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Anal, Chem. 1983, 55, 2196-2199
A-.
+ DI+., (AD),, (AD)s,...
Tuning the photon source a t the wavelength of a specific complex could achieve the selectivity of the promotion.
ACKNOWLEDGMENT The author thanks F. W. Roellgen, U. Giessman, and S. S. Wong from Bonn University for helpful discussions. Registry No. N,N,N',N'-Tetramethyl-l,4-benzenediamine, 100-22-1. LITERATURE CITED
165 165 165 165 m/e Flgure 1. Molecular region of the FAB-MS spectrum of TMPD: (a) solution in glycerol, (b) solution in glycerol-NH,Br, (c) solution In gly-
cerol-quinone, (d) solution in Me,SO-quinone. Relative recorder settings are as follows: (a) 1; (b) 0.2; (c) 0.6; (d) 0.5. complexes not dissociated in solution can be brought to an excited dissociated state by irradiation in the CT absorption band. This band is located in the visible-near-UV range and thus is easily accessible. Moreover its position is directly related to the ionization energy of D. Simultaneous photoexcitation of the glycerol drop during FAB can lead t o a selective promotion reaction according to the reaction
(1) Benninghoven, A.; Slchtermann, W. Anal. Chem. 1978, 50, 1 180- 1 184. (2) Kambara, H. Org. Mass. Spectrom. 1982, 17, 29-33. (3) Martin, S. A.; Costello, C. E.; Biemann, K. Anal. Chem. 1982, 5 4 , 2362-2368. (4) Busch, K. L.; Cooks, R. G. Science 1982, 278, 247-254. (5) Day, R. J.; Unger, S. E.; Cooks, R. G. Anal. Chem. 1980, 52, 353-354. (6) Foster, R. "Organic Charge Transfer Complexes"; Academic Press: London and New York. 1969; Oraanic Chemistry, a Series of Monographs, Voi. 15. (7) Field, F. H. J . fhys. Chem. 1982, 86, 5115-5123.
E. De Pauw Institut de Chimie Universit6 de LiBge, B6 B 4000 Liege I, Belgium RECEIVED for review April 14, 1983. Accepted July 11, 1983.
AIDS FOR ANALYTICAL CHEMISTS Time-Optimized Thin-Layer Chromatography in a Chamber with Fixed Plate Lengths Ronald E. Tecklenburg, Jr., Rose M. Becker,' Eric K. Johnson, and David Nurok* Department of Chemistry, Indiana University-Purdue Indianapolis, Indiana 46223
University at Indianapolis, P.O. Box 647,
Thin-layer chromatography (TLC) has many advantages over high-performance liquid chromatography (HPLC) for the separation of simple mixtures. These include the fact that multiple samples can be run simultaneously, that solvent properties do not interfere with solute detection, and that the volume of solvent used per sample is generally 1t o 2 orders of magnitude less than in HPLC provided that a suitable development chamber is used. A limitation of TLC is that solvent path length is limited t o about 20 cm with conventional TLC plates and to about 10 cm with high-performance plates. These path lengths are often not long enough for difficult separations. Such separations can often by accomplished by continuous development whereby solvent is allowed to evaporate off the end of the TLC plate. It has been recommended that binary solvents comprising a weak solvent such as hexane and a stronger solvent such as acetone be used for these separations (1). Apparatus for such developments-the Regis Short Bed/Continuous Development (SB/CD) chamber-has been available for several years and has been used for a variety of separations. It has recently been shown that continuous developments with binary solvents can be time-optimized (2). The suitability of 'Present address: Dow Chemical Co., Midland, MI 48640.
using the Regis SB/CD chamber for time-optimized separations is evaluated below.
