Anal. Chem. 1993, 65, 1615-1621
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Preparation of Open Tubular Columns for Reversed-Phase High-Performance Liquid Chromatography Antonio L.Crego, Jose C. Diez-Masa, and Manuel V. Dabrio' Instituto de Qutmica Orglrnica General (C.S.I.C.), J u a n de la Cierua, 3 28006-Madrid, Spain
A procedure for the preparation of a thin layer of silica gel with chemically bonded c18 moieties on the internal wall of fused-silica capillaries is developed. The preparation of the silica layer is based on the hydrolytic polycondensation and gelling of tetraethyl orthosilicate (TEOS) in a pHfixed water-methanol solution. It is demonstrated that the most critical step of this method is the formation of a thin, homogeneous film of the pregelling solution of TEOS on the capillary wall, which is achieved by using a static technique. In a subsequent step, complete gelling of the film is achieved in a stream of nitrogen at 100"C.Finally, the bonding the Cle is carried out at elevated temperature. Some of the parameters controlling the porosity and thickness of the silica layer (such as preconditioning of the fused-silica wall, time and temperature of the pregelling process, and pH at which hydrolysis takes place) and the yield of the bonding reaction are studied. It is demonstrated that although the method is developed on 50-prn4.d. capillaries, it is easily modified for achieving 10- and 5-pm4.d. columns. Values of reduced plate height around 0.4 were obtained for 10-and 5-pm4.d. columns. Using 5-pm columns 0.5 X 106 plates m-l and 1000 plates s-l were obtained. The reproducibility of the method in terms of k' (RSD= 12-14%) and reduced plate height (RSD = 4-5% ) from column to column is quite good. Some applications of these open capillary columns for the separation of PAHs are presented. INTRODUCTION Open tubular columns (OTCs) were proposed for gas chromatography (GC) by Golay1 in 1959. Since that time, OTCs have replaced packed columns (PCs) in most analytical applications because a higher plate number can be obtained with OTCs for similar analysis time and inlet pressure. Twenty years later, the possibility of obtaining similar advantages using OTCs in high-performance liquid chromatography (HPLC) has been theoretically demonstrated.z4 The packing materials used in reversed-phase HPLC (RPHPLC) have large surface area and small particle size, resulting in high phase ratio and thus suitable capacity factor for the solutes. In contrast, the smooth inner wall of glass or fused-silica capillaries, even though silanized using highyield reaction conditions, gives rise to phase ratio values around 350 times smaller than those of commercialRP-HPLC packings. In such Conditions, the capacity factor for most of (1)Golay, M.J. E. In Gas Chromatography;Coates, V. J., et al., Eds.; Academic Press: New York, 1958; pp 1-13. (2)Knox, J. H.; Gilbert, M. J. Chromatogr. 1979,186,405. (3)Knox, J. H. J. Chromatogr. Sci. 1980,18,453. (4)Guiochon, G. Anal. Chem. 1981,53,1318. 0003-2700/93/0365-1615$04.00/0
the solutes separated becomes so small that OTCs cannot be used for analytical purposes. Therefore, the surface area of the inner capillary wall has to be increased and the yield of the subsequent silanization reaction optimized in order to improve phase ratios for OTCs. To increase the surface area, Nota et al.,5Ishii et al.,6r7 and Tsuda et al.8 treated the inner wall of soda-lime glass capillaries with several alkalis. Other procedures, such as that reported by Jorgenson and Guthrie? who used phosphate buffer (pH 7) solution and electric field (3-kVdc) across the walls of a capillary tubing for etching ita internal surface, were used to increase the surface area and the reactivity of the borosilicate glass wall, but the improvement achieved in terms of solute retention was rather poor. The use of polymeric stationary phases to increase retention utilizing a liquid-liquid chromatography (LLC) approach in OTCslO generally caused low efficiencies because of the small diffusion coefficient of the solute in the stationary phase used. Instability of the columns due to the unavoidable bleeding of the stationary phase was also observed for this type of column. Despite these drawbacks, OTCs using polymeric stationary phases is the approach most often employed at the present Recently, Gohlin and Larssonle have reported the use of immobilized poly(dimethyloctadecy1siloxane) (PMSC18) as stationary phase in OTCs with good efficiency and stability. Another approach to increase the phase ratio in OTCs is to lay down a thin layer of porous silica of large surface area on the inner wall of the capillary and chemically bond a monomeric phase on it. Tock et al.17 used a dynamic method to coat the capillary wall with a liquid layer of poly(ethoxysilane) (PES) which was subsequently converted into silica gel using gaseous ammonia. The porosity of the extremely thin silica layer obtained by these authors was so low that they were unable to bond a substantial amount of alkylsilane, and therefore, the columns could only be used in the liquid-solid chromatography (LSC) mode. Tock et al.18 modified the previous method by using a static procedure in (5)Nota, G.; Marino, G.; Ballio, A. J. Chromatogr. 