measured values. These differences are much greater than the error associated with the method. We have also examined some cellulose ethers containing carboxymethyl groups. After hydrolysis the resonances of the -O-CH2COOH group fall between 4.2 ppm and 4.7 ppm, i.e., in the window between the 1-CH bands and bands of the remaining -CH groups of the anhydroglucose unit, though there may be overlap from the wing of the large resonance associated with the latter. However, this is not too serious and some idea of the carboxymethyl content can be obtained, though it is probably not as accurate as that obtained by titration.
Finally, the method can be used to determine the MS of substituents introduced into starches. In this case, hydrolysis is generally more rapid, so shorter times are possible, and the amount of degradation appears to be less. ACKNOWLEDGMENT
The author is grateful to P. L. Wright for preparing some of the materials used in this work and for the Zeisel determinations, and to Mrs. L. J. Phillips for running many of the spectra.
RECEIVED for review May 8, 1972. Accepted July 20, 1972.
Thermodynamic Measurements by Frontal Chromatography Liquid Surface Adsorption Effects J. F. Parcher and C. L. Hussey Department of Chemistry, The Unicersity of Mississippi, University, Miss. 38677 ELUTIONCHROMATOGRAPHY has been used successfully to measure thermodynamic properties of hydrocarbons at infinite dilution. The results are accurate and easily obtainable for nonpolar systems; however, the measurements are less accurate and more difficult in systems which involve more than one retention mechanism, i.e., concurrent solution and adsorption. Several recent studies (1-5) have dealt with this subject and these theories are satisfactory for systems involving nonpolar solutes. Systems of this type are less complex, because of the absence of potential adsorption at the liquid-solid interface, and the retention volume can be determined from Equation l (1-6)
VNm = KLmVL + K L S m A L S
The various retention mechanisms must be identified and evaluated, or eliminated, in order to obtain thermodynamic solution data for the system. Numerous authors (4) have proposed techniques for correction of elution data to correct for solute adsorption. An alternate approach is to evaluate the separate sorption equilibria. Since all of the partition and adsorption coefficients of alcohol-hydrocarbon systems are functions of concentration, the sorption isotherms must be measured over a finite concentration range. This can be done in the static system or with frontal chromatography. The frontal analysis technique involves the integration of an equation of the form,
(11 i=l
where VNmis the infinite dilution net retention volume, KLm is the infinite dilution bulk liquid partition coefficient, K L S m is the infinite dilution Ldsorption coefficient for the gas-liquid interface, VL is the volume of liquid phase, and A L S m is the total surface area of the liquid phase. Both KLm and K L S m are usually constant for very low concentrations. It has been often postulated (I, 4 , 7) that the isotherms for adsorption of solutes at the gas-liquid interface are Langmuir type, which would indicate that the retention volume should vary with sample size, at finite concentrations. Chromatographic systems involving polar solutes and nonpolar solvents are far more complex and, consequently, less well understood. In particular, the role of liquid surface adsorption of solutes such as alcohols on hydrocarbon solvents is uncertain.
where Qt is the amount of solute sorbed per unit area in phase i , C is the concentration (mole/l.) of solute in the gas phase, Y is the mole fraction of solute in gas phase, Ai is the surface area of phase i , and n is the number of distinct interfaces contributing to the retention of solute. Usually, the desired information is Q L = f ( c ) and the measured variable is VN =
f(4. Integration of Equation 2 is not simple, due to the complexity of the system. Gas phase nonideality, nonequilibrium, and the so-called “sorption effect” are a few of the complicating factors which must be taken into account. Numerous authors (8-10) have suggested procedures for integrating Equation 2 , and we have used the general procedure given in reference IO. EXPERIMENTAL
(1) J. R. Conder, D. C. Locke, and J. H. Purnell, J . Phys. Chem., 73, 700 (1969). (2) D. F. Cadogan, J. R. Conder, D. C. Locke, and J. H. Purnell, ibid., p 708. (3) D. F. Cadogan and J. H. Purnell, ibid., p 3849. (4) H. Liao and D. E. Martire, ANAL.CHEM., 44,498 (1972). (5) V. G. Berezkin, J. Chromarogr., 65, 227 (1972). (6) R. L. Martin, ANAL.CHEM., 33, 347 (1961). (7) J. R. Conder, J. Chromatogr., 39, 273 (1969). 188
The gas chromatograph used in this study was a Beckman GC-45 with a thermal conductivity detector. The basic gas
(8) J. R. Conder and J. H. Purnell, Traits. Faraday SOC.,64, 1505 (1968 ). ( g j Ibid, p 3100. (10) C. J. Chen and J. F. Parcher, ANAL.CHEM., 43, 1738 (1971).
