AMI. m m .
iaaa, 65, 1451-1455
1451
Packed Column Supercritical Fluid Chromatography with 220 000 Plates Terry A. Berger' and William H. Wilson Hewlett-Packard Company, 2850 Centerville Road, Wilmington, Delaware 19808
Up to 250 000 plates and 298 plates/s were simultaneously observed in nonprogrammed packed column supercritical fluid chromatography. Up to 400 000 or even 667 000 plates appear possible. In programmed runs, substantially higher apparent efficiencies were observed. These results were obtained in spite of repeated assertions in the literature that efficiency in packed column supercritical fluid chromatography has a practical upper limit of 20000 theoretical plates. The simultaneous high efficiency and high speed appear to substantially exceed all previous reports using packed columns and dense fluids. INTRODUCTION A controversy exists over the effect of column pressure drop on efficiency in packed column supercritical fluid chromatography (SFC). Most studies conclude that pressure drops greater than 20 bar cause severe efficiency lossesl-3 in SFC. With such small allowable pressure drops, efficiency is limited to approximately 20 OOO theoretical plates. Several theories294~5have been advanced relating retention gradients to losses in chromatographic efficiency. Therefore, both theory and practice appear to indicate that large pressure drops must be avoided. Consequently, many SFC practitioners avoid pressure drops greater than 20 bar. Under such a limitation, packed column SFC could never become a highefficiency technique. However, previous work12J3in this laboratory employing single columns with severe pressure drops and large density gradients failed to produce the losses in efficiencysuggested. Reduced plate heights as low as 1.78 were obtained13 with total efficiency limited to 30 O00 plates. In related work,14 a theory was developed which predicted that retention gradients cannot improve efficiency compared to steady-state,
-
(1) Schoenmakers, P. J.; Uunk, L. G. M. Chromatographia 1987,24, 51. (2) Schoenmakers, P. J. In Supercritical Fluid Chromatography; Smith, R. M., Ed.; RSC Monograph Series; Royal Society of Chemistry: London, 1988, Chapter 4. (3) Janssen, H. G.; Snijdem, H.M. J.; Rijks, J. A.; Cramers, C. A.; Schoenmakers, P. J. J. High Resolut. Chromatogr. 1991, 14, 438-445. (4) Mourier, P. A.; Caude, M. H.; Rosset, R. H. Chromatographia 1987, 23, 21. (5) Poe,D. P.; Martire, D. E. J. Chromatogr. 1990,517, 3. ( 6 ) Halaz, I.; Endele, R.; Asehauer, J. J. Chromatogr. 1975,112,37-60. (7) Kraak, J. C.; Poppe, H.; Smedes, F. J. Chromatogr. 1976,122,147158. (8) Dewaele, C.; Venele, M. J. High Resolut. Chromatogr. 1980, 3, 273-276. (9) Snyder, L. R.; Dolan, J. W.; Van Der Wall, S. J. J. Chromatogr. 1981,203,3-17. (IO) Verzele, M.; Dewaele, C. J. High Resolut. Chromatogr. 1982,5, 245-249. (11) Lauer, H. H.;McManigill, D.; Bored, R. D. Anal. Chem. 1983,55, 1370. (12) Berger, T. A.; Deye, J. F. Chromatographia 1990, 30, 57-60. (13) Berger; T. A.; Deye, J. F. Chromatographia 1991, 31, 529-534. (14) Blumberg, L. M.; Berger, T. A. J. Chromatogr. 1992,596, 1-13. 0003-2700/93/0365-145 1$04.00/0
uniform conditions but also cannot cause serious losses in efficiency. The present work contains experimental support for this new theory. A number of standard liquid chromatography (LC) columns were connected in series to generate large pressure drops and high efficiencies. There are no recent reports in the literature where long columns have been proposed for generating high efficiency. Capillary GC exhibits enviable speed, sensitivity, and resolution. It is desirable to have a technique with similar attributes for nonvolatile and labile molecules. High efficiency has long been a goal in LC. However, LC is limited by large pressure drops. Numerous authors68have concluded that, with standardbore columns, "high" efficiencies (variously d e f i e d as 50 OOO or 60 OOO plates) could only be achieved using large particles and long analysis times (assuming pressure limita of 300 or 350 bar). Few workersgJ0 have generated more than 50 OOO plates using standard-bore LC columns. High plate counts are generally achieved a t the expense of speed (plates/second). Since in routine workspeed is at least as important as efficiency, most analysts compromise, choosing modest LC efficiency with modest LC speed. Supercritical and near-critical fluids have much lower viscosities than normal liquids,11 so pressure drops on new columns (2O-cm-long, 5-pm particles) seldom exceed 20 bar in SFC (at optimum linear velocity). Since most chromatographs have an upper pressure limit of at least 400 bar it seems reasonable that multiple columns could be connected in series to generate high plate counts if pressure drop were not a problem.
