Anal. Chem. 1993, 65, 1615-1621

Preparation of Open Tubular Columns for High-Performance Liquid Chromatography

1815

Reversed-Phase

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 pH- fixed 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 dem- onstrated 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 chroma- tography (HPLC) has been theoretically demonstrated.z4

The packing materials used in reversed-phase HPLC (RP- HPLC) 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 high- yield reaction conditions, gives rise to phase ratio values around 350 times smaller than those of commercial RP-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.;

(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.

Academic Press: New York, 1958; pp 1-13.

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.,5 Ishii 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-kV dc) 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 improve- ment 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(dimethyloctadecy1si- loxane) (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.

(7) Ishii, D.; Tsuda, T.; Takeuchi, T. J. Chromatogr. 1979, 185, 73. (8) Tsuda, T.; Tsuboi, K.; Nakagawa, G. J. Chromatogr. 1981, 214,

(9) Jorgenson, J. W.; Guthrie, E. J. Chromatogr. 1983,255, 335. (10) Takeuchi, T.; Kitamura, H.; Ishii, D. HRC & CC, J . High Resolut.

(11) Jorgenson, J. W.; Guthrie, E. J.; St. Claire, R. L., 111, J. Pharm.

(12) Fabrot, A.; Folestad, S.; Larsson, M. HRC & CC, J. High Resolut.

(13) Folestad, S.; Larsson, M. In 8th International Symposium on

(14) Dluzneski, P. R.; Jorgenson, J. W. HRC & CC, J. High Resolut.

(15) van Berkel, 0.; Kraak, J. C.; Poppe H. J. Chromatogr. 1990,499,

(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.

(18) Tock, P. P. H.; Boshoven, C.; Poppe, H.; Kraak, J. C.; Unger, K.

(19) Halasz, I. 2. Anal. Chem. 1968,236, 15.

Chromatogr. 1979,175, 105.

283.

Chromatogr. Chromatogr. Commun. 1983,6,666.

Biomed. Anal. 1984, 2, 191.

Chromatogr. Chromatogr. Common. 1986, 9, 117.

Capillary Chromatography; Sandra, P., Ed.; 1987; Vol. 2, p 1112.

Chromatogr. Chromatogr. Commun. 1988,11, 322.

345.

K. Chromatographia 1987, 24, 617.

K. J. Chromatogr. 1989,447, 95.

0003-2700/93/0365-1615$04.00/0 0 1993 American Chemical Society

1616 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. How- ever, the method reproducibility was poor and it was unsuitable for preparing columns with 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 silanol groups (up to 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 polycon- densation of tetraethyl orthosilicate (TEOS),21 also used to produce porous silica packings for HPLC.22-24 Although a similar reaction was already used by Tock et al.17J3to prepare OTCs, several modifications were carried out in the present work to achieve poly(ethoxysi1ane) formation inside small- diameter 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 CIS bonded 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

(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,

(24) Unger, K. K.; Scharf, B. J. Colloid Interface Sci. 1976, 55, 377.

97-234.

253, 658.

v/h

121

O Y , I I I , , 0 5 10 15 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 com- pounds 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 (2) h + Bu-' + C

The limit of the function u/h when u - is 1/C. Figure 1 depicts variations of u/h with 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

1 - =

ANALYTICAL CHEMISTRY, VOL. 65, NO. 11, JUNE 1, 1993 1617

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 3uOp can 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

1 + 6 k f + llk'' and c,= 2kf -- d? D m c, = 96(1+ k')2 3(1+ k')' 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 pulse- driven stopped-flow injection (PSI) system, designed by Manz and Sim0n,~5 may 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 fused-

silica tubings of 5-, lo-, and 50-pm nominal i.d. (Polymicro Technologies), tetraethyl orthosilicate (TEOS), ethanol, ammo- nium hydroxide (all from Merck, Darmstadt, Germany), and dimethyloctadecylchlorosilane (Fluka, Buchs, Switzerland). Pol- yaromatic hydrocarbons (Fluka) dissolved in methanol or me- thylene 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.

different injection systems 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 injection loop) with a homemade splitter 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 Instru- mentos S.A., Barcelona, Spain) manometer (maximum pressure 10 atm, precision f0.25%).

