Anal. Chem. 1988. 60. 1659-1662
part, on the availability, specificity, and thermal stability of the enzyme. The formate dehydrogenase reaction was adapted to a chromatographic method to allow for the determination of formate in natural water samples at submicromolar concentrations in the presence of fluorescent interferences; these interferences limit the sensitivity of batch techniques. However, if the concentrations of formate are fairly high (21p M ) and fluorescent interferences are relatively low, then the method could easily be adapted to a simple batch method (9). ACKNOWLEDGMENT We thank B. F. Taylor and P. J. Walsh for their valuable input into early phases of this research and E. S. Saltzman and D. L. Savoie for assistance with ion chromatographic analyses. Registry No. Formate, 71-47-6; water, 7732-18-5; formate dehydrogenase, 9028-85-7. LITERATURE CITED (1) Barcelona, M. J. Gsochlm. Cosmochim. Acta 1980, 4 4 , 1977-1964. (2) Hordljk, K. A.; Cappenberg, T. E. Appl. fnvlron. Mlcroblol. 1983, 4 6 , 361-369. (3) Gottschalk, G. Bacterial Metabolism, 2nd ed.;Springer-Verlag: New York, 1966. (4) Chameaes, W. L.;Davis, D. D. Nature (London) 1983, 304, 427-429. (5) Mopper, K. Dynamic Processes in the Chemistry of the Upper Ocean; Plenum: New York, 1986;pp 137-157.
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(6) Lawrence, J. F.; Frei, R. W. Chemhl Derivatization in Liqukl Chromatography; Elsevier: Amsterdam, 1976. (7) Dawson. R.; Llebezeit, G. Marine Organic Chemistry; Elsevier: Amsterdam, 1961;pp 445-496. (8) Saltzman, E. S.; Savoie, D. L.; Zlka, R. G.; Prospero, J. M. J . Geophys. Res., C : Oceans 1983, 88, 10987-10902. (9) Boehrlnger Mannhelm. Mthods of Enzymtic Food Ana/ys/s; Boehrlnger Mannheim GmbH Blochemica: Mannheim, West Germany,
1984. (IO) Keene, W. C.; Galloway, J. N. J . Geophys. Res., D : Atmos. 1986, 91 14 466-14474. (11) Schutte, H.; Flossdorf, J.; Sahm, H.; Kula, M. Eur. J . Biochem. 1978, 62,151-160. (12) Sahm, H.; Wagner, F. Arch. Mlkrobbl. 1973, 90, 263-266. (13) Sueter, C. H. A Practical Guide to Enzymology; Why: New York, 1965;pp 1-28. (14) Zika, R. G. Marlne Orgenic Chemktry; Elsevier: Amsterdam, 1981;pp 299-325. -.. ._. (15) Koyama, T.; Thompson, T. 0. J. Oceanogr. Soc. Jpn 1964, 2 0 , 209-220. (16) Parkes, R. J.; Taylor, J.; JorckRamberg, D. Mar. 8/01. 1984, 83, 271-276. (17) Billen, 0.; Jolris, C.; Wijnant, J.; Gillain, G. Estuarine Coasta/,f&r. ~ c i . 1980, 1 1 , 279-294. (18) Hanson, R. 6.; Snyder, J. Mar. Chem. 1979, 7 , 353-362.
RECEIVED for review December 14,1987. Accepted March 9, 1988. This work was supported by the United States Office of Naval Research (N00014-85-C-0020). Ship time was provided by the National Science Foundation (OCE86-13940). Additional funding for graduate student support was provided by the National Oceanographic and Atmospheric Administration.
Preparation and Evaluation of Dry-Packed Capillary Columns for High-Performance Liquid Chromatography Giancarlo Crescenthi,* Fabrizio Bruner, Filippo Mangani, and Guan Yafeng’
Istituto di Scienze Chimiche, Universitci di Urbino, Piazza Rinascimento, 6, 61029 Urbino, Italy
A dry-packing method to prepare fused-slllca columns of 0.250-mm 1.d. packed wlth 5-pm particles Is presented. Several C,, packlng materlals (Spherlsorb ODS1, Spherkrb ODs-2, Hypersil ODs) are studied, and the evaluatlon of column performance Is carried out by means of the reduced plate helghtheloclty equatlon and the separatlon Impedance. A comparison between dry-packed and analogous slurrypacked columns Is also carried out. It Is shown that the dry-packlng method ylelds columns of analogous or better efflclency and requlres a simpler apparatus and a shorter packing time.
