In-Depth Characterization of Slurry Packed Capillary Columns with 1.0

Anal. Chem. 2004, 76, 5777-5786

In-Depth Characterization of Slurry Packed Capillary Columns with 1.0-µm Nonporous Particles Using Reversed-Phase Isocratic Ultrahigh-Pressure Liquid Chromatography Kamlesh D. Patel,† Anton D. Jerkovich,‡ Jason C. Link, and James W. Jorgenson*

Department of Chemistry, University of North Carolina at Chapel Hill, Venable Hall, CB#3290, Chapel Hill, North Carolina 27599-3290

Fused-silica capillary columns packed with 1.0-µm nonporous C18 bonded particles are evaluated with isocratic ultrahigh-pressure liquid chromatography (UHPLC). Improved UHPLC techniques have demonstrated column efficiencies as high as 730 000 plates/m and run pressures over 6800 bar (100 000 psi) for packed 10-µminner diameter (i.d.) columns. Columns as large as 150 µm have been tested with UHPLC and show no flowinduced heating effects on separation efficiencies. van Deemter plot analysis for column i.d.s ranging from 10 to 150 µm shows an increase in column efficiency with a decrease in column i.d.. Reduced parameter analysis further illustrates a decrease in reduced parameter A term and C term values with decreasing i.d. However, reduced parameter C term values for columns evaluated with UHPLC are an order of magnitude larger than C term values for larger particles at conventional pressures. Retention factors for moderately retained compounds are observed to increase with column i.d., suggesting an increase in packing density. Highly ordered packing arrangement at the column wall is seen for packed beds extruded from large-diameter columns. The use of small stationary-phase spherical supports, sized 1.5 µm in diameter and below, is gaining popularity in capillary chromatography. Theory predicts an increase in efficiency with decreasing particle size due to reduced eddy diffusion and resistance to mass-transfer contributions to band broadening.1 The commercial availability of highly monodisperse, nonporous silica particles has enabled improved efficiencies in both capillary electrochromatography and liquid chromatography (LC).2-11 With conventional LC systems, the use of small particles has been * To whom correspondence should be addressed. E-mail: [email protected]. † Current address: Sandia National Labs, P.O. Box 969, Livermore, CA 94551. ‡ Current address: Novartis, Bldg. 401 A220, One Health Plaza, Rt. 10, East Hanover, NJ 07936. (1) Snyder, L. R.; Kirkland, J. J. Introduction to Modern Liquid Chromatography, 2nd ed.; John Wiley & Sons: New York, 1979. (2) MacNair, J. E.; Lewis, K. C.; Jorgenson, J. W. Anal. Chem. 1997, 69, 983989. (3) MacNair, J. E.; Opiteck, G. J.; Jorgenson, J. W. Rapid Commun. Mass Spectrom. 1997, 11, 1279-1285. (4) MacNair, J. E.; Patel, K. D.; Jorgenson, J. W. Anal. Chem. 1999, 71, 700708. 10.1021/ac049756x CCC: $27.50 Published on Web 09/02/2004

© 2004 American Chemical Society

limited to short, fast separations of simple mixtures.3,5,8,12,13 To work with longer columns, pumps that can achieve higher pumping pressures than conventional pumping systems are required. The pressure drop (∆P) necessary to flow mobile phase at the optimum linear velocity (uopt) through the packed bed increases at a rate inversely proportional to the particle diameter cubed, assuming constant mobile-phase viscosity and column length.1,2 For instance, flow at uopt requires 125 times greater liquid pressure at the head of a column packed with 1-µm particles than for a column of equal length packed with 5-µm particles. The benefits are a 5-fold increase in theoretical plates (N) and a 5-fold decrease in analysis time. In our laboratory, we have developed ultrahigh-pressure liquid chromatography (UHPLC) to take advantage of smaller particles in long capillary columns.2,4 Using our UHPLC system, we have successfully achieved over 200 000 theoretical plates (440 000 plates/m) for a simple organic compound (k′ ) 1) using pressures as high as 4900 bar (72 000 psi) with 1.0-µm particles.4 The initial obstacle in the development of UHPLC was the construction of a leak-free pumping system, injector, and column that can withstand extreme pressures and operate successfully with common chromatographic solvents. A constant-pressure isocratic and a constantflow gradient LC system capable of pressures as high as 7500 bar (110 000 psi) and 5100 (75 000 psi), respectively, have been previously described in detail.2,4 Work from our laboratory and others5-7,14,15 has shown that UHPLC is a viable and useful chromatographic technique. Our experiences show no undesirable effects on the separation related (5) Wu, N.; Collins, D. C.; Lippert, J. A.; Xiang, Y.; Lee, M. L. J. Microcolumn Sep. 2000, 12, 462-469. (6) Wu, N.; Lippert, J. A.; Lee, M. L. J. Chromatogr., A 2001, 911, 1-12. (7) Lippert, J. A.; Xin, B.; Wu, N.; Lee, M. L. J. Microcolumn Sep. 1999, 11, 631-643. (8) Witowski, S. R.; Kennedy, R. T. J. Microcolumn Sep. 1999, 11, 723-728. (9) Colon, L. A.; Burgos, G.; Maloney, T. D.; Cintron, J. M.; Rodriguez, R. L. Electrophoresis 2000, 21, 3965-3993. (10) Dadoo, R.; Zare, R. N. Anal. Chem. 1998, 70, 4787-4792. (11) Seifar, R. M.; Kok, W. T.; Kraak, J. C.; Poppe, H. Chromatographia 1997, 46, 131-136. (12) Kalghatgi, K.; Horvath, C. J. Chromatogr. 1987, 398, 335-339. (13) Kalghatgi, K.; Horvath, C. J. Chromatogr. 1988, 443, 343-354. (14) Xiang, Y.; Yan, B.; Yue, B.; McNeff, C. V.; Carr, P. W.; Lee, M. L. J. Chromatogr., A 2003, 983, 83-89. (15) Cintron, J. M.; Colon, L. A. Analyst (Cambridge, U. K.) 2002, 127, 701704.

