Anal. Chem. 1985, 57,2239-2242
the average reduced plate height, 4.37, calculated from the four injections with the result obtained with pyrene in Table I1 showed a 20% decrease in efficiency in this elution system. Figure 6 shows a chromatogram of human urine after administration of the antibiotic Latamoxef. Paired-ion chromatography technique was used to adjust the retention time and suppress tailing on the chromatogram due to the carboxylic group in the molecule. Two epimeric isomers could be clearly separated from the endogenous urinary components in a reasonable analysis time. Registry No. Stainless steel, 12597-68-1; benzophenone, 119-61-9; biphenyl, 92-52-4; anthracene, 120-12-7; benzamide, 55-21-0; acetophenone, 98-86-2; (R)-latamoxef,64952-97-2; (S)latamoxef, 79120-38-0. LITERATURE CITED Ishii, D.; Asai, K.; Hibi, K.; Jonokuchi, T.; Nagaya, M. J . Chromatogr. 1977, 144, 157-168. Tsuda, T.; Novotny, M. Anal. Chem. 1978, 5 0 , 271-275. Scott, R. P. W.; Kucera, P. J. J. Chromatogr. 1979, 769, 51-72. Yang, F. J. J . Chromatogr. 1982, 236, 265-277. Hirata, Y.; Jinno, K. HRC CC,J . H@h Resolut. Chromatogr. Chromatogr. Commun. 1983, 6 , 196-199. Ishii, D.; Murata, S.; Takeuchi, T. J . Chromatogr. 1983, 2 8 2 , 569-577. McGuffin, V. L.; Novotny, M. Anal. Chem. 1981, 5 3 , 946-951. McGuffin, V. L.; Novotny, M. J. Chromatogr. 1981, 218, 179-187. Tsuge, S.; Hirata, Y.; Takeuchi, T. Anal. Chem. 1979, 57, 186-169. Henion, J. I n "Microcolumn High-Performance Liquid Chromatography"; Kucera, P., Ed.; Eisevier: Amsterdam, 1984; pp 260-300. Hirata, Y.; Novotny, M.; Tsuda, T.; Ishii, D. Anal. Chem. 1979, 5 1 , 1807-1 809. Tsuda, T.; Novotny, M. Anal. Chem. 1978. 5 0 , 632-634. Tsuda, T.; Nakagawa, 0. J. Chromatogr. 1980, 199, 249-258. Ishii, D.; Takeuchi, T. J . Chromatogr. Sci. 1980, 18, 462-472. Tijssen, R.; Bieumer, J. P. A.; Smit, A. C. C.; Van Kreveid, M. E. J. Chromatogr. 1981, 218, 137-165. Takeuchi, T.; Ishli, D. J . Chromatogr. 1981, 213, 25-32.
2239
(17) Gluckman, J. C.; Hirose, A.; McGuffin, V. L.; Novotny, M. Chromatographla 1983, 17, 303-309. (18) Novotny, M.; Alasandro, M.; Konishi, M. Anal. Chem. 1983, 5 5 , 2375-2377. (19) Novotny, M.; Karlsson, K. E.; Konishi, M.; Aiasandro, M. J. Chromatogr. 1984, 292, 159-167. (20) Karisson, K. E.; Wiesier, D.; Alasandro, M.; Novotny, M. Anal. Chem. 1985, 5 7 , 229-234. (21) Tsuji, K.; Binns, R. B. J. Chromatogr. 1982, 253, 227-236. (22) Novotny, M.; Hirose, A.; Wiesier, D. Anal. Chem. 1984, 56, 1243-1248. (23) Novotny, M.; Konishi, M.; Hlrose, A,; Gluckman, J. C.; Wiesier, D. Fuel 1985, 6 4 , 523-527. (24) Hirata, Y.; Novotny, M. J. Chromatogr. 1979, 186, 521-528. (25) Shelly, D. C.; Giuckman, J. C.; Novotny, M. Anal. Chem. 1984, 5 6 , 2990-2992. (26) Konaka, R.; Kuruma, K.; Nishimura, R.; Kimura, Y.; Yoshida, T. J. Chromatogr. 1981, 225, 169-176. (27) Hartwick, R. A.; Dezaro, D. D.I n "Microcolumn High-Performance Liquid Chromatography"; Kucera, P., Ed.; Eisevier: Amsterdam, 1984; pp 75-110. (28) Ryall, R. R.; Kessler, H. D. Am. Lab. (Fairfield, Conn.) 1982, 14, 49-56. (29) Ishii, D.; Hirose, A.; Hibi, K.; Iwasaki, Y. J. Chromatogr. 