Over the past several years much effort has been addressed toward increasing the speed of analysis and efficiency in high-performance liquid chromatography (HPLC), and recent trends have been mostly directed toward the use of narrowbore columns (1-12).In fact, microcolumns, as compared to conventional ones, are essentially characterized by higher efficiencies and lower flow rates and require minimum sample sizes. Thus, they are particularly suitable for the analysis of very complex mixtures and for direct interfacing to mass 1Permanent address: Laboratory of Chromatography, Dalian I n s t i t u t e of Chemistry a n d Physics, Chinese Academy of Sciences, P.O. Box 100, Dalian, People’s Republic of China. 0003-2700/86/0360-1659$01.50/0
spectrometers (13, 14) and flame-based chromatographic detectors (15-17), while providing a great potential for the analysis of biological fluids where the manipulation of very small sample volumes is often required. In addition, the routine use of hazardous or “exotic”solvents is feasible. HPLC microcolumns can be conventionally classified in three categories (18): open tubular (14-24),packed capillaries where the adsorbent is partially embedded into the column walls (3, 9,15,16,25), and narrow-bore slurry-packed columns where the adsorbent is tightly packed as in conventional HPLC columns (4, 5, 8, 10, 11). Though the first two types, in particular the open tubular columns, yield higher efficiencies and shorter analysis time, severe limitations to the full exploitation of their capabilities arise from the stringent requirements imposed on the experimental apparatus. Further, they are difficult to prepare and offer both limited sample capacity and column selectivity (5). Narrow-bore (capillary) columns suffer much less from these restrictions and seem to be at the moment the best compromise between open tubular and short microparticle-packed conventional columns. Fused-silica capillary columns of inner diameters ranging between 0.300 and 0.150 mm and of various lengths, packed with 5- and 3-pm particles, have been succesfully prepared by several groups (6,7,10-12).In all cases a slurry-packing technique has been employed. In fact, the dry-packing technique is usually confined to the preparation of columns containing particles larger than 20 pm, since 0 1966 American Chemical Society
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particles of smaller diameter show a high surface-to-mass ratio and have the tendency to form aggregates. In this paper we show that it is possible to prepare HPLC microcolumns with 5-pm particles by using a dry-packing procedure (26). Narrow-bore (0.250-mm i.d.1 fused-silica columns packed with Spherisorb ODs-1, Spherisorb ODs-2, and Hypersil ODS are described and compared with columns prepared according to a well-established slurry-packing technique. Evaluation of column performance is carried out by using the separation impedance and the reduced plate height/velocity equation. I t is shown that the dry-packing technique may yield columns of analogous efficiency with respect to the slurry-packing methods, while requiring a simplier apparatus and a shorter time.
