633
Anal. Chem. 1904, 56,633-638
quential anion determinations be performed by using the method developed for the seven anion determination since the seven anion method provided wider dynamic range and fewer potential interferences. Effects of Automated Sampling. The possibilities for sample contamination from the polystyrene vials used in the autosampler and carry-over from the probe and inlet line to the injection loop of the chromatograph were investigated. Several polystyrene vials were filled with the eluent used for chromatography and then analyzed after several hours of contact. The results obtained were below the minimum reporting limits for the seven anions of interest and indicated that no pretreatment of the vials was necessary. Evaluation of potential carry-over was investigated by analyzing six series of vials (three vials per series) with contents alternating between eluent and calibration standard. Immersion of the sampler probe in a rinse cell (continuously flushed with low conductivity water at a dropwise rate) during advancement of the autosampler carousel was sufficient to
cleanse the outside surface of the probe. Carry-over due to previous sample was eliminated by flushing the inlet line and sample injection loop with sample for a period of 1 min prior to injecting the sample into the eluent stream.
LITERATURE CITED Small, tiamish; Stevens, Timothy S.; Eauman, Wllllam C. Anal. Chem. 1975, 4 7 , 1801-1809. Pohl, C. A.; Johnson, E . L. J . Chromatogr. Sci. 1980, 18, 442-452. Rawa, J. A. In “Ion Chromatographic Analysis of Environmental Pollutants”; Mullk, J. D., Sawlcki, Eugene, Eds.; Ann Arbor Science: Ann Arbor, MI, 1979; Vol. 11, pp 245-269. Wetzel, Roy; Smith, Frank C., Jr.; Cathers, Elizabeth I n d . Res. Dev. 1981, 23 (I), 152-157. Stevens, Timothy S.; Turkelson, Vlrgli T.; Albe, Wllllam R. Anal. Chem. 1977, 49, 1176-1178. Stevens, Timothy S. I n d . Res. Dev. 1983, 25 (9), 96-99. Lipskl, A. J.; Vairo, C. J . Can. Res. 1980, 13,45-48. Koch, Willlam F. Anal. Chem. 1979, 51, 1571-1573. Jenke, Dennis Anal. Chem. 1981, 53, 1535-1536.
RECEIVED for review July 20,1983. Resubmitted January 9, 1984. Accepted January 12, 1984.
Determination of Monosaccharides as Aldononitrile, 0-Methyloxime, Alditol, and Cyclitol Acetate Derivatives by Gas Chromatography Gordon 0. Guerrant* and C. Wayne Moss
Division of Bacterial Diseases, Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia 30333
Mlxtures of neutral, alcohol, and amine monosaccharides were separated by gas chromatography after conversion to volatile derlvatlves. Neutral and amine sugars were derlvatired as aldononltrlle acetates whlle we simultaneously derlvatlzed alcohol sugars as alditol or cyclltol acetates. I n addition, neutral and amine sugars were derlvatlzed as 0 methoxlme acetates, with alcohol sugars as alditol or cyclitol acetates. Derlvatlratlon was facllkated by use of the catalyst 4-(dlmethylamlno)pyrIdlne. Stable derivatives were readily formed by both of the proposed methods, and a mixture of 28 sugars was analyzed that Included sugars contalnlng five through nlne carbon atoms. The usefulness of these methods was demonstrated by analysis of carbohydrates In whole bacterial cells.