1978, 157, 43-50. (30) Baker, D. R. LC Mag. 1984, 2 , 38-43. (31) Bristow, P.; Knox, J. Chromatographla 1977, 10, 279-289. (32) Sternberg, J. C. I n "Advances in Chromatography, Voi. 2"; Giddings, J. C., Keller, R. A., Eds.; Marcel Dekker: New York, 1966; pp 205-270. (33) Karger, B.; Martin, M.; Guiochon, G. Anal. Chem. 1974, 46, 1640-1647. (34) Snyder, L. R.; Kirkland, J. J. "Introduction to Modern Liquid Chromatography"; Wlley-Interscience: New York, 1979; pp 234-240. (35) Gluckman, J. C.; Novotny, M. I n "Microcolumn Separations"; Novotny, M., Ishii, D., Eds.; Eisevier: Amsterdam, 1985; pp 57-72. (36) Scott, R. P. W. J. Chromatogr. Sci. 1980, 18, 49-54.
RECEIVED for review March 4,1985. Accepted June 3,1985. Part of this paper was presented at the 33rd Annual Meeting of the Japan Society for Analytical Chemistry, Nagoya, Japan, Oct 10-14, 1984.
Modifier Effects on Retention and Peak Shape in Supercritical Fluid Chromatography Ann Lisbeth Blilie and Tyge Greibrokk*
Department of Chemistry, University of Oslo, P.O. Box 1033, Blindern, 0315 Oslo 3, Norway
The addltlon of organlc modlflers to supercrltlcal carbon dloxide reduced the retentlon and Improved the peak shape of polycyclic aromatic hydrocarbons, nltrated polycycllc aromatic hydrocarbons, polystyrenes, and a selected group of more polar compounds on comrnerclally avallable reversedphase C,, columns. 1-Alkanols reduced the retentlon more than branched alcohols and the Impact Increased wlth Increasing chaln length up to 1-hexanol. Addltlon of 1-10% modifiers was needed In order to elute large and polar compounds wlth reasonable retentlon and good peak shapes. The modlflers functloned as deactlvatlon agents by direct lnteractlons wlth resldual sllanol groups and also as modlflers of the elutlng power of the moblle phase. A reversed-phase column which contained few residual sllanol groups could be uilllzed without modlflers for compounds of Intermediate polarities.
Addition of polar modifiers to supercritical fluids has been reported by several investigators to have a considerable effect
on the retention characteristics in supercritical fluid chromatography (SFC). Small amounts of methanol, ethanol, or 2-propanol improved the resolution of polystyrenes in alkanes (1-6) and in carbon dioxide (7) and also improved the resolution of polycyclic aromatic hydrocarbons (PAH) in carbon dioxide (8, 9). On reversed-phase columns the addition of methanol or ethanol was needed in order to elute a mixture of ubiquinones and a mixture of vitamins A, E, and D2(10). The addition of alcohols also improved the peak shapes of aromatic peroxides (11) and of carotenoids (12). The retention of polystyrenes on reversed-phase columns was reported to increase with the addition of ethanol to supercritical hexane ( 5 ) ,while on silica contradictory results have appeared. In one report the retention was increased over the whole concentration range by the addition of 0-20% ethanol (6),whereas in another report the retention was greatly reduced by additions of less than 1% ethanol and increased by additions beyond 1% (5). Tetrahydrofuran (THF), which otherwise is known as a good solvent for polystyrenes, was found to have little modifier effect with hexane on silica and was recommended as solvent for large injection volumes (6).
