topic/ in chemic~lin~trument~tion
edited by
FRANK A. SETTLE.JR. Lexington. Mltitary VAfwitute 24450
An Introduction to Supercritical Fluid Chromatography Part 2. Applications and Future Trends Margo D. Palmieri National Institute of Standards and Technology, Gaithersburg, MD 20899 In recent v e m sunercritical fluid chro. matozreohv k. ~-h& ~ "~~-. ~ ~ underzone , l" the tran. aition from an experimental procedure to a viable separation alternative. Although SFC instrumentation and theory have received much attention, until recently relatively few application papers have heen published. The use of SFC is currently under investigation in many applications laboratories. As more people have turned to SFC to solve their separation problems, and as commercial instrumentation has become available, a greater number of applications have been reported in the literature. Part 1of this dcle dealt with the basic theory and instrumentation of supercritical fluid chromatography. In Part 2 selected applications and future trends in SFC will be discussed. ~~~~~
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with packed column LC. SFC is particularly useful when separating compounds containing long hydrocarbon chains, such as high molecular weight hydrocarbons, glycerides, carboxylic acids, polyglycols, polymers, and polymer additives. These compounds have high moleeuLar weights and thus are not sufficiently volatile for GC analysis. Derivatization of these compounds is often ineffective because the products may also not he sufficiently vola-
tile for GC analysis. Many of these compounds also lack chromaphoric and other functional groups that are used for detection in LC. To date, the use of SFC in the industrial analytical laboratory has not been extensively reported. Many of the SFC applicatigns reported in the literature have been related to specific industries that have analytes suitable for SFC analysis. Several SFC applications related to petroleum refining
Many SFC separations have been reported in the literature within the last five yearn. Recently Lee and Markides compiled an extensive list of SFC separations ( I ) . The areatest area of ap~licationfor SFC involves hose analytes that are difficult to separate bveither eas chromatomaohv (GC) or liauid Chester &i&romato&phy (LC):T: mated that up to 25% of all separation prohlems are not solvable by GC or LC (2).GC analysis cannot he carried out on samples in which the components are either thermally Labile or are not volatile under normal GC conditions. In LC, on the other hand, the available detection techniques frequently restrict the t w e s of analvtes that can he LC hcwctors such as determined. !&&dad ultraviolet-visible, electrochemical, fluorescence, or conductivity respond only to analytes t h a t contain specific functional groups. With SFC, GC detectors such as the flame ionization type can be used for detection of these analytes. Mass spectrometers and infrared soectrameters are also available for analysis and structure elucidation alter SFC separation. In LC, use 01 these detectors has been limiteddue todifficulties in connecting the liquid chromatograph to the detectors. In addition, the potential for higher separation efficiencies is greater with SFC than with LC. Separations of complex mixtures are easier with capillary SFC than
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than the required GC column temperature. Class separations of components in oils can also he carried out by SFC (14-16). The relative ratios of olefins.. saturated hvdrocarbons, and aromatics present in fueGhelp e rating for different determine the w types of fuels. Figure 2 shows SFC group separations of various types of fuels (16). Typically, group separations and determinations involve a gravity column separation using dye-impregnated silica gel, followed by colorimetric analysis. The colored bands are often not well separated and the absorbing species in the oil sample can interfere with the spectropbotometricdetermination. In the SFC analysis, the various classes are well separated, and spectral interferences seen in the colorimetric determination are avoided because detection is based on flame ionization detection. Research haa also been carried out on the separation of polycyclic aromatic hydrocarbons (PAHJ. Many PAH compounds are known to be carcinogenic and are classified as priority pollutants by EPA. PAH's can be found in fuels, fuel exhaust systems, lubricants, and as contaminants in many samoles. Not all PAH molecules are suffieientlv ;olatile for GC analysis, and in an elfart& improve on the LC separation of PAH's, researchers have turned to SFC (17-26). An ~
and petroleum product characterization have been published 13-16). Petroleum is a complex mixture of several different types of organic compounds; the more prominent components are long-chain hydrocarbons, aromatics, heterocyclic, and polycydic ammatic hydrocarbons. One important analysis involves the eharaderization of oetroLeum by determining boiling range frakions ( 4 4 ) . For example, lighter oils have a high percentage of smaller hydrocarbons with low boiling points while heavy oils contain primarily high boiling point organic eompounds. Knowledge of boiling range fractions is important in the processing of crude oil. Various boiling range fractionsare separated by distillation for use as different types of fuels. An example of the use of SFC in the determination of these fractions is shown in Figure 1.The separation of acrude oil sample is shown as a function of column temperature. The separation efficiency improves at higher column temperatures hecause the analyks do not elute from the column as rapidly as at lower temperatures due to the lower mobile phase densities. In the SFC separation, elution order is related to the boiling points of the components.The authors reported that the SFC results compared favorably with data obtained using the standard ASTM GC procedure for the analysis of petroleum fractions (4). The upoer limit of the temoerature ranee was greater for the SFC separation than"for the GC method, which is useful when characterizing oils containing high boiling fractions. In addition, decomposition of organic samples was avoided in the SFC methodbecause the SFC column temperature ia much lower
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Figwe 1. E M of temperature on mtentlm and resolution. Sample. PE-740. wim penlacoctane added as a marker to identify peak, linear pres S1uB program horn 2000 to 5500 psi in 20 min. R~pdntBdwim permisim horn ref 4.
