2235
Anal. Chem. 1985, 57. 2235-2239
brated in terms of polystyrene. ICP element-specific detection informs us that iron- and magnesium-containing compounds distribute widely in the molecular weight range larger than 500, while vanadium-containing compounds distribute in the molecular weight range smaller than those of other metal compounds. The elution time of the sharp peak that appeared in the UV chromatograms is almost consistent with that of the vanadium peak. This result on vanadyl compounds can be explained by the fact that vanadyl porphyrin compounds have molecular weights less than 600, although complexation to the asphalthene fraction of the oil could drastically increase their apparent molecular weight (19). Even though the chromatogram monitored at aluminum emission line is not clear because of its low sensitivity, aluminum-containing compounds also seem to be present in the molecular weight range around 300. The example demonstrated here strongly implies that the microcapillary LC-ICP system can provide a molecular weight categorization of organometallic compounds present in a shale oil, although more detailed discussions on the speciation are not performed because they are beyond the scope of this contribution. With the help of this technique, speciations of organometallic compounds in petroleum samples will be attempted effectively, since the method provides information on molecular weight and spectroscopic characterization along with elemental composition. Thus it is expected to widen application especially in the fields of oil chemistry and biochemistry, as many important substances have metal atoms in their functional sites.
CONCLUSION With the direct sample introduction system, i.e., the %ospray chamber" system proposed in this work, ICP has been
able to work as a detector for microcapillary LC without any change in spectrometric characteristics. The results of this instrumental investigation show the technique will allow us more precise studies on speciation and characterization of trace metals and organometallic compounds in petroleum and biological samples.
LITERATURE CITED Lawrence, K. E.; Rice, G. W.; Fassel, V. A. Anal. Chem. 1984, 56, 292. Ishii. D.; Asai, K.; Hlbl, K.; Jonokuchi, T.; Nagaya, M. J . Chromafogr. 1977, 144, 157. Novotny, M. Anal. Chem. 1981, 5 3 , 1294A. Novotny, M. Anal. Chem. 1983, 55, 1308A. Jinno, K.; Tsuchlda, H. Anal. Left. 1982, 15, 427. Jinna, K.; Nakanishi, S.; Tsuchida, H.; Hlrata, Y.; Fujimoto, C. Appl. Specfrosc. 1983, 3 7 , 258. Jinno, K.; Nakanishl, S. HRC CC J . H@h Resoluf. Chromafogr. Chromafop. Commun. 1983, 6 , 210. Jinno, K.; Nakanishi, S.; Nagoshi, T. Anal. Chem. 1984, 56. 1977. Jlnno, K.; Nakanlshi, S.; Nagoshi, T. Chromatographia 1984, 18, 437. Boorn, A. W.; Browner, R. F. Anal. Chem. 1982, 5 4 , 1402. Barrett, P.; Pruszkowska, E. Anal. Chem. 1984, 56, 1927. Hausler, D. W.; Taylor, L. T. Anal. Chem. 1981, 53, 1223. Schutyser, P.; Janssens, E. Spectrochlm. Acta, Part 6 1979, 346, 443. Browner, R. F.; Smlth, D. D. Anal. Chem. 1983, 55, 374. McGuffin, V. L.; Novotny, M. Anal. Chem. 1983, 55, 2296. Braetter, P.,Schramel, P., Eds. "Trace Element Analytical Chemistry in Medicine and Biology"; Walter de Gruyter & Co.: Berlin, 1980. Fish, R. H.; Komlenic, J. J. Anal. Chem. 1984, 56, 510. Fish, R. H.; Komlenic. J. J. Anal. Chem. 1984, 56, 2452. Sugihara, J. M.; Branthaver, J. F.; Wu, G. Y.; Weatherbee, C. Prepr. Am. Chem. SOC.Div. Pet. Chem. 1970, 15, C5.
RECEIVED for review March 12,1985. Accepted June 11,1985. This paper was presented in part at the 6th International Symposium on Capillary Chromatography held in Riva del Garda, Italy, in May 1985.
High-Performance Packed Glass-Lined Stainless Steel Capillary Column for Microcolumn Liquid Chromatography Masaharu Konishi,* Yoshio Mori, and Tameyuki Amano Shionogi Research Laboratories, Shionogi & Co., Ltd., Fukushima-ku, Osaka 553, Japan
A promlslng new glass-lined stainless steel tublng was successfully used as a packed narrow-bore column for hlghperformance llquld chromatography and examlned for Its potential as a microcolumn for routlne cllnlcal work. A 30 cm X 0.3 mm column with 40000 theoretlcal plates could be easily packed with 3-pm ODS particles by the same procedure used for conventlonal columns. The analytical system used ordlnary equipment with no modlflcation except a mlniaturized flow-through cell for detection. The effect of column dimension (length and Inner diameter) on column efflclency and use of the column for routlne clinical work are dlscussed.