0003-2700/65/0357-2239$01.50/0 0 1985 American Chemical Society
2240
ANALYTICAL CHEMISTRY, VOL. 57, NO. 12, OCTOBER 1985
This paper is a part of an investigation of the effects of different modifiers in chromatography with supercritical fluids on reversed-phase columns. The main part of the study was performed on microbore c18 columns, which apparently contained appreciable amounts of residual silanol groups. A comparative study included another column which had been specifically treated by the manufacturer to reduce the amount of silanol groups. EXPERIMENTAL SECTION The instruments including modified Waters Model 6000 A pumps have been described in detail elsewhere (13). The solvents were of HPLC grade S quality from Rathburn Chemicals (Walkerburn, U.K.) or of pro analysis quality from other suppliers if not available from Rathburn. The reference substances were obtained from several different commercial sources, except the nitro-PAH, which were synthesized in our laboratory (14-16). The nitro-PAH, which have been studied due to their mutagenic properties, had a purity of about 99.9%. The polystyrenes (A?, 800) were obtained from Supelco, Inc. (Bellefonte, PA). The CP-Spher Columns (250 X 1.3 mm) were obtained from Chrompack (Middelburg, Holland). NOVA-PAK CIBcolumns (150 x 3.9 mm), which are sequentially bonded and end-capped to remove residual silanol groups, were obtained from Waters Associates (Milford, MA). Injections of 0.1-1 p L were performed with a Valco CI.4 W injector, which was heated to 35-40 "C. Larger volumes were injected with a Rheodyne 7410 injector (at room temperature). All samples for the modifier experiments were dissolved in dichloromethane, except when mentioned otherwise. On the Perkin-Elmer LC-55 UV detector all of the steel tubing prior to the flow cell was removed in order to reduce peak broadening. The quartz windows in the flow cell were tightened with an extra set of springs. The modifiers were added to carbon dioxide with a Waters Model 590 microflow pump and a T piece of steel tubing. The simple mixing system resulted in retention times with a variation of less than 2 % . The amount of modifier is given as volume per volume of the liquid phases. When the system was transferred from one modifier to another, a purging time of 30-45 min was needed in order to obtain stable conditions on the column. To be on the safe side, each column was purged with the new mobile phase for ll/zh before use. The void volumes needed to calculate capacity factors were determined by using the dichloromethane solvent peaks. Asymmetry factors ( A , = b/a) were determined at 10% of the peak height. Column efficiencies were calculated as
&) 2
N,,, = 5.54(
RESULTS AND DISCUSSION Modifier Effects. When a group of PAH with two to six condensed rings were chromatographed with pure carbon dioxide on CP-Spher C18 columns, the capacity factors varied between 1 and 75 and the late eluting peaks were broad and asymmetric. A group of nitro-PAH showed a similar picture, but with even more retained and asymmetric peaks. When methanol was added to carbon dioxide, as a modifier of the mobile phase, the retention was reduced. The addition of modifier also improved the peak shapes considerably. For PAH and for nitro-PAH, 1% modifier was sufficient to obtain symmetric peaks. Higher concentrations of modifier continued to decrease the retention but had no effect on peak symmetries. The asymmetric peaks in pure carbon dioxide were not a result of low solubility, since injection of smaller loads (1/10) had no effect on the peak shapes or on the retention times. The effects of different alcohols as modifiers on the retention of a few selected PAH and nitro-PAH are demonstrated in Figure 1. The retention decreased with increasing chain length of straight-chain alcohols up to hexanol. Higher alcohols resulted in the same retention as with hexanol. A
l o g k'
BENZOlghil PERYL ENE
i
3-NOz-FLUORANTHENE
1
1,oo
w
Flgure 1. Modifier effects ( 2 % v/v) with supercritical carbon dioxide at 40 "C and 160 bar on CP Spher C,*. PS is polystyrene ollgomer. The samples were Injected in 1 pL of dichloromethane.
