The degree of substitution of the samples of Na-CMC was also determined by acid wash (1) and Wilson’s method (2) and tabulated in Table II. The results from RDPT were found to be in close agreement with the results obtained by these two other methods.
Table II. Comparison of the Results of DS from Different Methods, Degree of Substitution Sample no.
Acid wash method
Wilson’s method
1
0.43 0.31 0.30 0.25
0.37 0.26 0.27 0.23
2 3 4
RDPT
0.38 0.28 0.25 0.20
The concentration of COO- ions in each solution of the Na-CMC sample was estimated by the RDP technique and the equivalents of total carboxyl per gram ( A ) of the sample were calculated. From this procedure, the DS was calculated using Equation 1, and the data are presented in Table I.
CONCLUSIONS This study suggests that the quicker and simpler RDP technique may be recommended as an analytical method for the determination of the degree of substitution in CMC. This technique can also be used for the estimation of acid groups present in traces in other types of watersoluble polymers. Received for review October 24, 1972. Accepted January 18, 1973.
Analysis of Polynuclear Aromatic Hydrocarbons, Some Heterocyclics, and Aliphatics with a Single Gas Chromatograph Column D. A. Lane, H. K. Moe, and Morris K a t z l C e n t r e for R e s e a r c h
on
Environmental Quality, York University, D o w n s v i e w , Ontario, Canada
The combustion of fossil fuels for heat, power, and transportation, including internal combustion engines of automobiles and the diesel engines of buses, produce most of the organic fraction of the atmospheric particulate matter commonly found in urban centers. Many of the compounds constituting this organic fraction are known to produce cancers in the experimental animal. Hence, it is of interest to know what compounds exist and in what concentrations they occur. The currently accepted method of Sawicki et al. (1) uses column chromatography to separate the organic fraction of the particulate matter into about 50 subfractions. Each subfraction is spectrophotometrically analyzed for its polynuclear aromatic hydrocarbon content. Other methods such as fluorescence ( Z ) , thin layer chromatography ( 3 ) ,paper chromatography ( 4 ) , and high-pressure liquid chromatography ( 5 ) have been used with varying degrees of success. It has long been recognized that gas chromatography is, in principle, an ideal method for the qualitative and quantitative determination of air pollutants. By nature, 1 To whom a l l correspondence should
be directed
(1) E. Sawicki, R . C. Corey, A. E. Dooley, J. B. Gisclard, J. L. Monkman, R . E. Neligan, and L. A . Ripperton, Health Lab. Sci., 7, 31 (1970). (2) E. Sawicki, R . C. Corey, A. E. Dooley, J. €3. Gisclard. J. L. Monkman, R . E. Neligan, and L. A. Ripperton, Health Lab. Sci.. 7, 45 (1970). (3) E. Sawicki, R . C. Corey, A. E. Dooley, J. B. Gisclard. J. L. Monkman, R. E. Neligan, and L. A. Ripperton, Health Lab. Sci., 7, 60 (1970). (4) T. M . Spotswood, J. Chromatogr.. 2, 90 (1959). (5) G. J. Fallick and J. L. Waters, Amer. Lab., 1972 (8),p 21. 1776
A N A L Y T I C A L CHEMISTRY, VOL. 45,
gas chromatography is a simpler and more direct method for the separation and quantitative determination of compounds than are the spectrophotometric methods, provided, however, a suitable column can be found. Columns for specific compounds or narrow ranges of compounds have been developed (6-11). However, these columns cannot separate, for example, a mixture of benzo[k]fluoranthene, benzo[a]pyrene, and perylene, or a mixture of benzo[lz]fluoranthene, benzo[e]pyrene, and perylene. One packed column (12) achieves partial success but is unsatisfactory for the lower molecular weight compounds such as phenanthrene and anthracene. Carugno and Rossi (13) achieved fairly good separation of these compounds on a capillary glass column. Since a splitter was used, they could not carry out quantitative determinations accurately. A description is given below of a packed column, capable of being used for quantitative determination, which exhibits separations comparable to those obtained by Carugno and Rossi (13). Not only is this column suitable for the determination of polynuclear aromatic hydrocarbons, but it has been found suitable for the separation of some heterocyclics and aliphatics. It is, therefore, highly suited to the analysis of organic atmospheric pollutants. (6) R . M . Duncan, Amer. Ind. Hyg. Ass., J.. 30,624 (1969). (7) L. DeMaio and M .Corn, Anal. Chem., 38,131 (1966). (8) A. Liberti, G . P. Cartoni, and V. Cantuti, J. Chromatogr.. 15, 141 (1964). (9) A. Zane, J. Chrornatogr., 38,130 (1968). (10) J. Frycka,d. Chromatogr., 65, 341 (1972). (11) J. Frycka, J. Chromatogr., 65, 432 (1972). (12) K. Bhatia,Ana/. Chem., 43,609 (1971). (13) N. Carugnoand S.R0ssi.J. Gas Chrornatogr.. 5 , 106 (1967).
