries were filled with the separation efficiency of monolithic column for proteins, 30% (v/v) 3-(trimethoxylsilyl)propyl methacrylate in acetone, such as monomers, porogens, preparation temperature, column sealed, and left to react for 14 h at 50 ◦C. The modified cap- porosity and equivalent permeability. However, the effect of the illary was washed with acetone and dried by passing through inner diameter (I.D.) of the capillary column on the separation nitrogen. efficiency of proteins has not been studied to our knowledge. Subsequently, a typical polymer mixture was prepared Whether the influence of the I.D. of this new separation matrix [16,32,33]. Briefly, 20% (v/v) styrene, 20% (v/v) DVB, 6% is similar to that of the conventional packed capillary column (v/v) toluene, 54% (v/v) decanol and 1% ((w/v) with respect [30,31] is pending. In this paper, capillary monolithic columns to monomers) AIBN were mixed ultrasonically into a homoge- with four different I.D. were prepared by in-situ organic poly- nous solution and purged with nitrogen for 10 min. 45-cm long merization with styrene and divinylbenzene (DVB) in order to silanised capillaries with different I.D. were filled with the mix- make clear the effect of I.D. of the capillary column on the ture to a length of 20 cm, sealed with silastic, and then left to separation of protein. The performances of these columns are polymerize for 24 h at 70 ◦C. After the polymerization was com- evaluated in terms of plate height, flow resistance, separation pleted, the monolithic column was washed with methanol for impedance and apparent capacity factor. 12 h using HPLC pump to remove unreacted monomers and porogens. 2. Experimental An on-column detection window was made next to the end of the monolithic polymer by removing the polyacrylamide coating 2.1. Chemicals and materials using a razor blade after the preparation was completed, and columns of 20 cm effective length and 44 cm total length were Divinylbenzene (DVB, 80%, residual mainly 3- and 4- adopted for all the experiments. ethylvinylbenzene) and trifluoroacetic acid (TFA, for protein sequence analysis) were purchased from Fluka (Buchs, Switzer- 3. Results and discussion land). ␥-Methacryloxypropyltrimethoxysilane was from Acros (New Jersey, USA). Acetonitrile (HPLC gradient grade) was 3.1. Morphology of the monolithic column obtained from Fisher (Darmstadt, Germany). Styrene, 2,2- azobisisobutyronitrile (AIBN), decanol, toluene were obtained The obtained morphology of the polymeric rod from scan- from Shanghai Chemical Reagent Company and were of analyt- ning electron microscopy (SEM) is shown in Fig. 1. These ical reagent grade. Phosphorylase b from rabbit muscle, bovine micrographs demonstrate well-ordered polymeric clusters of serum albumin (BSA), actin from rabbit muscle, carbonic anhy- quasi-beads structure. All the columns contained large through- drase from bovine erythroytes, trypsin inhibitor from chicken pores with the sizes among 1–4 ␮m. The 100-␮m I.D. PS-DVB egg white and lysozyme from chicken egg white were all pur- monolith consisted of larger polymer globules, while the chased from Sigma (St. Louis, MO, USA). Deionized water 320-␮m I.D. PS-DVB monolith was composed of smaller cross- (18.2 M) was prepared using a Milli-Q system from Milli- linked polymer clusters of globules. Because the thermally pore (Bedford, MA, USA). Styrene and DVB were extracted initiated free-radical copolymerization of styrene is an exother- with 5% aqueous sodium hydroxide and water, dried over anhy- mic reaction, it was supposed that, compared with the larger drous magnesium sulfate, and distilled under vacuum before use. I.D. column, the smaller I.D. column may have a faster heat Fused silica capillaries with different I.D. were purchased from radiation during the polymerization and then keep a relatively Yongnian Optic Fiber Plant (Hebei, China). lower temperature in the inner space of the mold where the 中国科技论文在线 http://www.paper.edu.cn

