when the principle occurs in a very low concentration as i t usually does in biological work. With such a problem, t h e first step usually involves some method for enriching rm extract. With CDCD the effective partition ratio could be set at unity and many transfers applied with continual feed. Most of the unwanted inactiqe material would then be thrown off in either effluent while the active principle would be held largely in the train. LITERATURE CITED
(1) Alderweireldt, F., Verzele, M., Bull. SOC.Chim. Belg. 70, 703 (1961). ( 2 ) Barker, J. A , , Beecham, A. F., Australian J . Chem. 13, 1 (1960). ( 3 ) Craig, L. C., Craig, D., ‘Technique of Organic Chemistry,” A. Weissberger,
ed., 2nd ed., Vol. 111, p. 149, Interscience, New York, 1956. (4) Craig, L. C., Hausmann, W., Ahrens, E. H., Jr., Harfenist, E. J., ANAL. CHEM.23, 1236 (1951). (5) Craig, L. C., King, T. P., Federation Proc. 17, 1126 (1958). (6) Craig, L. C., Post, O., ANAL.CHEM. 21, 500 (1949). (7) Davis, hl. W., Hicks, T. E., Vermeulen, T., Chem. Eng. Progr. 50, 188 (1954). ( 8 j Hietala, P. K., Acta Chem. Scand. 14, 212 (1960). (9) Hill, R. J., Konigsberg, W., Guidotti, G., Craig, L. C., J. Bid. Chem. 237, 1549 (1962). (101 Hunter. T. G.. Nash. A. W.. Ind. ‘ E’ng. Cheri. 27, 836 (1935). (11) Jantzen, E., “Das fractionierte Distillieren und das fractionierte Verteilen, Dechema Monographie,” Vol. V, No. 48, p. 81, Verlag Chemie, Berlin, 1952.
(12) King, T. P., Craig, L. C., “Methods of Biochemical Analysis,” D . Glick, ed., Vol. X, p. 201, Interscience, iYew York, 1961. (13) Scheibel, E. H., “Technique of Organic Chemistry,” A.Weissberger, ed., 2nd ed., Vol. 111, p. 332, Interscience, Xew York, 1956. (14) Stene, S., Arkiv. Kemi, Mineral. Geol. A18, No. 18 (1944). (15) VonMetsch, F. A., Chem. Inqr. Tech. 31, 262 (1959). (16) Wilhelm, H. A., Andrews, M. L.,
Ind. Eng. Chem., Process Design Develop.
1, 305 (1962). (17) Wilhelm, H. A., Foos, R. A., Ind. Eng. Chem. 51, 633 (1959).
RECEIVEDfor review Yovember 8, 1962. Accepted January 29, 1963. Work supported in part by Grant No. 2493 from the National Institute of Arthritis and Metabolic Diseases, Public Health Service.
Q ua nt itut iive Gas Liquid Chro matog ra phic Estimatio n of Volatile Fatty Acids in Aqueous Media R. G. ACKMAN and R. D. BURGHER Fisheries Research Board of Canada, Technological Research Laboratory, Halifax,
b The quantitative iunalysis b y gas liquid chromatography of volatile fatty acids in aqueous solutions has been rendered difficult b y adsorption and the appearance of ghost peaks on subsequent injection of samples. The addition of formic acid vapor to the carrier gas eliminates these problems and gives reproduc;ble quantitative results in conjunctiori with a flame ionization detector. Excessive amounts of water under certain conditions may cause double peak formation, but the method is applicable to analyses with Silicone!, polyester, and Tween substrates on Chromosorb W support.
