lates to f 2 0 % for all compounds studied, while the correlation for structurally related compounds correlates to within f 2 % . The plot in Figure 3 of the mobility data of Table I for the nitrosamines compares favorably with correlation data of others (18) and tends to confirm the ionic assignments, although not providing a definite proof. In all cases, an injection of 10-8 gram of sample gave mobility spectra with a signalhoke ratio greater than 1000 indicating that quantities approximating gram are detectable. The changing relative abundance of the MH+ and MpH+ product ion peaks with concentration can serve as a means of identification as well as quantitate these compounds. A t the lowest concentrations, the MH+ product ion provides the most sensitive measure of the compound; a t the higher concentrations the MaH+ product ion peak does. J. Cohen, Franklin GNO Corp., West Palm Beach, Fla., personal communication, 1974.
(18) M.
The results obtained indicate that the plasma chromatograph can provide qualitative analyses for N-nitrosamines a t trace levels. Obtaining mobility spectra is a rapid and simple procedure that gives no evidence of compound decomposition. Further investigative work is presently being conducted to obtain reference mobility spectra on other N-nitroso compounds.
ACKNOWLEDGMENT The authors wish to thank J. G. Smith of the University of Waterloo for suggesting the study of these compounds. Received for review February 4, 1974. Accepted April 29, 1974. The research for this paper was supported by the Defence Research Board of Canada, Grant Number 9530-116, and the National Research Council of Canada, Grant Number A5433.
On-Column Preparation of Bonded Phases for High Pressure Liquid Chromatography R . K. Gilpin, J. A. Korpi, and C. A. Janicki McNeil Laboratories, Camp Hill Road, f t . Washington, Pa. 79034
Preparation of bonded phases for chromatographic application has become a wide area of interest. Initially, bonded phases were investigated for their application in gas chromatography ( 1 - 3 ) . However, with the emerging use of high pressure liquid chromatography (HPLC), bonded phases have shown even greater potential and widespread acceptance. Since the initial work, not only are commercially-prepared materials available for both GC and HPLC, but also a number of authors (4-13) have described various adsorbent modification procedures. Often these adsorbent modifications are tedious processes, and reaction conditions are very cumbersome (14). Hastings et al. (8) have described a partial in-situ reaction process for preparing bonded GC columns. However, a majority of their procedure was carried out external to the column and only the final polymerization was in situ. In this paper, a simple and yet very effective way of preparing bonded phases for HPLC use is described. A totally in-situ process has been used whereby all modifications Abei, F. H . Pollard, P. C. Uden. and G . Nickless, J. Chromatogr., 22, 23 (1966).
(1) E. W. (2) (3) (4) (5)
(6) (7) (8) (9) (10) (11) (12) (13) (14)
C. J . Bossart, /SA Trans., 7, 283 (1968).
I . Halaszand I Sebestian,Angew. Chem., E, 453 (1969). W. A . Aueand C. R. Hastings. J. Chrornatogr., 42, 319 (1969). J. J. Kirkland and J. J . DeStefano, J. Chromatogr. Sci., 8, 309 (1970). J. B . Sorrel1 and R . Rowan, Jr.. Ana/. Chern., 42, 1712 (1970). C. R . Hastings, W A . Aue, and J. M. Augl, J , Chromatogr.. 53, 487 (1970). C. R. Hastings. W. A. Aue, and F. N . Larsen, J. Chromatogr., EO, 329 (1971). J . J . Kirkland, J. Chromatogr. Sci., 9, 206 (1971) K . Unger and P. Ringe, J. Chrornatogr, Sci., 9, 463 (1971). D C Locke, J . T. Schmermund, and B . Banner, Ana/. Chem., 44, 90 (1972) K . Unger.Angew. Chem.. Int. Ed. Engl., 11, 267 (1972). R . K. Gilpin and M . F. Burke, Anal. Chem., 45, 1383 (1973) E. Grushka and R. P W. Scott, Ana/. Chem.. 45, 1626 (1973)
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are carried out directly in the packed column. This has been made possible by the use of Teflon-lined tubing. Both phenyl and octadecyl coatings on Corasil have been prepared. These on-column modified supports have been examined in light of existing commercially available packings .