Carbohydrates are important components of biologic materials and are present in organisms as polysaccharides attached to glycoproteins. The monosaccharide moieties of polysaccharides can be liberated by hydrolysis and subsequently analyzed to determine the individual neutral, alcohol, and amine sugars present. The identification of these constituent sugars can provide useful biologic information for recognition and differentiation of cells (1). Gas-liquid chromatography (GLC) has been used to determine small amounts of neutral sugars after conversion to volatile alditol acetate derivatives (2,3). However, the sample preparation procedure is timeconsuming for removal of excess borate used to reduce neutral sugars before acylation. Recently, reduced neutral and amino sugars were acylated without removal of borate by use of a
catalyst 1-methylimidazole(4,5). Also, by this method neutral and alcohol sugars cannot be differentiated since neutral sugars are reduced to alcohols before derivatization. Separations have been significantly improved by use of the fused-silica capillary column; recently 20 neutral and amino sugars have been determined in a single chromatographic analysis (6). The fused-silica column has also been used for the separation of trimethylsilyl (Me,Si) derivatives (7) and trifluoroacetyl (TFA) derivatives (8) of monosaccharides. However, multiple peaks from isomeric forms of sugars are produced with both Me3Si and TFA derivatives which complicate interpretation of chromatograms; also Me3Siderivatives are often unstable in storage (9). Aldononitrile acetate derivatives have been used (9-11) instead of alditol acetate derivatives because of easier preparation, greater stability, and good chiomatographic separation and for good mass spectra (12). Although the neutral pentose and hexose sugars readily formed aldononitrile acetate derivatives, the derivatizations of glucosamine (GlcN), galactosamine (GalN), and mannosamine (ManN) were not reproducible (10). Therefore, the hexosamine sugars were derivatized (10) as the 0-methyloxime acetates which were stable and could be readily chromatographed. Glycoproteins were analyzed (IO)by first separating the neutral and amine sugars of hydrolyzed glycoproteins using ion exchange chromatography. These respective sugar fractions were derivatized separately as aldononitrile acetates and 0-methyloxime acetates and then the two derivatives were combined for a single chromatographic analysis. Aldononitrile acetate derivatives of neutral sugars and glucosamine have been prepared by using 1-methylimidazole
This article not subject to U S . Copyright. Publlshed 1984 by the American Chemical Society
634
ANALYTICAL CHEMISTRY, VOL. 58, NO. 4, APRIL 1984
as catalyst and solvent (13). Another acylation catalyst, 4(dimethylaminolpyridine (DMAP) is effective for tertiary alcohols that are not readily acetylated with acetic anhydride and pyridine (14). The efficiency of catalyzed acylation of 2-propanol with either pyridine, 1-methylimidazole, or DMAP has been reported to have relative rates of 1:360:17000, respectively (15). Therefore, DMAP appeared to have good potential as a catalyst for acylation of sugars. In this paper we describe a procedure that uses DMAP for the simultaneous derivatization of neutral and amino sugars as aldononitrile acetates, with alcohol sugars as alditol or cyclitol acetates. In addition, an 0-methyloxime acetate derivatization procedure for mixtures of these sugars is described. Chromatographic separations are shown for 28 sugars including three amine sugars containing eight- and nine-carbon atoms, namely, octulosonic, muramic, and neuraminic acids. The methods have been designed for routine application by using solvent extraction, instead of evaporation, for removal of pyridine and acetic anhydride from reaction mixtures. The methods have been used to detect carbohydrates in hydrolyzed bacterial cells. EXPERIMENTAL SECTION Apparatus. The gas chromatograph used was a Perkin-Elmer Model 900 modified for fused-silica capillary-column operation by installation of a capillary-column conversion kit (PE No. 332-4002) plus a he-ionization-detector base from a Model 3920 gas chromatograph. The column was fused silica coated with OV-1, 0.11-pm film, 0.20 mm X 50 m (Hewlett-Packard No. 19091-60350). Helium was used as carrier gas at a column flow rate of 0.5 mL/min and a split ratio of 35:l. The initial column temperature was set at 175 "C for 4 min and then temperature programmed at 4 "C/min