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Anal. Chem. 1983, 55, 1462-1465
Table VI. Voltammetric Characterization-Oxidized Pterins in Urine neopterin xanthopterin biopterin E , mV std samp samp std samp std -600 0.77 0.80 0.62 0.60 0.73 0.70 -500 0.35 0.33 0.27 0.26 0.33 0.35 -400 0.11 0.10 0.07 0.06 0.10 0.10 a Currents normalized to that observed at -700 mV. limits because of decreased complexity of the chromatogram. Increased selectivity for easily oxidized compounds in a complex sample is illustrated by the dual-electrode detection of reduced pterins in urine. Figure 6 shows the simultaneous chromatograms obtained from urine samples with one electrode at +800 mV and the other electrode at +400 mV. This urine sample was acidified and filtered to remove proteins but was not subjected to the sample preparation procedure. It can be seen that the chromatogram obtained a t the lower potential is much less complex than the chromatogram obtained at +800 mV. In this manner, tetrahydropterins can be determined with lower detection limits and with less sample preparation.
CONCLUSION It has been shown that operating a dual-electrode amperometric detector in the parallel-adjacent configuration can offer advantages in versitility, peak identity/purity assessment, and selectivity. In addition, a method has been presented for the determination of pterins in biological samples. Although the determination of pterins in urine was illustrated, this approach is applicable to a wide range of biological samples. Registry No. Biopterin, 22150-76-1; dihydrobiopterin, 677987-9; tetrahydrobiopterin, 17528-72-2;6-hydroxymethylpterin,
712-29-8; dihydro-6-hydroxymethylpterin,3672-03-5; tetrahydro-6-hydroxymethylpterin, 31969-10-5;neopterin, 2009-64-5; dihydroneopterin, 1218-98-0;tetrahydroneopterin, 25976-00-5; pterin, 2236-60-4; dihydropterin, 17838-80-1;tetrahydropterin, 1008-35-1;pterin-6-carboxylic acid, 948-60-7; dihydropterin-6carboxylic acid, 17833-48-6;tetrahydropterin-6-carboxylicacid, 7449-02-7;xanthopterin, 119-44-8;dihydroxanthopterin,1131-35-7; erythro-neopterin, 39923-31-4;threo-neopterin, 2277-42-1.
LITERATURE CITED (1) Shoup, R. E., Ed. “Recent Reports on Liquid Chromatography/ Electrochemistry”; BAS Press: West Lafayette, IN, 1982. (2) Roston, D. A.; Shoup, R. E.; Kissinger, P. T. Anal. Chem. 1982, 5 4 , 1417A-1434A. (3) King, W. P.; Klssinger, P. T. Clln. Chem. (Winston-Salem, N.C.) 1980, 2 6 , 1484-1491. (4) Schieffer, G. W. Anal. Chem. 1981, 5 3 , 126-127. (5) Roston, D. A.; Kisslnger, P. T. Anal. Chem. 1982, 5 4 , 429-434. (6) MacCrehan, W. A.; Durst, R. A. Anal. Chem. 1981, 5 3 , 1700-1704. (7) Goto, M.; Sakural, E.; Ishil, D. J . Chromafogr. 1982, 238, 357-360. (8) Bratin, K.; Kissinger, P. T. J . Li9. Chromafogr. 1981, 4 , 321-357. (9) Alllson, L. A.; Shoup, R. E. Anal. Chem. 1983, 55, 8-12. (IO) Roston, D. A.; Klssinger, P. T. Anal. Chem. 1981, 5 3 , 1695-1699. (11) Shoup, R. E.; Mayer, G. S. Anal. Chem. 1982, 5 4 , 1164-1168. (12) Lunte, C. E.; Kissinger, P. T. Anal. Biochem. 1983, 129, 377-386. (13) Brautlgam, M.; Dreesen, R. Hoppe-Seyler’s 2.Physiol. Chem. 1982, 363, 1203-1207. (14) Thijssen, H. Anal. Biochem. 1973, 5 4 , 609-811. (15) Kaufman, S. J . Biol. Chem. 1987, 242, 3934-3943. (16) Bobst, A.; Vlscontini, M. Helv. Chlm. Acta 1986, 4 9 , 875-884. (17) Karber, L. G.; Dryhurst, G. J . Elecfroanal. Chem. Interfacial Elecfrochem. 1982, 136, 271-289. (18) Fukushlma, T.; Nixon, J. Anal. Biochem. 1980, 120, 176-188. (19) ’261, E. M.; Sherman, A. D. Prep. Biochem. 1977, 7 , 155-164. (20) Niederwieser, A.; Curtlus, H. C.; Gitzelmann, R.; Otten, A,; Baerlocher, K.; Blehova, B.; Berlow, S., Grobe, H.; Rey, F.; Schaub, J.; Scheibenrelter, S.; Schmltt, H.; Viscontinl, M. Helv. Pedlat. Acta 1980, 35, 335-342. (21) Dhondt, J. L.; Largllliere, C.; Adrouln, P.; Farriaux, J. P.; Dautrevaux, M. Clin. Chim. Acta 1981, 110, 205-214.
