peaks. Personal numerical experience of one of the authors and similar results published in reference (8) indicate that the extraction of information from a chromatograph trace is not yet completely satisfactory when done in the time domain.
function. The power spectrum for such a peak is clearly non-Gaussian however. The authors are currently working on a method t o permit handling of non-Gaussian peaks automatically by working with the Fourier Transform to characterize the peak shape. Our experience in working in the Fourier domain indicates that one is using the relevant information content of the time trace in a more meaningful manner. For example, the noise is readily evident. Previously published methods use least squares fitting of data in the time domain (6, 8) to resolve overlapped chromatograph
The authors are grateful to Goodyear Tire and Rubber Company for providing the sampled experimental chromatograph data used in this paper.
(8) A. H. Anderson, T. C. Gibb, and A. B. Littlewood, ANAL. CHEM., 42,434 (1970).
RECEIV~D for review July 24,1970. Accepted March 24,1971.
ACKNOWLEDGMENT
Gas Chromatographic Analysis of Phenol and Substituted Methyl Phenols Using Open Tubular Columns Jfin Hrivhak Institute of Chemistry, Komenskj. Unioersity, Bratislava, Czechoslovakia
Jiii Macak Department of Coke and Gas Technology, University of Chemical Technology, Prague, Czechoslovakia
The separation of phenol, cresols, and xylenols i s studied using di-n-decylphthalate, di(3,3,5-trimethyl cyclohexy1)o-phthalate, tricresylphosphate, and tri(2,4-xylenyl)phosphate as liquid phases coated on stainless steel open tubular columns at temperatures between 100 and 130 “C. The retention data of phenols are given. Phosphoric acid was used as tailing suppressor. The best separation was obtained on tri(2,4-xylenyl)phosphate liquid phase. The results of quantitative analyses based on peak high measurements were *1.2% average deviation.
THECOMPLETE SEPARATION of a mixture of phenol, methyl, and dimethyl phenols on conventional packed columns is very laborious. Long columns are usually used with impractically long analysis times. Many gas chromatographic analyses of phenols using packed columns have been published ( I ) . The nonpolar liquid phases and most of the polar liquid phases are unable to give sufficient resolution of some pairs of methylphenols. Di-n-decylphthalate (DDP), di(3,3,5-trimethyl cyclohexy1)ophthalate (TMCP), tricresylphosphate (TCP), and tri(2,4xyleny1)phosphate (TXP) are the most selective liquid phases frequently used for the chromatography of methyl phenols (2-6). The resolution of methyl phenols has been improved by using a packed capillary column with a mixture of liquid phases (7). (1) S. T. Preston, “A Guide to the Analysis of Phenols by Gas Chromatography,” Polyscience, Evanston, Ill., 1966. (2) V. T. Brooks, Chem. Ind. (London),1960, 1090. (3) W. Sassenberg and K. Wrabetz, 2. Anal. Chem., 184, 423 (1961). (4) A. R. Paterson, in “Gas Chromatography,” Instrum. SOC. Amer. Symposium, June 1959, H. J. Noebels, R. F. Wall, and N. Brenner, Ed., Academic Press, New York, N. Y . , 1961, pp 233-6. (5) J. Q. Walker, J . Gas Chromatogr., 2, 46 (1964). (6) J. Zulaica and G. Guiochon, J. Polymer. Sci.,4, 567 (1966). (7) C. Landault and G. Guiochon, ANAL.CHEM.,39, 713 (1967).
Difficulties resulting from the highly polar phenolic group can be avoided by chromatographing of derivatives such as alkoxy (&IO), trimethylsilyl ethers (11-14, and acetate esters (15-17). The use of derivatives instead of free phenols has some limitations. Sterically hindered phenols can react slowly and incompletely and hydrolysis should be taken also in account (16). As a result of the wall effect, which causes excessive tailing, attempts to use open tubular columns for separation of free phenols have been often unsuccessful. In the few papers dealing with the separation of phenols o n open tubular columns (15, 18-20), the use of surface active agents is often recommended for suppression of tailing effects. Symmetrical peaks are very important to obtain when precise quantitative analyses are required. In the work described in this paper, we have studied various liquid phases (DDP, TMCP, TCP, TXP) coated on a stainless (8) G. Bergman and D. Jentzsch, Angew. Chem., 70, 192 (1958). (9) W. Carruthers and R. A. W. Johnstone, Nature, 185, 762 (1960).