to 260 "C and held for 5 min. The injector and detector interface temperatures were 230 and 275 "C, respectively. The results were recorded on a Hewlett-Packard Model 3390A recording integrator and also a Perkin-Elmer Model 56 recorder. A DuPont Model 21-491B gas chromatograph/mass spectrometer (GC/MS) with chemical ionization was used to confirm the aldononitrile acetate derivatization of amino sugars. Sample preparation was aided by using a heated ultrasonic bath for derivatization and a vortex mixer for solvent extraction. Standards and Reagents. Carbohydrate standards were obtained from Sigma Chemical Co. and are listed (including abbreviations used in the figures): N-acetylmuramic acid (AcMur), N-acetylneuraminic acid (Ac-Neu),adonitol (Ado-ol),D(-)-arabinose (Ara), L-(-)-arabinitol (Ara-ol), 2-deoxy-~-glucose (De-Glc), 2-deoxy-~-ribose(De-Rib), erythritol (Ery), &D-(-)fructose (Fru), a-L-(-)-fucose (Fuc), a-D-(+)-fUCOSe, galactit01 (Gal-ol), D-(+)-galactosamine (GalN), D-(+)-galactose (Gal), Dglucoheptulose (Glc-Hep), D-(+)-glucosamine (GlcN), P-D-(+)glucose (Glc),myo-inositol (Ino-ol),2-keto-3-deoxyoctonate (KDO), D-mannitol (Man-ol), D-mannoheptulose (Man-Hep), Dmannosamine (ManN), D-(+)-mannose (Man), muramic acid (Mur), a-L-rhamnose (Rha), D-(-)-ribose (Rib), sedoheptulose anhydride (Se-Hep), D-sorbitol (Sorb-ol), D- (+) -xylose (Xyl). Spectral grade pyridine was distilled and stored over sodium hydroxide pellets, and methanol was dried by storage over 4A molecular sieves. Standards and solid reagents were vacuum-dried for 20 min at 60 & 5 OC at 250 mm/Hg, or less, using a water aspirator. The reagent for preparing aldononitrile derivatives contained 32 mg/mL of hydroxylamine hydrochloride (Sigma) and 40 mg/mL of 4-(dimethy1amino)pyridine (Sigma), in pyridinemethanol (4:l v/v). The reagent for preparing 0-methyloxime derivatives contained 66 mg/mL of 0-methoxylamine hydrochloride (Aldrich) and 66 mg/mL of D W in pyridine-methanol (2:l v/v). Procedure. Aldononitrile acetate derivatives were prepared by adding 0.3 f 0.05 mL of hydroxylamine reagent to a dry sample containing approximately 0.15 mg of each standard carbohydrate in a 13 X 100 mm screw-capped culture tube which was closed with a Teflon-lined cap. The sample was sonicated for 1rnin and heated to 75 f 5 "C in a water bath and held for 25 min. The tube was cooled, and 1 f 0.1 mL of acetic anhydride was added.
The tube was closed, sonicated for 1min, and reheated for 15 min. The tube was cooled, 2 A 0.2 mL of 1,2-dichloroethanewas added, and excess derivatization reagents were removed by two extractions with 1 f 0.1 mL each of 1 N HC1 followed by three extractions with 1 f 0.1 mL each of HzO. Extractions were performed as rapidly as possible to minimize hydrolysis. Each extractant was added, the mixture was agitated on a vortex mixer for 15 s, and the upper aqueous phase was removed and discarded. After the final extraction, the tube was centrifuged at 1500g for 3 min to separate any entrapped HzO which was then removed. We transferred the washed dichloroethane to a warm dry tube, avoiding the transfer of any water, and evaporated it to near dryness by using dry nitrogen and heating at 50 "C in a water bath in order to recover the derivatized sugars. The sample was reconstituted to 400 pL with ethyl acetate-hexane (1:1v/v), and 0.5 pL of this mixture was injected into the chromatograph for analysis. We prepared 0-methyloxime acetate derivatives in the same manner as the aldononitrile acetates, using 0.3 A 0.05 mL of 0-methoxylamine reagent. Bacterial cells that had been grown on a culture plate were prepared for carbohydrate analysis by transferring the cells to a 13 X 100 mm culture tube with 0.5-1.0 mL of water, centrifuging, removing the HzO, and then washing the cells once with HzO. The washed cells were hydrolyzed by heating to 75 "C and holding for 16 h with 0.2 mL of 3 N HCl in a nitrogen-filled tube, We extracted the free fatty acids with hexane; then the aqueous phase was evaporated to dryness at 65-75 OC by using the vacuum of a water aspirator while introducing dry nitrogen through a capillary at a flow of 50-100 mL/min. The dried residue containing the liberated monosaccharides was then derivatized by one of the procedures described above and reconstituted to a final volume of 100 gL for analysis by GLC. Throughout the derivatization procedures, conditions were maintained as anhydrous as was practical. Pipets and culture tubes were kept in an oven at 100 "C and removed