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Journal of Chemical Education
rnin F l p e 2. Chmmatoprams ot gaaolim, (A). k a p sene. (01. and diesel fuel (Cl. Saturates are amnuated 8X. Reprimed wlth permlssbn hom ret 16.
interesting PAH separationwas reported by Christensen using multidimensional SFC (26). In multidimensional chromatography, components are separated by multiple retention mechanisms in a single run. Two columns of different polarities were initially tested for retention of the PAH analyks. The separation order of various PAH's on the polar column was similar to that seen on normal phase HPLC, while the retention order on the nonpolar column was similar to that found in reversed phase HPLC. The same eluant, carbon dioxide, was used for both columns. In order to improve separation efficiency, the polar and nonpolar columns were coupled together; thus the analytes were separated by two different retention mechanisms in asingle chromatographic run. This procedure cannot be easily done in LC because different mobile phases, which are generally not miscible, must he used for each column. After the f m t separation, the effluent must be dried of solvent and redissolved in a different solvent before its introduction to the second column for further seoaration. ~olym&icprodumandprecursorscan be analyzed using SFC 127-35). Many of these compounds have high molecular weights and long carbon chains that cannot be easily analyzed using standard LC detection methods. Separation of the various molecular weight fractions of the polystyrenes shows the distribution of the molecular weight in the sample, an important quality control test in the polymer industry. Pbysical properties of plastics are strongly dependent on the molecular weight distributions of the polymer components. Figure 3 shows an SFC separation on a packed microcolumn of a mixture of polystyrenes (33).The numbers in Figure 3 refer to the degree of oligomerization of the polystyrenes. The molecular weight of the components of the sample range from 580 to 9000. Determination of various polymer additives can also he carried out by SFC. These additives are used as antioxidants,plasticizers, W stabilizers, and mold release agents in the polymer industry. By their very nature, polymer additives tend to he reactive and often de4 comoose when emosed to heat. Fieure " shows a separation of various types of polymer additives that were extracted from a polymer (34). Analysis for such residues in polymers is important in the packaging industry because the packaged product can become contaminated as a result of the leaching of these organic compounds from the polymer. Glycerides, carboxylic acids, aliphatic hydrocarbons, and long-chain carboxylic acid esters have been separated by SPC. Low volatility andlor poor temperature stability limit the range of molecular weights for compounda that can be analyzed at normal GC operating temperatures. Because these compounds lack spectral or electroactive functional groups, sensitive LC detection is difficult. Two major products containing long-chain organic compounds are waxes and oils. Both natural and synthetic waxes have been analyzed using SFC (36). Separations of the alkanes in Fischer Tropsch waxes have been attained for components up to 100carbonsin length (36). Difficultiesin the analvsis of lone-hvdrocarbon-chain carborylic acids and ;he; esters, commonly called fatty acids and fatty acid esters, respecrively, have hampered chemical analysis of wax.