C. Gu et al. / J. Chromatogr. A 1170 (2007) 15–22 17

Fig. 1. Scanning electron micrographs of the prepared monolithic columns. (A) 100 ␮m I.D. monolith; (B) 150 ␮m I.D. monolith; (C) 250 ␮m I.D. monolith; (D) 320 ␮m I.D. monolith. polymerization taking place, although the outer temperature be estimated [30] is the same. Furthermore, it was commonly received that a uηL lower temperature causes a lower rate of initiator decompo- K = p (1) sition and thus a less number of nuclei to be generated at early stages of polymerization. As a consequence, the glob- where u is the linear velocity of eluent, η the dynamic viscosity ules, which emerge from nuclei coalescence, have a smaller of eluent, L the column length, p is the pressure drop. size compared to those that would result at lower tempera- Results shown in Fig. 2 indicated that there was a decrease in ture [34,35]. And thus, a smaller I.D. leads to more uniformity K with the increasing of column I.D. It was supposed that intersti- of the monolithic matrix and with bigger polymer globules. tial structure of the pores formed during the polymerizing of the However, this hypothesis cannot be verified at this stage of polymer depends a lot on the I.D. of the capillary, and the struc- study. ture of the pores directly affect the permeability of the column. The significant disparity of the permeability drop a hint of the 3.2. Effect of column I.D. on specific permeability difference in pore structure among these columns with different I.D. which could also be primarily deduced from the SEM pic- A parameter used to indicate the influences of I.D. on col- tures of these monoliths. Unfortunately, it would be too difficult umn performances is flow resistance, which is closely related to measure the actual pore size distribution since the amount of to the column permeability. With the specific permeability K the monolithic material in the capillary is not sufficient for deter- (written as Eq. (1)), it is possible to determine how much pres- mination using standard methods such as mercury-intrusion sure is needed or how long columns can be used under given porosimetry and nitrogen adsorption porosimetry [35]. Addi- pressure, and the mobile phase velocity constrains can also tionally, size exclusion chromatography used for determine the 中国科技论文在线 http://www.paper.edu.cn

18 C. Gu et al. / J. Chromatogr. A 1170 (2007) 15–22

Fig. 2. Plot of specific permeability vs. column I.D. Fig. 4. Van Deemter plots for Carbonic anhydrase characterizing monolithic columns with different I.D. Mobile phase: 65% ACN, 0.1% TFA in water. pore size distribution of silica monolith is not suitable for the Detection: UV 214 nm. determination of pore size in the micrometer size range which is the typical of polymer-based monolith [35,36]. 3.3. Effect of column I.D. on column efficiency (Van What also has to be pointed out is that the same polymeriza- Deemter curves) tion mixture was used for the preparation of all the monolithic columns and all the other conditions were kept the same except To investigate the effect of the I.D. on the column efficiency, the inner diameter of the fused silica capillary, and it reflects the dependency of the height equivalent to a theoretical plate that the inner diameter does pose a significant effect on the pore (HETP) on the linear flow rate was measured with carbonic structure of the monolith. anhydrase. The HETP curves, depicted in Fig. 4, showed that Fig. 3 showed the plots of pressure drop against linear velocity columns with smaller I.D. appeared to perform better at the same of the mobile phase, and the trend was in agreement with that of linear flow velocity. The minimum plate height decreases from the permeability. Additionally, the back pressure against linear 34.99 ␮m (320 ␮m I.D. column) to 5.39 ␮m (100 ␮m I.D. col- velocity is a straight line with the regression factor R2 better than umn). It is obvious that more theoretical plates can be achieved 0.999 for every measured curve. It is of evidence that both the in less time, using the smaller diameter columns. stability and permeability of the monoliths are excellent under The Van Deemter equation (written as Eq. (2)) was used to high pressure. For the 100 ␮m I.D. monolith, to obtain a flow study the effect of I.D. on the molecular diffusion processes of velocity as high as 40 mm/s, a pressure drop of 22 Mp is enough. monolithic columns extensively [37]. Van Deemter functions While, for a traditional packed capillary column, this is almost were fitted to the measured HETP curves yielding the three impossible. parameters A, B and C, which represent the eddy dispersion, longitudinal diffusion and mass transfer, respectively. B H = A + + Cu u (2) The results of the parameters were summarized in Table 1. For the parameter A, the trend of the dependency was not clear, however the overall trend is rising with the increasing of diameter. This could be interpreted that flow rate distribution becomes narrow over the column cross-section when the column diameter decreases [38], and then the narrower flow rate distribution yields lower eddy diffusion. The most striking difference appeared in the B term which describes the axial dispersion. It decreased rapidly with the increasing of the I.D. Equation for the B term introduced by Knox takes into account of the fact that the magnitude of the axial dispersion depends on the time of the solute stays in the column and the diffusion coefficient of the solute in the station- Fig. 3. Plots of pressure drop against linear velocity of mobile phase. Mobile ary phase [39]. So this phenomenon may be interpreted by the phase: 65% ACN (v/v) in water. Columns: 320 ␮m I.D. (), 250 ␮m I.D. (), fact that the staying time of the solute in the column will become 150 ␮m I.D. (), 100 ␮m I.D. (᭹). longer, given a larger diameter. 中国科技论文在线 http://www.paper.edu.cn