NO. 9, AUGUST 1973
Table I. Data for Polycyclics Peak No. Compd name 1 Fluorene 2 9-Fluorenone 3 Phenanthrene 4 Anthracene 5
6 7 8 9 10 I
11 11
12 13 14 15 16 17 18 19 20
Acridine Carbazole Fluoranthene Pyrene 1 1 H-Benzo[a]fluorene 1 1 H-Benzo[b]fluorene Benzo[c]phenanthrene Benz[a]anthracene Triphenylene Chrysene Naphthacene Benzo[k]fluoranthene Benzo[a]pyrene Benzo[e]pyrene Perylene Dibenz[a,h]anthracene Benzo[ghi]perylene 7H-dibenzo[c,g]carbazole
Carcinogenic activity
-
+++ ++-
+++ + (?) -
+++ +c+
Re1 ret time wrt pyrene
0.345 0.579 0.612 0.625 0.665 0.720 0.948 1.000 1.088 1.101 1.256 1.274 1.281 1.284 1.304 1.552 1.639 1.650 1.686 2.223 2.346 2.456
Mol wt
166.22 180.19 178.23 178.23 179.21 167.21 202.26 202.26 216.29 21 6.29 228.29 228.29 228.29 228.29 228.29 252.32 252.32 252.32 252.32 278.35 276.34 267
Soh concn, Mp. "C
1 16-1 1 7 86 101 216.2 111 247 111 149-1 50
Resolution
1
0.634
209-21 0.5 68 162 196.5 254 357 } 21 7 175-1 77 } 178 273-274 } 269.70 277-279 157-1 58
1
0.9126
mg/ml
0.13 0.10 0.10 0.10 0.125 0.135 0.10 0.18 0.14 0.12
... 0.12
...
0.550 1.250 0.590 1.214
0.14 0.06 0.10 0.09 0.12 0.11 0.12 0.12 0.10
EXPERIMENTAL Several columns using Corning GLC-110 textured glass beads were prepared using OV-7 as the liquid phase. The performance of these columns was nonreproducible, and resolution was not satisfactory. Also, liquid phase coatings higher than about 0.170 were not practical. High-performance Chromosorb W of 80-100 mesh size was found to be a much more suitable solid support. Columns with OV-7 coatings of 0.5, 1, 3.15, and 5.25% were investigated and the optimum liquid phase coating was found to be 1%. The standard solution coating procedure (14, 15) was used and was found to be satisfactorily reproducible. A glass column of outside diameter 6 mm and of 5.4 m length enabled the preparation of a column with uniform packing, and minimal particle fracturing. The initial conditioning of a new column was done a t 280 "C for 24 hr while subsequent conditioning required only overnight baking a t 280 "C. One column has been used for over 3 months and no deterioration has been detected. At oven temperatures greater than 260 "C, Teflon ferrules proved to be unstable and were therefore replaced by Dexsil impregnated asbestos cord (16). A Varian Aerograph Model 1740 gas chromatograph equipped with flame ionization detector and a linear temperature programmer was used. The recorder used was a Varian Model 20 strip chart recorder. The gas chromatograph operating conditions were as follows: the helium carrier gas flow rate was 75 ml/min under a column head pressure of 35 psig, the hydrogen flow rate was 63 ml/min, and the air flow rate was 300 ml/min. The injector temperature was 250 "C and the detector temperature was 300 "C. Two temperature