EXPERIMENTAL SECTION A prototype of the Hewlett-Packard (HP) SFC with two pumps was used as the chromatographic system. The pumps were operated in the flow control mode (set to iO.OO1 mL/min). The pressure just downstream of the mixing point was monitored electronically and called the inlet pressure. A Rheodyne Model 7410 valve with 0.5- and 5.0-pL loops was used to inject samples. The columns were mounted in the oven of an HP Model 5890 gas chromatograph, which served as the chromatographic oven. A 1-m-longpiece of 0.17-mm-internaldiameter tubing connected the columns to the detector. A standard HP Model 1050 multiwavelength (photodiode array) detector was used with a high-pressure flow cell. The detector outlet pressure was monitored and controlled with a low dead volume electronicbackpressure regulator. Columns were standard LC columns, 4.6 X 200 mm, packed with 5-wm-diameter Hypersil silica particles from HewlettPackard, Waldbronn, Germany. The only pretreatment involved washing the column with pure methanol pumped at 0.5 mL/min for >30 min at room temperature. Methanol, tetrahydrofuran, and toluene were High Purity purchased from Burdick and Jackson, Muskegon, MI, and used without further preparation. Carbon dioxide was supercritical grade purchased from Scott SpecialtyGases, Plumsteadville, PA. Standards were purchased mostly as neat compounds from ChemicalServices,West Chester, PA. 0 1993 American Chemical Society
1452
ANALYTICAL CHEMISTRY, VOL. 65, NO. 10, MAY 15, 1993
RESULTS AND DISCUSSION Steady-State Chromatograms. Ten phenylurea herbicides were separated on a 4.6 X 200 mm, 5-pm Hypersil silica column using 2 mL/min methanol-modified carbon dioxide. Column transit time (to) was less than 1 min. The column produced -20 000 theoretical plates, the practical limit suggested by The first eluting peak produced >250 plates/s. Chromatograms were collected using various nonprogrammed combinations of outlet pressure, temperature, and modifier concentration. Combinations of outlet pressures between 80 and 200 bar, 1-20 72 methanol, and temperatures between 25 and 60 "C were used without significant changes in efficiencyor pressure drop (-20 bar). Supercritical, sub(near-) critical, and combinations of the two existed oncolumn, depending on specific conditions. As pointed out elsewhere,15these designations are irrelevant, all providing the same fluid characteristics (viscosity and diffusion coefficients) exploited with supercritical fluids. Most transitions from sub- to supercritical do not involve a phase transition. The efficiencies observed matched or exceeded the theoretically predicted value (hmin= 2 4 ) . Flows from 1 to 4.5 mL/min confirmed that the optimum efficiencywas obtained near 2 mL/min. Two additional identical columns were then installed in series with the first column, and the chromatogram was repeated. The three columns produced as many as 60 00070 OOO plates (h, = 1.7-2), with a pressure drop of less than 60 bar. Additional sets containing two columns each were sequentially added (Le., 5, then 7, then 9, then 11 columns). After each addition of two more columns, chromatograms were collected. No lossesin efficiencywere observed, and the total number of theoretical plates increased to 100 OOO, then 140 000-150 000, then 180 OOO, and finally to as many as 250 OOO. Early-eluting peaks produced up to 298 plates/s (>215 OOOplatesin17 h (inlet pressure 600 bar). Tsuda and co-workers" reported 880 OOO plates, from a loosely packed 10.3-m microcapillary, 47-pm i.d., with to= 106min (500kg/cm3,1800plates/bar). Karlsson and Novotnyls reported up to 226 OOO plates (h,= 1.8) at up to 114plates/s in 33 min on a 1.95-m micropacked LC column packed with 5-pm particles with 360 bar inlet pressure (630 plates/bar). This result suggests that either the pressure drop calculations of HalazG were in error or the smooth walls or loose packing of micropacked capillary columns reduce pressure drops compared to standard bore columns. Compared to standard-bore LC, packed column SFC produced far higher efficiencies in much shorter times. Micropacked LC columns produce equal or greater efficiencies but analysis times were 3->10 times longer while pressure drops were simultaneously 2.3-3.8 times larger. Microbore (16)Menet, H.G.;Gareil, P. C.; Rosset, R. H. Anal. Chem. 1984,56, 177&1773. (17)Tsuda, T.; Tanaka, I.; Nakagawa, G. Anal. Chem. 1984,56,12491252. (18)Karlsson, K.-E.; Novotny, M. Anal. Chem. 1988,60,1662-1665.
