Roughly the areas were proportional to concentrations, but at lower concentrations they were smaller than they should have been, taking the 10.0 mg/ml solution as reference. The discrepancy exceeds experimental error and may indicate that the samples were decomposing, and that decomposition was more serious at lower concentrations. It may be significant that the preliminary peak area was 2 4 x of the total amphetamine-metamphetamine peak areas at 2 mg/ml but only 1 3 x a t 10mg/ml. We do not exclude the possibility that air dissolved in the feed solution oxidizes the bases, with copper(I1) acting as catalyst. However, boiling the water beforehand and keeping the feed solution under nitrogen did not seem to reduce the preliminary wave. CONCLUSIONS
Ligand exchange chromatography on metal-loaded carboxylic resin columns gives satisfactory resolution of mixtures of amphetamine bases. A resin loaded with copper(I1) ions gives the best resolution and the sharpest bands, but the Cu (11) seems to promote some decomposition of the substrates that we have been unable to characterize or control. Nevertheless it is possible to detect and determine these bases at concentrations down to 0.1 mg/ml, using 0.5-ml samples. Probably this performance could be improved. From this work and previous work in our laboratory (5, 6), we may make certain generalizations about elution orders in metal ion-loaded columns. Strong bases are more strongly held than weak ones, and primary amines are more strongly held than secondary, tertiary, or heterocyclic amines. The
binding to the metal ion-loaded resin is weakened by: (a) methyl groups or other alkyl groups on the amine nitrogen; (b) methyl groups one carbon atom removed from the amine nitrogen; (c) hydroxyl groups more than two carbon atoms removed; (d) methoxyl groups (mescaline, for example, is less strongly bound than phenethylamine). It appears that binding is weakened by steric hindrance around the amine nitrogen and by hydrophilic substituents. The matrix of the ion-exchange resin has a large effect, as we have noted, and it can change elution orders (5). Relatively few of the drugs and biogenic amines that we have studied are strongly held by metal-loaded carboxylic resins, and the selectivity thus shown can be exploited analytically. Obviously, resolution can be improved and the method can be adapted to modern high-speed liquid chromatographic techniques (14). ACKNOWLEDGMENT
We gratefully acknowledge the help of Vernon Shaw, who was sponsored by the National Science Foundation’s Summer Research Participation Program for College Teachers, and of Jared Baker. Harold Heim, Dean of the University of Colorado School of Pharmacy, gave valuable advice. RECEIVED for review November 8,1971. Accepted January 7, 1972. Work supported by the National Science Foundation, Grant No. GP-25727. (14) J. J. Kirkland, Ed., “Modern Practice of Liquid Chromatography,” Wiley-Interscience, New York, N.Y., 1971,
Gas Chromatographic Analysis of Complex Deuterated and Tritiated Mixtures with Packed Columns Fabrizio Bruner, Paolo Ciccioli, and Antonio Di Corcia Laboratorio Inquinamento Atmosferico del C.N.R. and Istituto di Chimica Analitica dell’ Unioersith, 00185 Roma, Italy High efficiency packed columns of about 100 m in length and 130,000 theoretical plates have been employed to separate isotopic mixtures with tritium, as they are originated by the reaction between propene and HT in the presence of catalysts. Separations have been carried out at easily controllable temperature, such as 0 “C and -78 “C. Quantitative analysis can be made also for deuterated mixtures like C2H6C2H6Dand CaH7D-C3H8.
MANY ATTEMPTS have been made to exploit gas chromatography for the analysis of isotopic compounds, either with packed (1-9), or capillary columns (10-12). Careful thermo-
dynamic calculations on the isotope effect in gas chromatography were also possible because of the high resolving power of capillary columns (11, 12). However, in only one case was the problem of analysis of deuterated and tritiated mixtures completely solved with the almost complete separation of all deuterated and tritiated methanes (13). This technique, involving the use of etched glass capillary columns at liquid nitrogen temperature, is further complicated by the necessity of employing a deactivating gas, to be mixed with the carrier. More recently, some of us (8) succeeded in analyzing deuterated methanes by using high efficiency packed columns ;
(1) K. Wilzbach and P. Riesz, Science, 126,748 (1957). (2) P. L. Gant and K. Yang, J . Amer. Cl7em. SOC.,86,5063 (1964). (3) J. W. Root, K. C. Lee, and F. S . Rowland, Science, 143, 376 (1964). (4) W. A . Van Hook and M. E. Kelly, ANAL.CHEM.,37, 509 (1 965). ( 5 ) J. J. Czubryt and H. D. Gesser, J . Gas Chromatogr., 6 , 41 (1968). (6) F. Bruner and A. Di Corcia, J . Chromatogr., 4,5, 304 (1969). (7) A . Di Corcia and F. Bruner, ibid., 49, 139 (1970). (8) A. Di Corcia, D. Fritz, and F. Bruner, ibid., 53,135 (1970).