EXPERIMENTAL SECTION The Regis SB/CD chamber wm used for chromatography. The chamber is rectangular with four ridges along the floor,all parallel to its long axis. This allows a TLC plate to be inserted silica gel coated side up in five positions with the base of the plate resting against a ridge (or in position 5 on the wall) and the upper end of the plate propped against the opposite wall. The line of contact between the TLC plate and the glass cover of the chamber defines the line where solvent starts to evaporate. It is essential to allow about 1.5 cm of plate to extend beyond this contact line as this is the area from which solvent evaporates. The rate of evaporation can be roughly optimized by placing the chamber in a hood and raising the front to an appropriate level. In our laboratory an opening height of 18 in. was found satisfactory. With 26 mL of solvent in the chamber, the path lengths for positions 1through 5, from solvent source to the initial point of solvent evaporation, are 2.35 cm, 3.85 cm, 5.40 cm, 7.00 cm, and 8.30 cm. Analtech, Inc. (Newark, DE) silica gel plates, catalogue no. 47011, were cut into appropriate sections 10 cm wide before use. Plates were stored in the laboratory atmosphere overnight before use. The humidity reported ahead refers to the ambient laboratory humidity.
0003-2700/83/0355-2196$01.50/00 1983 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 55, NO. 13, NOVEMBER
For the experiment in whnch plates were stored at a controlled humidity the plates were spotted and then placed in a desiccator over 39% sulfuric acid for 15 h. This concentration of sulfuric acid maintains the relative humidity at 60%. Temperature in the laboratory was in the range of 23-25 "C. The steroids used in this study were obtained from Sigma Chemical Co. (St. L,ouis,MO). The solvents used were obtained from Aldrich Chemical Co. (Milwaukee, WI).
1983 2197
0
RESULTS AND DISCUSEIION The method of optimization has been fully described elsewhere (2). Familiarity with the original publication is assumed in the following discussion. The method is dependent on using a binary solvent such that the following relationship exists between solute capacity factor, 12, and the mole fraction, X, of the stronger component of the binary: In k 3- a In X, b (1)
+
where a and b are constants characteristic of each solute in the binary. It is necessary to determine the constants a and b for each solute by performing TLC at several mole fractions of the binary. It is also necessary to experiimentally construct a plot of tho solvent velocity constant, K vs. X,.Once this has been done, it is possible to calculate a t any mole fraction, X,, the time by continuous development, ti,necessary to yield a specified center-to-center spot separation, SD.This is
ti = (21L - L 2 ) / K
(2)
where ti is the development time in seconds, 1 is the length of plate in millimeters, usled for continuous development, L is the length of plate in millimeters, required for the same separation by conventional development, rind K is the solvent velocity constant The length L is a function of the diffeirence in R, values, AR,, of the pair of compounds to be separated, and of SD,the center-to-center spot separation required, swhereas the length1 1 depends on the product RfL, where R, refers to the spot of highest R, value. In the case of a mixture containing more than two components (not considered here) L would be determined for that pair of compounds most difficult to separate. The value of tl varies with solvent composition. The minimum of a plot to tivs. X , yield,a the solvent composition a t which the most rapid separation occurs. This value is referred to as ( t h , . Time optimization by continuous development requires a development chamber where any predicted plate length, I , can be used. In contrast, thie SB/CD chamber has five fixed positions so that for many separations the correct plate length can only be approximated. The analysis time in the SB/CD chamber can be optimized in the following way. At a given mole fraction, the analysis time, tl*, using a plate position of fixed length, 1*, is ti* =
(21*L - ( L * ) 2 ) / K
(3) The values of L and K are determined in exactly the same way as when using eq 2. The value of 1* corresponds to the length of one of the fiied plate positions in the SB/CD chamber. The minimum of the plot of ti* vs. X,will determine the solvent composition that will yield the shortest ,analysis time for a given plate position in tlhe SB/CD chamber. I t should be noted that this minimum analysis time will usually be at, or close to, the intersection of this plot witlh the tl vs. X,plot (obtained by using eq 2) provided that the same values of slope and intercept are used for both plots. At the mole fraction corresponding to the intersection of the two curves the values! of 1 and 1* are identical. At lower mole fractions the value of 1* > 1 and the compound of highest Rf will not reach the top of the plate although the required value of SDwill still be obtained. At higher mole fractions the value of 1 > I* and the required value of SDcannot be
SI Flgure 1. Plots of computed analysis time vs. mole fraction for a spot separation of 5 mm for hydrocortisone/corticosterone on silica gel with ethyl acetate/ 1,l ,2-trichlorotrifluoroethane. Slopes and intercepts are for a humidity of 60%. Each plot is computed by use of the slope and intercept corresponding to one of the four fixed positions in the SB/CD chamber.