EXPERIMENTAL Equipment. All HPLC work was performed on a Waters Associates Model 202 liquid chromatograph equipped with dual 6000 psi pumps, solvent programmer, and a UV detector (254 n m ) . Samples were introduced by a 25-pl Precision Sampling syringe. 5 Columns were maintained a t ambient temperature ( ~ 2 "C). Columns. All columns were prepared by the following procedure: Corasil I1 (37-50 km) beads were packed into 'k-inch 0.d. Teflon-lined stainless steel columns, 2 ft in length. Before packing, all tubing was rinsed with methanol and chloroform and dried. Columns were packed by a combination of gentle vibration and suction. A lh-inch 0.d. by 6-ft Teflon-lined stainless steel reservoir column filled (by suction) with either 10% octadecyltrichlorosilane or phenyltrichlorosilane in toluene was attached to the Corasil I1 column. The other end of the column reservoir was connected to the down-stream side of the LC pump. The up-stream side of the pump was connected to a solvent reservoir containing either dry or wet toluene. In those cases where dry toluene was used, the initially untreated Corasil columns were dried a t 150 "C for a t least 4 hours while passing dry nitrogen through them. The toluene-silane solution was pumped through the column a t a rate of 0.5 ml/min. After all the silanizing solution had been eluted from the column, a n additional 100 ml of toluene was pumped through. The column was rinsed with a t least 100 ml of acetonitrile followed by 100 ml of 80:20 acetonitrile:O.OlM ammonium carbonate. Before use, each column was conditioned with a t least 100 ml of a desired mobile phase composition. Multi-coatings were prepared by rinsing the silanized columns between each coating with at least 200 ml of distilled water. Columns were then dried a t 150 "C for a t least 12 hours with a stream of dry nitrogen. Additional coatings were carried out in a manner similar to the initial procedure.
A N A L Y T I C A L CHEMISTRY, VOL. 46, NO. 9 , A U G U S T 1974
a
1
J L 1
2
l
1
3
2
3
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l
'
l
'
1
2
3
4
5
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6
7
ntiiniion T i Y i . r n ~
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Figure 1. Octadecyltrichlorosilane-modified Corasil I I with dry toluene rinse. Peaks: ( 1 ) p-benzoquinone, ( 2 ) 1,4-naphthoquinone, (3) anthraquinone. (a) 1 coating, 30:70 acetonitrile:O.OlM ammonium carbonate mobile phase. ( b ) 2 coatings, 40:60 acetonitrile:O.OlM ammonium carbonate mobile phase
Figure 3. Octadecyl-modified Corasil I I from Waters Associates Peaks: (1) p-benzoquinone, ( 2 ) 1,4-naphthoquinone, (3)anthraquinone, 30:70 acetonitrile:O.OlM ammonium carbonate mobile phase
a
1
2 3
RilinllOl
TIMI,
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Figure 2. Octadecyltrichlorosilane-modified Corasil I I with wet toluene rinse (0.02% water). Peaks: (1) p-benzoquinone. (2) 1,4-naphthoquinone, (3)anthraquinone, 30:70 acetonitrile:O.OlM ammonium carbonate mobile phase
Bonding Studies. Octadecyl- and phenyl-modified Corasil samples were removed from in-situ modified columns. These samples were washed three times with methanol and placed in a 110 "C oven until dry. The samples were divided into two sets. One set of samples was retained as a control while the second set was subjected to continuous extraction in a soxhlet apparatus with toluene for 48 hours. These samples were again rinsed three times with methanol and dried a t 110 "C. The amounts of bound carbon on both sets of samples were determined by removing physically adsorbed organic matter in a dry nitrogen stream a t 150 "C for 18 hours and analyzing the dried samples for total carbon. All carbon analyses were performed by Huffman Laboratories, Inc., Wheatridge, Colo. Reagents. The toluene used was analytical reagent grade obtained from Mallinckrodt Chemical Works and contained 0.02% water. The dry toluene was prepared by refluxing over and redistilling from calcium hydride. Acetonitrile (distilled in glass) was obtained from Burdick and Jackson Laboratories. The 0.01M ammonium carbonate solution was prepared by dissolving 2 grams of ammonium carbonate (Mallinckrodt) in 2 1. of distilled water. Octadecyltrichlorosilane and phenyltrichlorosilane were obtained from Aldrich and used as received.
1
2
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3
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Figure 4. Phenyl-modified Corasil I I Peaks: ( 1 ) p-benzoquinone, ( 2 ) 1,4-naphthoquinone, ( 3 ) anthraquinone. Mobile phase 30:70 acetonitrile:O.OlM ammonium carbonate. ( a ) Commercially available from Waters Associates, ( b ) On-column modification with wet toluene rinse (0.02% water)
RESULTS AND DISCUSSION A test mixture of p-benzoquinone, 1,4-naphthoquinone, and anthraquinone was used to evaluate all columns. Representative chromatograms showing on-column octadecyltrichlorosilane- and phenyltrichlorosilane-modified Corasil I1 appear in Figures 1, 2, and 4b. Chromatograms obtained on commercially available octadecyl- and phenylmodified Corasil I1 columns appear in Figures 3 and 4a. For comparative purposes, all chromatograms shown, except Figure l b , were run a t a 30:70 acetonitrile:O.OlM ammonium carbonate mobile phase composition. The chromatogram shown in Figure l a was obtained on a single coating column prepared with 10% octadecyltrichlorosilane in dry toluene followed by a dry toluene rinse. Figure 2 shows the same components chromatographed on
ANALYTICAL CHEMISTRY, VOL. 46, NO. 9, AUGUST 1974