Column Preparation. In the preconditioning step, the fused- silica capillaries were subjected to one of the alternative treat- ments: (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 (E- 308, 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 cooled at 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 solution whose 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 following procedures: (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 capillary was 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 in- ternal diameter (d,) was determined by a dynamic method% 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 allowance for 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 coefficient of the solute in the mobile phase (D,) was estimated using the Wilke-Chang equation. Columns with d, = 50 pm for which uOp is not experimentally attainable were compared by

(26) Krejci, M.; Tesarik, K.; Rusek, M.; Pajurek, J. J . Chrornatogr. 1981,218, 167.

1618 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)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

vapor vapor basic vapor vapor basic basic basic none none basic none vapor basic basic none basic basic

16 80 9 8 40 3 16 40 3 8 80 3 16 40 9 16 80 3 8 80 9 8 40 9 12 60 9 12 60 3 8 80 9 8 80 9 8 80 9 10 80 9 7 80 9 8 80 9 8 60 9 9 80 9

12.0 0.00 0.5 0.09 2.0 0.12 4.5 1.00 0.0 0.02 14.0 0.00 2.0 0.20 0.0 0.02 clogged clogged 2.0 0.22 clogged 0.5 0.06 6.0 0.00 1.0 0.10 clogged 0.5 0.05 3.5 0.34