straight-chain alcohol decreased the retention more than a branched alcohol, and this effect on the eluting strength decreased with increased branching (primary > secondary > tertiary). Of the other modifiers that were examined hexane had little effect, while acetonitrile and the cyclic ethers dioxane and THF resulted in the largest reduction in the retention of PAH and nitro-PAH. The bulky methyl butyl ether had little effect, similar to hexane. The modifiers had a similar effect on the retention of a mixture of polystyrenes (M,800), as shown by one of the oligomers in Figure 1. The retention of the large polystyrenes was reduced over the concentration range of 0-5% methanol, whereas an increase from 5% to 10% had little effect. These results are in full agreement with earlier studies on the effects of methanol on polystyrenes in supercritical pentane on silica (4) and in carbon dioxide on a Cl8 column (7). In the present system tetrahydrofuran wm found to be an effective modifier, comparable to ethanol/propanol (Figure 1) and henceforth not recommendable as solvent for large injections. By injection of model compounds which gave symmetrical peaks with pure carbon dioxide, the use of T H F and carbon disulfide as injection solvents was found to result in significant band broadening with larger solvent volumes (Table I). A similar pattern was found with the other injector on another column. Since the retention of polystyrenes was reported to be increased by the addition of alcohols to supercritical hexane on reversed-phase columns (6),polystyrenes appear to be better solubilized in supercritical hexane than in supercritical carbon dioxide. Considering the high critical temperature of hexane, this suggestion is not unreasonable. On silica the retention is expected to decrease with small amounts of alcohols, due to deactivation of the adsorbent. High amounts of alcohols may increase or decrease the solubilization, depending on the "solvent strength" of the fluid. The final effect of adding a
ANALYTICAL CHEMISTRY, VOL.
Table I. Column Efficiency (N,2) and Asymmetry Factors ( A#) as a Function of Injection Golumes (Vi) and Solvents, Measured by Injecting 15 pg of Pentadecylbenzene (k'= 3.8) with Supercritical Carbon Dioxide at 40 "C and 170 bar on CP Spher C18 (250 X 1.3 mm)
L
A254 nm (rnAU1
vi = 1p L
vi= 0.1 pL solvent
N,jz
A,
NljZ
4
methanol acetonitrile methyl tert-butyl ether n-octane dichloromethane tetrahydrofwan carbon disulfide
5900 5900 6100 6300 6000 6000
1.3 1.3 1.3 1.3 1.3 1.3
5900 6300 5400 4900 4600 3700 3000
1.2 1.2
57, NO. 12, OCTOBER 1985 2241
0
10
20
40 tR (mini
30
1.1 1.2
1.1 1.1 1.0
A
A254 nrn lmAUl
6050-
40-
3020. 10B
C
I
180mAU
0
I I
,
'
0
,
2
4
6
8
10
12
14 t,(min)
Flgure 3. Nitro-PAH eluted with carbon dioxide at 40 OC and 180 bar on CP Spher C,B (A) and on NOVA-PAK C,8 (6): 1-nitronaphthalene
0
5
10
15
20 tRimin)
0 2 4 tR(rninl
Flgure 2. 13H-Dibenzo[a ,i]carbazole eluted with 0% (A), 1 % (B), and 10% (C) methanol in carbon dioxide at 40 OC and 160 bar on CP Spher CI8.