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Table I.
Corrected Infinite Dilution Activity Coefficients of n-Hexane and 2-Propanol at 50 "C n-Hexane 2-Propanol -Elution Frontal Elution (13)o Frontalh Elution (13) Elutionb 0.91 0.89 0.83 13.6 13.6 zt 0.7 13.4 =k 0.6 n-Cda6 0.90 5.28 5.5 i 0.1 5.5 i0.3 (n-CsH17)20 0.93 0.96 3.1 i 0.1 0.98 2.93 3.3 i.0 . 1 (n-CsHn)zCO 1.10 1.09 a Liao and Martire ( 4 ) have recently criticized these values and suggested that there is probably a positive error of up to 10% in the specific retention volumes used to calculate the activity coefficients. Each data point represents the average of 4-15 experiments on 2-6 liquid loadings. The deviations quoted are absolute deviations. Liquid phase
flow pattern was the same as previous equipment (10) except for an accurate flow meter (11) incorporated into the heated detector compartment of the chromatograph. The output of the detector was digitized by a Beckman 3111 Intercoupler and punched out on paper tape. The computations were performed in an IBM 360/40 computer. The columns used in this study were prepared from Varian Aerograph Chromosorb P, AW-DMCS. Liquid coatings consisted of n-heptadecane (Aldrich Chemical Company), di-n-octylether (K and K Laboratories), and di-n-octylketone (Aldrich Chemical Company). Dioctylether and n-heptadecane were chromatographically tested for purity while the dioctyl ketone was specified 98 % purity by GC-IR analysis of the manufacturer. n-Heptadecane columns were prepared in the following percentages: 0.73, 3.16, 7.49, 12.18, 19.29, 24.03%; dioctyl ether was prepared as 8.49 and 16.20%; and dioctyl ketone columns were made at 6.09 and 11.10%. In addition, a column packed with uncoated support was prepared. Each of the columns was analyzed to within 1 % relative deviation by a previous method (12). Each of the columns was tested by comparison of infinite dilution activity coefficients with previously reported literature values. These comparisons are shown in Table I.
0
1.o
3.0
20
SAMPLE SIZE(gI)
Figure 1. Variation of retention volume with sample size for 2-propanol in n-heptadecane at 50 "C Retention volumes are calculated as milliliters per gram of solid support
RESULTS AND DISCUSSION
The activity coefficient values reported in Table I are the values obtained when the sample size is extrapolated to zero. This is the usual approach to the elimination of sample size effects. Variation of the retention volume of an alcohol is usually observed when the solvent is nonpolar. Figure 1 is an example of this phenomenon for 2-propanol in heptadecane at 50' C. The usual interpretation of this type of curve is to divide the curve into a "solution dominated" region (dVN/ dbl > 0) and an "adsorption dominated' region (1, 2, 4 ) (dVN/dgl 5 0). The increase in V N with concentration or sample size, in the solution dominated region is due to the anti-Langmuir form of the bulk solvent partition isotherm. The decrease in VSv with concentration in the adsorption dominated region is due to the decrease in the adsorption coefficient because the isotherms are Langmuir. This explanation would suffice for either liquid surface or solid adsorption, as long as the isotherms are of the Langmuir type. In the adsorption dominated region, the increase in V.I. at a given sample size, increases with liquid load. If a constant sample size always produced a given C, then this trend is the opposite of what is expected for both types of adsorption, because the adsorption coefficients, and the area of the solid support A s s , would be constant and ALS would decrease with increasing per cent liquid (6). However, experiments at a constant sample size are hard to interpret because of the variation of the gas phase concentration (at a given sample size) (1 1) J. F. (12) J. F.