1970,46,103. (6) Hibi, K.; Tsuda, T.; Takeuchi, T.; Nakanishi, T.; Ishii, D. J. Chromatogr. 1979,175,105. (7)Ishii, D.; Tsuda, T.; Takeuchi, T. J. Chromatogr. 1979,185,73. (8)Tsuda, T.; Tsuboi, K.; Nakagawa, G. J. Chromatogr. 1981,214, 283. (9)Jorgenson, J. W.;Guthrie, E. J. Chromatogr. 1983,255,335. (10)Takeuchi, T.; Kitamura, H.; Ishii, D. HRC & CC, J . High Resolut. Chromatogr. Chromatogr. Commun. 1983,6,666. (11)Jorgenson, J. W.; Guthrie, E. J.; St. Claire, R. L., 111, J. Pharm. Biomed. Anal. 1984,2,191. (12)Fabrot, A.; Folestad, S.; Larsson, M. HRC & CC, J. High Resolut. Chromatogr. Chromatogr. Common. 1986,9,117. (13)Folestad, S.; Larsson, M. In 8th International Symposium o n Capillary Chromatography; Sandra, P., Ed.; 1987;Vol. 2,p 1112. (14)Dluzneski, P. R.;Jorgenson, J. W. HRC & CC, J.High Resolut. Chromatogr. Chromatogr. Commun. 1988,11,322. (15)van Berkel, 0.; Kraak, J. C.; Poppe H. J.Chromatogr. 1990,499, 345. (16)Gohlin, K.; Larsson, M. J. Microcol. Sep. 1991,3,547. (17)Tock, P. P. H.; Stegeman, G.; Poppe, H.; Kraak, J. C.; Unger, K. K. Chromatographia 1987,24,617. (18)Tock, P. P. H.; Boshoven, C.; Poppe, H.; Kraak, J. C.; Unger, K. K. J. Chromatogr. 1989,447,95. (19)Halasz, I. 2. Anal. Chem. 1968,236,15. 0 1993 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 11, JUNE 1, 1993
which a volatile solvent was used to introduce the PES into the capillary tubing and the solvent was evaporated under vacuum. The PES layer was subsequently treated with ammonia solution to convert it into silica. They achieved a layer with sufficient surface area and activity as to bond an important amount of octadecylsilane (ODS) moieties. However, the method reproducibility was poor and it was unsuitable for preparing columnswith inner diameters smaller than 10 pm. Fused-silica tubings have better mechanical and optical properties than glass tubings. That is why fused silica is becoming a material of choice for the preparation of OTCs. However, the chemical inertness of fused silica is a drawback when this material is to be used in the preparation of chemically bonded stationary phases for HPLC. Only a few silanolgroups (upto a maximum of 8-9 groups/IOo A2) remain on the silica surface, this amount depending on the previous thermal and chemical treatment of the surface. Their role, however, is very important since silanol groups are the only point to attach any chemical functionality to the inner wall of the capillary tubing. Two methods are traditionally used to increase the number of silanol groups on the silica surface. Alkaline hydrolysis (pH >8) causes a partial dissolution of the silica surface, increasing the number of active sites available for ulterior chemical reactions. Hydrothermal treatment using water vapor at high temperature may be another appropriate procedure to hydrolyze siloxane groups. The chemical reaction used in this work to prepare a silica layer on the fused-silica surface was the hydrolytic polycondensation of tetraethyl orthosilicate (TEOS),21also used to produce porous silica packings for HPLC.22-24 Although a similar reaction was already used by Tock et al.17J3toprepare OTCs, several modifications were carried out in the present work to achieve poly(ethoxysi1ane) formation inside smalldiameter capillaries. In this way, the silanol groups on the wall are able to take part in the initial step of the reaction, which should in principle contribute to anchoring and stabilizing the silica layer. The reaction is catalyzed by an acid or a base and takes place in two steps, hydrolysis and condensation. With acid catalysis the hydrolysis rate is higher than the condensation rate whereas with basic catalysis the opposite happens. The presence of an organic solvent (ethanol) in the reaction mixture enhances TEOS solubility and alters the water to TEOS ratio, which has a major effect on the final result of the reaction. In principle, in acidic medium, polycondensation tends to produce small particles of silica gel whereas in a basic medium it tends to produce large gel networks.21 The objective of this work was to develop a new method to prepare a layer of silica gel on the inner wall of small inner diameter (50, 10, and 5 pm) fused-silica capillaries using hydrolytic polycondensation and ulterior gelling of TEOS. The experimental conditions that maximized the amount of CISbonded on the silica layer were also studied. Particular attention was paid to the reproducibility of the preparation procedure in terms of k' and reduced plate height of the resulting columns. THEORETICAL SECTION The goal of chromatographic separation is to obtain a resolution (R,) greater than or equal to 1 in a reasonable (20) Guiochon, G. In HPLC. Aduances and Perspectiues; Horvith, Cs., Ed.; Academic Press: New York, 1980; Vol. 2, pp 1-56. (21! Brinker, C. J.; Scherer, G. W. Sol-Gel Science. The Physics and Chemistry of Sol-Gel Processing; Academic Press: New York, 1990;pp 97-234. (22) Unger, K. K.; Schick, J.;Krebs, K. F. J. Chromatogr. 1973,83,5. (23) Unger, K. K.; Schick, J.; Straube, B. Colloid Polym. Sci. 1975, 253, 658. (24) Unger, K. K.; Scharf, B. J. Colloid Interface Sci. 1976, 55, 377.