T
volatile fatty acids was one of the first applications of gas liquid chromatog,raphy ( I S ) , and interest has continued in this field (2, 6-8, 12, 19, 63, 25). Thermal conductivity detectors have relatively modest sensitivity, and also 1,espond to water, hence the development of several techniques (7, 9-11, 17, 62, 26), some rather complex, for the concentration and recovery of the volstile fatty acids from dilute aqueous solutions. I n some instances these procedures include conversion to less nolar derivatives as part of the process. Argon ionization detectors are desensitized by water (18) and therefore d s o normally require either recovery of the acids from aqueous solutions or modifications to the apparatus (68). The employment of HE ANALYSIS OF
N. S., Canada
flame ionization detectors in this field is particularly indicated since they are insensitive to water and applicable with very dilute solutions (3, 4, 18). I n this laboratory attempts were made to analyze the steam distillate of volatile fatty acids from the ozonolysis of unsaturated fatty acids ( I ) , the solutions also containing a very large proportion of formic acid, Kith a flame ionization detector. Initial use of a conventional silicone-stearic acid type column ( I S ) was very discouraging from the point of view of reproducibility and quantitative results, as were attempts to employ a polyester-phosphoric acid column (20). The presence of the formic acid, which itself gives no response in flame ionization detectors (18), although a base line disturbance appears in the region corresponding to acetic acid, prompted an investigation of the influence of formic acid on the evident reversible adsorption of the higher volatile fatty acids on the column. The terms “ghosting” or “repeater effect” have been employed to describe such effects (4, 26, 27”). The continuous addition of formic acid vapor to the carrier gas, employing three different types of substrates, suppresses adsorption of the weaker acids, permitting reproducible and quantitative analyses of aqueous solutions as dilute as 0.01% (w./w.) in volatile fatty acid. Since the inception of this study the use of steam as a carrier gas in the analysis of polar compounds has been sug-
gested (SO), and also ammonia in the carrier gas in the analysis of volatile bases (94, the latter instance being directly comparable with the addition of formic acid to the carrier gas in the analysis of volatile acids. The use of water vapor in the carrier gas ( I 6 ) , or of carbon dioside as a carrier gas (15), has also been recommended in analyses of polar compounds. EXPERIMENTAL
Apparatus. T h e gas liquid chromatography apparatus employed consisted of a Wilkens flame ionization detector (gold plated) operating in a thermostated oven built in this laboratory. T h e stainless steel injection port was of t h e type used in t h e Aerograph A-90, and all columns were of l/s-inch stainless steel tubing, 6 feet in length. The Wilkens electrometer was used with a Minneapolis-Honeywell 1-mv. span recorder fitted with a ball and disc integrator (Disc Instruments, Inc., Model K-1) counting at 860 counts per square inch of chart. Materials. T h e acids employed were freshly distilled under vacuum prior to use. All showed less t h a n 0.1% impurity by gas chromatography, evcepting t h e valeric acid which contained 3.0% isovaleric acid. Appropriate corrections were therefore made in t h e tabulated weights of these two acids. Pent-oxone (4methoxy-4-methyl pentanone-2) was selected as a water-soluble, nonacidic standard and employed without further purification (technical sample, courtesy of Shell Chemical Co.). Reagent grade VOL 35, NO. 6, MAY 1963
647
RESULTS
Table I. Illustration of Ghosting through Consecutive Injections of Solution of a Mixture of Volatile Fatty Acids" and Formic Acid-Waterb on a Polyester Column
Response relative to propionic acid in each chromatograph Acids Propionic Butyric Valeric Caproic
Experiment Sample First injection 1.00 0.93 0.53 0.25 Second injection 1.00 1.04 0.71 0.43 Formic acid-water First injection 1.00 2.6 3.5 4.0 Second injection 1.00 2.5 5.0 8.0 Third injection 1.00 2.0 4.9 9.0 a 0.001 ml. of approximately 1% of each acid in water-acetone solution. 0.001 ml. 1:1 mixture. Table II.