(9) R . J. Cvetanovic, F. Duncan, and W. E. Falconer, Cm. J . Chem., 41, 2095 (1963). (10) W. E. Falconer and R. J. Cvetanovic, ANAL. CHEM.,34, 1064 (1962). ( 1 1) F. Bruner, G . P. Cartoni, and A . LibeIti, ihid., 38, 298 (1966). (12) F. Bruner, G. P. Cartoni, and A. Liberti, in “Gas Chromatography 1964,” A. Goldup, Ed.. Institute of Petroleum, London, 1964, p 301. (13) F. Bruner, G. P. Cartoni, and M. Possanzini, ANAL.CHEM., 41?1122(1969).
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ANALYTICAL CHEMISTRY, VOL. 44, NO. 6, MAY 1972
these results encouraged us to try the analysis of isotopically substituted higher hydrocarbons with enough resolution to allow a good separation of isotopic molecules differing by one or two atomic mass units. Such a method of analysis would be of great help to those people involved in studies of reaction kinetics in the gas phase with isotopic substitution and similar topics. To date, the only way to achieve satisfactory results in the analysis of complex mixtures of partially deuterated and tritiated hydrocarbons is by mass spectrometry, but only computer-aided calculations allow a rapid and quantitative interpretation of their mass spectra. Furthermore, tritiated samples are often extremely diluted and the sensitivity of the mass spectrometer can be insufficient to ensure reliable results. A rather simple gas chromatographic technique for the analysis of these mixtures is therefore desirable; this paper reports some conclusive results in this field together with a critical evaluation of the technique for the separation of more complicated isotopic species. Several parameters exert a critical influence on the resolution of isotopic molecules and their mutual dependence must be evaluated to obtain the best results. They are: separation factor, capacity ratio, and column efficiency, that are linked together by the well known relationship ( 1 4 ) :
R is the resolution, a the separation factor, k’ the capacity ratio, and n the number of theoretical plates. Temperature and analysis time, which are also very important, do not appear in Equation 1, but are included in the values of a and k’. On the other hand, the isotope effect in gas chromatography for deuterium and tritium compounds is well represented by the relationship : lna
=
VRL B A In - = - - VRH T T2
+C
where VRL and VRH are the net retention volumes of the light and heavy species, respectively, and T is the absolute temperature, the values of the constants A , B, and C depend upon the nature of the adsorbent or the liquid phase. The same isotopic pair can thus show a different behavior on different fixed phases, as has been found experimentally ( 4 , I I ) and confirmed with theoretical approaches (15,16). Equation 2 shows that two counteracting isotope effects take place, the “inverse” being predominant at high temperatures while the “normal” one is effective at very low temperature. In the case of the present work, the inverse effect is exploited. This is shown in Figure 1, where a comparison of the ratios of retention volumes for some isotopic pairs us. 1/T is reported. The best results are obtained with Porapak Q and graphitized carbon black ; the choice of the latter is due to three reasons. First, the HETP of Porapak columns is not satisfactory, especially when a high length is required. Second, the maximum isotope effect for ethanes occurs at -20 O C , while (14) I. Halasz and E. Heine, in “Progress in Gas Chrornatography,” J. H. Purnell, Ed., Interscience, New York, N.Y., 1963, p 162. (15) W. A. Van Hook and J. T. Phillips, J. Phys. Chem., 70, 1515 (1966). (16) A. Di Corcia and A. Liberti, Trans. Faraday SOC.,66, 967 (1970).