v/h
121
O
Y 0
, 5
I
10
15
I
I
,
20
25
30
, 35
40
45
50
Reduced vdoalty (v)
Figure 1. Plot of ulh against u accordlng to the Knox equatkn for several types of columns with dtfferent values of A and the same value of B = 2 and C = 0.1.
amount of time. In a practical approach, there are two ways to make R, 2 1: (i) using highly selective phases (values of a as different from 1 as possible) and (ii) increasing N by increasing column length (15).The more selective the phases are, the lower the value of N that is required to achieve a given resolution. In column comparison,the concept of plate generation velocity (PGV) (plate per unit time, N / ~ R )could '~ be employed to take separation time into account. This concept was expressed mathematically by Guiochon20 as follows:
Since the values of Dm in liquids are 4 orders of magnitude smaller than in gases, this equation explains why HPLC requires packings with particle size on the order of 100 times smaller than GC. Likewise, to obtain similar values of N/tR, the internal diameter of capillary tubes should be 2 orders of magnitude smaller in HPLC than in GC. For purposes of comparison, values of N / ~ R have to be measured for compounds having similar values of k' in both chromatographic techniques. In HPLC, the difference in terms of PGV between PCs and OTCs can be analyzed using the u/h ratio. This value can be calculated using the Knox equation as follows: -U =
1
(2) h + Bu-' + C The limit of the function u/h when u is 1/C. Figure 1 depicts variations of u/hwith increasing values of v for several different columns having the same values of B = 2 and C = 0.1 and different values of A. For A = 0 (which is the case for an OTC), the value of u/h increases quickly with the reduced velocity whereas for A > 0 (for an PC) it approaches the limit much more slowly. The coefficient A could be regarded as a measure of the packing quality of a PC. As the value of A increases, the slope of the curve decreases, which indicates that, the better the packing quality of the columns, the faster they are. For OTCs the minimum h value is achieved at uop = (B/C)lI2. At that velocity (ulh),, = l/2C, that is, half the maximum PGV value which could be obtained with OTCs. Calculations also indicate that 90% of the maximum PGV value is achieved a t a reduced velocity of 3uoP, but in practice, this value of PGV cannot be attained using PCs, as can be deduced from Figure 1. In short, increasing velocity in a PC does not produce great benefits in terms of PGV whereas in OTCs there is a rapid increase in PGV up to values of u = 3uOp For a given separation (where values of k' and D, are defined), this velocity rate determines
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 11, JUNE 1, 1993
the maximum practical value of PGV which can be achieved by a column with a given d, (3)
The loss in efficiency (N) that occurs when a column is operated at 3uOpcan be offset by increasing column length 1.67 times. In this way, N plates can be obtained either with a column length equal to L, at linear velocity of the mobile phase equal to uop[ t = (1 ~ +~ kf)L/uop], ~ or with a column length of 1.67L, at linear velocity 3uop[retention time (1 k')1.67L/3uopl,that is, only 0.56th,. In practice, column length is limited by column permeability (k)and by the maximum inlet pressure delivered by the pump. The pressure cost (APlN) for the column20 is defined as
+
(4) Combining eqs 1 and 4 yields the relationship between pressure cost and PGV value
Equation 5 indicates that (i) an increase in PGV is accompanied by a proportional increase in the pressure drop needed to generate each theoretical plate; (ii) the higher the retention values (VI, the higher pressure cost to operate; (iii) the most economical velocity for the mobile phase in terms of pressure cost is the optimum value given by the Knox equation; (iv) since flow resistance (4) for OTCs (=32) is smaller than the values of 4 for PCs (50+1300), OTCs may be 15-30 times longer than PCs for a given pressure cost. The instrumentation required to work with OTCs is somewhat different from that used with PCs because of the difference in flow rate utilized in each type of column. To prevent extracolumn molecular diffusion of the sample, the linear flow rate of the mobile phase in the injection and detection systems should not be lower than the value in the column itself. A decrease in efficiency of less than 10% from extracolumn effects is usually considered as acceptable. The theoretical values C, and C, due to the column can be estimated using the Golay1 equation, where