RECEIVED for review February 8, 1983. Accepted April 22, 1983. We gratefully acknowledge A. Niederweiser of the University of Zurich for the gift of 5,6,7,8-tetrahydrobiopterin.
Poly(perfluoroalky1 ether) Stationary Phase for Gas Chromatography Subhash C. Dhanesar and Colin F. Poole” Department of Chemisrty, Wayne State University, Detroit, Michigan 48202
Efflclent packed columns havlng between 2000 and 2500 theoretical plateslm can be prepared wlth the poly(perfluoroalkyl ether) Fomblln YR. Its maximum allowable operating temperature Is a functlon of the stablllty of the llquld phase film, 255 OC for a 10% (w/w) loaded packing on Chromosorb P, and not by column bleed. The absolute retention of organic compounds on Fomblln YR Is generally an order of magnltude, or greater, less than on conventlonal nonfluorlnated llquld phases. Fomblln YR may be used to separate nonpolar and moderately polar organic compounds wlth good peak shape. Polar compounds such as alcohols, amlnes, and phenols, etc., produce asymmetric peaks.
Separations in gas-liquid chromatography occur because of selective interactions between the sample and the liquid phase. All components have essentially the same residence time in the gas phase. Thus, many liquids have been evaluated
as stationary phases for gas chromatography but only a few of these can be described as having unique separation properties (1). In general, there exists a large number of stationary phases having nearly identical separation properties, from which a small number can be identified as preferred phases characteristic of all others. Recently we have described the properties of some novel stationary phases for gas chromatography, namely, organic molten salts ( 2 , 3 )and substituted poly(pheny1ethers) (4-6). The former represent phases having ionic functional groups providing both different and stronger interactions with polar compounds than found for conventional liquid phases and the latter an attempt a t standardization by developing thermally stable, nonpolymeric phases having a defined chemical structure reproducible via synthesis. In this paper we wish to introduce a further development, the use of perfluorocarbon stationary phases for the analysis of organic compounds at temperatures lower than those required with conventional nonfluorinated phases. It is hoped that phases of this kind will eventually extend the molecular weight
O003-270O/83/0355-1462$01.50/00 1983 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 55, NO. 9, AUGUST 1983
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Table I. McReynolds' Constants for Fomblin YRa squalene capacity retention factor index value value test probe benzene 1-butanol 2-pentanone 1-ni tr opropane pyridine 1,4-dioxane 1-iodobutane 2-octyne 2-methyl-2-pent an
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20.7 12.2 18.9 20.7 35.0 21.9 120.0
637 584 628 635 690 654 807
23.4
648
ckhydrindane
Fomblin YR capacity retention factor index value value 622 722 716 776 741 708 792 841 770 961
0.8 1.3 1.2 2.1 1.5 1.2 2.4 3.3 1.5 6.9
McRevnolds' constants -1.5 138 88
141 51 66 -15 122
a 3 m X 2 mm i.d. glass column packed with 10% (w/w) stationary phase on Chromosorb P (100-120 mesh), isothermal 45 O C , and nitrlogen carrier gas flow rate 20 mL min-' .