just before use. Reagents were added quickly by using warm, dry Pasteur pipets and reagent containers. Sample tubes were kept tightly closed during derivatization. Reagents for derivatization were prepared in 3- to 5-mL quantities and stored at ambient temperatures. In order to work without a fumehood,the disagreeable odor of pyridine was eliminated by the immediate rinsing of glassware with 2 N HCl after use. For derivatization of sugars or hydrolysis of biologic samples, temperatures were maintained below 80 "C to prevent decomposition, discoloration, and the appearance of extraneous peaks in the chromatogram. RESULTS AND DISCUSSION Initially alditol acetate derivatives of neutral pentose and hexose sugars were prepared by the method of Englyst and co-workers (2). However, we considered this procedure to be too time-consuming for routine use since 2 h was required for reduction of aldose sugars to alditols, approximately 3 h for repeated evaporation of acetic acid-methanol, and then an additional 2 h for acylation. We then evaluated the methods of Mawhinney and co-workers (10) for preparation of aldononitrile acetate and 0-methyloxime acetate derivatives, except we used anhydrous conditions. We found that these derivatives could be prepared in approximately 2 h compared with approximately 7 h required for alditol acetate derivatization; however, the newer method of Blakeney and coworkers (4), taking about 2 h time for derivatization, was not evaluated. Moreover, we observed that the derivatives gave sharp well-resolved chromatographic peaks using a 50-m fused-silica capillary column. The 0-methyloxime acetate derivatives of many sugars show two chromatographic peaks due to the presence of anti and syn forms of the sugars (10). However, we consider this to be an advantage since it increases the reliability of sugar identification by comparisons of chromatographic retention times. We substituted DMAP for 1-dimethylamino-2-propanol which was used by Mawhinney and co-workers (10). For acylation of sugars using an acetic anhydride-pyridine mixture, we found that the presence of DMAP resulted in a
ANALYTICAL CHEMISTRY, VOL. 56, NO. 4, APRIL 1984
635
r
w
v)
z v)
w
Ee
1
0
Figure 1. Gas chromatogram of abbreviations and conditions.)
I
28
J
I
10
I
I
20
I
30
MINUTES
carbohydrates as aldononitrile and alditol (-01)
noticeable exothermal reaction indicating rapid derivatization. Also, sugars dissolved more readily in a pyridine-DMAP mixture than in pyridine alone. Therefore DMAP was added to the reagents used for the initial derivatization of sugars as aldononitriles and also as O-methyloximes. For acylation using DMAP, samples were heated for 15 min to ensure a complete reaction with low concentrations of compounds with sterically hindered hydroxyl groups. Hexosamine sugars were not derivatized as aldononitrile acetates by the procedures used by Mawhinney and co-workers (IO)or those used by Varma and co-workers (16). In our study using DMAP and 5 min for the oximation step, we found that the chromatograms of aldononitrile acetate derivatized hexosamine sugars contained a primary peak and smaller late eluting peaks for each sugar. When amino sugars were derivatized by acylation only, using acetic anhydride and DMAP, we noted that the late eluting peaks were identified a~ acylated sugars indicating that the oximation reaction was not complete. Increaaing the derivatization time for oximation from 5 to 25 min resulted in a single well-defined chromatographic peak for each hexosamine sugar. The chromatographic peak for GlcN was confirmed to be the aldononitrile acetate derivative by GC/MS from which the molecular weight of the derivatized parent molecule was found to be 386. In addition to the use of DMAP, existing methods (IO, II) were modified to improve their usefulness for routine work. Mawhinney and co-workers (IO) evaporated mixtures of pyridine, methanol, and l-dimethyl-2-propanol after converting the aldehyde or carbonyl functional group of sugars to the aldononitrile or O-methyloxime derivatives and before acylation of the hydroxyl or amine functional groups. With our procedure, we did not remove the reagents before proceeding with acylation, and thus the evaporation step was eliminated. Then, after the acylation step, we removed the combined derivatization reagents by solvent extraction as an expedient for preparing each sample for analysis by GLC. Use of 1,2-dichloroethane (density 1.26) as extracting solvent facilitates phase separation with easy removal of the aqueous phase. Heat was required to increase the evaporation rate of dichloroethane (bp 83 "C), but this solvent was selected rather than a lower boiling solvent since it gave more complete