c,
= 1 + 6 k f + llk'' and 96(1+ k')2
c,=
2kf 3(1+ k')
-d? D m d:
D,
can be used to calculate N . Such a value can be compared to the experimental value obtained in the same conditions. As a general rule, C, can be considered as negligible when compared with C, in OTCs. The use of a split injection system or the pressure pulsedriven stopped-flowinjection (PSI)system, designed by Manz and Sim0n,~5may help obviate extracolumn effects of injection. Efficiency losses in the detector can be reduced if on-column monitoring is used. EXPERIMENTAL SECTION Reagents and Chemicals. OTCs were prepared using fusedsilica tubings of 5-, lo-, and 50-pm nominal i.d. (Polymicro Technologies),tetraethyl orthosilicate (TEOS),ethanol, ammonium hydroxide (all from Merck, Darmstadt, Germany), and dimethyloctadecylchlorosilane(Fluka, Buchs, Switzerland).Polyaromatic hydrocarbons (Fluka) dissolved in methanol or methylene chloride were used as standards. The mobile phase consisted of HPLC-grade methanol (Scharlau, Barcelona, Spain) and Milli-Q (Millipore, Bedford, MA) water. Apparatus. The chromatograph assembled for this work used a Model 590 Waters (Waters, Mildford, MA) pump. Two (25) Manz, A.; Simon, W. J. Chromatogr. 1987, 387, 187.
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different injectionsystems were utilized depending on the column inner diameter. For columns with d, > 20 pm, a Model CI4W Valco (Valco Europe, Schenkon, Switzerland) valve (60-nL injectionloop)with a homemadesplitter was used. For capillaries with a d, 20 pm, a PSI systemz5(Valco Europe) was utilized. The column effluent was monitorized using a Model 440 Waters fixed-wavelength (254 nm) detector which was home modified for on-column detection. The detector signal was recorded with a Model 851 (Philips, Eindhoven, The Netherlands) recorder. When necessary, the pressure at the column inlet was measured using a Model DP precision gauge Bourdon (Bourdon Instrumentos S.A., Barcelona, Spain) manometer (maximum pressure 10 atm, precision f0.25%). Column Preparation. In the preconditioningstep, the fusedsilica capillaries were subjected to one of the alternative treatments: (a) rinse with ammonium hydroxide solution (pH 9) for 12 h or (b) flow with water vapor for 12 h. The water vapor was generated in a hermetically sealed reservoir to which one end of the capillary column was attached. The assembly (reservoir + column) was heated gradually to 300 "C in an oven. In both cases, after treatment, the column was washed with methanol at room temperature for 2 h and dried by purging with nitrogen at 200 "C overnight. The gelling solution was prepared by stirring 0.6 mL of TEOS, 0.8 mL of ethanol, and 0.3 mL of a catalytic solution (ammonium hydroxide at pH 9 or hydrochloric acid at pH 3) for 10 min and then the resultant mixture was filtered through a 0.22-pm pore size nylon membrane (MSI, Westboro, MA). At this point, the pregelling step was initiated. The capillaries, preconditioned as indicated above, were filled with TEOS gelling solution using He (the pressure was between 4 and 20 atm, depending on the tube inner diameter). The filled capillaries were sealed with a photosensitive monomeric glue (E308, Loctite, Madrid, Spain) and heated. Temperatures over the range from 40 to 80 "C and heating times between 6 and 16 h were tested. After the capillarywas cooledat room temperature, both ends of the tubing were cut off, and it was emptied by pushing the gelling solution with He at the same pressure used when the capillary was filled. The capillary wall was covered with a layer of gelling solutionwhose thickness could be controlled by modifying the temperature and the time of this pregelling step. The tubing was then heated at 100 "C for 12 h while purging with nitrogen at 4 atm. After heating, the capillary was washed with water for 2 h and dried at 200 "C for at least another 2 h under a nitrogen flow. The porous silica layer obtained was silanized following one of the followingprocedures: (a) the dried capillary was filled with pure ODS at 40-60 "C, hermetically sealed using the Loctite glue, and heated at 180 "C for 12 h; (b) the capillary was filled with a solution of 20% (w/v) ODS in xylene, sealed with the glue, and heated at 120 "C for 12 h. The capillarywas then emptied