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range of organic compounds that can be analyzed by gas chromatography and also permit the analysis of thermally labile compounds a t sufficiently low temperatures that decomposition is no longer a problem. Perfluorocarbons are uniquely suited to the above applications due to their weak intermolecular interactions (7). Perfluorocarbon-containing derivatives of organic compounds and perfluorocarbon chelates of metals have been used routinely in gas chromatography to separate substances of low volatility on organic liquid phases at temperatures generally less than the parent compound or a ciimilarly constituted hydrocarbon derivative (8,9).The rea8on being that in the liquid phase the interactions between the perfluorocarbon chain and the stationary phase are weaker than those between the hydrocarbon derivative and the stationary phase resulting in shorter retention times. This procesti obviously applies in reverse, and if we make the fluorocarbon component of the chromatographic system the stationary phase, then we would expect diminished retention of organic compounds in general compared to an organic liquid phase. Alternatively, it should prove possible to elute compounds from a perfluorocarbon stationary phase which are too well retained on a n organic phase to be eluted at the same temperature. The use of perfluorocarbon stationary phases is not new to gas chromatography. The period 11960-1968 witnessed several attempts to exploit fluorocarbons such as perfluoroalkanes, Kel-F oils, fluoroalkyl esters, etc. as general phases in gas chromatography (I, 10-14). Column efficiencies were generally poor for organic compounds, typical values ranging from 30 to 225 theoretical plates/m. In part this may be attributed to the use of poor grades olf support materials, although history shows that the silicone oil and polyester phases introduced a t the same time invariably produced superior columns. It was recognized that fluorocarbon phases produced unstable films leading to low efficiency and had low upper operating temperature limits due to either column bleed or contraction ojf the liquid phase f i i . They were widely used, however, as liquid phases on Teflon or diatomaceous supports for the analysis of corrosive and reactive chemicals [e.g., UFs, C102F,SF5C1, etc.] for which their low column efficiency was acceptable due to a lack of alternative materials with equivalent chemical inertness. In this paper we will describe the properties of a new perfluorocarbon stationary phase, Fomblin YR, and methods to prepare efficient column packings of reasonable thermal stability for the separation of organic compounds.
EIXPERIMENTAL SECTION Unless otherwise stated, all chemicals and solvents were general laboratory or analytical grade in the highest purity available. Test compounds were available as Thetakits (Anspec,Ann Arbor, MI).
C5 f(O-AF-CF*
1"- ( 0 - C F ,
)J-
Flgure 1. Structure of Fomblin YR.
The sample of fuel oil No. 1 was obtained from Marathon Oil (Detroit, MI). Fomblin YR was obtained from Montedison (New York, NY) and Silyl-8 and BSTFA from Pierce Chemical Co. (Rockford, IL). Column packings containing from 3 to 15% (w/w) of Fomblin YR on Chromosorb P (100-120 mesh) were prepared by use of the rotary evaporator technique and 1,1,2-trichlorofluoroethane as solvent for the stationary phase. The air-dried packings were sieved before use and packed into glass columns of 1 to 3 m in length with the aid of vacuum suction and gentle vibration. Prior to use the packed columns were on-column silylated by injection of Silyl-8 or BSTFA. For gas chromatographya Varian 3700 gas chromatograph with an on-column heated injector, temperature.programmer, and flame ionization detector was used. Separation conditions are given in the figure legends.
RESULTS AND DISCUSSION Fomblin YR is a synthetic, inert, functional fluid used as an engine lubricant and vacuum pump oil (15,16). It has an average molecular weight of 6000-7000, a pour point of approximately -20 OC, and a general structure given in Figure 1. Fomblin YR has good coating characteristics on Chromosorb P providing packings having between 2000 and 2500 theoretical plates/m after on-column silylation. Packings with a 10% (w/w) loading are thermally stable a t 255 "C and at the 3% (w/w) loading to 200 "C. Neither temperature represents the thermal stability of the phase itself as column bleed is not detected until temperatures in excess of 275 "C are reached. The upper temperature limit is established by contraction of the stationary phase film, exposing bare support, which leads to a loss of column efficiency and the introduction of peak asymmetry. The choice of column support and surface treatment is important to obtaining efficient column packings. All attempts to prepare packings with Chromosorb W and Gas Chrom Q supports resulted in columns having 200 and 300 theoretical plates/m. Persilylating Chromosorb P prior to coating also gave equally inefficient packings. Persilylated packings also show evidence of liquid phase film contraction at lower temperatures, 100 "C,compared to the results found for the same support silylated after coating. Both the choice of support, the method of surface deactivation, and the selection of the perfluorocarbon stationary phase are important to the preparation of efficient column packings. A limited number of experiments with perfluorokerosenes and Kel-F oils did not result in the preparation of efficient column packings using the techniques discussed above.