Silicone Oil
Substrate NPGA
1.00 1.25 0.28
0.03 0.01
Tween
Sonlinear Sonreproducible bisalnte dcidi
Nonlinear Sonreproducible I)isulst.es Lieids
Displaces acids
Displaces traces of Displaces traces acids of acids
Nonlinear sonreproducible Disulacvs ncids
Linear Linear Linear Reproducible Reproducible Reproducible May displace traces S o acids displaced No acids displaced of acids
Area Response for Butyric Acid, Relative to Pent-oxone as 1 .OO, for Solutions of Different Strengths
Average of three consecutive runs. Solution strength, g./lOO ml. Substrate Pent-oxone Butyric acid Silicone Oil XPGA 0.3800 0,4902 1.10 1.16 0,098 1.11 0.076 1.14 0.015 0.020 1.16 1.15 Relative retention time of acid (to pent-oxone) 0.6 1.7
acetone was employed in numerous solutions and showed no peaks on chromatographs in the fatty acid region. The formic acid added to the carrier gas was of 98+% concentration (Eastman Organic Chemicals). Chromosorb TV (80- to 100-mesh) was used in all columns without treatment, and all columns were packed using continuous vibration and aspirator vacuum on the precoiled column. Substrates (25% added in chloroform solution to the support, followed by evaporation with stirring on a steam bath) were respectively DC-550 Silicone Oil, containing 5% stearic acid, Tween (polyoxyethylene sorbitan monooleate) , and KPGA (neopentyl-glycol adipate) polyester. Procedures. Hydrogen and air flow rates t o t h e flame head were nearly constant in all experiments at
648
(Propionic acid)
Summary of Column Substrate Performance with and without Formic Acid Vapor in the Carrier Gas
Conditions and experiment Plain carrier gas Response \list u r i p Y t t t r r i Iniwtion of iurtric heidkater mixture Addition of formic acid vapor t o carrier gas Formic acid in carrier gas Response Mixture pattern Injection of formic acidwater mixture Table 111.
Relative response between chromatographs
ANALYTICAL CHEMISTRY
Tween 1.22 1.21 1.18 2.4
20 cc. per minute and 230 cc. per minute, respectively. Helium was employed as t h e carrier gas. Dependent on the desired analysis time the flow rates in cc. per minute normally employed for analyzing mixtures containing caproic and shorterchain acids were: Silicone, 6 to 8 a t 138" C., 16 to 24 at 105" C.; KPGA, 16 to 20 at 138" C., 40 to 60 a t 105" C.; Tween, 10 to 16 at 138" C., 30 to 60 a t 105" C. Injections of solutions were by Hamilton 10-pl. syringe fitted with a Chaney adaptor. When formic acid was desired in the carrier gas stream a cold-finger type trap containing the acid at a level just below the inlet tube was inserted in the gas line immediately prior to the injection port. Heavy wall rubber tubing was used for the connections.
Suppression of Adsorption. In Table I are summarized t h e results obtained from two consecutive analyses of a n aqueous solution of some volatile fatty acids, clearly illustrating t h e lack of reproducibility obtained under normal operating conditions on the polyester-phosphoric acid type colunin. Reversible adsorption of these acids is indicated by the displacements obtained with subsequent injections of formic acid-water mixture. This adsorption is suppreqsed by continuous addition of formic acid vapor to the carrier gas, since as summarized in Table 11, injection of the formic acid-water mixture after anal) mixtures of higher fatty acids on three substrates then gives no significant response, except in the case of the Silicone-stearic acid column \\-here 1.O% or leqs response, relath e to the preceding samplp, may sometimes be observed. With all columns a reduction in acid sample size of lo-? occasionally gave traces of ghosting in the firqt sample of lower concentration. Linearity