ANALYTICAL CHEMISTRY, VOL. 65, NO. 10, MAY 15, 1993
100
-
80-
60
40
-
1 5.60
1
lO.’W 15.00 Time (min)
2O.bo
25.bO
Flguro 2. Packed-column SFC chromatogram of Brazilian lemon oil exhibiting>200 000 theoretical platesobtainedusing 10 columns (each 4.6 X 200 mm 5-pm Hypersll silica) In serles, with 2 mL/mln of 5 % methanol In carbon dioxMe at 60 OC, 150 bar outlet pressure.
techniques are also severely limited by injection and detection problems and have not found a place in routine analysis. Lifetime. After 900 h of operation involving many hundreds of injections of numerous families of compounds, includingreal samples,other nonprogrammedchromatograms were collected. A Brazilian lemon oil was separated on 10 columns using 2.0 mL/min of 5 % methanol in carbon dioxide, as shown in Figure 2. The detector wavelength was 270 nm. Observed efficiencies bracketed 200 OOO plates with individual peaks sometimes exhibiting substantially higher values (i.e., -260 OOO plates, h, = 1.54). Thus, after fairly extensive use, column efficiency remained at the theoretical maximum while pressure drop only increased slightly (one fewer column was used in the second experiment). After 1500 h of operation, one of the columns began to show distorted peaks for some polar molecules. Parameter Programing. PAHs. Polycyclic aromatic hydrocarbons (PAHs) are often separated by either hightemperature GC or capillary SFC with a density program. They can also be separated by packed column SFC as shown in Figure 3. With seven columns in series, a standard mixture of 40 PAHs (Table I) was separated using a combination of pressure and composition programming. The initial conditions included 80 bar outlet pressure, 2 mL/min of 2% methanol in carbon dioxide, and 60 OC. Pressure and composition were programmed through several steps to 130 bar and 20% methanol. The solutes ranged from indene (CgH6, 2 rings) to coronene and rubrene (eight rings), and decylene (10 rings, MW = 532). Scraping8from the interior of a chimney used with a woodburning stove were analyzed for PAHs. Approximately 100 mL of toluene was added to 50 g of the residue. The mixture was sonicated, stirred, and then allowed to stand for several hours. Fifty milliliters of the liquid was decanted, filtered, and evaporated to dryness at room temperature. The residue was redissolved in 0.5 mL of toluene of which 5 pL was injected. A representative chromatogram collected at 254 nm is presented in Figure 4 and indicates that a large number (>80) of compounds was present. A blank run is also shown. Such complex chromatograms collected in this short time are generally only obtained in capillary GC. Efficiency and Resolution Using Pure Fluids. Much of the previously quoted literature’-5 focused specifically on the effect of pressure drops on peak shapes and efficiency. However, only pure fluids were considered as mobile phases. The results reported above were obtained with binary mobile phases. With binary fluids, composition is more important
1453
in determining retention than density or pressure.19 It is possible that solutes in binary fluids are less influenced by density or pressure gradients than in pure fluids. This paper is about potential efficiency losses associated with pressure and density gradients. However, efficiency is only a small part of chromatographicresolution. If the solutes are unretained (very low k’)at the density near the inlet, then that part of the column could act as nothing more than a transfer line. Apparent efficiency would remain high, but retention and resolution would be lower than if no gradient existed. Some workers have rejected the use of packed columns with significant pressure drops to avoid such situations. It is important to quantify the extent to which resolution can be degraded by density gradients to see if such caution is justified. Resolution (R) is a function of efficiency (n1/2),selectivity ((a- l)/a), and retention (k’/(k’ + 1)).