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to ultrahigh pressures as previously suggested.16,17 Not only have we demonstrated high efficiencies with simple compounds using a constant-pressure isocratic UHPLC system but we have also achieved the separation of a complex peptide mixture from a protein digest using a constant-flow gradient UHPLC system.4 Column efficiencies can be evaluated by plotting the plate height (H) versus linear velocity (u). These plots show the summed contribution for peak broadening described by the van Deemter equation:1

H ) A + B/u + (CMP + CSP)u

(1)

where the A term is the eddy diffusion coefficient, the B term is the longitudinal broadening coefficient, and the CMP and CSP terms correspond to the resistance to mass transfer from the mobile phase and stationary phase, respectively. Currently, the particles used with our UHPLC systems are nonporous. Hence, the stagnant mobile-phase resistance to mass-transfer term (CSM) is nonexistent. When comparing two different columns with different particle sizes and analytes, it is convenient to normalize the plate height and linear velocity using reduced parameter theory:

h ) H/dp

(2)

v ) udp/Dm

(3)

and

where h is the reduced plate height, v is the reduced linear velocity, dp is the particle diameter, and Dm is the diffusion coefficient for the analyte in the mobile phase.1 Until now, there has been no systematic study to characterize capillary columns packed with 1.0-µm nonporous silica particles using UHPLC. While several methods for a packing capillary column with small particles have been reported,2,7,18 there is no conclusive evidence suggesting a specific method produces more efficient columns. Further factors such as theoretical plate calculations, injector and detector contributions to broadening, flowinduced column heating, the ratio of column diameter to particle diameter (F), and packing density have not been explored in depth with regard to their effects on column efficiency using UHPLC. This paper discusses recent improvements in column packing methods for small particles used in our laboratory, as well as attempts to characterize our columns with respect to the factors listed above. EXPERIMENTAL SECTION Packing Apparatus. MacNair and co-workers initially developed the slurry packing procedure for capillary columns with small particles for UHPLC.2 A similar procedure was used for packing columns for experiments described in this paper. Improvements to the high-pressure packing system, column fitting, and packing technique have increased the success rate of well-packed columns for use with UHPLC. A 3400 bar (50 000 psi) pneumatic amplifier (16) McGuffin, V. L.; Evans, C. E. J. Microcolumn Sep. 1991, 3, 513-520. (17) Hala´sz, I.; Endele, R.; Asshauer, J. J. Chromatogr. 1975, 112, 37-60. (18) Colon, L. A.; Maloney, T. D.; Fermier, A. M. J. Chromatogr., A 2000, 887, 43-53.

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Figure 1. Axial section view of the newly designed UHPLC capillary fitting. After capillary is secured in the fitting, the entire fitting is connected to the packing reservoir or injection block high-pressure port with a gland.

pump (model DSHF-302, Haskel, Inc., Burbank, CA) and highpressure packing reservoir was constructed to make a packing station for column packing. The pneumatic amplifier pump used a standard factory piston seal made from ultrahigh molecular weight polyethylene. The pumps were connected to the packing reservoir with 3/8-in. XF6-style high-pressure fittings and tubing (HiP Inc., Erie, PA). A light microscope (Nikon, Tokyo, Japan) coupled with a high-resolution CCD camera and monitor (Sony Electronics, Inc., Burbank, CA) provided real-time inspection of the particles packing in the column. A PEEK sleeve secured on a microscope slide guided the capillary under the 100× oil immersion objective (Edmund Scientific, Inc., Barrington, NJ). Capillary Fitting. An improved and more dependable UHPLC capillary fitting has replaced our previous design.2 The new fitting, illustrated in Figure 1, is more compact and the column is more accessible than the old design. The body of the fitting is machined from 17-4 PH stainless steel (SS) rod (McMaster-Carr, Atlanta, GA) with a 59° sealing cone that mates with the high-pressure ports. The compression bolt is machined from 316 SS rod (McMaster-Carr) with a 1/16-in. through hole for a Teflon sleeve insert. A 1/8-in.-diameter D-2 air-hardened tool steel (McMasterCarr) stem with a 0.020-in. through hole is press-fitted into the compression bolt. The D-2 tool steel is harder than 316 SS, thereby better resisting deformation under compression.19 The ferrule used to fasten the capillary in the fitting is an 1/8-in.-diameter × 0.400in.-long PEEK (Boedeker Plastics, Inc., Shiner, TX) cylinder with a 380-mm through hole. As the compression bolt is screwed into the body, the PEEK ferrule is compressed against the inner walls of the fitting and the outer wall of the capillary. The capillary is firmly gripped along the entire length of the cylinder and sealed in place against high pressures. Column Packing. To prepare each column for packing, an outlet frit is made by tapping the end of the fused-silica capillary column (Polymicro Technologies, Inc., Phoenix, AZ) in a vial filled with bare nonporous silica particles (Eichrom Technologies, Inc., Darien, IL). For 10-25-µm-i.d. columns, 3.5-µm spherical particles are used for the frit material. A 10-µm tungsten wire pusher (Goodfellow, Berwyn, PA) is used to push an ∼100-µm-thick plug (19) Oberg, E.; Jones, F. D.; Horton, H. L.; Ryffel, H. H. Machinery’s Handbook, 24th ed.; Industrial Press: New York, 1992.