24 31 430 2

5 2

5

4

4

3 29

Mobile phase, water-methanol 60140; solute, ethylbenzene (D, = 0.53 X le5 cm2 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 experimental data 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 the 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 precondi- tioning, 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 (columns 9,10,12, 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 polycon- densation 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 (<60 A), preventing the formation of a tightly bonded ODS so that the amount of C18 chains bonded to the silica layer was very small. If the reaction was carried out a t lower temperatures (columns 3 and 5), different results were obtained depending on the pH solution. Short

~~~~ ~

(27) Staroverov, S. M.; Fadeev, A. Yu. J. Chromatogr. 1991,544, 77.

pregelling time and lower temperatures (columns 2 and 8) yielded excessively thin layers with low rentention values.

The effect of catalytic solution pH on the layer's chro- matographic characteristics is clearly demonstrated in Table I. None of the columns prepared in an acidic medium (columns 2-4 and 6) presented good efficiency although the layer thickness and the k' for these columna were higher than those obtained at pH 9. This result might be due to the fact that, at acidic pH, the hydrolysis reaction of TEOS predom- inated over the polycondensation reaction, giving rise to silica patches on the capillary wall which could have an inadequate pore size to bond CIS moieties. On the contrary, under alkaline conditions, although the reaction is somewhat slower than at acidic pH, the polymerization of TEOS predominates, pro- ducing a continuous, more porous layer suitable for a high- yield reaction with chlorosilane derivatives.

The type of capillary preconditioning treatment plays an important role in the chromatographic features of the columns obtained by this procedure. This is well illustrated by columns 7, 11, and 13, for which the gelling reaction was carried out at 80 "C and pH 9. These gelling conditions seemed to be a good compromise of time and pH for achieving high- efficiency columns. The two first columns, which were preconditioned using basic solution, presented rather good retention and efficiency values while the third column, which was preconditioned using water vapor, had very small retention values. This behavior could be related to the fact that the alkaline attack of the silica surface not only hydrolyzes siloxane bonds but it could also dissolve the tubing surface, increasing its heterogeneity.

In conclusion, the best conditions for silica layer formation seemed to be those used in the preparation of columns 7 and 11. Columns 15, 17, and 18 showed that varying these conditions worsen the method results. Specifically, lowering the temperature (17) brought about a sharp reduction in the layer thickness and retention whereas increasing (18) or decreasing (15) pregelling time either decreased the efficiency or reduced the capacity factor dramatically. (b) Silanization. In order to optimize the silanization

reaction for the capillary columns, 10 columns were prepared using the conditions of layer formation used for columns 7 and 11. Five of the columns were silanized using ODS dissolved in xylene, and the other five were silanized using pure ODS as indicated in the Experimental Section. Re- tention and efficiency tests for all the columns were performed as indicated in Table I. The columns silanized with a xylene solution of ODS gave rise to k' = 0.10 (RSD = 18.7%) and h = 3.44 (RSD = 10.5% 1, whereas the columns silanized using pure ODS brought about k' = 0.19 (RSD = 9.4% ) and h = 4.72 (RSD = 10% ). The difference in the value of k' for the two groups of columns indicates that use of pure reagent and somewhat higher silanization temperature yields a higher surface coverage of the silica layer by the C I ~ moieties. The different values of h observed between the two column groups could be due to the differences of k' obtained for the ethylbenzene (the test solute) in both column groups. The 12-h time period was sufficient for completion of the silanization reaction because experiments using longer re- action times (24 h) or successive silanization reactions did not yield significant increases in the retention of the standard.