Recent advances in high-performance liquid chromatography (HPLC) column technology have facilitated analysis of a wide range of compounds which cannot be subjected to gas-liquid chromatography (GLC). Separation power is in-
creased by the use of high-quality microparticles for bonded phase packings (3 or 5 pm), which allow faster analysis with shorter columns. It can also be increased by capillary GLC, and some elegant attempts have been made to decrease column size by miniaturizing the system (1-5). The major benefits of microcolumn analytical technology are increased sample detectability and decreased solvent consumption. Reduction of column diameter by a factor of 10, considering the diameter to be 2-4.6 mm for a conventional column and 0.2-0.5 mm for a microcolumn, decreases solvent consumption 102-103-foldand increases detectability in a sample of limited volume. Lower flow rates offer cost savings, when expensive chemicals such as deuterated solvents and elution systems containing coenzymes (6)are employed, and also allow combination with new universal detection systems for HPLC such as flame-based photometric detection (7,8)and mass spectrometry (9,10). Capillary columns, which are generally called microcolumns, can be classified into three major categories, packed microcapillary columns (2, II), open-tubular columns
0003-2700/85/0357-2235$01.50/0 0 1965 American Chemical Society
2238
ANALYTICAL CHEMISTRY, VOL. 57, NO. 12, OCTOBER 1985
(12-15), and narrow-bore columns (4,16,17). Microcolumns are useful for studying various compounds which are of biological (18-21) or energy-related (22,23)interest. To make microcolumn liquid chromatography (MLC) adaptable to routine work, individual components including the injector, column, detector, and connector should be easy to make, purchase, or adapt from commonly available products. Thus far, most MLC assemblies have required special techniques to prepare (4,24) and thus are difficult to use for routine work. This paper describes a new glass-lined stainless steel tubing (GLT) for HPLC columns. It is promising for MLC because it is easy to handle and yet offers structural strength against pressure or physical force. The more fragile Pyrex glass tubing or fused silica material is commonly used for MLC at present (4,5,16-23). However, GLT columns ranging from conventional (-4.6 mm i.d.) to microbore (-1.0 mm i.d.) types are now commercially available (SGE, Inc.) for use with conventional systems. The GLT column is believed to offer several advantages over traditional stainless steel tubing in cleanness, smoothness, uniformity, and inertness of the tubing interior, all of which raise column efficiency. This paper describes the physicochemical evaluation of packed columns using GLT, 0.3-0.5 mm i.d. and 10-30 cm long, and also discusses the potential of using GLT microcolumns in routine work.
T O detector
FST ( 5 0 p m 1.0.)
TO injector
V e s ~ e Iferrule
I
P&
( f . 5 m m 1.0.) SUS F e r d e
I
Porous PTFE
Flgure 1. Schematic diagram of the connection device between column and detector.
(26).The UV response was recorded at a wavelength of 229 nm.
RESULTS AND DISCUSSION Connection Hardware. The connection between the GLT column and the detector is shown in Figure 1. A 7 mm long PTFE tubing ('/la in. 0.d. and 0.2 mm i.d.) was inserted into a 13.5-mm long PTFE tubing (2.2 mm 0.d. and 1.5 mm i.d.) and the GLT column was inserted from the other end. A porous PTFE was placed between the GLT column and the inner PTFE tubing. The fitting assembly described was inserted into a '/le in. stainless steel union with the inner diameter spread to 2.2 mm. For connection to the detector, a EXPERIMENTAL SECTION 50-pm fused-silica capillary tubing was inserted through the The chromatographic system used in this study consisted of inner PTFE tubing of the fitting. The distance between the a Varian Model 8500 syringe pump, operating in the constant column end and the detector cell through the fused silica pressure mode, a JASCO UVIDEC-100-11ultraviolet detector, tubing was 16 cm. A Vespel ferrule (SGE, Inc., Australia) and in which the time constant was fixed at 0.5 s, with a 10-nL destainless steel ferrule were used to tie the capillary tubing and tection cell modified with a fused silica capillary (Gaskuro Kogyo, Inc., Tokyo, Japan), and a Rheodyne 7520 valve injector with a the GLT column respectively. To connect the GLT column 0.2-pL sample rotor. The column material was capillary tubing and the injector, porous PTFE and a 0.5 mm long PTFE of glass-lined stainless steel (10,15 or 30 cm long, 0.3 or 0.5 mm tubing ('/I6 in. 0.d. and 0.2 mm i.d.) were placed on the top i.d., and 1/16 in. in 0.d.) which was purchased from a commercial of the column to be connected. The mechanical dead volume source (GaskuroKogyo, Inc., Tokyo, Japan). The frit of the GLT in the injection valve associated with an inserted tube (7.7 mm packed column was a porous polytetrafluoroethylene (PTFE) X 0.13 mm i.d.) between the sample rotor and the column port (Umetani Seiki Co., Osaka, Japan) 1mm thick, 4 mm in diameter, was 0.1 pL. The system functioned satisfactorily without and 0.7-pm pore size, which was pressed to a thickness of 50 pm considerable band broadening which arises from dead volume with a micrometer before use (25). The column was packed with due to unsuitable fittings as described below. 