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Table 1 Calculated Van Deemter equation coefﬁcients for monolithic columns with different I.D. Column I.D. (␮m) Eddy dispersion, A (␮m) Longitudinal diffusion, B (␮m mm/s) Mass transfer, C (␮m/(mm/s))

100 4.0 0.7 1.8 150 3.1 3.0 2.5 250 21.6 10.4 1.9 320 15.9 29.5 3.1

The coefﬁcients were determined by ﬁtting against linear velocity from all columns in a given size to the Van Deemster’s equation. Two columns of each dimension were used.

There is an overall increasing trend for the C coefficient with the increasing of the I.D. except for the I.D. of 150 and 250 ␮m. However, among the three parameters, the C term is the lowest. This is in agreement with the theory that the convective mass transfer taking place in the monolithic column is an effective mass transfer mode which contributes a lot to the fast separation without sacrificing the efficiency at higher flow rate. Even though each of these effects are discussed individually, it should be pointed out that it is the combined nature of the A, B and C terms that determines the final performance of the column, and it is useless to attempt to attribute it only to one term. It was interpreted that the smaller diameter can provide more uniform polymer monolithic matrix and less flow resistance. The better performance may also be improved by the increasing of the effective diffusion.

3.4. Comparison of chromatographic properties under gradient elution Fig. 5. Monolithic columns of different I.D. for the separation of six stan- A mixture of six standard proteins was taken as separation tar- dard proteins with the same volume flow rate. Columns: (A) 100 ␮m get to evaluate the influence of the I.D. on the chromatographic I.D.; (B) 150 ␮m I.D.; (C) 250 ␮m I.D.; (D) 320 ␮m I.D. Column length: properties under gradient elution. The plots of the chromatogram 20/45 cm. Sample mixture introduced into the column: 0.3 mg/ml six standard proteins (0.05 mg/ml each). Mobile phase: (A) 10% ACN, 0.1% obtained at constant volume flow rate and linear flow velocity TFA in water. (B) 60% ACN, 0.1% TFA in water. Gradient elution pro- were shown in Figs. 5 and 6, respectively. It should be pointed gram: (A) 0–0.6–1.6–2.4 min, 15%–40%–62%–78% B. (B) 0–1.3–3.6–5.4 min, out that, the gradient program is set to keep the eluent volume in 15%–40%–62%–78% B. (C) 0–5.7–16.3–24 min, 15%–40%–62%–78% B. a direct proportion to the monolith volume. The resolution (R), (D) 0–3.5–10–15 min, 15%–40%–62%–78% B. Flow rate of mobile phase: 4.8 ␮L/min. Temperature: ambient temperature. Detection: UV 214 nm; 0.8 ␮L tailing factor (γ), peak width at half-height (Wh/2) and apparent injection. Peak 0: mark of t0; peaks 1 and 2: trypsin inhibitor; peak 3: lysozyme, capacity factor (k ) for two proteins were shown in Table 2. peak 4: BSA; peak 5: carbonic anhydrase; peak 6: actin; peak 7: phosphorylase. At a constant volume flow rate, with the increasing of the column I.D., Wh/2 became broader and γ became larger for tailing peaks (BSA) and smaller for leading peak (lysozyme). In I.D. at constant flow velocity as mentioned before. This resulted addition, the overall trends for R and k were both increasing. in an earlier elution of the band and an elution at a lower solvent As shown in Fig. 6, similar results were obtained at constant strength under gradient elution, and hence a broader band width flow velocity. The wider the column was, the broader the peak is produced [40]. In addition, changes of the flow rate led to widths at half height became. It should be mentioned that the changes of the system dead volume, and hence the changes of the volume flow rate increased with the increasing of the column gradient delay appear, although the gradient slop (%ACN) is kept