certain amount of an alcohol is then a combination of the deactivation effect and the solubilizing effect. Apparently the results that were reported on the effect of 0-20% ethanol (6) cannot be correct, as indicated by the more recent results on the same columns ( 5 ) ,illustrating the bifunctional effect of the alcoholic modifiers. A higher eluting power of supercritical hexane compared to supercritical carbon dioxide also explains the different effects of tetrahydrofuran. With supercritical hexane T H F acted as a relatively weak solvent, allowing large injection volumes (6),whereas with carbon dioxide THF acted as a relatively strong solvent prohibiting the use of large volumes (Table I). Even if the studies with supercritical hexane have been performed with a different instrumentation, the results suggest, in our opinion, that the use of supercritical hexane could be advantageous compared to carbon dioxide for the analysis of mixtures of polystyrenes. The effect of modifiers on compounds with polar groups was remarkable. Arachidonic acid was completely adsorbed with 5% acetonitrile in carbon dioxide but eluted with little retention as a symmetrical peak with 0.1% formic acid plus 5% acetonitrile. With these modifiers UV detection at 190 nm became possible (17). Another carboxylic acid, benzoic acid, also resulted in strong retention and severe tailing and even an ester, methyl stearate, resulted in considerable tailing, which disappeared with 1%methanol. Other compounds with polar groups, such as 2,7-dinitro9-fluorenone and 13H-dibenzo[a,i]carbazole(Figure 2) also
(12), 2-nitrofluorene (13),3-nitrofluoranthene (14), 1-nitrotriphenylene (15), 2-nitrotriphenylene (16), 1-nitrobenzo[e]pyrene (17), 6-nitrobenzo[a Ipyrene (la), g-nitrodibenz[a ,c]anthracene (19), 3-nitrobenzo[e] pyrene (20), 7-nitrobenzo[ghi]perylene (21), and 5-nitrobenzo[ghi]perylene (22).
showed severe tailing without the addition of alcoholic modifiers. Column Effects. Reversed-phase columns which give asymmetric peaks of polar compounds may be expected to contain high amounts of residual silanol groups. Accordingly, another CIS column, which had been specifically treated by the manufacturer to remove silanol groups, was examined. On the NOVA-PAK column the PAH and the nitro-PAH (Figure 3) eluted largely as symmetric peaks, without the addition of modifiers. The difference between the two columns is demonstrated by the effect of five different modifiers on benzo[ghi]perylene and on 7-nitrobenzo[ghi]perylene (Figure 4). The higher elution power of hexanol compared to methanol can be attributed to a better coverage of the active sites by a long lipophilic alcohol or to the increased solubility in hexanol compared to methanol of the large PAH and nitro-PAH. However, since straight chain alcohols reduced the retention more than branched alcohols, which we suggest is a question of easier access to sterically crowded active sites, the explanation based on a better coverage of the active sites is favored. The effect of different amounts of methanol on the retention of benzo[ghi]perylene and 7-nitrobenzo[ghi]peryleneon the two columns is shown in Figure 5. The difference between the two columns in the 0-2% range clearly illustrates the little contribution of silanol groups to the retention on the NOVA-PAK column. Of the more polar compounds, methyl stearate now eluted as a sharp, little retained peak, while benzoic acid tailed badly
2242
ANALYTICAL CHEMISTRY, VOL.
57,NO. 12, OCTOBER 1985
considerably, but the symmetries as well as the peak widths were far from optimal. In general, the NOVA-PAK column could be used with compounds of medium polarity without the addition of modifiers to the mobile phase. For compounds of higher polarity, better peak shapes were obtained with modifiers.