obtained as the fixed path length is too short for adequate separation. Thus the permissable solvent concentration range in the SB/CD chamber is such that 1* 2 1. We have found that one of the three shortest positions generally yields the lowest analysis time for a single pair of compounds. This includes difficult separations where a plate length of up to 39 cm (see Table 11) would be required for the same separation by conventional TLC. The two longer positions may however be of value for the separation of more complex mixtures. The slopes and intercepts (constants a and b in eq 1) vary with plate position. Thus even if the plate length were adjustable, different ti vs. X,curves would be obtained in each position. This is illustrated in Figure 1,which shows the variation of tiwith the solvent composition for the separation of corticosterone/hydrocortisone on silica gel by using ethyl acetate/l,l,2-trichlorotrifluoroethane as solvent and by using the slopes and intercepts found for positions 1 through 4. It should be emphasized that the plate length required for t Las defined in eq 2 varies with solvent composition, even though this variation can be slight. Thus the minimum of the lowest curve (i.e., (tJmh, the shortest analysis time) in Figure 1will in general not be attainable unless there is fortuitous agreement between the variable plate length 1 corresponding to the minimum of the curve and the fixed plate length l*. Furthermore, the curve that has the lowest minimum is not always an accurate indicator of which position of the SB/CD chamber will yield the shortest analysis time for a given separation. This is because the values of E and E* can be very different for a given slope and intercept. This is illustrated in Table I. The conditions for time optimized TLC appear to be dependent on humidity. Figure l was constructed a t a relative humidity of 60%. Similar figures were constructed at relative humidities of 21% and 23%. The sets of curves obtained at either of the latter two humidities are virtually identical with each other but are significantly different to that obtained at 60% relative humidity. The slope and intercept of position 3 yielded the lowest analysis time (19.7 min) of the four positions at 60% relative humidity whereas a t either 21% or 23% relative humidity, the slope and intercept of position 1 yielded the lowest analysis time (24.8 min) of the positions considered. Thus optimized continuous development in the SB/CD chamber using silica gel plates should be performed at a controlled humidity. Preliminary experiments indicate that small fluctuations in humidity can be compensated for by storing the TLC plates under controlled humidity until
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ANALYTICAL CHEMISTRY, VOL. 55, NO. 13, NOVEMBER 1983
___
Table I. Analysis Time and Plate Length in SB/CD Chamber Compared to Fully Optimized TLC for a 5 mm Separation of Hydrocortisone/Corticosterone Developed in Ethyl Acetate/l, 1,2-Trichlorotrifluoroethane SB/CD
position 1 2 3 4
I*,
mm 23.5 38.5 54.0 70.0
1 at ( tdrnim
(thpinr
min
( tl* ) F i n , min
38.6a 34.8 31.3 33.3
31.5a 26.5 19.7 24.1
108.8 27.0 30.8 46.9
mm
0 0
ri
a Values of ( tl)min and 1 at ( tl)rnin are calculated from the corresponding value of slopes and intercepts obtained for each of the four fixed positions in the SB/CD chamber.