programs were used, one for the polycyclics and one for the aliphatics. In the case of the polycyclics, the oven temperature was held at 170 "C for 2 min and then increased a t 2 "C/min to 190 "C a t which temperature the programing rate was changed to 4 "C/min. The oven was maintained a t a final temperature of 280 "C until the last compound had emerged. For the aliphatics, the oven temperature was held a t 150 "C for 2 min., then increased at 2 "C/min to 280 "C. The oven was held a t 280 "C until the last compound had emerged. All impurities from Hamilton Microsep F-138 septa were removed by conditioning the septa a t 250 "C for a 12-hr period. The upper temperature limit for the injector was about 250 "C since the septa were found to crystallize a t or above a temperature of 260 "C. Excessive tailing of the solvent peak was observed on a scale (14) "Columns and Column Technology," Chromatographic Specialties Ltd., Brockville,Ontario, Canada. (15) H. M . McNair and E. J. Bonelli, "Basic Gas Chromatography," 5th ed., Varian Aerograph, 1969,pp 65-66. (16) M. Berozaand M . C. Bowman, Anal. Chem., 43,808 (1971).
T I M E I N MINUTES
Figure 1. Chromatogram of a known mixture of polycyclics Sensitivitiesare in termsof A full scale. S8 = sensitivity of 8 X lo-" A full scale. S16 = sensitivity 16 X lo-" A full scale
sensitivity of 1 X A full scale (afs) when 1 pl or greater injections of benzene-polycyclic solutions were made. Other solvents were found to interact similarily with the stationary liquid phase. Carbon disulfide, although it is a good solvent for polycyclics, gave a negative signal which lasted for 5 min. This would preclude its use as a solvent when low molecular weight compounds are of interest. It was therefore decided to make solid injections. A Hamilton solid sampler (ss60) was used for the injections but was found to be unsatisfactory. Upon insertion of the sampler, the hollow needle cut a small plug of septum which remained in the needle. When depressed, the pointed plunger passed through the plug of septum material wiping off a sizable portion of the dried sample. The plunger was modified by melting the pointed tip forming a blunt end which would force any septum out of the needle when the plunger was depressed. Thus sample losses during the injection were greatly reduced but not to such an extent that quantitative analyses could be performed with this injection procedure. Many of the polynuclear aromatic hydrocarbons (PAH) were obtained from Aldrich Chemical Co. and Eastman Kodak Co. Those PAH compounds not available from the chemical supply houses were supplied by J. L. Monkman of the Air Pollution Control Division of Canada Department of Environment. An alkane mixture (No. 19254) was obtained from Applied Science Laboratories Inc.