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a single coating column prepared with 10% octadecyltrichlorosilane solution followed by a rinse with toluene which contained 0.02% water. These results are consistent with the rationale that an increase in available water should result in a polymeric coating of greater depth and an increase in retention as observed. This is further supported by comparison of data between one coating and two coatings of octadecyltrichlorosilane with a dry toluene rinse after each reaction step as shown in Figure 1, a and b. respectively. Retentions were significantly increased for the doubly coated Corasil column. By increasing the ratio of acetonitrile to 0.01M ammonium carbonate, the retention times were decreased to those obtained with a single coating of octadecyltrichlorosilane. Kirkland (9) has also found that increases in YO polymeric coating result in increases in compound retentions. Chromatograms comparing phenyl on Corasil I1 obtained from Waters Associates with an in-situ phenyltrichlorosilane-modified Corasil I1 column appear in Figure 4, a and b, respectively. The phenyl column was prepared by a single coating of 10% phenyltrichlorosilane in toluene followed by a 0.02% water in toluene rinse. In the cases of a single coating of phenyltrichlorosilane prepared with dry toluene rinses. retention volumes were less than those obtained with the commercial phenyl on Corasil I1 packing. These results closely parallel and support data obtained on the octadecyl columns. In general octadecyl and phenyl in-situ-modified columns, when compared to commercially available packings, gave similar retention behavior. In each case, single coats prepared with dry toluene produced lower retention volumes than those obtained with commercial packings. Two coats of a particular silane applied in the same manner gave retention volumes similar to commercial packings. However, with columns prepared from either octadecyltrichlorosilane or phenyltrichlorosi-
lane followed by a wet toluene (0.02% H20)rinse, retention volumes were greater than those Qn the commercial packings. Also, a column was prepared with phenyltrichlorosilane followed by a water-saturated toluene rinse. Little difference was found in results between columns prepared by the 0.02% H20 in toluene rinse and columns prepared by the water-saturated toluene rinse. Thus, after the addition of varying amounts of water, further increases have been found to have little effect on changes in retention volumes. An exhaustive extraction procedure similar to that described by Aue and Hasting ( 4 ) was carried out on both octadecyl- and phenyl- on-column modified Corasil. In each case, no significant difference in amounts of bonded carbon was found before and after extraction. In the case of the octadecyl column, 1.34% and 1.36% carbon was found initially and after extraction, respectively. For the phenyl modification, 0.79% and 0.74% carbon were obtained. These results would support the premise that the totally in-situ modified coatings are chemically bound to the silica surface.
CONCLUSION An in-situ preparation of bonded phases for use in HPLC has been developed. An advantage of this method is the ease and convenience of preparation. Also, the method provides a means of creating multi-layer coatings which should prove to be extremely useful for investigating the effect of bonded phase film thickness on chromatographic behavior. A detailed chromatographic study of this is now in progress. Received for review January 7, 1974. Accepted April 5, 1974.
Gas Chromatographic Determination of Inorganic Sulfide and Organic Thiol Compounds Using the Catalyzed Azide-Iodine Reaction Larry P. Atkinson and Joseph G. Natoli Skidaway institute of Oceanography, P.O. Box 13687, Savannah, Ga. 3 1406
The determination of sulfur-containing compounds in natural water, industrial effluent, and sediment samples is often difficult because of their low concentration or the presence of interfering compounds. Methods in use include the titrimetric analysis ( I ) , the methylene blue method ( Z ) , and gravimetric determination ( 3 ) . These methods determine HZS, HS-, and the common acid soluble metallic sulfides (except c u s ) . In our work on the anoxic sediment systems of Georgia salt marshes, we could not use the above procedures because high turbidity interfered with the colorimetric determination, and titrimetric methods lacked adequate sensitivity. We were also interested in the concentration of the organic thiol compounds that might be present in marsh (1 American Public Health Association, Standard Methods for Examination of Water and Waste Water, A.P.H.A.. 874 p (1971). (2) J. D. Cline, Limnol. Oceanog., 14, 454-458 (1969). (3) A. Nissenbaum, 6 . J. Presley. and I. R. Kaplan. Geochim. Cosmochim. Acta., 36, 1007-1028 (1972).
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sediment or industrial effluent, which are not determined by present methods. During our search for a suitable substitute method, we noted the interesting reaction 2NaNs Ip 2NaI 3Np in Feigl (4).This reaction is very slow but is catalyzed by sulfides, thiosulfates, thiocyanates, and other R-S and R-S-H compounds. Thioethers (R-S-R), disulfides (R-S-S-R), sulfones (R-SOzH), and sulfonic acids (R-SO3H) do not catalyze the reaction ( 4 ) . As discussed later, the complete reaction mechanism is not known. The reaction has been utilized in other studies on kinetics (5, 6),and it has been used to determine cystine in human blood serum ( 7 ) and sulfide in water (8). These
-
+
+
(4) C. Feigl, "Spot Tests in Organic Chemistry," Elsevier, New York, N.Y.. 1972. (5) W. L. Carpenter. Anal. Chem., 36,2352-2353 (1964). (6) W. E. Dah1 and H. L. Pardue. Anal. Chem., 37, 1382-1386 (1965). (7) R. D. Strickland, R. A. Mack. and W . A. Childs. Anal. Chem., 32, 430436 (1960). (8) J. Naumczyk, Gaz. Woda i Technika Sanifama, 45, 346-348 (1971).
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