of Response and Quantitative Results. The injection of 0.001-nil. sanipleb of propionic acid solutions varying in concentration from 0.05 to 5.0%, either in sequrnce or in random order, suggested t h a t response was linear n i t h amount of acid under the above experimental conditions with all columns evcepting the N P G d column a t 105" C. a t a carrier gas flow rate of 60 cc. per minute, where the upper limit of linear response !vas obtained with a 1.0% solution. Hon-ever, reducing the carrier gas flow rate to 40 cc. per minute again raised the upper linear responqe limit to that of a 5.0y0 solution. The range of linearity might also be extended upm r d s by replacement of the 67.5volt battery in the detector circuit by one of 300 volts (29). The grossly nonlinear peaks were always followed by a negative dip in the base line. Studies n ith butyric acid, relative to pent-osone as an internal standard, confirmed that the response was linear over a practical range of concentration for all three columns (Table 111). The slight increase in the relative response of the butyric acid at low concentration on the Silicone column may possibly be due to some adsorption of the pent-oxone in the substrate, since this material has a greatly increased relative retention time on this column, and a t all three concentrations the pent-oxone showed double peak formation (see below). Under these conditions there was therefore good reason to believe that the results of analyses of mixtures mould be quantitative. The experimental conditions in further experiments (Table IV) were
deliberately chosen to conform to practical conditions t h a t might arise in normal laboratory usage in that large amounts of water (0.005-ml. samples) were commonly used, and peak heights were kept at 30% or less of full scale deflection. Under these conditions peak areas averaged about 500 counts with a probable errclr in estimation of 5%, somewhat further increased by the slight tailing particularly noted with the Silicone and Tween columns. The addition of aceton? (30%) to the stronger mixtures cf acids was necessary to ensure that the caproic acid was in solution and this solvent concentration was retained in the more dilute solutions. Consequently the acetic and propionic. acid peaks were located on the tail of the acetone peak and in the case of the weaker solutions would have a larger potential error in estimation. Thl. use of smaller samples and a higher electrometer sensitivity to give greater peak area would undoubtedly improve the accuracy of the results given in Tables I11 and IF7. Relative Acid Response. T o compare results from Ihe two mixtures of acids at different cmoncentrations t h e response per unit a e i g h t of acid has been computed relative to isovaleric The latter acid acid (Table V). could be used in many cases as a n internal standard in essentially aqueous solutions since i t is rarer t h a n the straight-chain acids and has an appropriate retention time in the analysis of either the 5horter or longer chain-lengths of acids. The retention times, relative to butyric acid, were 1.47 for the Silicone column, 1.23 for the Tween coluinn, and 1.36 for the NPGA column. On the latter two columns pent-oxone may, depending on operating conditions, conflict with either acetic 0 " propionic acids, but at 105" C. on the Silicone column i t falls between isovalerc acid and caproic acid and could be used as a more general internal standard in mixtures of acids. The results indicated in Table V are in reasonable agreement for the three types of substrate, and compare favorably with previously .eported data (5). However, the results of the present study indicating