of frit material 0.3-0.4 mm into the column. This open space at the end of the column provided room for the insertion of a carbon fiber electrode for electrochemical detection as described previously.20 The particles are sintered in place with an electric arc using a homemade arcer.21 The frits for columns larger than 25µm i.d. are made in a similar manner except 4.5-µm particles and a 25-µm-sized pusher are used. For a column i.d. larger than 50 µm, the arcer is used to initially taper the i.d to ∼30 µm to reduce postfrit volume around the carbon fiber. The frits for these columns are then made in a fashion similar to the 30-50-µm columns. Once fritted, the column is assembled into the capillary fitting with a PEEK ferrule and the compression bolt torqued to ∼20-30 ft‚lb, depending on the maximum run pressure desired. The column is extended 2 in. past the fitting to ensure the column inlet is positioned in the stirred slurry chamber of the packing reservoir. The 1.0-µm nonporous silica C18 bonded particles, donated from Eichrom Technologies, Inc. (formerly Micra Scientific, Inc., Darien, IL) and size verified by SEM imaging, are suspended in 33% acetone/67% hexane solution. The prefiltered slurry concentration used is 10-30 mg/mL depending on the column i.d.. A higher slurry concentration is used to pack larger i.d capillaries. To suspend the particles completely and break up any loosely aggregated particles, the slurry is placed in an ultrasonicating bath for 10-15 min. The particles are then filtered through a 5.0-µm nucleopore polycarbonate membrane filter (Millipore, Bedford, MA) to remove large foreign particles and remaining aggregates. The filtered slurry is sonicated again for 10-15 min and introduced into the packing reservoir with a long syringe needle. The column inlet is inserted through the packing reservoir lid and the fitting tightened in place. Liquid pressure from the pump is gently applied using 33% acetone/67% hexane solution as the pushing solvent. As the packed bed forms, the pressure is increased with time to maintain a constant, even packing rate. Columns 30-40 cm long are generally packed in less than 12 h. After achieving the desired column length, the pump is turned off and the system is slowly depressurized (usually overnight) to atmospheric pressure by bleeding the pressure through a valve. The column is removed from the pressure reservoir, refitted to a pump, and flushed with acetone to remove the packing solvent. The column is also conditioned with mobile phase using pressures greater than the maximum run pressure. This is done to ensure that the bed is compressed as tightly as possible. Again, the column is slowly depressurized, making sure the packed bed is not disturbed. The inlet frit is made by heating a narrow section (∼5 cm from the front of the packed bed) with a homemade, resistively heated NiChrom wire loop while applying 680 bar (10 000 psi) mobile-phase liquid pressure. The column is clipped at the inlet frit and refitted into a capillary fitting with 0.5-0.7 in. extended past the fitting. Column Evaluation. Column performance was measured with a system similar to the isocratic UHPLC system that was previously described.4 The constant-pressure, pneumatic amplifier pump (DSHW-1373, Haskel, Inc., Burbank, CA) with a urethane seal was capable of generating a pressure of 7700 bar (113 000 psi) with a single piston stroke. A new injector block was designed and fabricated with ∼50% less volume than the previous design.

Manual hand valves in this injector were used in the same fashion as the previous injector to control the solvent flow during an injection and chromatographic run. Furthermore, unpumped dead volume in the pump head and connecting tubing was reduced to decrease piston stroke usage due to solvent compression at high pressures. The pneumatic amplifier pump output pressure was calibrated by plotting the regulated gas pressure delivered to the pump versus the measured liquid pressure using a 10 200 bar (150 000 psi) high-pressure transducer (Omegadyne, Inc., Stamford, CT). The amplification ratio for the pump was measured to be 1710:1. Five electroactive compounds, ascorbic acid (AA), hydroquinone (HQ), resorcinol (RES), catechol (CAT), and 4-methylcatechol (MCAT), provide test compounds for analysis of column performance under reversed-phase conditions. The mobile-phase composition used to elute the compounds is 10% acetonitrile/90% deionized water, containing 0.1% trifluoroacetic acid (Sigma Chemical Co., St. Louis, MO). Injections on the column are made using a static split injection technique.22 The sample chamber in the injector is filled with sample, and 14 bar (200 psi) liquid pressure is applied to the column for 5-7 s, injecting a small sample plug onto the column. Excess sample in the chamber around the column is flushed away through the waste ports controlled by manual valves. Immediately after flushing, high pressure is applied to the column to start the chromatographic run. The injection process takes less than 10 s, and the column reaches its target pressure in less than 1 s. Amperometric detection is accomplished by inserting an 8-µm-diameter carbon fiber electrode into the outlet end of the column.21 A 1.00-V difference is applied between the carbon fiber working electrode and the Ag/AgCl reference electrode by applying -1.00 V to the reference electrode. The current from the carbon fiber electrode is amplified with a current-to-voltage preamplifier (model SR750, Stanford Research Systems, Inc., Sunnyvale, CA) and filtered with a low-pass filter (model 3341, Krohn-Hite Corp., Avon, MA) set for 29 Hz. Chromatograms are collected on a PC computer with an AT-MIO-16X data acquisition board (National Instruments, Austin, TX) at 20 Hz. Chromatograms are displayed and saved to disk with a custom-written LabView 6.0 data collection program (LabView 6.0, National Instruments). Plate heights and retention times are calculated by an iterative statistical moments macro written in-house with (3σ integration limits.23 van Deemter curves are determined by fitting the H versus u data with a nonlinear curve fitting function in Igor 4.0 (Wavemetrics, Lake Oswego, OR). Two-Point UV Detection. For determining the extracolumn broadening effects in our UHPLC system, a two-point on-column detection setup was implemented The setup used a 150-µm-i.d. × 48-cm-long capillary packed with 1.0-µm nonporous silica particles with two variable-wavelength UV detectors (Linear UV 200, Reno, NV) outfitted with on-column capillary detection cells. The detectors were set to a wavelength of 235 nm and detector rise times of 0.3 s. Both detectors showed similar sensitivities and no significant difference in output signals. Two, ∼5-mm-long sections of the polyimide coating were removed from the capillary using fuming sulfuric acid to create detection windows. The windows were centered at 21.2 and 38.9 cm from the column inlet. During

(20) Kennedy, R. T.; Jorgenson, J. W. Anal. Chem. 1989, 61, 1128-1135. (21) Hoyt, A. M. J.; Beale, S. C.; Larmann, J. P. J.; Jorgenson, J. W. J. Microcolumn Sep. 1993, 5, 325-330.