(10)G. A. L. Smith and D. A. King, Chem. I d (London), 1964, 540. (11 ) S.H. Langer, P. Pantages, and I. Wender, ibid., 1958, 1664. (12) D. W. Grant and G. A. Vaughan, in “Gas Chromatography 1962,” M. Van Swaay, Ed., Butterworths, Washington, D. C., 1962, pp 305-14. (13) R. W. Freedman and P. P. Croitoru, ANAL.CHEM., 36, 1389 (1964). (14) R. W. Freedman and G. 0. Charlier, ibid., p 1880. (15) E. R. Adlard and G. W. Roberts, J. Inst. Petrol., 51, 376 (1965). (16) A. T. Shulgin, ANAL.CHEM.,36, 920 (1964). (17) 0. Mlejnek, Chem. Zuesri, 22, 591 (1968). (18) W. Averill, Gas Chromatography Applications, No. GCDS-001, The Perkin-Elmer Corporation, Norwalk, Conn., 1963. (19) E. D. Barber, E. Sawicki, and S. P. McPherson, ANAL.CHEM., 36, 2442 (1964). (20) L. S. Ettre, “Open Tubular Columns in Gas Chromatography,” Plenum Press, New York, 1965, p 91. ANALYTICAL CHEMISTRY, VOL. 43, NO. 8, JULY 1971
1039
Table I. Boiling Points (5) and Relative Retention Times of Phenol and Methyl Phenols Peak No. (See figures) 1 2 3
4 5
6 7 8 9 10
TB
Compound Phenol 2-Methylphenol 2,fSDimethylphenol 4-Methylphenol 3-Methylphenol 2,CDimethylphenol 2,5-Dimethylphenol 2,3-Dimethylphenol 3,5-Dimethylphenol 3,CDimethylphenol
I
Abbreviation P 2-MP 2,fSDMP 4-MP 3-MP 2,4-DMP 2,5-DMP 2,3-DMP 3,5-DMP 3,4-DMP
9
( "C)
181.9 191.O 200.7 202.3 202.6 211.2 211.5 217.7 221.7 226.9
TMCP, 110 "C 0.53
0.70 0.73 0.93 1.OO 1.27 1.34 1.60 1.92 2.13
TCP, 120 "C 0.60 0.70
TXP, 120 "C 0.58 0.69
0.55
0.55
0.93 1.00 1.10 1.15 1.44 1.67 1.91
0.93 1 .oo 1.10 1.16 1.42 1 .69 1.91
I ?
4
min.
DDP, 120 "C 0.54 0.75 0.82 0.95 1 .oo 1.33 1.36 1.65 1.83 2.07
10
Figure 1. Gas chromatogram of phenol and methyl Dhenols on DDP liauid . Dhase - at 110°C (See peak names, Table I)
Figure 2. Gas chromatogram of phenol and methyl phenols on TMCP liquid phase at 110°C (See peak names, Table I)
Figure 3. Gas chromatogram of phenol and methyl phenols on TCP liquid phase at 120°C (See peak names Table I)
min. 15
5
0
Figure 4. Gas chromatogram of phenol and methyl phenols on TXP liquid phase at 120°C (See peak names, Table I) steel open tubular column at temperatures between 100 and 130 "C. The undesirable effect of the metallic column wall was eliminated by using phosphoric acid additive in the liquid phase (21). EXPERIMENTAL
Apparatus. A Fractovap Model GI (Carlo Erba, Milano) gas chromatograph equipped with a flame ionization detector was used for these studies. Nitrogen was used as the carrier gas. The column, fabricated from stainless steel tubing, was 20 m long, and had an i.d. of 0.01 inch. Chemicals. The commercial grade liquid phases were tricresylphosphate (May and Baker) and di-n-decylphthalate (21) J. Hrivsrik, J . Chromatogr. Sci.,8, 602 (1970). 1040
ANALYTICAL CHEMISTRY, VOL. 43, NO. 8, JULY 1971
(C. Erba). Di-(3,3,5-trimethyl cyclohexy1)o-phthalate was prepared as described by Sassenberg and Wrabetz (3), and tri-(2,4-~ylenyl)phosphatefrom 2,4-xylenol (Aldrich Chemical Company) with the reaction of POC18. All phenols used for quantitative measurements were 99.6-99.9xproducts (by gas chromatography). Column Preparation. The plug method was used as the coating procedure. The solution (0.5 ml) of the liquid phase (95 mg) and orthophosphoric acid ( 5 mg, 85 w/w) in acetone (1.0 ml) was forced through the tubing with the aid of nitrogen at the velocity 0.8-1.2 cmisec. Prior to use, the columns were conditioned at 120 "C for 3 hours under carrier gas flow. The column, when once used was cleaned by purging with acetone ( 5 ml), distilled water ( 5 ml), and