Method Application to Smaller Diameter Columns. As discussed in the Theoretical Section, a better plate number and shorter analysis time can be obtained using OTCs with smaller inner diameters (510 pm). First, OTC preparation of 10-pm4.d. columns using exactly the same protocol developed for the 50-pm columns was attempted. The four columns prepared by the above-mentioned method became clogged during the silica layer preparation step. It was considered that, by decreasing the pregelling time, the less viscous gelling solution resulting could be easily pushed out

ANALYTICAL CHEMISTRY, VOL. 65, NO. 11, JUNE 1, 1993 1619

Table 11. Chromatographic Parameters for 10-rm4.d. Columns column preael time (h) total attempts success rate (%) dp (pm) L (cm) dCb (pm) k’ c u (opt) h (min) B C

series A 1 7 8 25 0.72 100 9.56 0.24 10.3 0.56 2.87 0.0273 2 0.67 120 10.00 0.21 10.8 0.58 3.11 0.0268

series B 3 4 5 6 7 8

X d RSDd (%)

6.5 8 75

0.24 190 10.72 0.13 12.8 0.40 2.49 0.0143 0.26 172 10.68 0.13 13.4 0.39 2.72 0.0151 0.35 151 10.70 0.16 12.6 0.43 2.69 0.0169 0.21 175 11.08 0.12 14.8 0.38 2.78 0.0138 0.32 166 10.96 0.14 13.1 0.38 2.46 0.0153 0.36 156 10.88 0.17 12.5 0.42 2.60 0.0167

0.14 13.2 0.40 2.62 0.0154 13.7 6.4 5.2 4.9 8.1

a Layer thickness error: i O . 0 6 pm for series A, f0.05 pm for series B. Internal diameter error, zk0.03 pm. Solute, phenanthrene. Mobile phase, water-methanol 40/60 (v/v). Values for series B only.

Table 111. Chromatographic Parameters for 5-pm4.d. Columns column pregel time (h) total attempts success rate (%) d p (pm) L (cm) dCb (pm) k’ u (opt) h (min) B C

series A 1

series B 2 3 4 5

series C 6 7 8 9 10 11

Xd RSDd (%)

7 6 17 0.66 20 4.08 2.2 5.8 1.62 4.69

0.23 35 4.94 0.65 8.3 0.81 3.36 6.5 8 50 0.30 30 4.60 0.80 8.3 0.85 3.52

0.32 33 4.76 0.82 8.3 0.86 3.59 0.27 45 4.66 0.70 8.5 0.80 3.42

6 8

0.14 100 5.12 0.30 11.9 0.46 2.76 0.10 110 5.20 0.24 12.2 0.44 2.70

75 0.16 80 5.08 0.31 10.7 0.45 2.44 0.09 86 5.16 0.23 11.5 0.41 2.38 0.12 83 4.92 0.26 12.0 0.45 2.72 0.12 120 4.80 0.30 11.7 0.46 2.72

0.1392

0.0493 0.0517 0.0521 0.0471

0.0194 0.0183 0.0212 0.0179 0.0189 0.0198

0.27 11.7 0.45 2.62 0.0193 12.6 4.6 4.2 6.3 6.2

Layer thickness error: f0.06 pm for series A and B, f0.04 pm for series C. Internal diameter error: f0.03 pm for series A and B, zk0.02 pm for series C. Solute, phenanthrene. Mobile phase, water-methanol 40/60 (v/v). Values for series C only.

of the 10-pm capillaries by using a moderate (between 4 and 20 atm) pressure of He. The success percentage and the chromatographic characteristics of the columns prepared using pregelling times of 7 (series A) and 6.5 h (series B) are summarized in Table 11. The success rate for 10-pm columns prepared with a pregelling time of 6.5 h was quite high. Columns with good chromatographic characteristics (h = 0.4) were also obtained. The same procedure was used to adapt the preparation protocol to 5-pm4.d. OTCs. A significant success rate (75%) was obtained when the pregelling time was decreased to 6 h (Table 111). Using such a short time, OTCs of good efficiency (hmin = 0.45 at uop = 11.6) with reasonable retention (k’ = 0.27) were obtained.

It is interesting to note that a pregelling time of 6.5 h (series B in Tables I1 and 111) gives rise to similar layer thickeness (df = 0.3 pm) regardless of the inner diameter of the column, while the values of k’ were around 4 times higher in the 5-pm columns than in the 10-pm columns, as would be expected from geometrical considerations (phase ratio), assuming that the silica layers have the same structure and chromatographic behavior in both cases. Although the 5-pm-columns in series C (Table 111) have a layer thickness about half that of the 10-pm columns in series B (Table 11), they exhibit a higher retention value than 10-pm columns. This result was expected since the internal volume of 5-pm columns is one-fourth that of the 10-pm columns: the phase ratio is 2 times larger. These results suggest that, to increase retention values, decreasing column d i h e t e r is better than increasing layer thickness.

Pregelling time appears to be an easy to manipulate variable to adapt the proposed method for preparing columns of different diameters. Pregelling time should become shorter as column diameter decreases so that the resulting silica layer’s

thickness remains about one-twentieth the column radius. The method would, in principle, be applicable to columns with inner diameters smaller than 5 pm. This point is currently under investigation in our laboratory.