es, oils, and other fats. Derivatization must be carried out prior to GC analysis, and m e derivatized analytes are not volatile. F i e 5shows the separation of various fatty acids in a wax sample (36).Fatty acida have been separated in such products as coconut oil, butter, soybean oil, and soap (37). Separations of fatty acids of the sameehain l e n d , differingonly in the number and position of double bonds, have been repottad (38).SFC separations of polyglycerides, another typical component of waxen and oils, have been pexformed (M) Figure . 6 shows the separation of various mono-, di-, and triglyceridea (44). RPaearchera have Eported separations of triglycerides witb the enme number of carbon atoms but with. varying degrees of msaturation (36,40,41). Produotrr sucb as vegetable ehortening ( 4 ~ ' 16) and cocoa butter (46) have been amlyzed by SFC for varioua triglycerides. Analysis of pesticides has benefited from the development of SFC tecbniquea. Many pesticides can be analyzed by GC. However, Figure 3. Ressuwmgamnsdelution of a m1xtue of palWmm8 of MW 580.2100.4250. and 9000 one set of pesticides, tbe earbamates, are Rapinted wlth permisslrm from ref 33. difficult to anamalyae by other chromatographic method8 because carbarnates are thermally labile and can decompose in the GC system. In addition, the Lack of a reactive funct'10nal group hampers sensitive analysii in LC. Determination of low level8 of peaticidea ia important for toxicological and environmentslstndiea.Figure I shows a separation of various earhamate and acid pesticides using fast capillary SFC (46). In fast wpillary SFC, very high eluant flow rates are used in order to achieve rapid elution of sample components. The low elution times come a t the expenss of sample reaolution, but in this ease adequate reeolntion of the sample components was attained. Metabolites of aldimb, a carbarnate pesticide, have also been separated by SFC (34). The potential of SFC for separation of biological compounds has come under intense m t i n y . Many compounds of biulogical origin tend to be hydrophilic and are often not soluble in standard SFC solvents. Thin lack of solubility has severely limited the types of biologicalcompoundsamettable to SFC analysis SFC procedures reported in the litmature for a e p a r a m biological molecules typically are useful for one particaddltlves. Reprimed with permission horn ref 34. ular compound. As desoribed previously, fatty acid separation and analysis has been strengths of SFC and to point out the varifrom other stsmids having a similar strucadvanced through the use of SFC. Some of ety of compounds that can be separated by ture (52). Some tberapeutic drugs,in particthe more nonpolar and hydrophobic biologiSFC. ular antibiotics. have been isolated br SFC. cal compmnds have been separated by SFC. Tetracycline, oxytetraeycline, valinomycin, Due to their hydrophobic nature, fat-soluAreas oi Future Rewwch for SFC erythromycin, and atenol can ell be eluted ble vitamins such as vitamin A, vitnmim K, off an SFC column (52). SFC can be used to and vitamin D are separable by SFC (47SFC hss shown the potential to compleindicate and monitm the presence of d r w 48). SFC has been used to separate compoment GC and LC methods of se~aration.As and their metabolites. For example, subdescribed in the nrevious d o n . certain nents of extra& and spices. These comstances of abuse or their metabolites can be tgpes ofcompoun& that are either.impo£s contain various fatty acids, fatty monitored by SFC. F i e 9 shows the SFC ble or dficult tn analyze by LC or GC are acid e&m, glyoerides, vitamins, aldehydes, separation of tetrahydrocannabinol(THC), amenable tn SFC analysis. SFC hss had limand ketones. Examples of food prcduets the active ingredient in marijuana, and its ited applicability as a result of its inabiliiy that have been i n j d onto the SFC chmmetabolites (34,62). Typically, analyseg for to separate hiphly polar and ionic commatograph include paprika (49). grapefruit THC in humans has been carried out using pounds, in pdcuiar-those compounds with oil (50). and annatta extract (51).Different immunoaaaays.SFC separation of THC and pmtaglandins, which are metablitas of extensive hydrogen bonding. Although its analytes eliminate the interference are semable manv of these comoounds fatty acids, have been separated by $FC. ~~-~~~~ - - ~ bv~ ~ ~ - ~ present in the immunoaesay. In the SFC HPLC, the availability of many types of deF i e 8 shows the separation of f n u prmseparationell the metabolitesare separated, taglaadins that differ by the functional tectorsand the potsntially higher resolution and the individual sample components can group on the cyclopentane ring (38). offered by SFC are desirable for separation be monitored without interference, to deterof complex sample mixtures. The inability Various drug8 have p r o w amenable to mine accurately the level of THC metaboSFC analysis. For many drugs, derivatizato separate polar compounds in due in large lites in the blood. tion ia required before GC anal@ because part to the lack of suitable polar supermitiThe various types of separations that can their high polarity limita volatility. Various be carried out by SFC have been described anti-inflammatory steroids sucb as corti: (Continwd on page A144) here to show the reader some of the sone and prednisolone have been separated ~