130
f20
110
-L
fa2
1
130
f20
f10
CV
"c
100
7U/T
1-__.__ 1 Figure 7. Variation of the logarithm of relative retention 25 26 40fT 27 times (log u ) of phenol and methyl phenols on TCP column with change of temperatures (No.identification in Figure 5. Variation of the logarithm of relative retention times (log u) of phenol and methyl phenols on DDP column with change of temperatures (No.identification in Table I)
130
120
Table I)
1iU
Figure 6. Variation of the logarithm of relative retention times (log u ) of phenol and methyl phenols on TMCP columii with change of temperatures (No.identification in Tabla I). acetone again ( 5 ml); after the evaporation of acetone, the column was reused and, using the described procedure, coated with another liquid phase (in order DDP TMCP, TCP, TXP). RESULTS AND DISCUSSION Relative retention times of phenol and methyl phenols on four liquid phases are listed in Table I. All measurements were done using corrections on the dead volume of the chro-
Figure 8. Variation of the logarithm of relative retention times (log u ) of phenol and methyl phenols on TXP column with change of temperatures (No.identification in Table I) matographic system. Data in Table I show only little differences in the separations of the closely related pairs. High efficiency columns are therefore necessary to obtain good resolution. Relatively best resolution is obtained on the column with TXP liquid phase. The elution of phenols on phthalates is less affected by hydrogen bonding than on phosphate liquid phases. Chromatograms of mixtures of phenol, methyl, and dimethyl phenols are presented o n Figures 1 to 4. As can be ANALYTICAL CHEMISTRY, VOL. 43, NO. 8, JULY 1971
1041
Table 11. Analysis of Synthetic Phenol Mixtures Sample Relative Amount (w/w) No. Component Present Found Dev. 1 2,6-DMP 14.7 14.5 -1.4 P 13.3 13.4 +0.8 2-MP 26.2 26.5 +1.1 4-MP 21.1 20.8 -1.4 3-MP 24.7 24.8 +0.4 2 P 11.3 11.2 -0.9 3-MP 25.7 25.6 -0.4 2,5-DMP 20.3 20.8 $2.5 3,5-DMP 28.1 28.1 0 3,4-DMP 14.6 14.5 -0.7 3 2,6-DMP 22.7 22.0 -3.1 4-MP 10.9 11.3 $3.7 3-MP 20.4 20.5 $0.5 2,4-DMP 21.8 22.2 +1.8 24.0 -0.8 2,3-MP 24.2 4 3-MP 20.1 20.2 +0.5 30.2 -2.6 2,5-DMP 31 .O 28.8 +2.4 2,3-DMP 29.1 +1.0 9.8 9.9 3,5-DMP 9.9 - 1.o 10.0 3,4-DMP Avdev &1.2% seen on the chromatograms, the peaks are symmetrical in all cases. Addition of phosphoric acid eliminated the metallic column wall effect and peak tailing. Many authors use different temperatures for the separation of phenol, methyl, and dimethyl phenols. We decided to investigate the effect of operating temperature on relative retention times at temperatures 100, 110, 120, and 130 “C. This effect is illustrated in Figures 5 to 8. It is evident from Figures 5 and 6 that especially for the TMCP liquid phase, the separation of 2-MP and 2,6-DMP is improved by increasing the column temperature. The resolution of closely related pairs (4-MP and 3-MP; and 2,4-DMP and 2,5-DMP) on a TMCP column is also better
than on a DDP column. Unfortunately, the TMCP liquid phase appeared to be stable only up to 110-115 “C. Figures 7 and 8 indicate that the separation on TCP and TXP columns is only slightly affected by temperature changes; moreover, the separations on TXP are better than on TCP column. With the exception of the TMCP liquid phase column, all columns had a good thermal stability up to 13G135 “C. The use of only one stainless steel open tubular column demonstrates the possibility to reuse such a column even after treatment with phosphoric acid. The columns used