Table 2 Resolution, tailing factor and peak width at half-height against column diameter Column I.D. (␮m) Lysozyme BSA RWh/2 (s) h γ k RWh/2 (s) h γ k 100 1.08 2.88 1854 1.10 8.72 1.57 3.42 4998 1.76 9.45 150 2.12 7.43 1120 1.01 36.60 2.62 10.63 3633 2.17 43.24 250 3.25 10.55 940 0.94 19.18 2.94 19.35 2087 2.25 23.15 320 2.65 21.17 588 0.83 14.89 2.91 35.71 1024 2.44 17.55

Other conditions were the same as in Fig. 5. R: separation resolution; γ: tailing factor; Wh/2: peak width at half-height; k : apparent capacity factor. 中国科技论文在线 http://www.paper.edu.cn

20 C. Gu et al. / J. Chromatogr. A 1170 (2007) 15–22

Fig. 7. Minimum separation impedance (Emin) for (A) lysozyme; (B) BSA; (C) carbonic anhydrase as a function of column I.D. Two columns of each dimension Fig. 6. Monolithic columns of different I.D. for the separation of six stan- were used. dard proteins with the same ﬂow velocity. Columns: (A) 100 ␮m I.D.; (B) 150 ␮m I.D.; (C) 250 ␮m I.D.; (D) 320 ␮m I.D. Columns length: 20/45 cm. Sample mixture introduced into the column: 0.3 mg/ml six standard pro- and the ﬂow resistance effects into consideration, columns with tein (0.05 mg/ml each). Gradient elution program: 0–0.7–1.8–2.7–6.0 min, smaller I.D. performed better. 15%–40%–62%–78%–78% B. Temperature: ambient temperature. Detection: UV 214 nm; 0.8 ␮L injection. Peak 0: mark of t0; peaks 1 and 2: trypsin inhibitor; peak 3: lysozyme, peak 4: BSA; peak 5: carbonic anhydrase; peak 6: actin; peak 3.6. Effect of column I.D. on loading capacity of monoliths 7: phosphorylase. for protein