EXPERIMENTAL SECTION Apparatus and Materials. A Shimadzu LC-5A pump (Shimadzu, Tokyo, Japan) operating in the constant pressure mode and a Jasco Uvidec 100 (Jasco, Tokyo, Japan) were used as delivery pump and UV detector at 254 nm, respectively. A homemade 20-nL cell was connected to the column by a thick-wall Teflon tube (11). A split injector was used, consisting of a Valco EC-14W four-port valve (Valco, Houston, TX) with a 60-nL internal loop, a 1/16-in.Swagelock union tee, and narrow capillary tubing for flow resistance. The split ratio was adjusted to 1:4. The column material was fused silica (Supelco, Bellefonte, PA), while Spherisorb ODS-1, Spherisorb ODS-2 (Phase Separation, Queensferry, U.K.) and Hypersil ODS (Shandon, Runcorn, England) were used as packing materials. Acetonitrile, water, and the other solvents were all of HPLC grade (Carlo Erba, Milano, Italy). The mixture used to test the column contained acetone, benzene, naphthalene, phenanthrene, and pyrene. Acetone was used to measure the dead volume. Injection was performed by turning the valve from the “load” to the “inject” position and back to the ”load” position after 4 s. Column Preparation. (a) Slurry Packing. Columns with an inside diameter of 50 cm X 0.250 mm were packed by following a procedure described in the literature (11,27). After a porous Teflon frit was fixed at the end of the column, the column was slurry-packed by using a Haskel air-driven liquid pump or a Shimadzu LC-5A pump. The packing pressure was initially set at 100 atm and then increased manually by 50 atm every 5 s up to 450 atm. Acetonitrile was used as slurry solvent and pumping medium. (b) Dry-Packing. The packing material was conditioned in a glass container with ethyl alcohol vapors for several hours. A 40-mg sample of the conditioned phase was put into a stainless steel reservoir (5 cm X 2.1 mm i.d.) that was connected on one side to a N2 gas cylinder equipped with a high-pressure regulator and on the other side to the fused-silica capillary containing a porous Teflon frit at its end. Connections were made by means of standard Swagelock reducing unions, and a metal frit was placed at the reservoir inlet. Some droplets of ethyl alcohol were placed on the frit before the reservoir was connected to the Nzcylinder, and the column was placed in an ultrasonic bath. Packing pressure and time depend upon column length, but typically for a 30-cm column, 5-7 atm and 15-20 min are required. EtOH vapors were used to reduce static charges and aggregation. The choice of this compound was based on factors such as polarity, vapor pressure, and toxicity. Columns of lengths up t o 70 cm were prepared according to this technique. All the columns were conditioned with the mobile phase for 4-5 h before use. Reduced plate height h, column permeability K , and column resistance cp were calculated by using the well-known equations introduced by Bristow and Knox (28)(Table I). The plate number has been calculated from the equation N = 5.54t,/ Wllz where t , is the net retention time of the compound and W l l zis the peak width at half peak height.
RESULTS AND DISCUSSION In Figure 1 the variation of the reduced plate height with the reduced velocity for three dry-packed columns of different lengths is reported. A reduced plate height of 2.35 a t the minimum of the curve (Y = 2.3-2.5) is obtained for the 42- and 70-cm columns, while a slightly higher value is obtained for
I
0
10
,
,
,
,
,
30
20
40
I
50
,
,
,
60
,
70
80
Y
Figure 1. Performance of three dry-packed columns of different length: (0)L = 29 cm, (e)L = 42 cm, (0)L = 70 cm; column diameter,
0.250-mm i.d.; packing material, 5-pm Spherisorb ODs-1; mobile phase, acetonitrile/water (3:1); compound, pyrene (k’ = 3.0).
. .
t
0
io
20
I 40
30
1
50
,
60
Y
Flgure 2. Performance of a column (42 cm X 0.25 mm i.d.) drypacked with Spherisorb ODs-1: (A)benzene (k’ = 0.5), (0)naphthalene (k’ = l . l O ) , (0)phenanthrene (k’= 2.1), (e)pyrene (k’ = 3.0); mobile phase, acetonitrile/water (3:1).