in this work varied in H.E.T.P. from 0.4 to 0.6 rnm. Quantitative aspects of this method were studied by analyzing synthetic mixtures of phenols. Table I1 summarizes the results obtained by analyses of four synthetic mixtures of pure compounds. The results were obtained by the peak height calculations (22) under carefully maintained analytical conditions. Peak height factors were calculated analyzing three model samples of phenols with 3-MP as the internal standard. The column with TXP was used at 130 “C and two injections were made from each sample. The found amounts of phenols presented in Table I1 are in good agreement with the actual composition of the samples and the deviations are in the range to be expected when peak heights are used for concentration measurements. ACKNOWLEDGMENT
The authors thank Carlo Erba S.P.A., Scientific Instrument Division, for the loan of the gas chromatograph used in this work. RECEIVED for review June 1, 1970. Accepted January 18, 1971 (22) L. S. Ettre, “The Interpretation of Analytical Results; Qualitative and Quantitative Analysis,” in: “The Practice of Gas Chromatography,” L. S. Ettre and A. Zlatkis, Ed., Interscience Publishers, New York, N. Y., 1967, p 403.
Fluorometric Determination of Some Primary Aromatk Amines with 2,6-Diaminopyridine Lawrence J. Dombrowski and Edward L. Pratt Sterling- Winthrop Research Institute, Rensselaer, N . Y. 12144 A sensitive fluorometric method for the determination of primary aromatic amines has been developed. The procedure requires diazotization of the amino group followed by coupling with 2,6-diaminopyridine (DAP) and reaction of the resulting azo dye with ammoniacal cupric sulfate to produce an intensely fluorescent derivative. The spectral characteristics of the fluorogen (excit. max. 360 mp; fl. max. 420 mp) were found to be essentially common among the various amines investigated. The procedure is highly sensitive in that amines in the 2-6 nanogram per milliliter can be determined with a relative standard deviation of 6%. COLORIMETRIC PROCEDURES have been used extensively in primary aromatic amine analysis (Z-6). Bratton and Mar(1) V. Levin, B. Nippoldt, and R. Rebertus, ANAL.CHEM., 39, 581 (1967). (2) C. Bratton and E. Marshall, Jr., J . Biol. Chem., 128, 537 (1939). (3) E. Sawicki, J. Noe, and F. Fox, Tuluntu, 8, 257 (1961). (4) M. Pesez and J. Bartos, Ed/.Soc. Chim. Fr., 1966, 3802. (5) W. Elbert and J. Noe, ANAL.CHEM., 33,722 (1961). (6) J. Stewart, T. Shaw, and A. Ray, ibid., 41, 360 (1969). 1042
ANALYTICAL CHEMISTRY, VOL. 43, NO. 8,JULY 1971
shall (2) have described a method for measurement of sulfanilamide which depends on the color intensity of the dye resulting on interaction of the diazotized amine and the reagent 1-naphthylethylenediamine. This procedure has been used by Nakamura (7) to study the enzymatic hydrolysis of monofluoroacetanilides in mammals. Pesez and Bartos ( 4 ) developed a colorimetric method based on the reaction between various aniline derivatives and the reagent p-dimethylaminocinnamaldehyde. Stewart (6) was able to detect mole per milliliter of primary aromatic amines by their color formation with 9-chloroacridine. Chromatography also has been used successfully in aryl amine analysis. A thin layer chromatographic (TLC) procedure presented by Ratney (8)provided a means of detecting 0.1 pg of p-aminophenol. The gas chromatographic method (7) T. Nakamura, T. Ueda, and K. Tanaka, Nippoti Nogei Kagaku Kaishi, 40, 13 (1966). ( 8 ) R. Ratney, J . Chromarogr., 26, 299 (1967).