and washed with acetone for 1 h, with methanol for another hour, installed on the detector, and finally washed with mobile phase until no drift in the baseline was observed. Chromatographic Parameters. The column average internal diameter (d,) was determined by a dynamicmethod%based on the measurement of the retention times of a nonretained solute at five different values of AF' and applying Darcy's law. Estimation error was calculated from the RSD of the five values, making allowancefor errors in the measuring instrument readings, and it was estimated to be f0.03 pm. The average silica layer thickness (df) was calculated by subtracting the values of d, measured before and after formation of the silica layer. The chromatographic retention of the columns was estimated from the capacity factor (k') of a test solute determined with a standard mobile phase. The dead time of the column (t,) was measured from the solvent peak, which did not result in any significant error under the experimental conditions of this work. The experimental values of N were obtained from the width of the peak at half-height. The quality of the columns was evaluated using the reduced plate height (hmin)at uop and the parameters B and C of the Knox equation. The diffusion coefficientof the solute in the mobile phase (D,)was estimated using the Wilke-Chang equation. Columns with d, = 50 pm for which uOpis not experimentally attainable were compared by (26) Krejci, M.; Tesarik, K.; Rusek, M.; Pajurek, J. J . Chrornatogr. 1981,218, 167.
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ANALYTICAL CHEMISTRY, VOL. 65, NO, 11, JUNE 1, 1993
Table I. Experimental Conditions and Chromatographic Characteristics of Silica Layers Formed on 50-pm4.d. Columnsa time temp dr h column precondit (h) ("C) pH (pm) k' (v = 50) 16 80 9 1 12.0 0.00 vapor 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
vapor basic vapor vapor basic basic basic none none basic none vapor
basic basic none basic basic
8 16 8 16 16
8 8 12 12 8 8 8 10
7 8 8 9
40 40 80 40 80 80 40 60 60 80 80 80 80 80 80 60 80
3 3 3 9 3 9 9 9 3 9 9 9 9 9 9 9 9
0.5 2.0 4.5 0.0 14.0 2.0 0.0
0.09 0.12 1.00 0.02 0.00 0.20 0.02
24 31 430 2 5 2
clogged clogged 2.0
0.22
5
clogged 0.5 0.06 6.0 0.00 1.0 0.10 clogged 0.5 0.05 3.5 0.34
4 4
3 29
Mobile phase, water-methanol 60140;solute, ethylbenzene (D, = 0.53 X le5cm2 s-l); silanization, pure ODS,180 "C, 12 h.
using the value of h at u = 50 as reference. The values of B and C were calculated by fitting the experimentaldata points to the Knox equation by the least squares method. For the 50-pm4.d. columns, the data points were fitted to the linear equation h = Cu in lieu of Knox's equation.
RESULTS AND DISCUSSION Optimization of t h e Preparation Method. In order to simplify the optimization procedure, the steps of silica layer preparation and silanization were considered separately. Fused-silica capillary tubing with 50-pm i.d. were employed in this first development phase. (a) Formation of the Silica Layer. The effects of several experimental variables including capillary wall preconditioning, temperature and time of pregelling, and the pH of the catalytic solution on the silica layer chromatographic properties were studied. In all cases, the .same silanization procedure (bonding reaction using pure ODS as described in the Experimental Section) was carried out to graft the CIS moieties to the silica layer in these experiments. The values of the average silica layer thickness (df),capacity factor (k'), and reduced plate height (h) for the columns obtained in several experimental conditions are given in Table I. The columns with no capillary preconditioning became clogged (columns9,10,12, and 16). This result would seem to indicate that the nontreated surface of the silica tubing was not able to support the silica layer prepared on it, so that the layer detached from the capillary wall, once formed, and the silica debris clogged the tube. The capillary preconditioning could increase the number of free silanol groups on its surface,which probably become involved in the first step of the polycondensation reaction anchoring the silica layer to the capillary wall. Longer time and higher temperature during pregelling (columns 1, 6, and 14) gave rise to thick silica layers which exhibited insigificant chromatographic retention values. In such conditions, the layer formed could have low porosity or a pore size too small (