the 29-cm column. However, hminvalues are similar to the values reported in the literature for the same material when a slurry-packing technique is used (6-8,II). Typically, columns with (80-90) X lo3theoretical plates/m can be easily prepared. Figure 2 shows the variation of the reduced plate height with the reduced velocity for compounds with different capacity ratios ( k 3. Within the experimental error, practically the same curve is obtained for naphthalene, phenanthrene, and pyrene, while higher and more erratic h values are obtained for benzene (k’ = 0.53), probably because of extracolumn factors. Extracolumn band broadening (18) can be evaluated by means of the equations OZtot = 02c01+ 02ext and 02ext= 02inj 02conn+ 02detwhere is the total variance of the system and Bzcol, OZext, 02, and 02comare variances of the band broadening due to the column, extracolumn, injection system, connection lines, and detector, respectively. From the equations above ( 1 0 , l l )the contribution of extracolumn factors to the total variance (OzeXt/Oztot) at a flow rate of 1.2 pL/min is calculated to be 11%for benzene. In Figure 3 a comparison of a dry-packed and two slurrypacked columns is reported. Column b was slurry-packed by us following the method described in the literature (II),while the data for column c are taken from the same reference. Packing material from the same batch was used for the dryand slurry-packed columns we prepared. The curves show again that the efficiency of the columns prepared with the method described is similar to that of slurry-packed columns. A difference exists with the data from the literature (c) at higher reduced velocities. Since differences in this region can be generally imputed to the C term of the Van Deemter equation, it can be reasonably assumed that the discrepancy
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 17,SEPTEMBER 1, 1988
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Table I. Column Performance Parameters packing material
packing method
Spherisorb ODS-1, 5 pm
col length, cm
(T
(p"
hha
Ea
28 42 70 50
0.714 0.688 0.685 0.732 0.751 0.643 0.662
1272 1545 1436 1145 737 1003 973
2.56 2.35 2.36 2.57 2.47 2.13 2.60
8466 8760 7998 7563 4496 4550 6237
dry packed slurry packed
Spherisorb ODS-2, 5 pm Hypersil ODs, 5 pM
loo*
dry packed dry packed
33 40
+ Cu.
a E = t J / P q; E = P / K = eh2;h = B / v +
*Reference 11.
h
1
4.0
2.0
1 1
LU
1 -0A
' -02
1
~ 0.0
~
" 02
' 04
"
" 0.6
~
' 0.8
B
1
1
I
1
I
1.0
20
3.0
4,O
50
I
I
I
7.0
80
9.0
I
l
1.o
logy
Figure 3. Plot of h vs log v for three columns: (a)dry packed (42 cm X 0.250 mm i.d.); (b) slurry packed (49 cm X 0.250 mm i.d.); (c) slurry packed (77) (1.0m X 0.250 mm i.d.). Packing material, compound, and mobile phase are as in Figure 1.
observed is due to batch-to-batch variations of the packing material. In an attempt to clarify this point and to learn if the dry-packing technique could be of general use, other columns were packed with Spherisorb ODS-2 and Hypersil ODS. Figure 4 shows the reduced Van Deemter curve for (a) naphthalene, (b) phenanthrene, and (c) pyrene, obtained on Spherisorb ODS-1, Spherisorb ODS-2, and Hypersil ODs. It can be seen that with Hypersil ODs, lower h values are obtained in the region of high reduced velocities with respect to Spherisorb ODS-1. Spherisorb ODs-2 shows even lower h values, not only in that region but for the whole range of reduced linear velocity. hminis equal to 2.04-2.07 for naphthalene and 2.10-2.20 for phenanthrene and pyrene in the v range of 2.2-4.8, so the analysis shows that time can be significantly reduced without affecting the resolution. Further, it can be safely assumed that the discrepancies observed in Figure 3 are due to batch-to-batch packing material. Overall, Spherisorb ODs-2 yields the best results with regard to the packing conditions used and to the test mixture chosen, but this does not mean that is a better packing material than the others. By properly changing the packing conditions, one might obtain similarly good columns with the other materials. In Figure 5 a typical chromatogram of the test mixture obtained with Spherisorb ODs-2 is shown. The column is 32.6 cm long, and 32 000 theoretical plates for naphthalene and 31,000 for phenanthrene and benzene are obtained at a flow rate of 1.5 pL/min (v = 2.8). Table I shows the column performance parameters for all columns tested. The total porosity 9 is the sum of the inter- and intraparticle porosities and can be calculated from the equation ET = F,t,/?L where F, is the volumetric flow rate and r the column radius. Spherisorb ODs-2 and Hypersil ODS show the lowest tT values. Where Spherisorb ODS-1 is concerned, we obtain consistently higher 9 values with either the dry or the slurry technique, which in conjunction with the excessive E values, may indicate the presence of a high proportion of fines in the column packing. High cp and E values may be also due to the
0
6.0
Figure 4. Performance of three dry-packed columns: (0)Spherisorb ODS-2, 5 pm (33cm X 0.250 mm i.d.); (0)Hypersil ODS, 5 pm (40 cm X 0.250 mm i.d.); (H) Spherisorb ODS-1,5 pm (42 cm X 0.250
mm i.d.). Compounds: (a)naphthalene, (b) phenanthrene, (c)pyrene; mobile phase, acetonitrile/water (3:l). A.U.F.S
i3
0
10
30
20
40
50
min
Flgure 5. Chromatogram of the test mixture obtained on a 33 cm X 0.250 mm i.d. column dry-packed with Spherisorb ODS-2, 5 pm. Mobile phase, acetonitrile/water (3:l);flow rate, 1.5 pL/min; (1)acetone, (2) benzene, (3)naphthalene, (4)phenanthrene, (5)pyrene.