R = n’”(k’/(l+ k’))((a- l)/c~) Longer retained peaks tend to be better resolved, but most of this gain occurs at low k’ values &e., 1-2) and is minimal above k‘ = 10. With very large pressure drops, the inlet must be at relatively high pressure and density compared to the outlet. Flow rate was changed to study the effect of pressure drops on both efficiency and resolution. Unlike earlier ~0rk8,*-3 outlet pressure was fixed and inlet pressure was allowed to vary. The natural variation in efficiency accompanying flow changes, embodied in the van Deemter equation, cloud interpretation. Nevertheless, if pressure drops or density gradients have a large impact on efficiency, a comparison at two flow rates should be adequate to demonstrate such an effect. Numerous gasoline8and diesel fuels were directly injected onto a stack of 10 columns in series (totallength 2 m). Such low-boiling, low-polarity samples are better analyzed by GC. Chromatograms of a gasoline sample, collected at 220 nm, were specifically chosen to study the effect of pressure drops on efficiency and resolution. At this wavelength, only olefins and aromatics absorb,greatly simplifyingthe chromatograms since the majority of the compounds present are aliphatic and are not detected. At 2 mL/min, all the gasoline peaks eluted in less than 30 min (k’< 3), using 70 bar (outlet) and 100 “C. With a 120-bar pressure drop, the inlet was at 190 bar. This corresponds to an inlet density of 0.45 g/cm3 and an outlet density of 0.12 g/cm3. Typical published relationships (i.e., refs 20 and 21) between density and retention indicate that retention should change as much as 100times in passing through the density gradient along the column. The ratio of inlet to outlet densities was 3.75. This results in a decompression and expansion of the mobile phase by 3.75 times as it moves through the column, similar to the GC case described by the James-Martin gas compressibility correctionfactor familiar in GC. Thus, the solutes slow down due to increasing partition ratios (up to 100 times) and simultaneously speed up (3.75 times) due to higher mobilephase velocities as they pass down the column. The 10 columns in series produced 90 000-120 OOO plates but all the peaks were symmetrical, as shown in Figure 5a. The same column produced up to 200 OOO plates with more polar solutes and binary fluids at the same flow rate but lower temperatures. The lower efficiency could have arisen from the density gradient, but several other causes are also possible. (19) Berger, T. A.; Deye, J. F. Anal. Chern. 1990,62, 1181-1185. (20) Wilsch, A.;Schneider, G. M. Chrornatographia 1986,8,234-252. (21)Schmitz,F.P.; Klesper,E. J.HighResolut. Chrornatogr. 1987,10, 519-521.
1454
ANALYTICAL CHEMISTRY, VOL. 65, NO. 10, MAY 15, 1993 3
'q 7
5.00
10.00
25.00
15h 20.00 Time (rnin)
30:OO
Flgure 3. Chromatogram of a standard mixture of 40 polycyciic aromatic hydrocarbons (PAHs) obtained using seven columns in series with programmed pressure and composition. See Table I for a numbered list of solutes. Conditions: 2.0 mL/min, 60 OC. Composition program: initially 2% methanol in carbon dioxide for 5 min, then 3%/min to 5 % , hold 5 min, then 1%/min to 20%, hold 10 min, and then 20%/min to 2%. Pressure program: initial 80 bar for 5 min, then 5 bar/min to 130 bar, hold 21 min, and then 20 bar/min to 80 bar. Pressure drop was 123-1 24 bar.