tha ; valeric and isovaleric acids give tl-e same response, in agreement with the current concept that paraffin branching has very little effect on response (6),suggest t h a t the previous results ma:< have been influenced by some adsorption of acids. The slightly lower relative response values for acetic acid on the Silicone column may indicate some retention on the column. Presimably the acetic acid present as an impurity in the formic acid would be adsorked to some small degree in a state of equilibrium with the formic acid, and this (equilibrium might
Table IV. Average Area Response, Relative to isovaleric Acid as 1 .OO, for Acids from 0.005-rnl. Samples" of Acid Mixtures of Different Strengths
Group I I1 I11
I I1 I11
Experiment
Acetic
Acid PropiIsoonic Butyric Valeric Caproic valeric
Column and conditions: Silicone Oil, 105" C.; helium, 16 cc./min. Acid concn. (g./lOO ml.) 1.0096 0.8496 0.8399 1.0286 1.0206 Av. relative response 0.38 0.60 0.73 1.03 1 .09 Std. dev. (4 runs) 10.01 f O . O 1 f O . O 1 fO.04 10.07 Acid concn. (g./lOO ml.) 0.3072 0.2676 0.3257 0.4150 0.4458 Av. relative response 0.26 0.48 0.74 1.07 1.22 Std. dev. (3 runs) f 0 . 0 1 f 0 . 0 2 f 0 . 0 2 fO.02 1 0 . 0 3 Acid concn. (g./lOO ml.) 0.101 0.085 0.084 0.103 0.102 Av. relative response 0.37 0.60 0.74 1.02 1.10 Std.dev.(4runs) 1 0 . 0 2 ztO.03 zt0.04 ztO.02 zt0.02 Column and conditions: NPGA, 105' C.; helium, 40 cc./min. Acid concn. (g./lOO ml.) 0.3072 0.2676 0.3257 0.4150 0.4458 1 .19 Av. relative response 0.36 0.51 0.75 1.07 Std. dev. (4 runs) 1 0 . 0 1 fO.00 1 0 . 0 1 1 0 . 0 2 4 ~ 0 . 0 5 Acid concn. (g./lOO ml.) 0.061 0.53 0.065 0.083 0.089 0.75 1.03 1.17 Av. relative response 0.35 0.51 Std. dev. (3 runs) 10.02 10.00 10.01 10.03 10.03 Acid concn. (g./lOO ml.) 0.012 0.011 0.013 0.017 0.018 0.78 1.06 1.19 Av. relative response 0.36 0.53 Std. dev. (2 runs) 1 0 . 0 8 1 0 . 0 1 1 0 . 0 3 =!=0.07 zkO.00
Column and conditions: Tween, 105' C.; helium, 30 cc./min. Acid concn. (g./lOO ml.) 1.0096 0.8496 0.8399 1.0286 1.0206 Av. relative response 0.47 0.64 0.79 1.03 1.12 Std. dev. (4 runs) ztO.03 f O . 0 2 1 0 . 0 4 f 0 . 0 2 1 0 . 0 2 Acid concn. (g./lOO ml.) 0.101 0.085 0.084 0.103 0.102 I1 Av. relative response 0.50 0.62 0.76 1.03 1.09 Std. dev. (3 runs) 5 0 . 0 1 zk0.02 fO.O1 f 0 . 0 5 1 0 . 0 2 I11 Acid concn. (g./lOO ml.) 0.061 0.053 0.065 0.083 0.089 0.74 1.08 1.22 Av. relative response 0.38 0.49 Std. dev. (4 runs) 10.02 f O . O O 10.03 f0.03 10.08 Excepting Silicone Oil, group I, where sample size was 0.001 ml. I
0.9992 1.00
...
0.3848 1.00
...
0.100 1.00
...
0.3848
1.00 ...
0,077
1.00 ...
0.015 1.00
...
0,9992 1.00 ...
0.100 1.00
...
0,077 1.00 ...
Q
S
S
Figure 1 . Double peak phenomenon A. onic
0.005 ml. 1% acid
propiin water a t
3
l05O C. B. 0.005 ml. 1% propionic acid in 9070 acetone a t 138' C. C. 0.005ml. 1 % propionic acid in 70% acetone a t 138' C. D. 0.005 ml. 2 1% propionic acid in 0 water a t 138' C. Tween $j
I
column with formic acid vapor in helium carrier gas flowing a t 60 cc./min. a t 105' C. and 16 cc./min. a t 138' C. S is acetone peak, numerals are C-numbers.
w
E
0
z:
=
J
3
d
-
+TIME VOL. 35, NO. 6, M A Y 1963
649
Table V.
Area Response, Relative to Isovaleric Acid as 1 .OO, of Equal Weights of Acids
Substrate Silicone Oil NPGA
Tween Literature (3) (Tween)
Group I I1 I11 I I1 I11 I I1 I11
Acetic Propionic Butyric 0.38 0.33 0.37 0.45 0.44 0.45 0.47 0.50 0.48 0.45
0.71 0.69
0.71 0.73 0.73 0.76 0.75 0.73 0.71 0.73
0.87
0.88 0.88 0.88 0.88 0.92 0.94 0.90
0.88 0.86
Valeric
Caproic
1.00 0.99 0.99 0.99 0.96 0.99 1.00 1.00 1.00 0.91
1.06 1.06 1.07 1.02 1.01 1.03 1.10 1.07 1.05
Isovaleric 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.oo
...