I
acetate derivatives. (See Experimental Section for other
removal of residual water from the sugar derivatives which resulted in better storage stability. Standards were stable for at least 6 months when stored at 4 "C. The composition of an O-methyloxime acetate standard containing 27 sugars was unchanged after 10 weeks of storage at ambient temperature, in a partially closed culture tube from which the solvent had evaporated. In selecting sugars for standards, only one of either the D or L form was used since preliminary studies showed that a-D-(+)-fucoseand a-L-(-)-fucose could not be separated under the condition of this study. A derivatized sugar standard was prepared by combining mixtures of sugars. Individual sugars were weighed on a Kahn microbalance and the primary mixture consisted of 1.6 mg each of 21 sugars dissolved in 200 MLof water; a 5-WLaliquot was removed and evaporated to dryness with heat and vacuum. The dried aliquot was derivatized and reconstituted to 200 MLfor analysis by GLC. Ribose, 2-deoxyribose, erythritol, muramic acid (a nine-carbon amino sugar), and KDO (an eight-carbon sugar) were derivatized together. Acetylmuramic acid and acetylneuraminic acid were derivatized separately. The latter seven sugars were handled in the same manner as described, except that derivatives were prepared immediately after we weighed the sugars since instability was observed when these sugars were stored as aqueous solutions at 0 "C. A chromatogram of a mixture of 28 neutral, alcohol, and amine sugars derivatized by the aldononitrile acetate procedure is shown in Figure 1. Alcohol sugars did not react with hydroxylamine or O-methoxylamine reagents in the first derivatization step; they reacted only with acetic anhydride in the second step to form alditol or cyclitol acetates. The peaks shown in the chromatogram in Figure 1represent 2-4 ng of each sugar injected into the chromatographic column. GlcN and KDO coeluted; there were two peaks for fructose; possibly because of its existence in both the furanose and pyranose forms. The aldononitrile acetate derivatives of muramic acid and acetylmuramic acids coeluted since both form the same derivative. The alcohol sugars present as alditol acetates are erythritol, adonitol, arabinitol, mannitol, sorbitol, and galactitol and the cyclic sugars were inositol and sedoheptulose anhydride as cyclitol acetate.
636
ANALYTICAL CHEMISTRY, VOL. 56, NO. 4, APRIL 1984
L
I
10
0 Figure 2. Gas
20
MINUTES
I
2
30
chromatograms of 27 carbohydrates as 0-methyloxime and alditol acetate derivatives.
1 t I
1
I,L
I 1
U
I 0
10
20
30
MINUTES Figure 3. Gas
chromatogram of whole cells of group D Streptococcus as aldononitrile and alditol acetate derivatives; (U) unidentified.
A chromatogram of 27 of the 28 sugars derivatized by the 0-methyloxime acetate procedure is shown in Figure 2. Again, the alcohol sugars were present as the alditol and cyclitol acetate derivatives. With the resolution of the fused-silica column all peaks including sugars which produced two peaks were resolved with the exception of arabinose and fucose. The major peaks for these two sugars coeluted at 13.33 min; however, the minor peak for fucose eluted a t 12.12 min and the minor peak for arabinose eluted a t 12.41 min (peaks A and C in Figure 2). Acetylneuraminic acid is not present in the chromatogram shown in Figure 2; it eluted with a retention time of 35.1 min.
These methods were used to determine the carbohydrates present in whole bacterial cells of a group D strain of Streptococci. A chromatogram of the aldononitrile acetate derivatives is shown in Figure 3 and a chromatogram of the 0methyloxime acetate derivatives is shown in Figure 4. Identification of the sugars present in this organism is based on retention times of the component sugars derivatized by both procedures. The identities of the sugars are indicated above each peak and unknown peaks are designated by U. The peaks eluting within the first 6 min of the chromatogram are not sufficiently resolved for identification and some are reagent related. In Figure 3, the peaks eluting with retention
ANALYTICAL CHEMISTRY, VOL. 56, NO. 4, APRIL 1984
I 0
I
I
I
I
10
I
20
637
I 30
MINUTES
Figure 4. Gas chromatogram of whole cells of group D Streptococcus as 0-methyloxime and alditol acetate derivatives.