Table I. Polycyclic Aromatic Hydrocarbons in Mixa 1 indene 2 indan 3 durene 4 azulene 5 1-methylnaphthylene 6 2-methylnaphthylene 7 1,3-dimethylnaphthylene 8 1,4-dimethylnaphthylene 9 1,5-dimethylnaphthylene 10 2,3-dimethylnaphthylene 11 2,6-dimethylnaphthylene 12 fluorene 13 anthracene 14 phenthrene 15 2-methylanthracene 16 9-methylanthracene 17 fluoranthrene 18 pyrene 19 phenylnaphthylene 20 9,10-dimethylanthracene
21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
1,2-benzofluorene 2,3-benzofluorene 9,lO-benzophenanthrene 1,2-benzanthrene 2,3-benzanthrene benzo[alpyrene benzo[elpyrene perylene binaphthyl 9-phenylanthracene 7,i2-dimethylbenz[alanthracene 3-methylcholanthrene 1,2-benzberylene pentacene 1,2,5,6-dibenzanthracene 1,2,3,4-dibenzanthracene coronene p-quaterphenyl decylene rubrene
Listed in elution order.
Decreasing the flow rate to 1 mL/min resulted in the pressure drop falling to -60 bar. The inlet pressure dropped to 130 bar and the inlet density dropped to 0.29 g/cm3. The outlet pressure and density remained the same as before. This decrease in the pressure drop and the density gradient should result in an improvement in resolution and efficiency if density gradients produce the problems predicted. When the flow was decreased from 2 to 1 mL/min, the apparent partition coefficients (k') of early-eluting peaks approximately doubled, as shown in Table 11. This indicates that the average density changed significantly with flow. The value for k'/(k' + 1) increased more than 40%. Selectivity (a = k'2/k'l) increased 7 - l o % , while (a - 1 ) / a (from the resolution equation) increased 15-20%. Overall, efficiency degraded by 33 % while resolution of the early-eluting peaks increased 50-150% when the pressure
120
100
Chimney Extract 80
60
40
20
0
Blank 0
-a
IC
l " " 1 " " / " " 1 " " / " " ,
->O.OO
5.00
10.00
15.00
20.00
25.00
30.00
Flgure 4. Chromatogramof a concentratedextract of scrapings from a chimney used with a wood-burning stove showing the high resolution obtainablewith the seven columnsconnectedin series. Same program and conditions as in Figure 3.
drop was decreased by 50 % , as can be seen in Table I1 and Figure 5b. Since the efficiency a t 2 mL/min was up to 60% of theoretical, the effect of density gradients on efficiency must be modest (i.e., 140% ). The gain in resolution appears to be mostly due to the increase in apparent k' values. While such losses in resolution were significant at the higher flow rate, they were not catastrophic. Resolution between such solutes can always be enhanced by further decreasing outlet density and/or decreasing the flow rate. With later eluting peaks (at 2 mL/min k' = 2.35), resolution actually decreased slightly when the flow rate was dropped. It is still widely thought that a phase transition accompanies a change from gaseous t o supercritical conditions,so pressures
ANALYTICAL CHEMISTRY, VOL. 85, NO. 10,MAY 15, lQQ3 1455 *U
1
I 5 m .
11.50
1
12.50
13.W
CONCLUSIONS
, L
I lSh
12.00
2Q!W
22,'W
24,'W
n m imlni
Figure 5. Qasoiine separated at (a, top) 2 mL/min, pressure drop 123-124 bar, and (b, bottom) 1 mL/min, pressure drop 81-82 bar. Conditions: pure carbon dioxide at 100 O C , 70 bar outlet pressure; column 4.8 X 2000 mm, 5-pm HypersU slllca.