(22) Guthrie, E. J.; Jorgenson, J. W. J. Chromatogr. 1983, 255, 335-348. (23) Hsieh, S.; Jorgenson, J. W. Anal. Chem. 1996, 68, 1212-1217.

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a chromatographic run with the five test compounds, data from both detectors were collected at 20 Hz into a single custom-written LabView program. Because of the relatively low signal-to-noise ratio, the chromatographic peaks were fitted with Gaussian fits using Igor 4.0 to determine peak widths. Flow-Induced Column Heating Effects. To investigate possible flow-induced heating effects on plate height and retention factors on columns used with UHPLC, 100- and 150-µm-i.d. capillary columns were partly enclosed in an adiabatic environment created by a vacuum jacket. The vacuum jacket was constructed from a 1/4-in.-diameter × 30-cm-long glass tube fitted with Swagelok fittings. The side port of the Swagelok tee at the front of the vacuum jacket was connected to a vacuum pump (model 18, Edwards, Inc., Carwely, England) with thick-walled flexible tubing. A 0-2000 mTorr pressure gauge (Kurt J. Lesker Co., Clariton, PA) was connected in-line with the tubing and vacuum pump. After connecting the capillary fitting assembly to the injection block of the UHPLC system, the column was threaded through the vacuum jacket. The capillary was pulled taut to ensure that the capillary did not touch the inner walls of the jacket and was held in place with Vespel ferrules, which sealed the system to 80 mTorr. Plate height and k′ for the five test compounds under diathermic and adiabatic conditions were measured in an unbiased fashion at different run pressures for both columns. Packing Bed Structure Elucidation. After generating sufficient data for van Deemter analysis, the outlet frits for the different diameter columns were clipped. The columns were connected to the high-pressure pumps and 680 bar (10 000 psi) liquid pressure was applied. The pressure pushed on the front of the packed bed to extrude it from the outlet end as a continuous cylinder of particles. Sections as long as 3 cm could be collected and placed on carbon-coated, aluminum specimen stubs for analysis with a scanning electron microscope (SEM) (model JSM6300FV, JEOL, Tokyo, Japan). The extruded segments could be positioned on the stub with a fine pair of tweezers without causing any detectable damage or disturbance. Segments from the outlet, middle, and inlet for each different diameter were analyzed by SEM to visually assess differences in packing structure. Safety Considerations. Working with extremely high pressure can be potentially dangerous if proper precautions are not used.24 All tubing and fittings in our systems are rated higher than the maximum pressure at which the systems are operated. Although no damage or injury has resulted from column failures, a number of precautions are used to prevent such an occurrence. Each UHPLC system is enclosed in a 3/4-in.-thick plywood safety box which includes a Lexan window. All fittings and columns are also positioned in such a manner that in the event of a sudden failure of a capillary fitting no one is likely to be harmed.

Figure 2. Chromatogram of five test compounds: hydroquinone (HQ), resorcinol (RES), catechol (CAT), and 4-methylcatechol (4MCAT) with ascorbic acid (AA) as the dead-time marker. The column was a 10 µm × 43 cm long packed with 1.0-µm NPS particles. The inlet pressure was 7100 bar (103 000 psi) with a linear velocity of 0.39 cm/s. Number of theoretical plates for each test compound is listed in parentheses.

RESULTS AND DISCUSSION Capillary Fitting. The previous capillary fitting design2 was reliable up to 3450 bar (50 000 psi). At higher pressures, the risk of a column failure is much greater. Column failures usually occur when the ferrule does not grip the capillary sufficiently, allowing the column to be ejected from the fitting. In the current design, the ferrule is twice as long as in our earlier design and is made

from PEEK rather than polyimide polymer. Doubling the length and using a stronger polymer provides a higher gripping strength. This fitting design has repeatedly been used at pressures above 6800 bar (100 000 psi). After the first use, the PEEK ferrules cannot be easily removed from the fitting due to compression of the PEEK. However, the ferrule can be reused numerous times by reaming the capillary hole with a 380-µm drill bit to reopen it so a new capillary column can be threaded through and retightened. Ferrules can be completely removed from fittings by chucking the fitting in a lathe and drilling the ferrule out. Column Packing. When the efficiency of a series of different columns is studied, reproducible and well-packed columns are necessary for representative van Deemter plots. With the oncolumn monitoring of the column packing with a CCD camera/ microscope, the packing quality for each column could be visually evaluated in real time. For 1.0-µm nonporous silica C18 particles, a 33% acetone/67% hexane solvent mixture has provided a better suspension of particles and packing speed than any other solvent system tested. This is partly due to the low solvent viscosities of 0.31 cP for acetone and 0.30 cP for hexane.25 With this particular solvent mixture, pressures above 3100 bar (45 000 psi) cause the particles to form small aggregates while packing. This limited our maximum packing pressure for these particles. However, to produce tightly packed columns, it is critical to flush the columns at liquid pressures greater than the maximum run pressure. With the CCD camera/microscope, we have observed the bed to compress further with increased liquid pressure. If the column is slowly depressurized, the bed does not expand, suggesting that added pressure causes the particles to pack tighter by collapsing voids. Slow depressurization is necessary to prevent reintroduction of gaps or additional voids in the packed bed. Once the column inlet is fritted, the column can be depressurized rapidly without disturbing the packed bed. Column Evaluation. Figure 2 shows a chromatogram of the five electroactive test compounds obtained on a 10-µm-i.d. × 45cm-long column packed with 1.0-µm nonporous silica particles and run at 7000 bar (103 000 psi), 2.3 times greater than uopt. Our method for calculating the number of theoretical plates for

(24) Xiang, Y.; Maynes, D. R.; Lee, M. L. J. Chromatogr., A 2003, 991, 189196.