3 / 1
3
!
be upset through the water injected with the sample clearing the column of the adsorbed acetic acid. On the other hand, the retention time of this acid is very short on this column and a mechanical or systematic error in estimation is possible. Of the three sets of results for each substrate, those for the NPGA column are considered the most reliable, since the tailing of the peaks was minimal and the isovaleric peak was well separated from the butyric and caproic acid peaks. Double Peak Formation. I n analysis of t h e mixtures of t h e six acids with formic acid in t h e carrier gas a t 138' C. there appeared t o be a very satisfactory peak for each acid except acetic. On all three substrates this acid often appeared t o have two components, although no contaminant was present. Further experimentation indicated a new phenomenon which may be called double peak formation. Any volatile acid, or other material with short retention time, may give two peaks if the amount of water in the injected sample exceeds approximately 0.001 ml., although this is most marked with acetic acid. The effect of the amount of water on the double peaking of propionic acid is indicated by Figure 1. The double peak formation does not affect quantitative response, the sum of the two peaks remaining constant, and may be reduced or eliminated a5 shown by reducing the column temperature or decreasing the amount of water in the sample. I n addition to the occurrence with acids, this effect may also be observed with other materials in dilute aqueous solutions. Thus the pent-oxone solution on the Silicone column with formic acid vapor in the carrier gas, as mentioned previously, showed double peaking, and benzaldehyde in the absence of formic acid vapor in the carrier gas also gave double peaks on the polyester-phosphoric acid column. This may be a general phenomenon associated with analysis of aqueous solutions which does not appear to have been previously noted. DISCUSSION
6
A I5CI
I40
'30
TIME (MIN.)
'20
110
Figure 2. Comparison of analyses of 0.001-ml. samples of 1% each C2-Ca acid mixture with (bottom) and without (top) formic acid vapor in carrier gas Silicone Oil col., temp. 138' C., helium flowing a t 8 cc./min.
650
ANALYTICAL CHEMISTRY
S is acetone peak, numerals a r e C-numbers
-4 curious feature of the results (Table I) of analyses Tvithout formic acid vapor in the carrier gas, but with subsequent injection3 of formic acidwater mixture, is that the amount of response for the various ghost acids is inversely in order relative to the response from the original solution. This may arise from the higher volatility of the lower fatty acids, which would bleed more rapidly off the column than the higher acids once the water vapor from the initial injection has passed through. The formic acidwater mixture may then liberate a certain proportion of the remaining
3 I 2
Figure 3. Separation of acids with 0.001-ml. sample of 1 %each C2-Ceacid mixture NPGA col., temp. 105' C., formic acid vapor in helium flowing a t 60 S is acetone peak, numerals a r e C-numbers
cc./min.
adsorbed material, which would be roughly in the proportions shown, and subsequent injections of formic acidwater would continue the process. Alternatively, after the temporary displacement of the acids partial readsorption may take place, but in order of polarity, leading to th. same results. Since it was evident that formic acidwater mixture readily displaced other acids from the colurnn, even though phosphoric acid had keen added to the polyester to suppress tailing of the peaks, adsorption must continue to take place even in the presence of the latter acid. Varying results obtained with the other columna employed in this study indicate that the column is primarily responsible for the adsorption, although there may be some contribution from injection port deposits (27). The use of formic acid vapor in the carrier gas should also reduce the latter effect. The drastic shortening of retention time when formic acid vapor is employed with the Silicone Oil column (Figure 2) is not observed with either the Tween or NPGA columns. [n addition these latter substrates yidd much lower amounts of acids when formic acid vapor is added to the carrier gas subsequent to normal ustige, although the injection of formic acid-water mixture still displaces moderate amounts of acids unless formic acid has been employed in the carrier gas. The hydrophobic character of the Silicone Oil,
