Table 111. Comparison of Kegfor Vanadium-Indicator Reaction from Potentiometric and Kinetic Measurements (All at 25’ C)
HSOa concn, M 0.33 0.67 1.00 1.50 2.00
Formal potentials, volts Vanadium couple Indicator couple 0.936 1.087 0.986 1.073 1.008 1.06 1.030 1.045 1.056 1.03
Keq(potent) -350 -30 -1.6 ~ 1 . 8 4 . 4
its effect on the relative rates of interaction of vanadium(1V) and vanadium(V) with the oxidized and reduced forms of the indicator, respectively. Based on our knowledge of the predominant species present in sulfuric acid media (7-12), the most likely path for reaction 2 is Ce(SO&
+ VO(S0,) .HzO F? Ce(SO4)2-
+ VO(S0,) .HzO+
(4)
with a n equilibrium constant of 3.14 X lo6, in molar sulfuric acid solution, calculated from the formal potentials of the redox couples involved. Since both the forward and back(12) W. A. Waters and J. S. Littler, in “Oxidation in Organic Chemistry,” K. Wiberg, Ed., Academic Press, New York, 1965, p. 186ff.
kl M-1 sec-1
10.4 7.4 5.7 4.0 2.9
kz M-’ sec-l
0.12 0.67 2.45 14.4 29.6
Keq(kinetic) 87 11 2.3 -0.3 NO.1
ward rate constants of reaction 3 were measured in this study and since the formal potentials of the vanadium and indicator couples as a function of sulfuric acid concentration are available in the literature (6, IO), the equilibrium constants for reaction 4 obtained in our kinetic study are compared in Table I11 with those calculated from potentiometric data. Because the difference in formal potentials of the two couples is so small, the moderate uncertainty in the measured potentials produces a n especially large error in the calculated Kpot values. For this reason, our Kkin values calculated from the rate constant ratio are thought to be more reliable for this system.
RECEIVED for review April 7, 1967. Accepted June 5 , 1967. An Alfred P. Sloan Fellowship was awarded the senior author.
Separation of Amino Acids by Gas Chromatography Using New FIuoro Derivatives Glenn E. Pollock Exobiology DiGision, Ames Research Center, NASA, Moffett Field, CaW
IN THE COURSE of our research along other lines, it became necessary t o investigate a gas chromatographic technique for qualitatively identifying amino acids. A review by Weinstein ( I ) has thoroughly covered this field.
94035 W
z
4
EXPERIMENTAL
Several N-trifluoroacetyl (N-TFA) amino acid n-butyl esters were synthesized ( 2 , 3). Identical procedures were used for the N-pentafluoropropionyl (N-PFP) and N-heptafluorobutyryl (N-HFB) derivatives. G a s chromatography was carried out on a Perkin-Elmer 800 instrument, using Carbowax 20M, 0.02-inch X 150-foot columns and a linear pressure programmer. RESULTS AND DISCUSSION
The retention times of the high-boiling esters were long, resulting in analysis times well over 1 hour, while the retention time of the N-pentafluoropropionyl (N-PFP) derivative was significantly shorter. Because of the pronounced effect of N-PFP derivatives, we prepared N-heptafluorobutyryl (N(1) Boris Weinstein, “Methods of Biochemical Analysis,” Wiley, 1966, p. 203. (2) W. M. Lamkin and C. W. Gehrke, ANAL.CHEM., 37, 383 (1965). (3) I. Halasz and K. Bunnig, Z . Anal. Chern., 211, 1 (1965).
1 194
ANALYTICAL CHEMISTRY
Figure 1. Separation of the N-TFA, n-butyl esters of 14 amino acids Carbowax 20M, 0.02-inch X 150-ft column Temperature: Isothermal at 100” C for 2.5 min. Raise to 170” C at 4 “ C/min Gas: Helium, 9.5 Ib (about 10 mlimin) for 25 min. Then raise linearly to 20 Ib (28 mljmin) at rate of 7.74 Ib/min. These conditions are identical for Figures 1 and 2. Figure 3 had no carrier gas flow rate increase
HFB) derivatives and found that their retention times were even shorter. In Table I are shown the retention times, relative retention times, and per cent time reduction for each amino acid under isothermal conditions using a Carbowax 20M column. Table I shows that the N-PFP and N-HFB derivatives have
I
5.0 r
Figure 2. Separation of the N-PFP derivatives Conditions same as for Figure 1 TIME, mln
Figure 3. Separation of the N-HFB derivatives significantly lower rention times than N-TFA derivatives. From these values and the relative retention time values, it is strongly indicated that a similar type separation might occur with the new derivatives. Mixtures of 14 amino acids were made and the derivatives were chromatographed on a 20M column. Figures 1 and 2 show the separation of the NTFA and N-PFP derivatives, respectively, with exactly the same program. These 14 amino acids as N-PFP derivatives require about 3 6 z less time than the N-TFA derivatives. The actual separation is about the same. Figure 3 shows the N-HFB derivatives with the same temperature program. The results are almost the same as the N-PFP, but with one important difference. In the N-PFP and N-TFA chromatograms, pressure programming was necessary to reduce the time of analysis for lysine and ornithine. This pressure program began at 25 minutes and increased carrier gas pressure from 10 to 20 pounds. For the N-HFB derivatives, carrier gas pressure programming was unnecessary. Separation of the N-HFB derivatives is almost identical to that of the N-PFP and N-TFA derivatives. Alanine and isoleucine resolution suffers slightly, but is adequate.