times of 17.5 and 18.0 min (present between GlcN and GalN) have respective retention times close to sorbitol and mannosamine. In Figure 4,peaks for sorbitol and mannosamine were absent; therefore, two unidentified compounds were presumed present. In Figure 4, an unknown peak eluted just after Rha at 13.2 min near the retention time of the major peak of both arabinose and fucose; however the minor peaks of these sugars are absent. As shown in the standard in Figure 2, the minor peak [C] of arabinose eluted after the minor rhamnose peak [B], and that of fucose [A] eluted before [B]. In addition, arabinose and fucose were not present as the aldononitrile acetates in Figure 3. These unidentified peaks are believed to be sugars that were not included in the standards of this study, and the sugars discussed could have been misidentified by the chromatographic separations obtained with a single derivative. Alternate methods for confirmation of the presence of small amounts of sugars in complex mixtures are not readily available. Two unknown peaks were encountered in the initial analysis of sugars in these Streptococci which were subsequently identified as free palmitic and stearic acids. The proposed methods have been modified to remove interfering fatty acids from hydrolyzed bacterial cells by extraction with hexane before evaporation and derivatization of the sample. Possible thermal instability of sugar derivatives was observed as indicated by buildup of carbonaceous deposits on the glass insert in the injector of the chromatograph. Therefore, chromatographic operating temperatures were maintained as low as possible to minimize decomposition. Some bacterial cells that were tested contained unidentified components with retention times greater than those of the standards used in the study. When peaks were still eluting in the last several minutes at the end of the chromatographic program, a longer temperature-hold time was required to remove these peaks. Also during the longer maximum temperature-hold time, we found that several large injections of methanol, 7 KL,were beneficial in decreasing the background noise of subsequent analyses. In addition, methanol injections were made each day as part of the initial startup program before proceeding with chromatographic analysis. We obtained the best chromatographic separations by using high
instrument sensitivity and injecting small amounts of sugars. There are several advantages to the proposed procedures for analysis of monosaccharides. A wide variety of sugars may be determined simultaneously ranging from five-carbon atoms through nine-carbon atoms and consisting of neutral, alcohol, and amine sugars. A procedure was developed for derivatization of amine sugars as aldononitrile acetates along with neutral and alcohol sugars. Use of parallel derivatization methods to prepare aldononitrile acetates and 0-methyloxime acetates facilitates confirmation of identity by observing different retention times for the two derivatives. Low concentrations can be determined since good chromatographic response was obtained with a final sugar concentration of 0.1 Kg/pL. The proposed procedures have been simplified over most existing procedures and the derivatives are stable. A possible disadvantage is that reasonable precautions must be taken to maintain anhydrous conditions. Reagents and glassware should be dry and aqueous extractions of derivatized samples should be performed without delay.
ACKNOWLEDGMENT The authors are grateful to R. R. Facklam and L. M. Teixeira for providing the bacterial cells and to M. A. Lambert for preparing the cells for analysis. Registry No. Ac-Mur, 10597-89-4;Ac-Neu, 131-48-6;Ado-01, 488-81-3; Ara, 10323-20-3;Ara-01, 7643-75-6; De-Glc, 154-17-6; De-Rib, 533-67-5;Ery, 149-32-6;Fru, 53188-23-1;Fuc, 51348-50-6; Gal-01,60&66-2;GalN, 7535-00-4;Gal, 59-23-4;Glc-Hep, 5349-37-1; GlcN, 3416-24-8;Glc, 28905-12-6; Ino-ol,87-89-8;KDO, 1069-03-0; Man-ol,69-65-8;Man-Hep, 3615-44-9;ManN, 14307-02-9;Man, 3458-28-4;Mur, 1114-41-6; Rha, 6014-42-2;Rib, 50-69-1;Se-Hep, 469-90-9; Sorb-01, 50-70-4; xyl, 25990-60-7; a-D-(+)-fucose, 31178-76-4.
LITERATURE CITED (1) Sharon, N.; Lis, H. Chem. Eng. N e w s 1981, 59, 21-44. (2) Englyst, H.; Wiggins, H. S.; Cummings. J. H. Analyst (London) 1982, 107, 307-318. (3) Lehrfeld, J. Anal. Biochem. 1981, 115, 410-418. (4) Blakeney, A. B.; Harris, P. J.; Henry, R . J.; Stone, B. A. Carbohyd. Res. 1983, 113, 291-299. (5) Henry, R. J.; Blakeney, A. B.; Harris, P. J.; Stone, B. A. J. Chromatogr. 1983, 256, 419-427. (6) Oshima, R.;Kumanotani, J.; Watanabe, C. J. Chromatogr. 1982, 250, 90-95.