Column Reproducibility. Tables I1 and I11 give the experimental values for some chromatographic parameters representing the quality of the 10- and 5-pm-i.d. OTCs, respectively. A quite acceptable reproducibility for both column retention (RSD = 12-14% ) and column performance (RSD = 4-5 % ) was obtained using the proposed preparation method. The experimental data were fitted to the general and the simplified (A = 0) form of the Knox equation. The column behavior displayed a better fit to the latter form, as predicted by theory. The values of B obtained were greater than the theoretical values of 2, maybe because they were calculated from a set of experimental values where only a few flow velocities lower than uop were used. Figure 2 gives the data points for h against u for 10-pm columns of series B (Table 11) as well as drawing of the Knox curve calculated using average values for B and C (Table 11). The fits of the experimental values for each of the columns to the Knox curve were quite satisfactory. A similar plot for six 5-pm columns is given in Figure 3.

Performance Values. In order to assess the role played by the thickness of silica layer on the efficiency of the OTCs prepared by this method, we have compared the three series of 5-pm columns obtained in this work in terms of mean efficiency and mass-transfer capacity. To establish a fair comparison, the composition of the mobile phase was modified so that approximately the same value of k’ was obtained for the solute test in the columns of the three series. The plots of the mean reduced plate height for the columns of each

1620 ANALYTICAL CHEMISTRY, VOL. 65, NO. 11, JUNE 1, 1993

h

1.6 /

Table IV. Comparison of Some Theoretical and Experimental Performance Data for Column C-1 la

mobile phase 30/70b mobile phase 40/6W

params theor exP theor exp k' 0.10 0.30 B 2 2.39 2 2.72 C 0.015 0.017 0.023 0.020

(opt) 11.7 11.0 9.3 11.7 h (min) 0.34 0.40 0.43 0.46 NItR (U = uOp) 706 596 332 462 NltR (U = 3uOp) 1256 1007 591 828

Solute, phenanthrene. Mobile phase, water-methanol. D, = 0.52 X cm2 8-l. D, = 0.46 X le5 cm 5-l.

,

0.8 i

O e 6 1 0.4

Om2 0 i 0 10 20 30 40 50 60 70 80 90 100

V

-Averag. + 8.3 X 8.4 8 -5 x B-6 * 8-7 A 8-8

Figure 2. Plot of h agalnst u for the OTC 10-fim4.d. column, series 6 solute, phenanthrene (k' = 0.14); mobile phase, water-methanol (40160, v1v). For the meaning of the polnts and the continuous Ilne, see text.

h

2.5 1

1.5

0 0 10 20 30 40 50 60 70 60 BO 100

V

-Average + C-6 % C.7 C-8 x c-9 c-10 A c-11

Figure 9. Plot of h agalnst u for OTC 5-pm-1.d. column, series C; solute, phenanthrene (k'= 0.27); moblle phase, water-methanol (401 60, v1v). For the meaning of the points and the continuous Ilne, see text.

h

/ 7 1

O i ' ' " " ' I 0 10 20 30 40 50 60 70 80 90 100

V C-6 + C-7 C-8 c-9 x c-10 c-11

A A-1 X 8-2 8-3 8-4 #S B-6 Flgure 4. Plot of average value of h agalnst u for the three 5-fim cokrmnserlesshown InTabie 111. Themobilephase(water-methanol) composltlon was adjusted In order to obtaln a slmllar k' (-0.26) for the test solute (phenanthrene) In all of them: serles A, 12/88 (v1v); serles B, 30170 (v/v); series C, 40160 (v/v). series against the reduced velocity are given in Figure 4. The best efficiencies were obtained for series C, whose columns had the smallest layer thickness values (Table 111). These columns also presented the smallest values of C, which indicates that they have much better mass transfer than the other 5-pm columns. Consequently, as theory predicts (eq 31, greater values of PGV (faster columns) can be obtained with series C columns than with those of the other two series.

2

O,OOOI A.U. I

10 IS 14 I2

Time ( minutes FIgum5. Hlgh-effidencyseparatianofPHAs. Solutes: (l)naphthalene, (2) biphenyl, (3) fluorene, (4) phenanthrene, (5) anthracene, (6) flwxanthene. Column length, 120 cm; Ld., 5 m; statlonary phase, bonded CIS; mobile phase, waterlnethanol(30170, vlv); flow rate, 3 nL1mIn; inlet pressure, 27 atm; sample volume, 10 pL.

The theoretical value of C, calculated from the Golay equation for k' = 0.26 is 0.022. The values of C obtained experimentally for series B and C columns are very close to this value (Table III), and the small differences observed could be due to experimental errors. For columns of series A, however, the difference was significant, which indicates that at high layer thickness values, the term C, plays an important role on efficiency.