3- or 5-pm SpherisorbODs-2 (Phase Sep., Hauppauge, NY) using Evaluation of Packing Technique. Choice of the slurry a slurry-packmethod. The 0.3 g/mL slurry of Slurry Mix (Chemco Scientific Co., Osaka, Japan) was transferred into a 0.5-mL resor the packing solvent, i.e., the polarity, density, and viscosity, ervoir with a 1-mL syringe, and the system was pumped with and of the packing parameters is crucial to slurry-packed 100% methanol at 500 kg/cm2,with the pressure maintained until HPLC columns. Packing conditions were examined with an adequate amount of solvent (at least 30 times the column various combinations of slurry-packing solvents using acetovolume) was eluted. Next, the column was washed with 20 times nitrile-acetonitrile, isopropyl alcohol-methanol, and Slurry its volume of 100% water to keep the packed bed stable. The Mix-methanol. Slurry-Mix, which seems to contain a solvent down-flow method was employed during the packing procedure. of high density and low viscosity, is a commercially available Another type of column was made from 1.0 m of a 250-pm mixture of solvents specially prepared as a slurry solvent. A fused-silica capillary tubing (FST). A packing technique described constant slurry concentration and a packing pressure were elsewhere (17)was employed to pack the 5-pm particles. Column chosen to standardize the packing conditions as described in efficiencies were determined by plotting Van Deemter curves (reduced plate height vs. linear velocity) for biphenyl in 85:15 the Experimental Section. The combination of acetonitrileMeOH/H20. The sample used for column evaluation contained acetonitrile as a slurry and a packing solvent was suitable for benzamide, acetophenone, benzophenone, biphenyl, and anpacking FST column with 5-pm particles (17),but the effithracene in order to provide solutes with various chemical ciency of the GLT column in terms of reduced plate height properties and capacity factors. For sample injection, the valve of below 4 could not be obtained for a 30 cm X 0.3 mm column. was turned from the "load" to the "inject" position, then was The combinations of isopropyl alcohol-methanol and methreturned to the "load" position to inject part of the sample (apanol-methanol did not give better results. However, the Slury proximately 50 nL) since the volume of the rotor was too large Mix-methanol combination led to a remarkable increase in to inject when a shorter column was used. The wavelength used column efficiency. Observation of the slurry suspension for detection was 254 nm. To test an actual biological sample, urine was collected from showed that the packing particles in the Slurry Mix had a patient 1to 2 h after intravenous injection of 1g of Latamoxef, dispersed well and no sedimentation occurred for at least 30 78-[ [2-carboxy-2-(4-hydroxyphenyl)acetyl]amido]-7cu-methoxy- min, while in the other solvents sedimentation usually occurred 3-11(l-methyl-1H-tetrazol-5-yl)thio]methyl)-l-oxa-l-dethia-3-ce- quickly. Thus, the Slurry Mix is a recommendable slurry phem-4-carboxylic acid disodium salt. A 1-mL sample of the urine solvent for the GLT column. was filtered with 0.45 pm of membrane filter (Gelman Science, The effect of water as a conditioning solvent on a packing Inc., West Germany) and 0.2 pL of the filtrate was introduced bed was also investigated. The stabilities of the packing beds to the GLT column. The column used for the separation was 10 were tested with the packed 30 cm X 0.3 mm GLT column cm X 0.3 mm i.d. filled with 3-pm SpherisorbODs-2. The solvent with 3-pm particles by pumping 85:15 MeOH/H20 a t 200 system was 30% MeOH in pH 6.2 phosphate buffer containing kg/cm2 for 3 h. A 3-mm vacancy was found a t the top of a 10 mM tetrabutylammonium hydroxide as a paired-ion reagent
ANALYTICAL CHEMISTRY, VOL. 57,
NO. 12,
OCTOBER 1985
2237
Table I. Characteristics of Columns with Different Length'
L,cm
ozcOll nL2
a2inj,nL2
10 15
26 200 46 700
62 5
30
53 400
g2det,
nL2
9 9 9
625 625
a'conm
nL2
576 576
576
azt0tal,nL2
4atota1, d
a&/ ac01
27 400 47 900 54 600
0.66
0.21 0.16
0.88 0.93
0.15
"Sample: biphenyl (k' = 2.36). 6r
2.l 0
.2
.4
. 6
.8
1. 0
. '4 . '6 . '8
8.'0 . '2