As to the miniaturized HPLC system, capillary columns with constant [41]. This effect is much significant in capillary-column diameters from 50 to 320 ␮m are often used. For these capil- mode [42]. lary columns, loading capacity is one of the major limitations as compared to the usual dimension HPLC columns, especially 3.5. Effect of column I.D. on separation impedance for the separation of complicated macromolecules samples. To determine the analytical loading capacity of the monolithic The column’s separation impedance E, a widely used crite- columns with different diameters, breakthrough curves (shown rion for evaluating the efficiency of a column, combines both in Fig. 8) were measured by using BSA for frontal analysis the band broadening and the flow resistance effects [43].Itwas [13,45,46]. Quantitative information about loading capacity in calculated by Eq. (3). different chromatographic beds is represented by the mass of H2P E = uηL (3) where u is the linear velocity of eluent, η the dynamic viscosity of eluent, L the column length, p the pressure drop and H is the height equivalent to a theoretical plate. Generally, the smaller the E is, the better the performance of the column is. E is dimensionless and its best expected value for a well-packed column is 2000 [44]. Three proteins were chosen to investigate the effect of the column diameter on the Emin under the isoelution condition of 65% ACN and 0.1% TFA in water. As the plots shown in Fig. 7, there is a nearly linearly increasing trend in the Emin with the increasing of the column in the range of 150–320 ␮m. The values of Emin for BSA are about from 40,913 to 241 for the column I.D. from 320 to 100 ␮m. It was interpreted that both the increasing of H and the decreasing of K resulted in the rapid increasing min ␮ of E as described by Eq. (3). These illustrated once again Fig. 8. Breakthrough curves of monolithic columns: A (100 m I.D.), B min (150 ␮m I.D.), C (250 ␮m I.D.), D (320 ␮m I.D.) determined with 0.20 mg/ml that the I.D. had a significant effect on the performance of the BSA in 0.1% TFA in water. Flow rate 5 ␮l/min, detected at 214 nm with UV column. It reconfirmed that taking both the band broadening detector. 中国科技论文在线 http://www.paper.edu.cn

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Table 3 that the tested loading capacity is enough for most microanaly- Calculated loading capacity for the columns with different I.D. sis. These results will be helpful in practical use of monolithic