Parcher and C. L. Hussey, ANAL.CHEM., 44,1102 (1972). Parcher and P. Urone, J . Gus Clirornutogr., 2 , I84 (1964).
with the liquid load. At high coatings, the liquid phase contains a significant portion of the solute and C (constant gl) decreases as VL increases. Both the adsorption coefficients, KLs and K:;,s,increase with decreasing C and this accounts for the variation of V, with VLat low sample sizes. The usual extrapolation procedure (11 -,0) will yield a reproducible retention volume which is corrected for adsorption effects, for Langmuir isotherms. However, the uncertainty in the extrapolation increases as the adsorption effects increase, and the functional relation of C and sample size is uncertain. Thus, elution studies are of limited utility for systems involving concurrent adsorption and solution. The exact nature of the adsorption present in the alcoholhydrocarbon systems is uncertain. In 1963, Martin ( 6 , 13) observed that KLSmcould be correlated with the rate of decrease of the surface tension, y , with the mole fraction of solute in the liquid phase, x p , i.e., (dy/dx.J. Pecsok and Gump (14) measured (dyldx.?) for methanol in squalane and reported values of -2464 and -1903 at 30 and 50 " C , respectively. These values were then used to calculate KLSm values of 473.0 x lo-" cm and 201.1 X 1 0 P cm. The retention volume calculated using KLa = 9.15, KLSm = 473.0 X 10-6 cm, V L = 0.6 gram, A L S = 2.5 m2/gram (14) would be more than twice the retention volume using KL only. Pecsok and Gump (14) did not report any retention volumes; however, this large adsorption effect (even at 6x loading) has not
(13) R. L. Martin, ANAL.CHEM., 35, 116(163).
(14) R. L. Pecsok and B. H. Gurnp, J . Pliys.
Cliem., 71,2202 (1967).
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I-
I
O
I
0 d
4 0
c
0
0.1
0.2
0.3
0.4
V O L U M E O F L I Q U I D ( r n l l grn)
0
10
20
30
40
Figure 4. Amount of 2-propanol sorbed in n-heptadecane at 50 “Cas a function of concentration of 2propanol in the gas phase
1 / F,
Figure 2. Specific retention volumes of 2-propanol in n-heptadecane at various liquid loads
0 = 3 pmoles/ml A = 2 pmoles/ml 0 = 1 pmole/ml
A Data of Liao and Martire (4) ( 1 2 Propanol in n-ClsHa8) 0 This Work FLis the weight fraction of liquid phase,
FL =
Wliquld/W’dtd
0
A
a
A
1.o
0
2.0
3.0
CONCENTRATION lwrnoles/rnl) Figure 3. Measured partition isotherms of 2-propanol in n-heptadecane at 50 “C for various liquid loadings A
=
24.03%
0 = 12.18% 0 = 3.16% = 0.00 (uncoated support)
A = 19.29% 0 = 7.49% 0 = 0.73%
been observed experimentally. On the contrary, the retention volume of methanol increases linearily with liquid load on an inactive support (15). Martire (16) was the first to point out this discrepancy, and no satisfactory explanation has been given to date. Martire ( 1 7 ) later measured the specific retention volume of n-propanol in n-heptadecane and reported that the extrapolated specific retention volumes were constant for 10 and 15 % coating at 30 “C,and concluded that solute adsorption at the (15) P. Urone and J. F. Parcher, ANAL.CHEM., 38, 270 (1966). (16) D. E. Martire, in “Progress in Gas Chromatography,” J. H. Purnell, Ed., Interscience, New York, N. Y . , 1968, p 93. (17) D. E. Martire and P. Riedl, J. Plzys. Chem., 72,3478 (1968). 190