“
3
4
5
6
7
L,03 1
Figure 1. Separation factors of some isotopic pairs on different adsorbents
_ _ _ - _ Porapak Q packed
-.-.-._
Silica gel capillary Graphitized Carbon Black Sterling FT 0 c&/czD6 A C3Hs/C3HJh A C~HS/CJ& w C~H,S/C~H~T
for Sterling FT about the same value is obtained at -78 “C, a more convenient temperature to keep constant. This is particularly important if, as in our case, experiments last many hours. Third, in the maximum isotope effect region, analysis time is much larger with Porapak, as has been shown previously (8). Column efficiency must be such that isotopic pairs differing by one or two a.m.u. are conveniently resolved. For this aim, an order of magnitude of IO5 theoretical plates is required even if the maximum isotope effect is available. On the other hand, analysis time must be limited to a normal working day, say about 8 to 10 hours. Thus, optimum analytical conditions arise from a compromise among the values of all the parameters mentioned above, and the choice of the appropriate adsorbent among the graphitized carbon blacks is based on their surface area which plays an important role on the working temperature and analysis time. In Figure 2, the values of log k’/T for various packing materials are plotted against l / T . It is interesting to note that the slopes of the straight lines obtained are always the same for the same isotopic pair, showing that by changing the surface area passing from Graphon (100 mz/g) to Sterling FT (12 mz/g), only the entropic factor is affected. Experiments showed also that separation factors are scarcely affected passing from Graphon to FT. The small amounts of liquid phase added have the scope to eliminate peak tailing, and retention volumes are strongly reduced with respect to the pure adsorbent, as has often been reported in the literature (17). On the base of the above considerations, we decided that optimum analytical conditions could be achieved using Sterling F T deactivated with 0.2% squalane for all the isotopic systems considered, and with a working temperature (17) G. Alberini, F. Bruner, and G. Devitofrancesco, ANAL.CHEM., 41,1940 (1969) and citations therein. ANALYTICAL CHEMISTRY, VOL. 44, NO. 6, MAY 1972
895
'I
H.E.T.I?
2.0
4.0
6.0
8.0
10.0
U (cm/sec) Figure 3. HETP us. ferent columns Figure 2. Plots of log k'/T us. 1/T for ethane and propane on different adsorbents Dotted lines: propane Solid lines: ethane 0 Graphon 0.1% squalane m Sterling FT 0.2% squalane 0 Graphon 0.1 squalane 0.1 % glycerol A Graphon 2% squalane
+
+ +
+
ethane and propane on dif-
+
Solid lines: 105-m Sterling FT 0.2%squalane m ethane at 20 "C propane at 20 "C Dotted lines: 15-m Sterling FT 0.2% squalane ethane at -40 "C propane at 20 "C
+
+
of -78 "C for ethane, 0 "C for propane, and +45 "C for butane. EXPERIMENTAL Column Packing. The Carbon Black-Sterling FT, obtained from Cabot Corp., Billerica, Mass., is sieved to 40 to 60 mesh; the material is then treated with a very dilute solution of squalane in ether, with gentle stirring until the solvent is completely evaporated. The total amount of squalane in the solution is 0.2 % w/w with respect to the carbon black. Copper tubing (4-mm i d . , 6-mm 0.d.) is used as column material. The column with a total length of 105 meters is built by sealing together seven columns, each 15 meters long. Particular attention has been paid so that all the columns should have the same permeability and efficiency characteristics. Each 15-meter column is packed with 170 =t 1 gram of Sterling FT, after preliminary experiments showed that this amount would yield the best efficiency and permeability. All the columns are tested separately and give the same flow rate with the same pressure drop within 5 % . Propane is used to measure column efficiency and the results for different columns are the same, with a reproducibility of 10%. A high increase of column performance and permeability is noted if the columns, once packed and coiled, are emptied again and the packing material is resieved. A loss of about 5 of the packing was found in this case after sieving. This operation is repeated for all the 15-m columns. Two columns are then soldered together and the resulting column is tested, and so on to the desired length. Apparatus and Materials. The column is placed in a 30liter Dewar container. A thermostat-cryostat (Lauda, West 896
u plots for
ANALYTICAL CHEMISTRY, VOL. 44, NO. 6, MAY 1972
Germany) is used for temperatures other than 0 "C of -78 "C. Temperature constancy is better than i.l "C in all the temperature region investigated. About 10% of the gas flow at the end of the column is diverted to a FID, while the rest is passing through an ionization chamber, equipped with a proper electrometer Model 610B (Keithley Instruments, Cleveland, Ohio), when tritiated molecules have to be analyzed. A double channel recorder (Leeds & Northrup Model XL 682) is used in this case. Pure hydrogen is used as carrier gas. Column terminals are connected to the inlet systemdetectors assembly by means of stainless steel tubing 1-mm i d . , 2-mm 0.d. Nu appreciable dead volume is observed. Injections are made using a 0.5-ml gas-tight Hamilton syringe and no particular problems arise from the high inlet pressure which did not exceed 15 kg/cm2 in routine experiments. C2D6, C2H3D3,C3D8,C3H6D2,and deuterated butanes are from a commercial source (Merck, Sharp and Dohme, Montreal, Canada); tritiated samples were kindly furnished by F. Cacace (Laboratorio di Chimica Nucleare del C.N.R., Universith di Roma, Roma, Italy) by treating propene with HT in the presence of catalysts. We prepared C3H7Dand CZHSD from C3H7Brand CzHSBr,by means of the Grignard reaction and treatment with DzO (Merck, Darmstadt, Germany). The amount of specific radioactivity in the sample was 3.6 mCi/mmole; 0.5 cc was injected each time. The same sample, which is a random mixture of tritiated hydrocarbons, is used for ethane, propane, and butane. There was experimental evidence that higher hydrocarbons and methane were also present in the sample, but this has not been further investigated, The sample contains a very strong amount of propane, which is used as a retention time test to assign the number of tritium atoms.