(25) Lide, D. R., Ed. CRC Handbook of Chemistry and Physics, 73rd ed.; CRC Press: Boca Raton, FL, 1992.


Table 1. Comparison of the Calculated Values for the Number of Theoretical Plates for Each Test Compound on a 10-µm × 45-cm Column at 3060 bar (45 000 psi) Near Optimum Linear Velocity

HQ RES CAT MCAT

stat moments

Gaussian fit

fwhm

331 000 286 000 289 000 244 000

333 000 304 000 298 000 260 000

346 000 311 000 305 000 257 000

chromatographic peaks is by an iterative statistical moment method.23 This technique provides an accurate representation and unbiased theoretical plate value for a chromatographic peak. Table 1 displays the calculated number of theoretical plates by three methods: iterative statistical moments ((3σ), Gaussian fit ((3σ), and full width at half-maximum (fwhm) at uopt (3100 bar (45 000 psi)) for the five compounds on the 10-µm-i.d column described above. When compared to fwhm, the iterative statistical moments plate count values are 5 and 3% lower for hydroquinone and catechol, respectively. For resorcinol and 4-methylcatechol, which are slightly tailed, the plate count values by iterative statistical moments are 10% lower than both Gaussian fit and fwhm. These last two methods do not fully account for the slight peak tailing, resulting in artificially higher N values. To represent the plate count for peaks fairly, without bias, the iterative statistical moments approach is the preferred method and is the method used in the remainder of this work. Extracolumn Broadening Effects. The measured number of theoretical plates reported in this paper is for the entire chromatographic system, not just for the column. Additional broadening due to the injector or detector can significantly affect the overall efficiency, especially with the narrow peak widths in UHPLC chromatograms. To calculate broadening due only to the column, the contributions from the injector and detector have to be subtracted. Our past experiences have shown the electrochemical detector has negligible contribution to the overall peak broadening.20,23,26 To experimentally determine column efficiency without injector and detector contributions to peak broadening, we have devised a setup using two separate UV detectors for simultaneous oncolumn detection at two points on the large-i.d column. For this experiment, 150-µm-i.d. columns were used because of the necessary path length for UV detection through the packed bed. Any contribution to peak broadening from the injector and detector will be present in both detectors. By taking the difference of the measured variance of a peak from each detector, the peak broadening contribution from the injector and detector is essentially canceled out. The plate height between the two detector points, Hcalc, can be calculated as

Figure 3. Van Deemter plots for hydroquinone on a 150-µm × 48.8cm column from dual UV detection system. Detection 1 at 21.2 cm (b), detector 2 at 38.9 cm (9), and calculated 17.7 cm (2) columns are shown.

Figure 3 shows the van Deemter fits for hydroquinone measured for each detector and the resulting “calculated column length” between the two detectors. The calculated column length is 17.7 cm and has a Hmin ) 2.3 µm, whereas the Hmin values for the 21.2- (detector 1) and 38.9-cm-long columns (detector 2) are 2.5 and 2.4 µm, respectively. The difference between the two van Deemter plots from each detector is small even though they are detected 17.7 cm apart on the column. This suggests that the column is uniformly packed and has a constant average packing density throughout the entire column length. In comparing the three van Deemter plots, the two measured plate heights, which include band broadening due to our static split injection, indicate only a slight decrease in overall efficiency from the calculated 17.7-cm column. Results from a 75-µm-i.d. column also show trends similar to the 150-µm column but had a lower signal-to-noise ratio. From this study, we conclude that our injector and detector contributions to the overall peak broadening are acceptably small. No further subtraction or correction is necessary for a fair representation for column efficiency, and measured single detector values can be used directly. Flow-Induced Heating Effects. Another concern of particular importance in UHPLC is frictional heat generated from the flowing mobile phase through the packed bed at extremely high pressures. Slow heat dissipation could cause significant radial temperature gradients resulting in poor column efficiencies due to localized variations in analyte capacity factors and solvent flow velocities.17 Such effects would be most significant in largediameter columns.27 The rate of heat generation or the power that can be generated by flowing mobile phase through a packed bed can be calculated as

power ) F∆P

(5)

where σDet12 and σDet22 are the measured temporal variances and tr(Det1) and tr(Det2) are the retention times at the first and second detectors, respectively. L is the length between the two detection points.

where F is the flow rate and ∆P is the pressure drop. Flowing solvent at 1.3 µL/min through a 150-µm-i.d. × 50-cm-long column packed with 1.0-µm particles over a 100 000 psi pressure drop would generate 15 mW of heat. The heat generated must be dissipated through the fused-silica capillary walls to the surroundings. Work in the field of capillary electrophoresis has shown fused-silica capillaries can dissipate heat to the surroundings very

(26) MacNair, J. E. Ph.D. Thesis, University of North Carolina, Chapel Hill, 1998.

(27) Martin, M.; Guiochon, G. J. Chromatogr. 1975, 110, 213-232.