evidenced by the low carrier gas flow rates, would suggest that this substrate does not cover active basic sites in the support, whereas Tween and NPGA would have much greater covering power. Adsorption on the support with these latter substrates must then take place after transfer through the substrate and would be much less. In both cases the constant presence of formic acid would prevent adsorption of the weaker acids which are present in a lesser amount. The double peak formation with the volatile fatty acids may be due to excessive amounts of water vapor sweeping through the column and clearing active adsorption sites in the support, which are then free to adsorb part of the following acids which are in turn displaced by the formic acid vapor. It is significant that the first peak tails somewhat, while the second peak of the two is essentially symmetrical. In the analysis of very dilute solutions where large samples may be employed care must be taken to use conditions such that both peaks are visible and are included in the calculations. The use of completely inert supports (14, H),or even of a less basic type of support, might eliminate double peak formation with acids, but in practice with the columns employed in the present study appropriate operating conditions of low temperature confer added advantages in improving peak separation as well as eliminating double
peak formation with acids. The formation of double peaks with pent-oxone in the presence of formic acid vapor on the Silicone column (and with benzaldehyde on the polyester-phosphoric acid column with plain carrier gas) suggest, however, that a more complex general explanation involving the substrate may be necessary rather than the simple one advanced for the case of the acids. Formic acid is corroqive toward stainless steel, but with the XPGA and Tween columns the action of the acid becomes effective after a few minutes, permitting the use of plain carrier gas except when actually performing an analysis. The amount of formic acid passing into the column depended on the carrier gas flow rate b u t did not exceed approximately 0.5 ml. in 8 hours. There was some indication that with the Silicone column a higher level of formic acid vapor improved performance, hence actually bubbling the carrier gas through the acid is recommended in this case. The NPG*\ polyester n-as employed since the alcohol and acid components would yield only high-boiling products if decomposition occurred due to attack by the formic acid, although this did not apparently take place. Excellent separations of acids were obtained (Figure 3). The separation of isovaleric acid from butyric acid was somewhat less satisfactory with the Tween column, and heavy initial bleeding occurred. The recommended temperature limit for this substrate is 150" C. Analyses of acids up to nonanoic were performed on both NPGA and Tvieen columns a t high carrier gas flow rates a t 105' C. No estimate of column life or upper temperature limit could be made since performance did not change after several hundred analyses under the various conditions employed. The Silicone-stearic acid column was somewhat less efficient than the others with definite tailing observed. Although the stability of this type of column in ordinary usage has been criticized (19), many workers have satisfactorily used Celite 545 as a support and it is possible that a column with performance superior to that employed in the present study could be prepared. When using formic acid vapor in the carrier gas the addition of the stearic acid or other acid to the Silicone Oil would seem unnecessary. With the apparatus employed it is impossible to recover peak materials for alternative analysis to establish quantitative results. However the presence of formic acid vapor does not appear to interfere with linearity of response over a practical range of acid concentrations, and with this addition the reproducibility of diverse mixtures VOL. 35. NO. 6, MAY 1 9 6 3
651
of acids, and of a n acid with a neutral internal standard, coupled with the failure of the formic acid-water injection to displace acids, all indicate that the responses observed are in fact due to the entire sample and therefore quantitative. LITERATURE CITED