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ANALYTICAL CHEMISTRY, VOL. 55, NO. 9, AUGUST 1983
5------_(i_-
-7----
I
0
IO
20 TIME ( m n l
M
40
io
40
TiUEImn)
Jo
Flgure 3. Separation of fuel oil No. 1. Same column as given for Figure 2: temperature program 60 to 190 " C at 5 "6 min-' and then
held isothermally at 190 "C.
Flgure 2. Separation of C5 to ClS and C18to GZ4 n-hydrocarbons: column 3 m X 2 mm i.d. containing 10% Fomblin YR on Chrornosorb P (100-120 mesh) and nitrogen carrier gas flow rate 30 mL min-I; temperature program 35 i o 195 " C at 10 "C min-' and then held
isothermally at 195 "C. The polarity and select,ivity of a stationary phase can be characterized by its Mc Reynolds' constants values. For Fomblin YR these values me summarized in Table I. Several trends may be noted. First of all, the absolute retention of the test probes is substantially lower on Fomblin YR than squalane. Values of the Mc Reynolds*constants for 2-octyne and cis-hydrindane are not reported due to their impractically long retention times on squalane under conditions giving reasonable retention on Fomblin YR. In general, it can be seen that under the same experimental conditions the retention of all probes on Fomblin YR is better than an order of magnitude less than on squalane. This lack of retention for the test probes intoduces some experimental error in the values quoted for the Mc Reynolds' constants. However, these errors we not believed to be great and the values quoted serve to indicate the general properties of the phase. As would be anticipated, the Mc Reynolds' constants characterize the phase as being nonpolar with some low selectivity for the retention of compounds having hydrogen bonding and dipolar functional groups, presumably due to the presence of ether oxygen atoms in the stationary phase backbone. Fomblin YR provides good separations of alkanes at temperatures much lower than those found for conventional liquid phases. Figure 2 shows a temperature programmed separation of C5 to CI6 and CI8to CZ4alkanes. Higher molecular weight hydrocarbons C36,CS8,and CM can be eluted in a few minutes on the 10% (w/w) loaded column at 255 "C. Figure 3 shows the separation of a complex hydrocarbon mixture, fuel oil No. 1. Again the sample is conveniently eluted a t relatively low temperatures. Figure 4 shows the separation of decene (bp 181 "C), decane (bp 174 "C), and decyne (bp 174 "C). Several features can be seen from the chromatogram. First of all, the order of elution is not according to boiling point. Although in this example the alkene elutes before the alkane, in other cases the alkane may coelum or elute ahead of the alkene. The greater retention of alkynes with elution as an asymmetric peak is characteristic of these compounds and some polycyclic aromatic hydrocarbons as well. Both of these systems are
I
1
I
0
5
IO
TIME (inin)
Figure 4. Separation of decene, decane, and decyne. Same column as given for Figure 2. Isothermal at 45 " C . a-electron donors and presumably can form charge-transfer complexes with the electron deficient ether oxygens, the
ANALYTICAL
I
I
0
8
4
TIME (min)
Figure 5. Separation of naphthalene, chloro-, bromo-, lodo-, nitro-, and cyanonaphthalene, and phenanthrene: same column as glven for Figure 2: temperature program 120 to 180 OC at 4 OC min-'.