Hcalc ) (σDet22 - σDet12)L/(tr(Det2) - tr(Det1))2

(4)


5781

Figure 4. Comparison of Van Deemter fits for 150-µm column between adiabatic and diathermic environments for hydroquinone (b, O), catechol (2, 4), and 4-methylcatechol ([, ]). Solid markers denote adiabatic conditions.

efficiently,28 and 15 mW of heat dissipated over 50 cm is not expected to be a problem. Most of the past work in UHPLC work with 1.0-µm particles has been with relatively small 30-µm i.d., where the ratio of the inner wall surface area to volume is relatively large. It is unknown how larger i.d. columns are affected by flowinduced heating and whether they could be used in UHPLC without compromising separation efficiency. This effect must be addressed before column efficiencies of large diameters can be compared and evaluated. If flow-induced heating is a significant factor, the A, B, and C van Deemter coefficients would be larger for the diathermic system. In a large-bore column, the heat transfer from the core of the column to the walls is not as efficient, causing greater radial temperature differences to develop. Localized higher temperature will increase mobile-phase velocities and lower retention factors in that region. Radial temperature gradients would cause additional peak broadening and result in lower efficiencies. In the adiabatic system, the column is insulated with a vacuum jacket so virtually no heat transfer with the surrounding is possible. Thus, no radial temperature gradients can occur, and heat generated accumulates axially down the column’s length (axial temperature increase). Comparing the separation efficiencies and retention factors of electroactive test compounds in an adiabatic versus diathermic environment would show the effects of flow-induced heating. Figure 4 shows the van Deemter fits for the compounds tested on a 100-µm-i.d. column under the adiabatic and diathermic systems. Although the fits under diathermic conditions have slightly higher values, analysis using a confidence interval of one standard deviation show an overlap between the adiabatic and diathermic fits. Figure 5 shows the trend for k′ values for 4-MCAT at different pressures for adiabatic and diathermic conditions in a 150-µm-i.d column. Comparing the measured retention factor values between the two conditions, slightly lower k′ values are observed for columns under adiabatic conditions than diathermic conditions. A lower overall retention factor for adiabatic conditions suggests that the internal temperature of the column is slightly higher, causing the peaks to be less retained. From these experiments, evidence of the flow-induced heating effects in 150-µm-i.d. columns is just beginning to be noticeable. For our purposes, this study shows flow-induced heat generation (28) Jorgenson, J. W.; Lukacs, K. D. J. High Resolut. Chromatogr. 1981, 4, 230.


Figure 5. Calculated average k′ values for 4-methyl catechol under adiabatic (b, solid line) and diathermic (9, dotted line) conditions.

Figure 6. Van Deemter plots for hydroquinone using different column inner diameters. Data from two separate columns for each i.d. were combined for each fit. Each trace is labeled with its corresponding diameter.

with small particles at or below 150-µm i.d. used with UHPLC has a negligible effect on column efficiency. van Deemter fits for diameters larger than 150 µm were not tested with UHPLC because of the decreasing capillary wall thickness. The 180-µm capillaries failed repeatedly because the walls would fracture at the fitting. When the vacuum jacket was sleeved on the 180-µm capillary, the column would twist, creating stress at the fitting causing it to fracture. It is still unknown how large a diameter can be used in UHPLC before thermal gradients do cause appreciable effects on chromatographic performance. Effects of Column Diameter on Efficiency. The effect of the column inner diameter on column efficiency with 1.0-µm nonporous silica particles using UHPLC was investigated. Columns used in this study had inner diameters from 10 to 150 µm and column lengths between 39 and 53 cm. To test reproducibility, two separate columns for each diameter were packed and evaluated. For analysis, a single van Deemter curve is fitted to the two separate column data sets for each i.d. The data points collected for two columns at each column diameter were virtually superimposable. Rather than averaging the end results of two separate columns, the data from each column was combined for a single van Deemter fit. With more data points, the van Deemter fits were better. Figure 6 displays the van Deemter curves for hydroquinone for each different diameter tested. For the van Deemter curves, hydroquinone and catechol provide the best curves and results for making evaluations and therefore were used

exclusively when making conclusions about specific trends. Because of the slight tailing nature of peaks for resorcinol and 4-methylcatechol, the efficiencies and fits for the data sets are not as good. The trend observed in Figure 6 suggests that decreasing column diameter, or alternatively F, the column inner diameterto-particle ratio, leads to an increased column efficiency. Since only one particle diameter (1.0 µm) is evaluated, the term F and column i.d. can be used interchangeably. Also from Figure 6, it is noticed that the uopt occurs at slightly higher linear velocity and the C term slope of the fits becomes shallower with decreasing i.d. The relationship with i.d. and efficiency with 1.0-µm particles corresponds well with previously reported trends for larger particles packed in capillary columns.8,23,29 Initial findings by Wu et al. have shown that 1.5-µm nonporous silica particles packed in 29- and 100-µm-i.d. columns exhibit similar trends.6 Knox and Parcher have proposed and tested experimentally that the packed bed can be divided into two distinct regions: a densely packed core and a less densely packed wall disturbed region.30 The differences in packing densities near the wall and the core lead to locally higher flow velocities near the walls because of the lower flow resistance. With a larger i.d. column, the transcolumn variations in the velocity profile are expected to be greater. Similarly, retention differences due to packing density differences would result in a localized variation in the amount of stationary phase. Since the velocity of an analyte band is dependent on the mobile-phase velocity and analyte retention, the differences between the two regions would result in band broadening. As the column i.d. becomes smaller, the band broadening associated with these types of transcolumn differences is reduced.20,31 Another perhaps more important effect is diffusional relaxation.20,31 As the column diameter decreases, the analyte molecules are more likely to experience all possible flow paths equally in the column. The average time required for a solute molecule to diffuse from the column center to the walls is proportional to the column diameter squared.32 Diffusion relaxation essentially averages the effect of the differences that might be present in the packed bed. The Einstein equation can be used to give an idea of the average time it takes for an analyte molecule to diffuse from the core of the column to the wall (tD):

tD ) r2/2γDm

(6)

where γ is the tortuosity factor, which accounts for the obstruction to diffusion presented by the particles. For a 10-µm-i.d. column with γ ) 0.9 and Dm) 6.6 × 10-6 cm2/s, tD is 0.02 s. For a column with 150-µm i.d., tD would be 4.7 s. This treatment demonstrates the vast difference in time required to average the effects of different flow regions in 10- versus 150-µm-i.d. columns.20,32 The relationship of i.d. with efficiency can be further analyzed by plotting the reduced parameter A, B, and C term coefficients for the different column diameters. Figure 7 shows a plot of the (29) Kennedy, R. T.; Oates, M. D.; Cooper, B. R.; Nickerson, B.; Jorgenson, J. W. Science (Washington, D. C.) 1989, 246, 57-63. (30) Knox, J. H.; Parcher, J. H. Anal. Chem. 1969, 41, 1599-1606. (31) Bristow, P. A.; Knox, J. H. Chromatographia 1977, 10, 279-289. (32) Knox, J. H.; Scott, H. P. J. Chromatogr. 1983, 282, 297-313.