Teflon frit, to the breaking of the particles near the frit surface and/or a possible collapse of the frit structure. Spherisorb ODs-2 and Hypersil ODS show similar tT and (c values, but the reduced plate height and consequently the column performance E are consistently lower for the former. Finally, the E values for the dry-packed Spherisorb ODS-2 and the slurry-packed Spherisorb ODS-1 are the same. All the columns were retested after 1 and 2 months of storage, and no appreciable change in their chromatographic properties was found. In conclusion, a dry-packing technique for the preparation of fused silica packed columns with 5-pm particles has been
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described, which requires a much simpler and less costly apparatus than the traditional slurry-packing technique. Very similar and in some cases better efficiencies are obtained, while the overall packing time is much shorter (=1 h). Another advantage is that the columns can be stored under dry conditions before being used. Further studies are needed to evalute the method capabilities with regard to 3-pm particles and to phases other than CIS. ACKNOWLEDGMENT We thank A. Fabbri for experimental assistance. LITERATURE CITED Scott, R. P. W.; Kucera. P. J . Chromatogr. 1978, 125, 251. Ishii, D.; Asai, K.; Hibi, K.; Jonokuchi, T.; Nagoya, M. J . Chromatogr. 1977, 144, 157. Tsuda, T.; Novotny, M. Anal. Chem. 1978, 5 0 , 271. Takeuchi, T.; Ishii, D. J . Chromatogr. 1980, 190, 150. Yang, F. L. J . Chmmtogr. 1982, 236, 265. Hkata, Y.; Jinno, K.; HRC CC,J . High Resolut. Chromatogr . Chroma togr. Commun. 1982, 5 . 102. Takeucki, T.; Ishii, D. J . Chromatogr. 1982, 238, 402. Gluckmann J. C.; Hlrose, A.; McGuffin. V. L.; Novotny, M. Chromatographia 1983, 17, 303. Tsuda, T.; Tanaka, I.; Nakagowa, G. Anal. Chem. 1984, 56, 1249. Konishi, M.; Mori, Y.; Takeuchi, A. Anal. Chem. 1985, 5 7 , 2235.
-
(11) (12) (13) (14) (15) (16)
(17)
(18)
Borra, C.; Han, M. H.; Novotny, M. J . Chromatogr. 1987, 385, 75. Borra, C.; Wisler. D.; Novotny, M. Anal. Cbem. 1987, 59, 339. Tsuda, T.; Keller, G.; Stan, H. J. Anal. Chem. 1985, 5 7 , 2280. Alborn, H.; Stenhagen, G. J . Chromatogr. 1985, 323, 47. McGuffin, V. L.; Novotny, M. Anal. Chem. 1981, 5 3 , 946. McGuffin, V. L.; Novotny, M. J . Chromatogr. 1981, 218, 179. Gluckman, J. C.; Novotny, M. J . Chromatogr. 1985, 333, 291. Novotny, M. In Microcolumn High-Performance Liquid Chromatogfa phy; Kucera, P., Ed.; Elsevier: Amsterdam, Holland, 1984; pp
-
194-259. (19) Ishii, D.; Taeuchi, T. J . Cbromafogr. Sci. 1980, 18, 462. (20) Tijssen, R.; Bleumer, J. P. A.; Smith, A. C. C.; Van Kreveld. M. E. J . Chromatogr. 1981, 218, 137. (21) Yang, F. J. J . Chromatogr. Sci. 1982, 2 0 , 241. (22) Jorgenson J. W.; Guthrie. E. J. J . Chromatogr. 1983, 255, 335. (23) Tsuda, T.; Nakagawa, G. J. J . Chromatcgr. 1983, 268, 369. (24) Takeuchi, T.; Ishii, D. J . Chromatogr. 1983, 279, 439. (25) Hirata, Y.; Novotny, M.; Tsuda, T.; Ishii, D. Anal. Chem. 1979, 5 1 , 1807. (26) Bruner, F.;Crescentini, G.; Mangani, F.; Yafeng, G. Presented at the 8th International Symposium on Capillary Chromatography, Riva del Garda, Italy, May 19-21, 1987. (27) Hirata, Y.; Jinno, K. HRC CC,J . Higb Resoluf.Chromafogr. Chromatogr. Commun. 1983, 6.196. (28) Bristow, P. A.; Knox. J. H. Chromatographia 1977. T U , 279.