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micelles in supermiticalfluids may improve SFC separation of polar molecules. SFC separations of ionic compounds through the addition of ion pairing reagents have recently heen reported (54). In ion pairing chromatography, organic ionic compounds which are opposite in charge to that of the analvw are added to the eluant. Throueh el&trostatic interaction..~ the ionic analyt& form neutral eomplenes with the ionic additive; the complexes typically are more soluble in hydrophobic solvents such as carbon dioxide and can he separated by SFC. Column Technology Another area holdine notential for SFC dweloument is the evnththesisand testinn of new ciumn phases. h i k e CC and LC,;Olar stationary phases have not been widely developed for SFC,due in part to problems associated with the preparation of uniform and stable f i s in capillary columns. AnaIytes that are strongly absorbed to nonpolar stationan, nhaaes arenotretained as stroneIv. on MI& ohand ean he eluted u~~-~~~ s& weaker nonpolar SFC solvents. New column phases tailored for specific types of separations will also be developed. One such cLaas of columns, termed chiral columns, have special stationary phasea that have been designed to separate optically active wmpounds. Other applicationsof diral phase6 indude the separation of structural and positional isomers. The development of chiral SFC coluhlns will probably he based on existing chiral d u m n 8 in GC and LC. Researchers have reported SFC separations using packed (55) and capillary (56) chiral phase columns. Another type of column containing a liquid crystal stationary phase has been intmduced and used to separate PAH isomers (57). This type of phase was originally introduced and developed for GC capillary columns. Synthesi. of new stationary phaae support materials for packed column SFC will he anotherarea of research Several types of LC columns have been tested for separation notential in SFC. Research aimed at imboved deactivation of active sites on the silicasupport will continue ta he carried out on silica materials. The feasibility of using other stationary supports that do not contain active sites will also he tested. Organic polymers and inorganic compounds should he tested for their utility asstationaryphase support materials for SFC packed columns. For example, the preparation and testing of alumina phases for SFC has been recently reported (58). The use of small column diameters will continue tmhe investigated for both packed and oapillary columns. In the past several years, SFC column diameters have decreased over fourfold: from 100to 25 pm for capillary wlumns and from 4.6 nun to less than 1mm for packed columns. Some SFC work is being carried out using wide-bore capillary columns packed with small particles. These columns can produce highly efficient separations,such as that shown in Figure 3. With the decrease in column diameter, improvements in sample introduction and detector cell design must be carried out in order to maintain maximum efficiency, reproducibility, and sensitivity. Sample Preparation Sample. preparation is another means of ~
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Figure 5. SFC-FIDchromatogram of an acid montan wax. The cohain length and moleculsr weights of individual even-numbered carboxylic acids are shown on the chromatogram. The peaks between the evennumbered carboxylic acids were identified by SFGMS to be oddnumbered carboxylicacids. Reprinted with permission horn ref 36.
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cal fluid mohile phases. Many polar compounds are not soluble in carbon dioxide and nitrous oxide, the most common supercritical fluid solvents. The more polar supercritical solvents, such as ammonia and methanol, are either corrosive to the chromatographic system or are formed at very high temperatures. At the high temperatures. manv of the nolar solutes can decompose.'ln addition, fir capillary SFC systems, polar columns have not been readily available for separarions of polar molecules. Further research on various aspects of chromatographic conditions is required in order to broaden the separation capabilities of SFC.