638
Anal. Chem. I98qS56,638-642
(7) Chaplin, M. F. Anal. Blocbem. 1982, 723,336-341. (8) Bryn, K.; Jantren, E. J . Chromatogr. 1982, 240, 405-413. (9) Manius, 0. J.; Liu, T. M. Y.; Wen, L. F. I. Anal. Blochem. 1979, 99,
365-37I. (10) Mawhinney, T. P.; Feather, M. S.; Barbero, G. J.; Martinez, J. R. Anal. Blochem. W80. 101, 112-117. (11) Marier, R. L.; Milllgan, E.; Fan, Y. D. J . cm. Mlcrob/o/. 1982, 16, 123-128. (12) Dmltriev, 8. A.; Backlnowsky, L. V.; Chizhov, 0. S.;Zolotarev, E. M.; Kochetkov, N. K. Carbohyd. Res. 1971, 19, 432-435. (13) McGlnnls, G. D. Carbohyd. Res. 1982, 708, 284-292.
(14) Stegllch, W.; Hofie, 0. Angew. Chem., Inl. Ed. Engl. 1069, 8 , 981. (15) Wachowiak, R.; Connors, K. A. Anal. Chem. 1979, 51, 27-30. (16) Varma, R. S.;Varma, R.; Allen, W. S.; Wardl, A. H. J . Chromatogr. 1974, 93,221-228.
RECEIVED for review November 8, 1983. Accepted January 3,1984. Use of trade names is for identification only and does not imply endorsement by the Public Health Service or by the U S . Department of Health and Human Services.
Effect of Pore Diameter on Diffusive Sample Retention in Gas Chromatography Hikoyuki Kaizuma Department of Chemistry, The Konan University, Okamoto, Kobe 658, Japan
Conslderatlon was glven to dlffuslve sample retentlon In relation to the pore dlameter and the dlffuslblllties of sample and carrier molecules. Three klnds of mlcrobead slllca gel havlng dlfferent pore slze were used as packlng materlal. Inert gases, He, Ne, and Ar, were Injected Into carrier flow as the sample. Hydrogen and nltrogen gases were used as carrlers. A constant term, O , In the hyperbollc equatlon which was prevlously offered by the author can be related to the pore dlameter. I t Is also affected by the dlffuslblllty and/or the mean free path of the carrler gas. As the ratlo of the mean free path of the carrler gas agalnst the pore dlameter of the pcklng gets larger, the dlffuslve sample retentlon becomes more effectlve.
The practice of gas chromatographic separation usually adopts a rather fast flow rate above the optimum where the HETP becomes lowest. It is seldom operated below the optimum because of the following shortcomings: The lower flow rate takes longer time for the migration and, as the result, the peak becomes broader. It is considered that nonsorbed samples migrate with the carrier at the same velocity and retention times of nonsorbed samples should become the same (I). This is true for a nonporous column, while the result is quite different for packed columns of porous material. The retention time of nonsorbed sample in porous column differs from sample to sample and the difference becomes larger as the flow rate decreases (2). Since the samples are nonsorptive, the factors affecting the migration in the column would be the diffusibilities of the sample molecules and the micropores in the packing material. During the process of migration, sample molecules diffuse into the micropores on the packing and are trapped for a while and return back to the flow by diffusing out from the pores. Then, in a steadily flowing carrier, a sample having a smaller diffusibility prolongs the migration by such diffusive displacement and results in longer retention time. In the case of nonporous packing material, the relationship among the retention time (tRo), the interdiffusion coefficient of the sample into the carrier (Drfi),and the peak spreading (go, the standard deviation of the peak width) is given by eq 1, where y is an obstructive factor (3). tRO
=-
2yDm
(1)
0003-2700/84/0356-0838$01 SO/O
On the other hand, when a nonsorbed sample is applied on a column of porous packing, the length of the column is equal to that of the former, the diffusive displacement of the solute occurs between the flowing phase and the stagnant phase in the pores. Retention time of the latter (tR) would be longer than that of the former (tR"). A relation similar to eq 1 may be arrived at for tR
where Q is the standard deviation of the peak spreading and y' is the obstructive factor including intrapore diffusion. D is the apparent diffusion coefficient. The R value can be expressed by using these two retention times
(3) where td = t R - tR". The term td means the time consumed in intrapore diffusion and has following relation which is derived from eq 1and 2:
where 6 is defined as 6 = y ' ( ~ ' ) ~ / y uThe ~ . time ratio, h-J/tRo in eq 3 can be expressed by using above relation as
- Drii - 6D
6D Again, a new term Dm0 is defined by eq 6 D m 0 = Dlii - 6D
(5) (6)
Taking the relation into eq 5, eq 7 is obtained
(7) Substituting eq 7 into eq 4, one will find that 0 1984 American Chemical Society