RECEIVED for review August 12,1987. Accepted March 1,1988. This work has been carried out with the financial support of the Ministry of Public Education.
Separation Efficiency of Slurry-Packed Liquid Chromatography Microcolumns with Very Small Inner Diameters Karl-Erik Karlsson
Analytical Chemistry Department, Pharmaceutical Research and Development, A B Hassle, S 43183, Molndal, Sweden Milos Novotny* Department of Chemistry, Indiana University, Bloomington, Indiana 47405
Fused silica caplllarles of different dlameters were slurrypacked wlth an ldentlcal sorptlon material and evaluated chromatographically. The extent of band broadenlng was evaluated fluorometrlcally at dwerent column lengths, together wlth poroslty measurements obtained from a fluorescencequenchlng marker. Klnetlc analysls of the plate-helght vs veloclty data reveals that the Inner dlameter Is a very lmportant varlable In the preparatlon of such packed columns. Extremely hlgh efflclencles were obtained wlth columns of the smallest diameter (44 pm).
Microcolumn liquid chromatography (LC) continues to attract considerable attention because of the unique detection and application possibilities that various miniaturized columns facilitate (1-4). Among various microcolumn types, slurrypacked capillary columns (5-7) are becoming increasingly attractive due to their capabilities in resolving complex mixtures (8-10) and in effecting a relatively easy coupling to miniaturized detectors (2, 11, 12), capillary gas chromatography (13), and mass spectrometry (14). The current success of slurry-packed capillaries can be largely attributed to their reproducible technologies. Fused silica capillaries are typically used because of their flexibility and easy formation of an on-column optical cell (6,11) through removal of the polymeric overcoat. In a typical column
packing procedure, 200-300 pm i.d. fused silica tubing, approximately 1 m long and terminated with a frit (6, 15), is packed under pump pressure with a suitable slurry of the column sorption material and solvent. High reproducibility of the column packing procedures has been recently demonstrated with reversed-phase materials (16 ) , adsorbents and polar stationary phases (17),and the columns of interest to biomacromolecular separations (18). This article provides further insights into the microcolumn separation processes, through a unique experimental design in which different segments of a slurry-packed capillary have been evaluated for their contributions to band broadening and porosity variations. The role of column diameter in its performance characteristics is strongly suggested. The results are contrary to the common view that particle size is the sole unique characteristic of a totally packed microcolumn. The van Deemter curves obtained with the smallest diameter (44-pm i.d.) used in this work are likely to represent a “kinetic record” for a packed LC column a t this time. EXPERIMENTAL SECTION Apparatus. A Shimadzu LC-5A pump was used both for the mobile-phase delivery (acetonitrile,at constant pressure) and for the column packing procedure. The detector, a Kratos FS 970 LC fluorometer, was modified for on-column detection with an entrance slit width of 0.5 mm. The slit was mounted directly on the 2 7 ~steradian mirror. New holes were drilled through the cell housing, which allowed the
0003-2700/88/0360-1662$01.50/00 1988 American Chemical Society