Conditions same as indicated for Figure 1
If time were the only consideration, this finding would be significant since the reduction in time of analysis (35 to 3 6 z ) is desirable. Additional advantages of these new derivatives are: The column temperature can be reduced if time can be lengthened; a recent publication by Clarke et al. ( 4 ) on the chromatography of amines indicates that these same acyl groups significantly increase compound detectability (sensitivity) with an electron capture detector. These authors report that the N-TFA derivatives of the amines d o not capture well and that sensitivity with these derivatives shows no advantage over flame ionization detection. With N-PFP and N-HFB amines, however, sensitivity for electron capture detection is several hundred times greater.
(4) D. D. Clarke, S . Wilk, and S . E. Gitlow, J. Gas Chromatog., 4, 310 (1966).
Table I. Comparison of Gas Chromatographic Parameters of N-TFA, N-PFP, and N-HFB, n-Butyl Esters of Several Amino Acidsa Amino acid
N-TFA RTb,min
N-PFP RT, min
N-HFB RT, min
N-TFA RRTb
N-HFB RRT
N-PFP RRT
Isothermal, 100' C., He 9.5 Ib (10 ml/min), Carbowax 20M, 0.02 inch Valine a-Amino-n-butyric Isoleucine Alanine Norvaline Leucine Norleucine
11.29 15.66 15.84 17.79 22.50 26.60 32.41
7.19 9.63 9.50 10.91 13.26 15.37 18.62
6.66 8.67 8.77 9.61 11.86 13.81 16.71
Glycine Proline
9.95 12.02
7.19 8.51
6.50 7.49
Methionine Aspartic Phenylalanine Glutamic
8.18 10.17 11.47 17.49
0.348 0.483 0,488 0.548 0.694 0.820 1,000
Time reduction, N-PFP N-HFB
x 150 ft
0.386 0.517 0.510 0.586 0.712 0.825 1.000
0.398 0.518 0.524 0.575 0.709 0.826 1,000
36.4 38.5 40.1 38.7 41.1 42.3 42.6
41.1 44.7 44.7 46.0 47.3 48.1 48.5
0.845 1.000
0.868 1,000
27.8 29.2
34.7 37.8
33.1 28.0 31.2 35.6
37.9 35.7 29.1 44.2
140" C., He 9.5 lb 0.827 1.000
Isothermal, 170" C; He 9.5 Ib (10 ml/min); Carbowax 20M, 0.02 inch x 150 ft 5.48 7.33 7.90 11.27
5.08 6.54 6.99 9.77
0.461 0.581 0.655 1,000
0.485 0.650 0.701 1,000
0.520 0.669 0.715 1.000
170" C; 19.2 Ib (25 ml/min) Ornithine Lysine
63.5 72.2 25.33 9.26 7.05 0.711 0.691 0.691 62.4 71.4 35.62 13.40 10.20 1,000 1,000 1.000 a Pentafluoropropionic and heptafluorobutyric anhydrides purchased from K & K Laboratories, Plainview, N. Y. Synthesis of N-PFP and N-HFB identical to N-TFA derivatives. * R T = retention time; RRT = relative retention time. Norleucine, group 1 ; proline, 2; glutamic, 3; lysine, 4.
VOL. 39, NO. 10, AUGUST 1967
1195
The sensitivity varies greatly with electron capture, depending upon the functional groups of the compounds. Clarke et al. ( 4 ) found that hydroxyl groups amplify detection enormously so that sensitivity for hydroxy amino acids might be increased several thousandfold. Electron capture detection of N-2, 4-dinitrophenyl methyl esters of several amino acids was reported by Landowne and Lipsky ( 5 ) to be excellent.
It is felt that the fluoroderivatives would be of greater use in analysis because of their higher volatility, and the sensitivity of the N-PFP and N-HFB derivatives would appear t o approach the results with N-2,4-dinitrophenyl derivatives. Further work on these derivatives will be to investigate their feasibility for use in packed columns and perhaps t o evaluate sensitivity using electron capture detection.