gas-liquid interface was negligible. However, in another paper ( 4 ) , it was reported that the extrapolated specific retention volumes of n-propanol in n-octadecane varied significantly with the liquid load and that the data for n-heptadecane were in error. This would indicate that adsorption processes contribute significantly to the retention volume of alcohols in hydrocarbons. In a study of alcohol association, we measured the specific retention volume (extrapolated to zero sample size) of 2-propanol on heptadecane, dioctyl ether, and dioctyl ketone at 50 “C. More columns were prepared for the heptadecane since, according to current theory, it was the most likely to show liquid surface adsorption (16). The results are presented in Figure 2, and the extrapolated values of the specific retention volumes show no dependence on liquid load (relative deviation of 7 % for six liquid loadings). The extrapolated specific retention volumes for 2-propanol in dioctyl ether and dioctyl ketone were also constant, within 2% deviation, in the range from 6 to 16% liquid coating. This would indicate that the extrapolation procedure corrects for all adsorption effects; however, this is in direct conflict with Liao and Martire (4). We feel that the agreement between our results and the results of Martire and Riedl (17), as shown in Table I indicate that the original data presented for this system (17) are probably correct. In order to resolve this and other conflicting reports in the literature, we measured the sorption isotherms of a series of coated supports using frontal chromatography. The sorption isotherms for 2-propanol in n-heptadecane coated on Chromosorb P AW-DMCS are shown in Figure 3. This is a plot of the amount of 2-propanol absorbed per gram of uncoated solid support, Q , as a function of concentration of alcohol in the gas phase, C. Q would be the sum of the amounts of 2-propanol in the bulk liquid, at the gas-liquid interface, and at the liquid-solid interface, i.e., Q = Q L QLs QSS. Each of these Q, values will vary with C; however, the function dependence is determined by the form of the individual isotherms. Figure 4 is a graph of Q, at a constant C, as a function of the weight of liquid phase per gram of uncoated support for three different values of C. The graphs are linear and the curve extrapolates to the zero liquid load values for Q. This indicates that for M 5 C 5 3 X 10-3Madsorption at the
ANALYTICAL CHEMISTRY, VOL. 45, NO. 1, JANUARY 1973
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+
gas-liquid interface is negligible and that solid support adsorption is predominant at very low liquid loads, even with a relatively inert support. It is improbable that liquid surface adsorption is present because this should produce a curve which did not extrapolate to the point for the uncoated support. The amount of liquid surface adsorption will vary with the structure of the alcohol-i.e., primary alcohols would be expected to concentrate on the surface more than secondary or tertiary alcohols. Thus the results of this study cannot be indiscriminately extended to n-propanol or any of the other lower molecular weight alcohols.
The three independent phases of these studies--i.e., behavior of methanol on squalane with a completely inert support (15),constancy of V , values for columns of different liquid phase surface area (Figure 2), and the linear variation of Q at a given concentration, C, with the weight of liquid phase in a column-preclude the existence of significant liquid surface adsorption for 2-propanol on n-heptadecane at 50 O C . RECEIVED for review May 18, 1972. Accepted August 7, 1972. This work was supported by a Frederick Gardner Cottrell Grant from the Research Corporation and Grant No. GP-27999 from the National Science Foundation.