Figure 7. Reduced A, B, and C term trends for hydroquinone (b) and catachol (9) for different column i.d.s. Diffusion coefficient of Dm ) 6.6 × 10-6 cm2/s was used for both compounds.

reduced van Deemter equation coefficients for hydroquinone and catechol versus column i.d.. The improved efficiency with decreasing i.d. is due to the decrease in A and C term coefficients. Eddy Diffusion (A Term). The reduced A term data decreases nearly linearly with decreasing F, as would be expected. The more highly retained compound, catechol, has a larger reduced A term and a steeper slope as illustrated in Figure 7. Even at 150-µm i.d., the calculated reduced A term values are 0.6 and 0.7, respectively, for hydroquinone and catechol, lower than the value of 1 typically seen in larger bore columns.33 Previously reported results by Hsieh and Jorgenson with 5-µm porous particles in capillary columns and, recently, 4.5-µm nonporous particles by Witowski and Kennedy also show a similar trend.8,20,23 Their results also show reduced A term coefficients less than 1 for packed capillary columns. Longitudinal Diffusion (B Term). The calculated reduced B term has values similar to the accepted value of ∼2 for a wellpacked column.33 In agreement with prior literature and research, Figure 7 demonstrates that the B term coefficient has no strong dependency on column i.d.. Hydroquinone and catechol do have slight negative slopes. The slight negative decrease with column i.d. is suggestive of an increasing tortuosity factor (γ) related to an increased packing density observed with increasing column i.d.,20 as discussed below. (33) Kennedy, G. J.; Knox, J. H. J. Chromatogr. Sci. 1972, 10, 549-556.


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Resistance to Mass Transfer (C Term). Values determined for the reduced C term for hydroquinone and catechol versus F are shown in Figure 7. The trend shows a dependence with F below 75. However, for F greater than 75, a maximum limiting value of C ) 0.4 and C ) 0.5 is approached for hydroquinone and catechol, respectively. Reduced C term values determined for UHPLC columns at ultrahigh pressures are larger than typical values observed for capillary columns packed with larger particles and operated at conventional pressures. Such large reduced C term values in otherwise highly efficient columns have never been seen before. The value generally accepted for the reduced C term for well-packed porous particles has been experimentally determined to be ∼0.1-0.2.33 Values for nonporous particles are expected to be even lower because of a lack of stagnant mobilephase term (Csm). Knox estimated that C ) 0.003 for unretained solutes for nonporous particles.33 Witowski and Kennedy experimentally determined values of C ) 0.05 for 4.5-µm nonporous silica particles where F ) 11.1.8 For our experiments, the maximum reduced linear velocity never exceeded v ) 7 primarily due to a pressure limitation of our system. Witowski and Kennedy used v > 30, where the C term is the dominant contribution to band broadening. In an analogous experiment, a van Deemter curve with vmax > 30 was obtained for a short, 21.2-cm column packed with 1.5-µm particles on our isocratic system. For this column, reduced C ) 0.4 was found, which suggested that the trend found with longer columns at lower linear velocities is valid. Furthermore, inspection of the van Deemter data on 1.5-µm nonporous particles of Wu et al. also reveals large C term values for a 29- and 100-µm i.d. columns.6 The extreme pressures used with columns packed with 1.0µm particles could play a role in elevated C term values even at moderate linear velocities. A diffusion coefficient value of 6.6 × 10-6 cm2/s is used to calculate reduced linear velocity values for all analytes. This value was experimentally determined by the stopflow method at atmospheric pressure.2 Accurate diffusion coefficients values for small organic compounds in an acetonitrile/ H2O solvent at elevated pressures are unknown. From the WilkeChang estimation method for estimating the diffusion coefficients in binary liquids, the diffusion coefficient of a liquid solute is inversely related to the solvent viscosity.34,35 The viscosity of water increases to a value of 1.4 cP at 6800 bar (100 000 psi).36 Assuming a similar viscosity trend with a ACN/H2O solvent mixture, the diffusion coefficient should decrease proportionally. However, a decrease in the diffusion coefficient can only account for a small fraction of the abnormally high C term values. Nonetheless, accurate diffusion coefficient data at high pressures will be needed for accurate υ values for determining correct reduced parameter values. Column Packing Density. The most likely explanation for the large C term values is a result of a lower particle packing density than expected. Increasing the column porosity (decreasing the packing density) would have a direct effect on the CMP. The analyte molecule in the mobile phase must traverse a greater distance to interact with the stationary phase on the surface of the particle. UHPLC columns are slurry packed using a method (34) Li, J.; Carr, P. W. Anal. Chem. 1997, 69, 2530-2536. (35) Wilke, C. R.; Chang, P. AICHE J. 1955, 1955, 264-270. (36) Fo ¨rst, P.; Werner, F.; Delgado, A. Rheol. Acta 2000, 39, 566-573.