Eluants For SFC to become a widespread analytical separation technique, the solvation problem of analytes in common superuitical fluid mohile phases must be addressed. The emergence of new pure SFC solvents is not Likely, due to the limited number of suhstances that form sunercritieal fluids at low temperatures. Some increased use of available polar solvents such as ammonia, water, and alcohols may be seen when corrosion due to the supercritical solvent is eliminated by replacing stainless steel with inert materialssuch as titanium and organic polymers. Increasingly, temperatures above 200 OC have been used in SFC separations. At these elevated temperatures, s greater number of compounds can be tested as potential SFC Journal of Chemical Education
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A major thrust of mohile phase research
will involve the alteration of pure SFC solvents. Addition of various polar organic compounds to supercritical carbon dioxide, in order ta increase the polarity of the mobile phase, is being tested for a variety of samples and column types. Research thus far has been carried out using low-moleeular-weight alcohols, acetonitrile, formic acid, etc., as mohile phase additives. Recently, research on the addition of micelles tosupercritical fluid mohile phases has been published (53). A micelle is an aggregate of organic compounds that are polar at one end and hvdronhohic at the other end. The aegregace is iormed so that the hyd&phobTc and polar portions of the molecule are grouped together. For the normal micelle, the hydrophobic end of the molecules is on the interior of the aggregate, and the polar end is on the surface. The reverse micelle has a polar interior and a nonpolar surface. In liquids and supercriticalfluids,the molecules constituting the micelle orient themylves around a larger polar molecule so that the pblar end of the micelle is next to the polar end of the large molecule. The hydrophobic ends of the molecules in the micelle are now pointed out into solution. Such reverse micelles have polar interiors containinz the solute and hvdronrohic exteriors. . . ~ i polar e molecule reverse micelle aggregate is now soluble in nonpolar solvents. Further investigation of the properties of
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improvingand broadening SFC capabilities. Derivatization is a well-known sample preparation technique and bas been used to increase sample volatility in GC and enhance sample separation and dekction in LC. In particular, simple derivatizations of compounds can be performed to enhance the sample solubility in supercritical fluids. Derivatiiation of polar groups may make the sample less polar and thus more amenable to SFC analysis. The separation of several silylated oligosaccharides by SFC is shown in F i e 10 (59). Because of their many hydroxyl groups and extensive hydrogen bonding, these oligsaccharides are too polar to be a n a b e d directly by SFC. Their derivatives are not amenable to GC analyais duetotheirlowvolatility.I n F i i e 10oliosaccharides with a degree of polymerization ranging from 2 to 18 were separated after ailylation of the hydroxyl groups. Once derivatized, other compounds should be amenable to SFC analysis. Future Roles of SFC An increase in the use of supercritical fluid extraction coupled with SFC or GC will be seen. The ease of adjustment of solvent strength in SFC extraction provides a selective separation method in sample preparationandinseparations. Samples can be concentrated onto GC or SFC columns before fmal analysis simply by decreasingthe pressure of the system so that the solvent is no longer asupercriticalfluid but agas. Aselective method for d e t e r m i n i caffeine from coffee beana by supercritical fluid extraction SFC has been reported (60). Ground coffee h e m were extracted using supercritical carbon dioxide, and the extract was then introduced into the chromatographic system and analyzed. Supercriticalfluid extraction has also been usedto extract PAWS (6145) and other petroleum products (65). Finally, SFC islikely toplay an increasing role in process analytical chemistry. Thin area of analytical chemistry is concerned with on-line analysis of chemicalsin various stages of manufacturing.In process analytical chemistry, an analytical instrument is attached to a process line. Small amounts of sample are shunted from the process stream and are analyzed by the analytical inatrument. On-line analysis provides faster sample resultsbecause the samples are analyzed immediately instead of being sent to a lab,oratory for analysis. The faster analytical results are received, the more quickly plant operators can make changes in process conditions. As on-line SFC analysis of a pipe still sidestream in a petroleum refinery has been reported in the literature (66). Conclutlon When SFC wasfmt introduced,there was hope in the analytical community that it might solve several important separation problems. While SFC has shown impressive separation capabilities, it bas not gained widespread amptance as aresult of some of the limitations described above. For SFC to become a routine analytical technique, it must he shown that SFC can separate a large number of compounds that cannot be easily separated by GC or LC. While SFC cannot displace either LC or GC as an important separation technique, it must build its own niche for separating many different types of compounds. By providing unique separationsfor various analytes (particular-
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Figure 6. Capillary superulticai fluid chmmamgraphic proflie of a mlxtvre of monc-, dl-, and Wtglycerides Numbered chmmatographic peaks are Identified in ref 44 Reprinted with permission fmm ref 44.
ly biological compounds), SFC ran complement GC and LC, which will increase its acceptance as an analytical technique. SFC bas made some major advances in the last 10 years, and the emergence of commercial SFC equipment bas made SFC availableto a large number of acientists. The intense research presently being carried out assures that developmentwill continue in this interesting area of chromatography.