immediately before use. Plates stored at a relative humidity of 60% gave substantially the same results at an ambient relative humidity of 54% as a t an ambient relative humidity of 60%. It is recommended that if this procedure is followed that the composition of the mobile phase be restricted, such that I* 1 1 + 5 mm. This allows for the possibility of small changes in R, due to small fluctuations in temperature or humidity. Small changes in Rf do not have a significant effect on S D . The analysis times quoted in the above paragraph are of course hypothetical as they would only be obtained if the plate length could be optimized. As noted earlier this length can be very different from the length of the corresponding fixed position for which slope and intercept were measured. It is clear from the above discussion that the SB/CD chamber has two drawbacks, namely, that the slope and intercept values (a and b in eq 1) differ according to which position is used and that the user has five fixed plate lengths to choose between. It is common to find that the optimum length is only approximated by one of these fixed lengths. The latter can be overcome by adjusting the solvent level. However, preliminary experiments indicate that this leads to further changes in slope and intercept values. In spite of the above shortcomings the SB/CD chamber is of use in continuous development provided conditions are optimized. This is illustrated by the series of analyses reported in Table 11. The agreement between predicted and experimental analysis time is within 10% for all the separations. Moreover, for three of the five analyses reported, development time in the SB/CD chamber is significantly less than that required for conventional development. The advantage of working under fully optimized conditions where both plate length and mole fraction are completely variable, as compared to partially optimized conditions as in the SB/CD chamber, is illustrated by the two right-hand columns of Table 11. These express (tJmin and (tl*Imin as percentages of (tL)min, the minimum analysis time by conventional development. The latter corresponds to the minimum of the tL vs. curve where t L = L'/K. For the separations listed, the time required under fully optimized conditions is between 24% and 35% of that required for conventional development. A lesser reduction in analysis time is found for the SB/CD chamber where the time required is between 27% and 87% of that required for conventional development. As noted earlier in this report the reduction in time in the SB/CD chamber is significant in three of the five cases considered. The largest reduction in analysis time is for progesterone/pregnenolone where analysis time is reduced from 147 min for conventional development to 40 min by optimized development in the SB/CD chamber. Two of the pairs separated are reported at different values of S,. In both instances, the higher SDrequires a substantially
x,
La2 E QgE w
rl
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Anal. Chem. 1983, 55,2199-2201
longer time in the SB/CD chamber. Shorter times could possibly be obtained in positions other than position two. This would of course entail measurement of slope and intercept values in each of the new positions. I t should be rioted that for the separation of progesterone/pregnenolone there is a 10% difference between the f d y optimized time and the time in the SB/CD chamber even though there is an insignificant difference between 1 and I*. This is because there is only a very slight dependence of plate length on mole fraction in this region for this particular separation. The pairis listed in Table I are difficult t o separate by use of the solvents listed. It should be noted that for four of the five separations, conventional development requires a TLC plate in the range between 26 cm and 40 cni in length wherem position 2 of the !SB/CD chamber (as shown in Table I) requires an overall plate length of about 5 cm. Thus apart from considerations of shorter analysis time, the SB/CD chamber
allows difficult separations to be performed that would not normally be obtainable by conventional TLC. I t should be noted that for several of the solute pairs in Tables I and I I , l > l*. This inequality is permissible because (tl*)minand (tJmi,, occur a t different mole fractions. Registry No. Hydrocortisone, 50-23-7;corticosterone, 50-22-6; progesterone, 57-83-0;pregnenolone, 145-13-1;estrone, 53-16-7.
LITERATURE CITED (1) Perry, J. A. J . Cbromatogr. 1979, 165, 117-140. (2) Nurok, D.;Becker, R. M.; Sassic, K. A. Anal. Chem. 1982, 54,
1955-1959.
RECEIVED for review March 8, 1983. Accepted July 18, 1983. This work was presented in part a t the 3rd Biennial Symposium on Advances in Thin Layer Chromatography in Parsippany, NJ, Dec 1982. The work was supported by grants from the Research Corporation and the Society for Analytical Chemists of Pittsburgh.