column packed without conditioning with water, but no vacany was found when a column had been conditioned with enough water after the packing process. It is noteworthy that water is an effective conditioning solvent for keeping the packing bed of the GLT column stable. Evaluation of Column Performance. Column efficiencies in terms of reduced plate height vs. linear velocity are compared in Figure 2 for compounds with different capacity factor, k'. A 30 cm X 0.3 mm i.d. column, packed with 3-pm CI8bonded-phase silica gel particles, was used for the evaluation. For biphenyl and anthracene, which have k'of 2.36 and 4.00, the van Deemter curves were similar over a wide range of linear velocity. Reduced plate heights of 2.47 and 2.43 were measured for biphenyl and anthracene at a linear velocity of 0.272 and 0.378 mm/s, respectively. For biphenyl, an average reduced plate height of 2.60 was obtained over the linear velocity range from 0.224 mm/s (0.95 pL/min of volumetric flow rate) to 0.697 mm/s (2.95 pL/min). For benzophenone, a compound with a relatively small k' ( E 1)showed an entirely different Van Deemter curve. The reduced plate height reached optimum at a linear velocity of 0.22 mm/s (0.95 pL/min), and the slope of the curve at a higher flow rate was significantly higher than the others. This indicates that the response of the detector used would be too slow to operate at a high flow rate when a compound having a smaller k'value (51) is used. The effect of the time constant on column efficiency for compounds of different k'has been discussed elsewhere (27). The performances of columns of different dimensions are compared in Figure 3. The columns, all packed with 3-pm ODS particles, were 0.3-0.5 mm i.d. and 10-30 cm long. Theoretically, the column diameter should have no effect on efficiency, and comparable levels have been observed with 1.0 and 4.6 mm i.d. columns (28). However, in practice, column performance is considered to depend on its inner diameter (41,because the smaller the internal diameter, the smaller the flow-channeling effects will be. As expected, we found that column with smaller inner diameters were significantly more efficient (Table I). Column length also affected efficiency, which decreased with decreasing length. Thus, maximum performance came from
.'0
1 .' 2
1 .' 4
L I N E A R V E L O C I T Y , U (mm/sec)
L I N E A R VELOCITY, U (mdsec)
Flgure 2. Performance of GLT microcolumn packed with 3-pm particles: (X) benzophenone (k' =: 1.03); (0) biphenyl (k' = 2.36);) . ( anthracene (k' = 4.00).
1
Figure 3. Performance of GLT microcolumn packed with 3-pm particles in different dimensions: (+) 30 c m X 0.5 mm;).( 10 c m X 0.3 mm; (0)15 c m X 0.3 mm; (X) 30 c m X 0.3 mm. The sample was biphenyl (k' = 2.36).
the 30-cm column, Although the efficiency should be the same when particles of the same diameter are used as the column packing, length effects arise due to extracolumn factors like band broadening resulting from the analytical system, injection, connection, and detection. Extracolumn band broadening of the GLT packed columns, evaluated from eq 1 and 2 (29, 301, is shown in Table I, where u2col,u2inj, u2conn, u'total
u2ext
=
jni'"
=
u'col
+ u2ext
+ U2conn + u2d&
(1) (2)
uZdet,and u2extare variances of the band broadening contribution due to the column, injection, connection, detection, and extracolumn,and u2totalis the total variance of the system (17, 31-35). The variances of the band broadening were calculated with biphenyl as a sample at a flow rate of 2 pL/min in the tested columns. The contribution of the extracolumn band broadening to the total variance (uZext/uztotal) was calculated to be 4.4, 2.5, and 2.270 for the lo-, 15-, and 30-cm columns, respectively. For the samples in which k'is larger than biphenyl, the extracolumn effects seem to be negligible even in a 10-cm column. The maximum tolerable extracolumn contribution to dispersion must be less than 75% of the contribution of the column, if a 20% loss in resolution can be tolerated as shown in eq 3 (30). That means that the volume of the detector could be increased to 100 nL, which is 10 times larger than the flow cell used here, and the volume of injection could be increased to 200 nL, since the resulting contribution of uextto ucolwould become 66%, which is still acceptable. Vex. I0.75Uc,l
(3)
In Table 11, the efficiencies of columns with various dimensions are compared a t their optimum flow rate. The 30 cm X 0.3 mm column packed with 3-pm particles gave a plate number of 40600 (135000 plates/m), which is the highest value for a short packed column obtained thus far. A column packed with 5-pm particles was less efficient (h = 3.43), although particles with a larger diameter are easier to pack (3-5, 36). With the same 5-pm particles, an FST-packed column of 100
ANALYTICAL CHEMISTRY, VOL. 57, NO. 12, OCTOBER 1985
2238
Table 11. Comparison of Column Efficiency at Optimum Conditions"
H
N
type of column
length, cm
i.d., mm
d,, wm
plates
platesjm
w
h
GLT GLT GLT GLT GLT
30 15 30 10 30 100
0.3 0.3 0.3 0.3 0.5 0.25
3 3 5 3 3 5
40 600 16 800 17 400 9 065 24 400 71 000
135000 112000 58 000 90 650 81000 77 000
7.39 8.93 17.2 11.0 12.3 13.0
2.41 2.98 3.43 3.67 4.09 2.60
FST
"Sample: biphenyl (k' = 2.36). 83 !