High Resolution Field Ionization Mass Spectrometry of Bacterial Pyrolysis Products H. R. Schulten’ and H. D. Beckey Institut fiir Physikalische Chemie der Uniuersitiit, 53 Bonn, Germany
H. L. C. Meuzelaar and A. J. H. Boerboom FOM-Instituut coor Atoom- en Molecuufysica, Amsterdam/ Wgm., The Netherlands MASSSPECTRA OBTAINED by Field Ionization Mass Spectrometry (FI-MS) are generally characterized by the presence of prominent molecular ion (“parent ion”) peaks and of only a few minor fragment ion peaks ( I ) . FI-MS is therefore, in principle, a suitable technique for the analysis of multicomponent mixtures of volatile organic compounds. This was demonstrated by Beckey et al. in 1964 (2) in a small comparative study of the qualitative and quantitative analysis of a seven-component hydrocarbon mixture by Gas-Liquid Chromatography (GLC) and FI-MS. More complex mixtures were studied by Schuddemage and Hummel (3). These authors analyzed pyrolyzates of synthetic polymers by FI-MS and their results illustrate the striking simplicity of FI-MS spectra of multicomponent mixtures as compared to the corresponding Electron Impact Ionization (EI) mass spectra. It should be pointed out, however, that the interpretation of spectra obtained by low resolution FI-MS of multicomponent mixtures is inevitably complicated by the fact that molecular ions of different elemental composition may share the same nominal m/e value. High resolution FI-MS therefore greatly widens the scope of the method since it enables the determination of the elemental composition of the observed ions by accurate mass measurements. Forehand and Kuhn ( 4 ) performed high resolution FI-MS of To whom inquiries should be addressed. _
_
~
(1) H. D. Beckey, “Field Ionization Mass Spectrometry,” Pergamon Press, Oxford, and Akademie Verlag, Berlin, 1971. (2) H. D. Beckey, H. Knoppel, G. Metzinger, and P. Schulze, “Advances in Mass Spectrometry,’’ Vol. 3, W. H. Mead, Ed., The Institute of Petroleum, London, 1966, p 35. (3) H. D. R. Schuddemage and D. 0. Hummel, “Advances in Mass Spectrometry,” Vol. 4, E. Kendrick, Ed, The Institute of Petroleum, London, 1968, p 857. (4) J. B. Forehand and W. F. Kuhn, ANAL.CHEM., 42, 1839 (1970).
the condensable phase of cigarette smoke, and proposed probable elemental compositions for more than 50 components. Since a group at the FOM-Instituut voor Atoom- en Molecuulfysica at Amsterdam was engaged in a series of studies on the identification and classification of bacteria by PyrolysisGLC (5) and Pyrolysis-MS (6), these results prompted us to investigate the feasibility of high resolution FI-MS analysis of the extremely complex mixtures obtained by pyrolysis of bacterial samples. Although GLC “fingerprints” of bacterial pyrolyzates have been published by several authors (5, 7-13), only Simmonds (1.3) has attempted a systematic chemical identification of the components of these pyrolyzates through direct coupling of a quadrupole mass spectrometer to a GLC system (GLC-MS). He compiled a rather extensive list of identified products and tentatively assigned them to specific classes of biological compounds such as proteins, carbohydrates, lipids, nucleic acids, and porphyrins from which they probably originated. The purpose of the study reported here is to explore the potentials of high resolution FI-MS for the analysis of extremely complex multicomponent mixtures as well as to perform a general survey of the chemical nature of bacterial py-
(5) H. L. C. Meuzelaar and R. A. in’t Veld, J. Clironzatogr. Sci., 10, 213 (1972). (6) H. L. C. Meuzelaar and P. G. Kistemaker, ANAL.CHEM.,in press. (7) V. I. Oyama and G. C . Carle, J . Gas Chromatogr., 5 , 151 (1967). (8) E. Reiner, Nature, 206, 1272 (1965). (9) E. Reiner, J. Gas Chromatogr., 5, 65 (1967). (10) E. Reiner and W. H. Ewing, Nature, 217, 191 (1968). (11) E. Reiner, R. E. Beam, and G. P. Kubica, Amer. Rec. Resp. Dis., 99, 750 (1969). (12) E. Reiner, J. J. Hicks, R. E. Beam, and H. L. David, ibid., 104, 656 (1971). (13) P. G. Simmonds, Appl. Microbiol., 20, 567 (1970).
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