14. Schwarte.H. E:Bmrmlee, R. G. J. Chmmatogr. 1986.. 353,77. 15. Campbsll. R M.; Djordjevic,N. M; Markid-, K E.; Lee, M L.Anal. Chom. 1988.60.356. I& Nmrla. T. A,; Rawdrm. M. G. A d . Chem. 1984.56, 1767. 17. Morin,P.; Caude,M.;Riehd.H.;Russet,R Analyais~ 1987.15,117. 18 Lee,M.L.: Gostea,S. R.;Markid-, K.E.; Wise, S.A. P o l m l . A m m t . Hydrocarborn: C k m . Chomct. Coreinog. h t . Symp. 9th. 1988.13. 19. Jackwn, W. P.; Kbng, R C.; Lee, M. L. Poly%ml. Ammot. Hydm. Carbon (Pop. h t . Symp.18th. 1985. RM~ 20.
1. Markid-, K. E.: k, M. L.. Eds. SFC Applicotia~; Brigham Young University: PPPP, UT,1988. 2. Cheateq T. L. J. Chromfogr.Sei. 198624. 226. 3. Fuhr. B. J.; Hollossay, L. R.; Reiohsrt, C.; k. S. W.; Hayden. A. C. S. Am. C k m . Sac. Div. h o l Chem., 1587.32,W. 4. Sohv~H,E.:Bm&8,RG.:Boduaeyruu.M.M.; Su.F. Anal. C k m . 1981.59.1393. 5. Schv&. H. E.; Hidgins, J. W.; Bm&e,R. G. LCOC 1986.4.639. M.;Loo,M. 6. Nishoika,M.;Whiting,D.G.,Campbpu,R. L.Anal. Chem. 1)8(,58,2251. 7. J e d . D.M. Chmmatogr. Sci. 1979.11: Chramfagr. Pet. Awl. 273. S Snyder, T. R: Saundsrs, D.L. Chromaton. Sei. 1919, 11; Chramafogr. Pet. Anal. 215. 9. h m , R Chmmotop. Sci. 1979.11: Chmmtogr.Pet. Anal. 323. 10. Gouv.T. K: Jentoft, R. E. C h r a m t o p . Sri. 1979.11: Chmmtagr.F'et. A d . 313. 11. Altgclt.K. H. Chmmfogr.Sci. 1 ~ , 1 1 : C h r a m f o p . Pof. Anal. 287. 12. Altgelt. K. H.; Jeaell, D.M.; Latham, D.W.; Selueky, M. L. Chramfogr. S c i 1979, 11: Chromotogr. Pet. &I. 185. 13. Wright. B. W.; Smith, R. D. Chmmtogmphio 1984, 18,542.
Jinn~K.;Hoshioo,T.;Hondo.T.;Seito.M.;Senda,M.
Anal. Chem. lSS.58.2696. 21. Jinno. K.; Saito, M.; Hoodo, T.; sends, M. c h r o m t o gmphin 1986.21.219. 22 Jaekmn,W.P.;Mar!sidesK.E..Lee,M.L.HRCCC.J. High Re.oluf. chromtogr. Chmmfagr. C o r n " . 1986,9,213. 2 8 Weat, W. R: Lrr. M. L. HRCCC. J. High Resolut., Chramfop. Chramtogr. C a ~ n u n 1986.9.161. . 24. Jentofft, R E.; Gouv, T. H. Anal. C k m . L976.48, 2195. 25. Furit.. K.; Sbimokobo. I.; Nalureims, F. Nippan Kogobu Kalahi 1976,8.1348. 26. Chrhienaen, R G. HRC-CC. J. High Reaaiut. Chmm t o g r Chromatop. Commun. 1985.8.824. 27. SehmiU. F. P.: Iryendslrer. D.: hyendecker. D.: GemmelB. J. Chmmntogr. 1987.3%.1. 28. Sehmita. F. P.: Klsaoer. E. Mobmmol. Chem. Rooid c o m m i r as1.2, i 3 5 29. Sehmita, F. P.: Hiker, H. Makromol. C k m . R e i d commun. LOBE, 7,69. 80. SEhmita, F. P,Klaspsr, E.Polym. Commun. 1983.24. 31. Sebmita, F. P.: Khspcr, E. h l y m . Bull. I98l,S, 11. 32. Hartmann, W.;Kleawr.E J.Polym. Sci.PolymLefl. 1977,15,718
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