C3H75
a
b
b
10
‘qH4’6
I
I
I
I
255
260
265
270
I
530
540
550
I
560 570 t i m e (min)
/
460
Figure 4. Separation of partially tritiated and deuterated propanes (a) Column: 60-m Graphon
+ 0.1 z Squalane
Temperature: 16 “C. Inlet pressure (hydrogen) 15 kg/cm2 Flow rate: 210 ml/min (b) Column: 105-m Sterling FT 0.2 squalane Temperature: 16 ’C. Inlet pressure (hydrogen) 10.5 kg/cmz Flow rate: 140 ml/min
+ z
RESULTS AND DISCUSSION
Column Performance. The efficiency of the 15-m columns is about 20,000 theoretical plates within the optimum flow rate range. The number of theoretical plates is increasing linearly with column length, but the slope of the straight line obtained is less than 1, showing a slight loss of efficiency. The maximum number of theoretical plates was 130,000 for the 105-m column, using propane as a test. Plots of HETP us. u for 15-m and 105-111 columns are shown in Figure 3. By increasing column length, the minimum HETP is shifted toward the low linear gas velocities, and this unavoidable phenomenon, due to the carrier gas compressibility, is in agreement with what has been observed elsewhere (7, 14). The main feature of the curves is that they show a deep minimum, while at rather high gas velocity, their slope is smoother. This is in agreement with what Myers and Giddings (18) found some years ago, studying the behavior of high length packed columns. However, this paper had only a theoretical scope and at our best knowledge, to date, no analytical applications were found for these results. The characteristic concave “downness” found by Giddings is repeated in our case, and besides the theoretical interest, it is evident that such behavior, which allows high column performance at the higher gas velocities, is very convenient for practical gas chromatographers. The points in Figure 3 result from several measurements and the error in H and U measurements does not exceed the point thickness. (18) M. N. Myers and J. C. Giddings, ANAL.CHEM., 37, 1453 (1965).
,
I
480
.
,
/
500
500
,
l
520
I
/
540
~
,
,
560 580 time (min)
Figure 5, a. Separation of partially tritiated and deuterated butanes Column: 105-m Sterling FT Temperature: 45 “C. Inlet Flow rate: 300 ml/min
+ 0.2 zsqualane
pressure (hydrogen): 20 kg/cmZ
5, b. Separation of partially deuterated and tritiated ethanes
+
Column: 105-m Sterling FT 0.2 squalane Temperature: -78 “C. Inlet pressure (hydrogen) 18 kg/cm2 Flow rate: 650 mlimin
So we could perform the analysis of ethanes at -78 “C in a reasonable time at U = 9 cmisec, still having a column efficiency of 70,000 theoretical plates. Column permeability is quite satisfactory because with a pressure drop of 10.5 kg/cm2, not prohibitive for commercially available gas chromatographs and for syringe injection, we could obtain a linear gas velocity of 3.9 cm/sec at 0 “C. This is probably due to the homogeneity of column packing and to the mesh range used, which was found as the best compromise between column efficiency and permeability. The use of hydrogen as carrier gas was very helpful for permeability, also yielding the minimum shift toward higher gas velocities with respect to any other carrier gas, with a much lower pressure drop (7). No appreciable loss of efficiency is observed for the use of the hydrogen carrier. Isotopic Separations. The first experiments for the separation of deuterated and tritiated propanes were performed using a 60-m column packed with Graphon coated with squalane 0.1% w/w and the results are shown in Figure 4, a. The analysis is not completely satisfactory, because a good dosage of the relative amounts of the components is not easy, even though a certain separation is observed. Moreover, almost no separation occurs between CSHa and C S H ~ D .Better results could be obtained only by lowering the temperature and making the column much longer. However, both of these procedures would lead to a prohibitive increase of analysis time. ANALYTfCAL CHEMISTRY, VOL. 44, NO. 6 , MAY 1972
897
I
T , "C 45
30 20
16 0 - 78
Table I. Separation Factors for Some Isotopic Pairs C ~ H ~ O / C ~ H C~H~/C~DP, $T CsH6/CtHjD C~HP,/CZH~T C ~ H ~ / C ~ DC3HdC3HaD2 B CaHdC3HiD C ~ H B / C ~ H ~ T 1.006 ... ... ... ... 1.004 ... ... ... ... 1io67 1 .'Oi3 ... 1.009 ... 1.049 ... ... 1.069 1.015 ... ... ... ... ... ... ... ... ... 1.011 ... 1.052 ... ... 1.079 1.022 1.008 1.016 ... 1.088 1.012 1.015 ... ... ... ...