Table IV compares some performance values calculated using the Golay equation and eqs 1 and 3 for column C 11 (Table 111), neglecting the contribution of the stationary phase, with the values obtained experimentally. There is good agreement between both values. The small differences observed between them could be attributed at fist glance to the contribution of the extracolumn effects and the stationary phase (C, term) to peak broadening. However, it should be noted that the experimental values for N / ~ R are greater than the theoretical ones at higher values of k' (mobile-phase composition, 40/60), where the contribution of the stationary phase should have been the greatest of the two condition studied. Therefore, it seems that the differences observed between the theoretical and the experimental values of the parameter studied could be due to experimental errors and/

ANALYTICAL CHEMISTRY, VOL. 65, NO. 11, JUNE 1, 1993 1621

velocity was around ~ O U , , and column was only 30 cm long. To achieve 12 500 plates in 57 s, as was the case for the phenanthrene peak in Figure 7, with a good PC (A = 2, B = 1.5, C = 0.02, d, = 3 pm, 4 = 700) for the same value of k’ would require a ulh ratio of 7.7 (eq l), which according to the Knox equation is attained a t u = 78 and h = 10.12. The velocity of the mobile phase in such a case would be 1.2 cm s-l, and the column length needed to obtain 12 500 plates would be 38 cm. Under these conditions, the pressure needed to achieve the separation would be approximately 3.5 X 104 atm.

A decrease in efficiency was observed in our columns by increasing k’, as predicted theoretically. Gradient separations could be expected to be more appropriate than isocratic separations in this type of column. However, in our opinion, the main problem for the widespread use of 5- and sub-5-pm columns is the unavailability of sufficiently sensitive detection systems able to match the requirements of illuminatedvolume and time constant required by the small-diameter OTCs. This is the reason why columns with an i.d. < 5 pm could not be tested even though, according to theoretical calculations, the most suitable internal diameter values can be expected to be in the range of 1-3 pm.

5

.j

5 4 3 2 I O

Time ( minutes )

Flguro 6. Separation of PHAs at maxlmum value of PGV flow rate, 9 nL/mln (close to 3u,,); inlet pressure, 82 atm. Solutes and other conditions as In Figure 5.

5

4

3

T

1 o,oooi A.U.

I 60 40 20 d

Time ( seconds Flguro 7. Fast Separation of PHAs. Solutes: (1) naphthalene, (2) biphenyl, (3)fluorene, (4) phenanthrene, (5) anthracene. Column length, 30 cm; l.d., 5 pm; stationary phase, bonded CIS; mobile phase, water- methanol (40/60, v/v); flow rate, 40 nL/mln; Inlet pressure, 10 atm; sample volume, 10 pL.

or errors in the calculation of the diffusion coefficient (&). These resulb also suggest that, in our experimental conditions, the contribution of the equipment and the mass-transfer resistance from the stationary phase to the band broadening were negligible in first approximation.

The high efficiency which can be achieved with OTCs is well illustrated by Figure 5, which presents the separation of a polyaromatic hydrocarbons test mixture carried out with a 5-rcm column (C 11) at flow rate close to uOp. The efficiency measured in any peak of the chromatogram is around 0.5 X lo6 plates m-l. Figure 6 depicts the same separation at flow rate close to 3uOp in order to obtain the maximum value of PGV. In these conditions, lo00 plates s-1 has been obtained for the separation.

Using short OTCs, fast separations can be obtained with a small inlet pressure. This is illustrated in Figure 7, where a good resolution of polyaromatic hydrocarbons is achieved in 60 s using a 5-pm column. In this separation the reduced

ACKNOWLEDGMENT

This work was supported by CICYT (Project PB 88-034). The authors gratefully acknowledge M. van Tilburg (Valco Europe) for the loan of a prototype of the PSI system. Thanks are given to Dr. J. L. Oteo (Instituto de Ceramica y Vidrio, C.S.I.C.) for fruitful discussion about silica gel preparation. The authors further thank Mrs. C. Marin PBrez and Mr. J. M. Campos for technical assistance.

GLOSSARY coefficient of the Knox equation coefficient of the Knox equation coefficient of the Knox equation (C = C , + C,) mobile-phase plate height coefficient stationary-phase plate height coefficient particle size in PC or column internal diameter in

column internal diameter silica layer thickness particle size in PC diffusion coefficient of the solute in the mobile phase diffusion coefficient of the solute in the stationary

reduced plate height (h = H/d) permeability of the column capacity factor column length number of theoretical plates column pressure gradient resolution dead time retention time linear velocity of mobile phase selectivity viscosity of the mobile phase reduced velocity of the mobile phase column flow resistance

OTC

phase

RECEIVED for review October 14, 1992. Accepted February 12, 1993.