2
1
li
cL_LL
0 1 2 3
-
0
2
4
Time (min)
-i
0
1
10
20
+
..
6 -
U I
W I iW
5
a
4-
0 W
u
3 0 W
=
2-
ob
50
100
;N:ECTiON
150
200
VOLUME (nt)
Figure 5. Effect of Injection volume on column efficiency. For sample and conditions. see text.
Figure 4. Separation of a mixture of aromatic compounds: (A) 10 cm X 0.3 mm, flow rate 7.6 pL/min; (6) 15 cm X 0.3 mm, flow rate 9.8 pL/min; (C) 30 cm X 0.3 mm, flow rate 2.95 pL/mln; (1) benzamide, (2) acetophenone, (3) benzophenone, (4) biphenyl, (5) anthracene.
cm X 0.25 mm gave excellent efficiency (h = 2.60) of the same order reported earlier (17). In addition to the column listed in Table 11,5- and 60-cm columns with 0.3 mm i.d. were also packed with 3-pm ODS but they were not as efficient as the columns in Table 11. There is no theoretical evidence that the efficiency of the FST column is better than the GLT column of similar dimensions. From a technical viewpoint in the packing procedure, it is easy to tap a slurry packer, changing its position in order to control the packing speed continuously,since the fused silica tubing is quite flexible and transparent which allows visual inspection of the packing process. Thus, the packing technique or condition should be optimized according to the column dimensions. Typical chromatograms obtained from GLT-packed columns with 3-pm ODS of different lengths are shown in Figure 4. Solvent Effect in GLT Column. The solvent effect in a GLT-packed column was investigated in order to estimate the effect of injection volume when the GLT column is used in routine drug analysis. The solvent effect in a narrow-bore column has already been discussed (4) with a unique sample introduction technique to enhance peak resolution. In MLC, the injection volume is severely limited because of extracolumn band broadening. This is the main inconvenience when the system is used routinely. When the injection solvent containing a sample is much weaker than a mobile solvent, it may be possible to inject a large volume of sample since on-column focusing of the injected solute zone is obtained due to reduced solubility of the sample solutes in the mobile solvent. Figure 5 shows the effect of injection volume on column efficiency. 1-Methyltetrazol-5-thiol (MTT) was selected for paired-ion chromatography, a technique extensively used to separate drugs. A 10 cm X 0.3 mm column with 3-pm ODS particles was used to test the effect. MTT was dissolved in water at
ltll
1
P 0
5
10
15
Time(min) Figure 6. Chromatogram of human urine after administration of Latamoxef: column, 10 cm X 0.3 mm packed with 3-pm ODs: flow rate, 2.1 pL/min; operating pressure, 48 kg/cm2. Keys are (1) R epimer and (2) S eplmer of Latamoxef, respectively. For conditions, see text.
50 pg/mL and the mobile solvent was 30% MeOH in pH 6.2 phosphate buffer containing 10 mM of tetrabutylammonium hydroxide. Various volumes of sample, 50-200 nL, were introduced into the GLT column by changing the injection time with the volumetric flow rate at 3.1 pL/min (50 nL/s). Operating pressure was 70 kg/cm2. Reduced plate heights measured after the injection of 50, 100, 150, and 200 nL of MTT solution were 4.53,4.07,4.63, and 4.26, respectively. The results indicated that introduction of a large volume of sample up to 0.2 pL did not influence column efficiency. Comparing
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