For these reasons, we decided to use Sterling FT with a surface area about seven times lower. The results obtained working with the 105-m Sterling F T column at 0 "C are shown in Figure 4, b, and the analysis is now decisively good for tritiated samples. All the compounds present can be easily evaluated quantitatively with an error less than 10%. The separation is, of course, lower for deuterated samples, but also in this case the method allows checking of the sample purity. The separation factor decreases slightly passing from C3H1T4-C3H5T3 to pairs of lower mass so that a better separation can be obtained between C3HT7and C3H2Ts; the same is true for CzD6 and C2D5H. The separation of isotopic butanes, shown in Figure 5 , a, is performed on the same column at 45 "C. Practically no separation occurs between the monodeutero species and the fully hydrogenated compound, while a fairly good separation is observed for the tritiated molecules differing by one tritium atom. A further decrease of temperature would probably help because of the corresponding increase of the separation factor, but in this case the analysis time would be extremely high. The separations performed represent the extreme performance limit of the column presently used, and better results could be obtained only by increasing the column length by a factor of 2 or 3. But this would make serious trouble because of the high pressure drop. With tritiated butanes, we obtained a still appreciable analytical result with the separation of one tritium atom differing butanes. The separation of isotopic ethanes of Figure 5 , b was performed at -78 "C, and the choice of this temperature was suggested by the considerations pointed out before, Le., very large isotope effect and not excessive analysis time. The separation obtained between C2H5D and CzHs is analogous to that between CH, and CH3D previously reported (7), and allows the analysis of one component with respect to the other one also if the relative amount is about 10%. A better separation is obtained among the tritiated molecules,
898
ANALYTICAL CHEMISTRY, VOL. 44, NO. 6, MAY 1972
allowing analyses of mixtures with few per cents of one component with respect to the next. In Table I, the separation factors for the isotopic species investigated are reported, in order to show the difficulties of the analyses performed. Conclusions. The results show that gas chromatography can be successfully used as an analytical tool for deuterated and tritiated mixtures of hydrocarbons. The main difficulty presented by the analyses reported here is the high volatility of the substances examined, which to date hindered their separation by gas-liquid chromatography with capillary columns. With the use of capillary columns, separations of deuterated forms of aromatic hydrocarbons differing by one mass unit were also performed in the past (19, 20), but the sophisticated technique of glass capillary columns, available only in few laboratories around the world, is such that a wide use of these columns for this purpose is practically unfeasible. Packed columns can be prepared by every gas chromatographer without particular problems, so that this method of analysis can have a large use. Moreover, a preparative separation of these substances is possible with packed columns. As a side result, this work shows that it is possible to make very efficient packed columns, which can be used for the separation of complex mixtures, e.g., essential oils, with a number of theoretical plates similar to that obtained with capillaries. This can make the mass spectrometric analysis of trace components much easier, because of the higher capacity of packed columns. ACKNOWLEDGMENT
The authors wish to thank G . Crescentini, M. Galantucci, and C. Canulli, for experimental assistance, and F. Cacace and G . Ciranni for tritiated samples. RECEIVED for review August 11, 1971. Accepted January 6, 1972. Work supported by Consiglio Nazionale delle Ricerche. (19) F. Bruner and G. P. Cartoni, J. Chromarogv., 10,396 (1963). (20) A. Liberti and L. Zoccolillo, ibid.,49, 18 (1970).