Table 11. Effect of Pressure Drop on Resolution and Efficiency Using Three Randomly Selected Peaks in a Gasoline SampleP no. k' a a- l / a k'/(l + k') N R 2 mL/min 1
89 400
0.375 1.64
2
0.616
3
0.810
1
0.678
0.390
0.381
10.4 119 900
1.32
0.242
0.448
5.06 109 200
1 mL/min 60 900 1.81 2
1.23
3
1.74
0
0.448
0.552
16.8 79 200
1.41
GC) was not demonstrated since the detector was inappropriate for very low density fluids. High-pressure (from 1 to 50 and from 1 to 80 bar) gas chromatography with carbon dioxide was performed as long ago as 196522-24 and showed that, in GC with certain carrier gases (including carbon dioxide), retention was inversely related to pressure. Temperature could be lowered by increasing pressure while retaining constant retention. However, significant increases in density are required to provide modest increases in solvating power. Since solute binary diffusion coefficients are inversely proportional to density, speed of analysis drops significantly for small decreases in temperature. There is little to recommend such a trade-off. Gas chromatography offers superior speed, efficiency, sensitivity, detector options, and cost compared to SFC. SFC should generally be considered only when some solute characteristic prevents the use of GC.
0.291
0.635
12.9 81 700
Pressure drop was varied by changing flow rate. Optimum flow
as near 2 mL/min. Conditions: pure carbon dioxide a t 100 OC, outlet pressure 70 bar, pressure drop a t 2 mL/min 123 bar, pressure drop at 1 mL/min 61-62 bar. Column: 4.6 X 2000 mm, 5-pm Hypersil silica.
lower than 75-80 bar are generally not used. However, GC and SFC are a continuum. Only practical instrumental problems make it difficult to easily make a transition from one technique to the other. The 70-bar outlet pressure used above is actually below the critical pressure of carbon dioxide, so the outlet fluid was a gas (not supercritical). Thus, most of the column performed SFC, but part performed high-pressure GC at the same time. Such effects were further exaggerated by using outlet pressures as low as 45 bar (inlet at 165 bar). Both retention and resolution of the very light hydrocarbons increased but the later eluting, heavier components required programming to higher pressures to elute with good peak shapes. The even greater "extreme" of using atmospheric outlet pressures (Le., (22) Desty, D. H. In Adoances in Chromatography; Giddings, J. C., Keller, R. A., Eds.; Marcel Dekker: New York, 1965; Vol. 1, Chapter 6. (23) Sie, S. T.; Van Beersurn, W.; Rijnders, G. W. A. Sep. Sci. 1966, 1,459-490. (24) Wicar, S.; Novak, J. J. Chrornatogr. 1974, 95, 13-26.
Packed-column SFC is capable of generating very high efficiencies in relatively short times. A 4.6-mm-i.d., 2.2-mlong column packed with 5-pm particles produced a speed vs resolution trade-off similar to those obtained with widely used GC capillary columns. While achieving such high efficiencies, the ability to dramatically change retention through parameter programming was maintained. The efficiency of packed-column SFC is not limited by column pressure drop, despite suggestions to the contrary.'" It appears likely that 400 OOO plates are possible with common hardware. Density or retention gradients do not appear to limit efficiency either. Contrary to previous observations1.2.5 both large and small k' values appear to produce similar efficiencies. Contrary to previous 0bservations1~2~~ the outlet pressure did not appear to limit efficiency provided a single mobile phase existed. The minimum acceptable outlet pressure5 also appeared to be unrelated to k'. Previous theoretical treatments of retention gradients do not describe the observed results. The observed results do appear to support a more recent theoretical treatment.14 Problems with pressure or density gradients have been exaggerated and ignore the significant advantages that columns producingsuch gradientsprovide. Very long columns allow high efficiencies for the separation of more complex samples more rapidly than LC. The loss of resolution between light, low-polarity solutes due to density gradients has been overemphasized. Such solutes are better analyzed by GC and, thus, only represent a limitation at the edge of useful conditions in SFC. Higher boiling and/or more polar compounds, which are more representative of the kinds of solutes for which SFC offers a contribution, are more highly retained and are, thus, more easily resolved. In this work, all the columns employed the same stationary phase. Additionalselectivity adjustment might be obtainable by packing one or more of the columns with different stationary phases. All the compounds eluted in this work were of relatively low to moderate polarity. In a companion work a number of pesticide families were eluted on long packed columns with results similar to those reported above. The pesticides included families difficult or impossible to elute in GC, such as triazines, phenylureas, sulfonylureas, carbamates, and organophosphorous pesticides.
RECEIVEDfor review February 12, 1992. December 30, 1992.
Accepted