CHEMISTRY,
VOL. 55, NO. 9, AUGUST 1983 1465
shapes. The nitro- and cyano-containing derivatives show definite peak broadening and represent the upper polarity limit of functional groups which may be chromatographed on Fomblin YR. Compounds containing alcohol, amine, phenol or acidic groups do not chromatograph well on this phase. This may be due to their low solubility in the stationary phase (Fomblin YR is virtually insoluble in moderately polar and polar organic solvents) or due to interaction of the solute with active sites on the support which are poorly masked by the phase. As a final example of the chromatographic properties of Fomblin YR, Figure 6 shows a separation of Arochlor 1254. Separation into groups of isomers, presumably containing similar numbers of chlorine atoms with little selectivity for isomers containing the same number of chlorine atoms is obtained. This is in keeping with general observations concerning the separation of chlorocarbon compounds. Good separations of Freons and perfluoroalkanes have also been obtained on Fomblin YR. Fomblin YR is the first perfluorocarbon stationary phase to provide reasonably thermally stable column packings having acceptable column efficiencies for general chromatographic use. Its most remarkable property is the ability to separate organic compounds at temperatures substantially lower than those obtained with conventional liquid phases. However, polar compounds are generally eluted with poor peak shape, which is the principal limitation on the general applicability of this phase. R e g i s t r y No. Fomblin YR, 25038-02-2.
LITERATURE CITED (1) Balulescu, 6. E.; Ille, V. A. "Stationary Phases in Gas Chromatography"; Pergamon Press: Oxford, 1975. (2) Pacholec, F.; Butler, H. T.; Poole, C. F. Anal. Chem. 1982, 54, 1938. (3) Pacholec, F.; Poole, C. F., Chromatographia, In press. (4) Poole, C. F.; Butler, H. T.; Agnello, S. A,; Sye, W.-F.; Zlatkis, A,; Holzer, G. J . Chromatogr. 1981, 217, 39. (5) Dhanesar, S. C.; Poole, C. F. J . Chromatogr. 1982, 252, 91. (6) Dhanesar, S. C.; Poole, C,F. J . Chromatogr. 1982, 253, 255. (7) Reed, T. M. I n "Fluorine Chemistry"; Simons, J. H., Ed.; Academic Press: New York, 1964; Vol. 5, p 133. (8) Poole, C. F.; Zlatkls, A. Anal. Chem. 1980, 52, 1002A. (9) Zlatkis, A., Poole, C. F., Eds. "Electron Capture-Theory and Pratlce in Chromatography"; Elsevier: Amsterdam, 1982. (IO) Ellis, J. F.; Forrest, C. W.; Allen, P. A Anal. Chlm. Acta 1960, 22, 27. (11) Campbell, R . H.; Gudzinowlcz, B. J. Anal. Chem. 1981, 33, 842. (12) Lyzyt, I.; Newton, P. R. Anal. Chem. 1983, 35, 90. (13) Juvet, R. S.;Flsher, R. L. Anal. Chem. 1986, 38, 1860. (14) Pappas, W. S.;Million, J. G. Anal. Chem. 1988, 40,2176. (15) Laurenson, L. Vacuum 1980,30, 275. (16) Laurenson, L.; Dennis, N. T. M.; Newton, J. Vacuum 1979, 29, 433. I -
L
7
0
1
1
I
20
10
TIME (mid
Figure 6. Separation of Arochlor 1254 colurnn 1 m X 2 mm i.d. 10% (w/w) Fomblin YR on Chromosorb P (100-120 mesh) and nitrogen carrier gas flow rate 30 mL min-I; temperature program 55 to 220 OC at 6 OC min-'.
electron density on the ether oxygen being influenced by the neighboring electron-withdrawing fluorine atoms. Figure 5 shows the separation of some naphthalene derivatives and phenanthrene. Naphthalene and the halogencontaining derivatives are well-separated with normal peak
RECEIVED for review February 4, 1983. Accepted April 18, 1983. Work in the authors' laboratory is supported by the Camille and Henry Dreyfus Foundation, AB Hassle, and the Environmental Protection Agency. Although the research described in this article has been funded in part by the United Sates Environmental Protection Agency under assistance agreement No. R-808854-01-0to C.F. Poole, it has not been subjected to the Agency's required peer and administrative review and, therefore, does not necessarily reflect the view of the Agency and no official endorsement should be inferred.