k'
80,O70,O/ A
60,O50,O40,o-
30020,o10,o-
Y
Figure 4. Modifier effects on the retention of benzo[ghi]perylene (A) and 7-nkrobenzo[ghl]perylene (E) at 40 "C and 180 bar on CP Spher C, (0)and NOVA-PAK C,8 (0). The amount of modifier was 2%
Registry No. P.S., 9003-53-6; methanol, 67-56-1; ethanol, 64-17-5; 1-propanol, 71-23-8; 1-butanol, 71-36-3;1-pentanol, 7141-0; I-hexanol, 111-27-3;I-heptanol, 111-70-6;I-decanol, 112-30-1; 2-propanol, 67-63-0; 2-butanol, 18-92-2;2-pentanol, 6032-29-7; tert-butyl alcohol, 75-65-0; methyl tert-butyl ether, 1634-04-4; diisopropyl ether, 108-20-3;tetrahydrofuran, 109-99-9;dioxan, 123-91-1;acetonitrile, 75-05-8;chloroform, 67-66-3;hexane, 11054-3; benzo[ghi]perylene, 191-24-2;7-N02-benzo[ghi]perylene, 81316-88-3; 3-NQ2-fluoranthene,892-21-7;fluoranthene, 206-44-0; pentadecylbenzene, 2131-18-2; n-octane, 111-65-9; dichloromethane, 75-09-2; carbon disulfide, 75-15-0; '3H-dibenzo[a,i]carbazole, 239-64-5; I-nitronaphthalene, 86-57-7;2-nitrofluorene, 607-57-8; I-nitrotriphenylene, 81316-78-1;24trotriphenylene, 81316-79-2;1-nitrobenzo[e]pyrene, 91259-16-4; 6-nitrobenzo[a]pyrene, 63041-90-7; 9-nitrodibenz[a,c]anthracene,83314-29-8; 3-nitrobenzo[e]pyrene, 81340-58-1; 5-nitrobenzo[ghi]perylene, 81316-87-2; carbon dioxide, 124-38-9.
LITERATURE CITED
(v/v).
Sie, S.T.; Rijnders, G. W. A. Sep. Sci. WS7, 2, 729. Jentoft, R. E.; Gouw, T. H. J. Chromatogr. Scl. 1970, 8, 138. Nieman, J. A.; Rogers, L. B. Sep. Scl. 1975, 10, 517. Klesper, E.; Hartmann. W. Eur. Po/ym. J. 1978, 14, 77. Hirata, Y. J. Chromatogr. 1984, 315,31. Hirata, Y.; Nakata, F. J. Chromatogr. 1984, 295, 315. Gere, D. R. Application Note AN 800-3; Hewlett-Packard Co.: Avondale, PA, 1983. (8) Jentoft, R. E.; Gouw, T. H. Anal. Chem. 1976, 48, 2195. (9) Gere, D. R. Application Note AN 800-1; Hewlett-Packard Co.: Avondale, PA, 1983. (10) Board, R.; Mc Manigill, D.; Weaver, H.; Gere, D. R. Presented at the 1982 Pittsburgh Conference on Analytical Chemlstry and Applied Spectroscopy. (11) Gere, D. R.; Stark, T. J.; Tweeten, T. N. Appllcation Note AN 800-4; Hewlett-Packard Co.: Avondale. PA, 1983. (12) Gere, D. R. Applicatlon Note AN 800-5; Hewlett-Packard Co.: Avondale, PA, 1983. (13) Greibrokk, T.; Blllie, A. L.; Johansen. E. J.; Lundanes, E. Anal. Chem. 1984, 56, 2681. (14) Svendsen, H.; Rsnningsen, H.-P.; Sydnes, L. K.; Greibrokk, T. Acta Chem. Scand., Ser. B 1983, 37, 833. (15) Johansen, E.; Sydnes, L. K.; Grelbrokk, T. Acta Chem. Scand., Ser. B 1984, 838, 309. (16) Iversen, B.; Sydnes, L. K.; Greibrokk, T. Acta Chem. Scand., Ser. 8 in press. (17) Dshl, J.; Greibrokk, T., unpublished results. (1) (2) (3) (4) (5) (6) (7)
k' I
1
70,O
60.050,o40,o-
30,O20,o10,o-
0
2
4
6
8
10 % M d H
Flgure 5. Effects of 0-10% methanol in carbon dioxide on the retention of benzo[ghi]perylene (A) and of 7-nitrobenzo[ghi]perylene (B) at 40 OC and 180 bar on CP Spher C,, (0)and NOVA-PAK C18
(0).
even on this column, without modifiers. 2,7-Dinitro-9fluorenone and 13H-dibenzo[a,i]carbazoleeluted with capacity factors comparable to those obtained with 10% methanol on the CP Spher CI8 columns. The peak shapes had improved
RECEIVEDfor review March 11,1985. Accepted May 20,1985.