Trace Determination of Low IVlolecular Weight Aliphatic Amines in Air by Gas Chromatography Kazuhiro Kuwnta,* Erniko Akiyama, Yoshiaki Yamazaki, Hiroyasu Yamasaki, and Yoshio Kuge Environmental Pollution Control Center, 62-3, 1 Chome, Nakamichi, Higashinari-ku, Osaka City 537, J a p a n
Yoshiyuki Kisa Applied Physics and Chemistry, Facu1t:y of Engineering, Hiroshima University, Shitami, Saijo, Higashihiroshimn City 724, J a p a n Low molecular weight alliphatic amines have received much attention as odorous substances in studies of air pollution. Gas chromatography (GC) is widely used to determine such traces of the amines (1-5). However, consideralble difficulty is encountered in storing and analyzing amine samples in extremely low concentration levels because of their adsorption on solid surfaces and because of the presence of interfering organic substances. Although a number of the GC columns so far reported are discussed with respect to the resolution of C1-C4 aliphatic amines and the accuracy of the determined values (5), many of the columnin are not suitable for separation of the Cz-C3 amine isomers. The recently reported columns ( 4 , 5) offer good separation for the Cz-C3 isomers. The preparation of the columns, however, is substaiitially tedious, and interferences of water injected are often observed in the analysis. Recently, the Sep-PAK C18 (SP) cartridge has been often used to enrich and cleanup trace componeinta from food (6-91, environmental samples (10-13), biological samples (14-181, and other samples with substantial savings of the number of steps and total time. To date, the cartridge has, however, not been applied t o analysis of air samples. In this study, aL new GC analytical column, alkalized SEPABEAD GHP-1 (GHP-I.), was prepared to determine the Cl-C4 aliphatic amines without interferences of water, and handy SP cartridges impregnated with phosphoric acid were used to perform trace determination of ithese amines in air samples.
EXPERIMENTAL SECTION Reagents and Materials. Hydrochloridesof the C,-C, amines, isobutylamine, n-butylamine, and diethylairnine were of special grade from Tokyo Kasei Kogyo (Tokyo, Japan), and sec-butyl-
amine and tert-butylamine were of special grade from Wako (Osaka, Japan). The other reagents used were of commercially available special grade. The water used was prepared by redistilling deionized water. SEPABEAD GHP-1 (GHP-1) (60-80 mesh) was spherically shaped organic porous polymer (styrenedivinylbenzene copolymer) from Mitsubishi Chemical (Tokyo, Japan). Sep-PAK CIS (SP) cartridge was from Waters Associates (Mildord, MA). The stock solutions containing 1000 pg/mL of an amine were made by dissolving an amine hydrochloride or a free amine in redistilled water. Standards of lower concentrations were made by neutralizing a part of the stock solution with 1-2 N potassium hydroxide solution, if required, and by appropriately diluting the solution with redistilled water. Preparation of the Analytical Column. A 10-g amount of GHP-1 was washed with 100 mL of methanol for 3 h in a Soxhlet extractor. The GI*-1 was mixed with 30 mL of methanol solution containing 1.0 g of potassium hydroxide, and the mixture was dried under reduced pressure at less than 50 "C in a rotary evaporator. The analytical column was made by packing the material into a 2 m X 2 mm i.d. glass tube and by conditioning at 200 "C for 2 days. During the conditioning,10 pL of water was injected 15-20 times to ensure preparation of stable column. Prior to use of the column, 10 p L of 1 N potassium hydroxide solution was injected twice at 170 "C. Apparatus. A Hewlett-Packard (Avondale, PA) 5830A gas chromatograph with a nitrogen-phosphorus flame ionization detector (NP-FII)) was employed. The working conditions were as follows. The injection port temperature was 180 "C, and the column temperature was isothermal at 140 "C for 3 min and then programmed from 140 to 170 "C at 10 "C/min and isothermal at 170 "C for 10 min. The NP-FID temperature was 250 "C. The carrier gas was nitrogen at 45 mL/min. On-column injection was used. Preparation of the Sampling Tube. A SP cartridge was washed with 4-5 mL of methanol. Then, 3-4 mL of 0.3% phosphoric acid in methanol was forced through the cartridge,
0003-2700/83/0355-2199$01.50/0 0 1983 American Chemical Society