RESULTS AND DISCUSSIONS Polycyclics. A typical chromatogram of 20 common polycyclic hydrocarbons is shown in Figure 1, and in Table I are the data pertinent to the chromatogram. Two microliters of the benzene-polycyclic solution were used in the preparation for the solid injection. Quantities of PAH
ANALYTICAL CHEMISTRY, VOL. 45, NO. 9, AUGUST 1973
1777
as low as 10 ng have been injected on column and were easily detected. Assuming a linear detector response to all compounds under consideration, the detection limit was estimated to be in the order of 3-6 ng. This is comparable to the limits of fluorescence measurements done in this laboratory on a Farrand fluorescence spectrophotometer and those of an electron capture detector as reported by Duncan (6). Direct quantitative determinations are possible for all compounds shown with the exception of the phenanthrene/anthracene, benz[a]anthracene/chrysene, and benzo[a]pyrene/benzo[e]pyrene pairs. However, these pairs may be resolved and determined by the numerical method of Mori (17). Direct, qualitative determinations of these compounds in, for example, air pollution extracts are readily achieved. Most of these compounds have been tentatively identified in the aromatic portion of a n air extract. The resolution data shown in Table I are calculated by the standard formula
where A t is the time difference between the two peak maxima and w b l and wb2are the peak widths a t base. A value of R = 1.0 means a separation of about 9070, while a value of R = 1.5 means that the separation is practically complete. The resolution data shown in Table I are calculated by in Table I. Unfortunately, direct numerical comparison could not be made between our results and those of other workers. Hence, only a subjective comparison of the chromatograms presented could be made. Our separation of the groups-phenanthrene, anthracene; benz[a]anthracene, chrysene, and naphthacene; and benzo[lt]fluoranthene (BkF), benzo[a]pyrene (BaP), benzo[e]pyrene (BeP), and perylene-is better than that achieved by previous methods (6-8, 12) and is equivalent to that of Carugno and Rossi (13) who used a 65-m glass capillary column coated with SE-52. Although Zane (9) achieved almost total separation of phenanthrene and anthracene on graphitized carbon black a t 415 "C, his column is suitable for only the low molecular weight compounds. (17) Y . Mori, J. Chrornatogr., 66, 9 (1972)
Our column will completely separate BkF, BaP, and perylene. Complete separation is also obtained for BkF, BeP, and perylene. The problem of BaP and Bep separation still remains. A linear plot of corrected retention time us. melting point was obtained for the compounds with the formula ClsHlz (benz[a]anthracene, chrysene, and naphthacene). The corrected retention times of the two structural isomers, benzo[c]phenanthrene and triphenylene, were then determined. These are designated I and 11, respectively, in Table I. The former compound is of particular interest because of its high carcinogenic activity and occurrence in the urban atmosphere. In general, it appears that compounds with an overall bent structure are eluted before compounds with a linear structure. Thus, for example, phenanthrene is eluted before anthracene, 11H-benzo[a]fluorene before 11H-benzo[b]fluorene, and benz[a]anthracene is eluted before naphthacene. Aliphatics. The same column has also been used for the separation of a mixture of 12 n-alkanes ranging from nundecane to n-dotriacontane. Total separation of these compounds was achieved and, hence, both qualitative and quantitative determinations can be carried out. Aliphatics from C11 to C33 inclusive have been tentatively identified in the aliphatic fraction of an atmospheric extract by means of the relative retention data. This column, therefore, appears to be well suited to the analysis of the organic fraction of atmospheric particulate matter where submicrogram quantities of the individual components are involved.
ACKNOWLEDGMENT The authors wish to thank J. L. Monkman of the Air Pollution Control Division of Canada Department of Environment a t the Environmental Health Centre, Ottawa for supplying several of the commercially unavailable chemicals. Received for review October 11, 1972. Accepted February 20, 1973. This work was partially supported by the National Research Council of Canada (Special Project Grant No. S-13) whose financial assistance is gratefully acknowledged.
In Situ Coating of Supports with Stationary Liquids for High-Performance Liquid-Liquid Column Chromatography J. J. Kirkland and Charles H. Dilks, Jr. E. 1. du Pont de Nemours & Co., Inc., Biochemicals Department, Experimental Station. Wilmington, Del. 79898
The preparation of high-performance columns for liquid-liquid chromatography (LLC) requires that the partitioning phase be placed on the support homogeneously and that the support be uniformly packed into the column. In the past, packings for most high-performance LLC columns have been carried out by solvent evaporation or solvent filtration techniques, which also have been widely used for the preparation of high-performance col1778
umns for gas chromatography ( I ) . Packings made by these pre-coating techniques then are loaded into the column by dry-packing methods (2). For this pre-coating approach, an optimum stationary phase loading has been found, which is about 1% by weight of stationary. liquid for most . (1) J. J. Kirkland, "Modern Practice of Liquid Chromatography," Wiieyinterscience, New York, N.Y., 1971, Chap. 5. ( 2 ) J. J. Kirkland,J. Chromatogr. Sci., IO, 129 (1972).
A N A L Y T I C A L CHEMISTRY, VOL. 45, NO. 9, A U G U S T 1973