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Table 2. Summary of the Series of van Deemter Curves for the Different i.d. Columns i.d. (µm)

Hmin (µm)

uopt (cm/s)

Φ

10 15 30 50 75 100 150

1.3 1.5 1.8 2.0 2.1 2.2 2.5

0.18 0.17 0.15 0.14 0.13 0.13 0.12

907 ( 24 934 ( 56 848 ( 27 905 ( 27 990 ( 66 837 ( 12 800 ( 57

Figure 8. Trend for the measured average k′ for 4-methylcatechol with column i.d. Each fit is labeled with its corresponding diameter. Plots for 15- and 75-µm i.d. are omitted for clarity.

that is significantly slower than conventional “slam-packing” techniques commonly used with larger particles. A greater packing pressure than currently used for 1.0-µm particle might be necessary for more dense packing structure. As a measure of packing density, the column flow resistance factor (Φ) can be calculated for the different column inner diameters using the following relationship:37,38

∆Pdp2 Φ) uLηi

(7)

where i is the interstitial porosity for the column, usually 0.4 for randomly packed spheres, and η is the solvent viscosity. Table 2 shows the averaged Φ value for each diameter. The values calculated range from 800 to 1000. It is generally accepted that values between 500 and 1000 are expected for well-packed columns.1 The consistently high values for all columns are an indication of densely packed columns; however, there is no trend in Φ increasing or decreasing with column diameter changes. In addition, since the fritting technique also may have a role in the column flow resistance, more work investigating different packing and fritting techniques is necessary to fully understand their relationships with packing structure, column porosity, and C term broadening. Retention Factor versus Column Diameter. From previous work in our laboratory,2 average k′ has shown to increase linearly with increasing pressure. This is thought to arise from an increase in solvent density and an associated change in analyte distribution coefficients and the free energy of phase transfer. From the series (37) Giddings, J. C. Anal. Chem. 1965, 37, 60-63. (38) Knox, J. H. J. Chromatogr. Sci. 1980, 18, 453-461.

Figure 10. End-on view of a (a) 50- and (b) 150-µm packed capillary.

Figure 9. SEM images of extruded sections of packing bed for (a) 75-, (b) 30-, and (c) 10-µm-i.d. capillaries.

of columns evaluated, Figure 8 shows the relationship of average k′ with column diameter for the most retained test compound, 4-methylcatechol. The data clearly show an increase in retention factor with column diameter at any given pressure. This trend is also suggested by prior results from our laboratory and in initial findings with 1.5-µm nonporous silica particles.6 The k′ can be expressed as

k' ) K(Vs/Vm)

(8)

where K is a partition coefficient of the solute, Vs is the volume of the stationary phase, and Vm is the volume of the mobile phase.1

An increase in the Vs/Vm ratio is a direct result from increasing packing density with increasing F. SEM images in Figure 9a-c show extruded sections of packing from 75-, 30-, and 10-µm column diameters, respectively. From Figure 9, the surface of the 75-µm-i.d. column’s extruded section shows large regions of ordered close-packed arrangement. On the surface, defects can be noticed where particles do not fit well or are even missing. As the column diameter decreases, the packing arrangement becomes less ordered and more randomly packed as seen in Figure 9. The greater curvature of the wall with smaller diameter columns prevents the particles from packing as successfully in a close-packed arrangement. These images coincide with observed k′ trend in Figure 8, suggesting that larger i.d. columns can pack more densely and thus exhibit increased k′ values. In Figure 9, only the surface layer is seen, so the packing arrangement in the core of the column is unknown. Figure 10 is an end-on view of a 50- and 150-µm packed column to visualize the cross section of the packed bed. Near the capillary walls, a highly ordered arrangement of the particles can be seen, but the internal core structure is difficult to visualize. In the 150-µm-i.d. column, the ordered layer extends seven to eight particle diameters into the column before it becomes more randomly Analytical Chemistry, Vol. 76, No. 19, October 1, 2004

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packed. In Figures 9 and 10, an inverse of the wall effect seen by Knox30 is observed with column diameters larger than 75 µm; that is, the outer layers of particles are more densely packed than the interior regions of the packed bed. This is not only due to the large F but also the high monodispersity in the particles. Unfortunately, in both the processes of extrusion and cutting of the column for visual examination, the column packing might be disturbed, and it is unknown to what extent any disruption changes the arrangement of the particles in the packed structure. Summary. Fused-silica capillaries with internal diameters ranging from 10 to 150 µm and packed with 1.0-µm nonporous silica C18 bonded particles were evaluated at ultrahigh pressures. Improvements in the UHPLC fitting allowed for pump running pressures of over 6500 bar (100 000 psi) with no large heating effects in the capillary. In addition, efficiencies as high as 730 000 plates/m were achieved using these 1.0-µm particles with negligible broadening effects from the UHPLC injector and electrochemical detector. van Deemter analyses on columns with internal diameters ranging from 10 to 150 µm showed increasing efficiency with decreasing column diameter. Analysis of retention factor data demonstrated that packing density tends to increase as column i.d. increases, which may be due to the more shallow curvature at the capillary wall. Furthermore, the van Deemter A term coefficient was shown to increase with increasing column i.d.,

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while the B term coefficient was shown to only change slightly with increasing column diameter. The van Deemter C term coefficient exhibited an increase with column internal diameters up to 75 µm and leveled out at a maximum value of 0.4-0.5 above column i.d.s larger than 75 µm. Such large C term values are much larger than typical experimentally determined values of 0.1-0.2. More work is necessary to fully understand the larger than normal van Deemter C term values resulting under UHPLC conditions. More specifically, investigations into the packing structure of small, monodisperse particles may provide the key to fully understanding chromatographic parameters under UHPLC conditions. ACKNOWLEDGMENT This work has been supported in part by NIH Grant GM39515 and the Waters Corp. The authors thank Tim Barder at EiChrom Technologies for the donation of the 1.0- and 1.5-µm particles. The authors also thank Harlan Magnum, Freddie Pinero, Don Brewer, and James Perotti in the UNC Chemistry Department instrument shop for help in design and construction of various UHPLC components. Received for review February 12, 2004. Accepted June 18, 2004. AC049756X

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