Column I.D. (␮m) Loading capacity (␮g BSA) Total porosity, εT column for separation of biomacromolecules. 100 1.74 0.88 150 4.00 0.85 Acknowledgement 250 10.82 0.89 320 20.37 0.83 This work was financially supported by the National Natural Science Foundation of China (20571052). tested protein with respect to the whole column (mg BSA for each column), because this representation could provide direct References comparison of the actual columns with different I.D. The calculated results shown in Table 3 indicate that, for [1] M. Novotny, D. Ishll (Eds.), Microcolumn Separations, Elsevier, Amster- the 100 ␮m I.D. monolith, the loading capacity was estimated dam, 1985. to be 1.74 ␮g. For the 320 ␮m I.D. monolith, it was improved [2] Y. Saito, K. Jinno, T. Greibrokk, J. Sep. Sci. 27 (2004) 1379. approximate 12 times and yielded a 20.37 ␮g BSA loading [3] F. Svec, C.G. Huber, Anal. Chem. 78 (2006) 2101. [4] F. Qin, C. Xie, S. Feng, J. Ou, L. Kong, M. Ye, H. Zou, Electrophoresis 27 capacity. It could be concluded that with similar total poros- (2006) 1050. ity, the loading capacity of the monolithic columns is positively [5] N. Tanaka, H. Kobayashi, Anal. Bioanal. Chem. 376 (2003) 298. proportional to the stationary phase volume. In addition, a sharp [6] N. Tanaka, Anal. Chem. 73 (2001) 420. front in the sigmoidal-shaped breakthrough curve will indicate [7] S. Ito, S. Yoshioka, I. Ogata, E. Yamashita, S. Nagai, T. Okumoto, K. Ishii, the homogeneity of the separation medium and the enhanced M. Ito, H. Kaji, K. Takao, K. Deguchi, J. Chromatogr. A 1090 (2005) 178. [8] J. Masuda, D.M. Maynard, M. Nishimura, T. Ueda, J.A. Kowalak, S.P. mass transport over the column. From the breakthrough curves Markey, J. Chromatogr. A 1063 (2005) 57. shown in Fig. 7, it was found that the smaller the I.D. of the col- [9] E. Nagele,¨ M. Vollmer, Rapid Commun. Mass Sep. 18 (2004) 3008. umn, the sharper the front, which reconfirmed that the smaller [10] T. Le Bihan, H.S. Duewel, D. Figeys, J. Am. Soc. Mass Spectrom. 14 (2003) I.D. monolithic column may provide a more quick mass transfer. 719. By the way, the porosity of the monolith prepared in capil- [11] A.R. Ivanov, L. Zang, B.L. Karger, Anal. Chem. 75 (2003) 5306. [12] P. Coufal, M. Cihak, J. Suchankova, E. Tesarova, Z. Bosakova, K. Stulik, lary was examined by a flow method [47]. In detail, the mobile J. Chromatogr. A 946 (2002) 99. phase linear velocity was measured by an inert tracer and the [13] X. Gu, Y. Wang, X. Zhang, J. Chromatogr. A 1072 (2005) 223. volumetric flow rate was also measured. Then with the known [14] L. Cong, B. Huang, Q. Chen, B. Lu, J. Zhang, Y. Ren, Anal. Chim. Acta 569 (2006) 157. empty tube dimension, the total porosity εT can be expressed as Eq. (4): [15] K.M. Karlsson, L.E.M. Spoof, J.A.O. Meriluoto, Environ. Toxicol. 20 (2005) 381. V [16] C. Legido-Quigley, N.D. Marlin, V. Melin, A. Manz, N.W. Smith, Elec- ε = × 100% (4) trophoresis 24 (2003) 917. T πr2c [17] K.W. Ro, R. Nayak, D.R. Knapp, Electrophoresis 27 (2006) 3547. [18] E. Klodzinska, D. Moravcova, P. Jandera, B. Buszewski, J. Chromatogr. A where εT is the total porosity, V is the volumetric flow rate of 1109 (2006) 51. mobile phase, r is the inner radius of the empty column, c is [19] S. Eeltink, F. Svec, Electrophoresis 28 (2007) 137. the linear velocity of mobile phase, which was determined by [20] J. Ou, J. Dong, X. Dong, Z. Yu, M. Ye, H. Zou, Electrophoresis 28 (2007) unretained compound thiourea. 148. [21] M. Szumski, B. Buszewski, J. Sep. Sci. 30 (2007) 55. [22] M. Barut, A. Podgornik, P. Brne, A. Strancar,ˇ J. Sep. Sci. 28 (2005) 4. Conclusions 1876. [23] L. Rieux, H. Niederlander,¨ E. Verpoorte, R. Bischoff, J. Sep. Sci. 28 (2005) Polymer-based monolithic columns are mostly used for 1628. the separation of macromolecules. In this work poly(styrene- [24] M. Bedair, Z. El-Rassi, Electrophoresis 25 (2004) 4110. divinylbenzene) monolithic columns with different I.D. were [25] W. Walcher, H. Toll, A. Ingendoh, C.G. Huber, J. Chromatogr. A 1053 (2004) 107. prepared and used for separation of standard proteins. The effects [26] M. Petro, F. Svec, I. Gitsov, J.M.J. Freˇıchet, Anal. Chem. 68 (1996) 315. of the I.D. on various column parameters were studied. It was [27] K.M. Gooding, F.E. Regnier (Eds.), HPLC of Biological Macromolecules. found that the smaller the column diameter is, the higher the Methods and Applications, Marcel Dekker, New York, 1990. permeability. As to the separation efficiency, it was evident that [28] G. Glockner, Gradient HPLC of Copolymers and Chromatographic Cross- more theoretical plates can be achieved in less time using the Fractionation, Springer, Berlin, 1991. [29] M. Petro, F. Svec, I. Gitsov, J.M.J. Frechet, Anal. Chem. 68 (1996) 315. smaller diameter columns. It is interpreted that the interstitial [30] R.T. Kennedy, J.W. Jorgenson, Anal. Chem. 61 (1989) 1128. structure of the pores formed during the polymerizing of the [31] F. Svec, T.B. 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