( 5 ) R. A. Landowne and S. R. Lipsky, Nature, 199, 141 (1963).
RECEIVED May 8, 1967. Accepted June 5,1967,
Regarding Dependence of Retention Volume upon Carrier Gas in Gas-So Iid Chromatogr a phy SIR:A functional dependence of retention time upon carrier gas has been suggested recently by Hoffmann and Evans (1) for the gas-solid chromatography (GSC) of various hydrocarbons. The dependence has been postulated in terms of a desorption hindrance mechanism with a carrier gas of a given collision cross section offering hindrance to desorption and migration into the bulk gas phase. This would cause a longer retention time for this gas than that resulting when a carrier gas of smaller collision cross section is used a t the same outlet flow velocity. The possibility of direct involvement of such carrier gases as He, Ar, N?, and H? in the partition mechanism is extremely disturbing because G S C otherwise offers a particularly simple procedure for measurement of various thermodynamic quantities associated with physical adsorption. Several workers (2-8) have reported the use of G S C to evaluate heats of adsorption, but they have not noted carrier gas effects upon retention volume. However, in using gas chromatography to study the interaction of sorbate and inert (carrier) gas molecules during the adsorption-desorption process, the raw chromatographic data require modification to arrive a t a quantity which is related to the process of interest. Corrections are necessary for such extraneous variables as differences in carrier gas viscosity which affect the average carrier gas flow rate for a constant flow rate a t the outlet pressure. The parameter which would vary only with differences in the partitional behavior of a particular sorbate-sorbent pair a t a set temperature is the specific retention volume, V , (9),
(1) R. L. Hoffmann and C. D. Evans, ANAL.CHEM.,38, 1309
(1966). (2) R. A. Beebe, P. L. Evans, J. C . W. Kleinsteuber, and L. W. Richards, J. Phys. Chem., 70, 1009 (1966). (3) R. L. Gale and R. A. Beebe, J. Phys. Chem., 68,555 (1964). (4) R. S. Petrova, E. V. Khrapova, and K. D. Shycherbakova, in “Gas Chromatography, 1962,” M. van Swaay, Ed., Butterworths, Washington, 1960, p. 18. (5) S. Ross, J. K. Saelens, and J. P. Olivier, J . Phys. Chem., 66, 696 (1962). (6) S. A. Greene and H. Pust, J. Phys. Chem., 62,55 (1958). (7) A. V. Kiselev in “Gas Chromatography, 1964,” A. Goldup Ed., Elsevier, Amsterdam, 1965, p. 238. (8) D. H. James and C. S. G. Phillips, J. Clzem. Soc., 1954, 1066. (9) A. B. Littlewood, C. S. G. Phillips, and D. T. Price, J. Chem. Soc., 1955, 1480. 1 196
ANALYTICAL CHEMISTRY
rather than the retention time t R ‘ , as implied in (I). I n fact, V , is related linearly to the distribution constant, which will change in magnitude from carrier gas to carrier gas only if the latter influences the partition equilibrium. Because we are engaged in a detailed study of the thermodynamics of gas-solid chromatography, the results of Hoffmann and Evans (1) have led us to perform a series of experiments analogous to theirs to establish whether carrier gas affects the partition equilibrium. However, the resulting data have been converted to specific retention volumes before comparing the various carrier gases. EXPERIMENTAL
Gaseous samples of n-butane were injected into a 4-foot X 0.d. stainless steel column packed with 60- to 80-mesh Analabs Type F-1 activated alumina, specific surface area 200 mZ/g. A gas-tight syringe was used; the tests were performed with a modified Barber-Colman Series 5000 gas chromatograph operated a t 100.0 f 0.3 O C. Retention data for each carrier gas (helium, nitrogen, argon, and hydrogen) were taken after the column had been activated for 2 hours a t 350” C in contact with the gas to be used. Following the entire series of tests, a complete redetermination of all retention volumes was made; no significant deviation from the original data was evident. Samples were injected over a range of 1-10 pl; the data for the lower volume are reported because the retention volume is constant from 1 to 5 pl, and decreases slightly for higher sample volumes. At least three measurements a t different column inlet pressures were made for each gas. Specific retention volumes a t the column temperature, VgT,were calculated by VU* =
(t’R ’
f .F a .Tc/Ta -
Vd)
l/w
(1)
with the standard specific retention volume, V,, expressed by V,
=
V g T(273/Tc)
(la)
where t l R is the retention time from injection point to point of maximum butane concentration, Fa is the volume flow rate a t ambient temperature and pressure, T, and T, are the absolute column and ambient temperatures respectively, w is the weight of alumina used, V dis the total system dead volume, and f is the James-Martin gas compressibility factor (IO), (IO) A. T. James and A. J. P. Martin, Biochem. J., 59, 679 (1952)