(1) Ackman, R. G., Retson, M. E.,
Gallay, L. R., Vandenheuvel, F. A., Can. J. Chem. 39,1956 (1961). (2) Dahmen, E. A. M., Chim. Anal. ( P a r i s )40.430 (1958). (3) Emery, E. k - K o e r n e r , W. E., ANAL.CHEM.33,146 (1961). (4) Erwin, E. S., Marco, G. J., Emery, E. M., J . Dairy Sci. 44,1768 (1961). (5) . , Ettre. L. S.. in “Gas Chromatoeraphy,” 3rd International Symposium, p. 307, N. Brenner, J. E. Callen, M. D. Weiss, eds., Academic Press, Kew York, 1962. (6) Fukui, K., Nagatomi, H., Murata, I
,
S., Bunseki Kagaku 11, 432 (1962); C.A. 57, 978b (1962). (7) Gehrke, C. E., Lamkin, W. M., J. Agr. Food Chem. 9, 85 (1961). (8) Hankinson, C. L., Harper, W. J., Mikolajcik, E., J . Dairy Sci. 41, 1502 119581. (91 Hunter, I. R., J. Chromatog. 7, 288 (1962). (10) Hunter, I. R., Ng, H., Pence, J. W., Ibid., 32, 1757 (1960). (11) Hunter, I. R., Ng, H., Pence, J. W., J . Food Sci. 26, 578 (1961). (12) Hunter, I. R., Ortegren, V. H., Pence, J. W., ANAL. CHEM.32, 682 (1960). (13) James, A. T., Martin, A. J. P., Biochem. J . 50, 679 (1952). (14) Jowett, P., Horrocks, B. J., Nature 192.966 (1961). (15) Karmen, A.; McCaffrey, I., Bowman, R. L., Ibid., 193, 575 (1962). (16) Knight, H. S., ANAL. CHEM.30, 2030 (1958). (17) Kumeno, F.. J . Aar. Chem. SOC. ’ J a v a n 36. 181 (1962). ” (18) ‘Lovelick, J: E., ‘ANAL. CHEY. 33, 162 (1961). (19) McInnes, A. G., in “Vapour Phase
Chromatography,” p. 304, D. H. Desty, C. L. A. Harbourne, eds., Butterworths, London, 1957. (20) Metcalfe, L. D., Nature 188, 142 119601. (21) Prevot, A,, Cabeza, F., Rev, Franc. Corps Gras. 8,632 (1961). (22) Ralls, J. W., ANAL.CHEM.32, 332 (1960). (23) Raupp, G., Angew. Chem. 71, 284 (1959). (24) Saroff, H. A., Karmen, A., Healy, J. W., J. Chromatog. 9,122 (1962). (25) Smith, B., Acta Chem. Scand. 13, 480 (1959). (26) Smith. E. D.. Gosnell. A. B.. ANAL. ‘ CHEM. 34,438 (1962). ’ (28) Ibid., (27) Swoboda, p. 646.P. A. T., Chem. Ind. (London) 1960, 1262. (29) Wilkens Instrument and Research, Inc., rlerograph Research Notes, Summer issue, 1961. (30) Ibid., May 1962.
RECEIVEDfor review August 31, 1962. -4ccepted February 18, 1963.
Paper Chromatography of Substituted Trinitrobenzenes DAVID M. COLMAN lawrence Radiation Laboratory, University of California, livermore, Calif.
b A rapid classification of the dominant chromatographic functional group in 18 substituted trinitrobenzene compounds can b e made from R p values by using two paper partition chromatographic systems. Symmetrical trinitrobenzene is used as a reference for the functional group classification. Supplementing the RF data with the results of various heat, light, and spray tests permits a complete identification of any one of the 18 compounds.
I
AN EARLIER communication (1) the chromatographic spectra of 14 substituted trinitrobenzenes in 10 different solvent systems were reported. I n this report a chromatographic method using two solvent systems, derived from the 10 systems studied, is described which, when coupled with simple heat, light, and spray tests, permits the identification of any one of 18 substituted trinitrobenzene compounds. The use of lJ3,5-trinitrobenzene as a reference allows a degree of classification as to the dominant chromatographic functional group in the compound from the RF value. Generally, special tests are required for the final identification of the compound. This procedure has been of value in the rapid identification of explosive
components. It has also served as a method for determining the purity of samples of any of the 18 compounds.
17174th St., Berkeley, Calif.). Spraying apparatus. Streaking and self-filling pipets. Long wave a n d short wave ultraviolet sources. Whatman S u m b e r 1 chromatographic paper, 18- x 21-inch sheets. Reagents. Polar immobile solvent, 25y0 formamide (Eastman Kodak White Label) in acetone (v./v.). This solution should be made just before
EXPERIMENTAL
Apparatus. Chromatographic tanks are of a n all glass construction. Impregnating troughs are all glass construction (Kensington Scientific Co.
N
652
ANALYTICAL CHEMISTRY
U
0
O,’t-
U
0
0.0
‘ 1 A U 1 1 1 I
I
I
1
1
PH3 PH2 PH P PH PH PH2 PH2 PH PH PH2 PH P$ OH (OH)3 CH3 NH2 OCH3 (OCH312 (OH02
COOH
NH2
OH
OH
OW3
(NHJ2
CI
1
1
P C13
P+ CH3
’
1
PH P (CH3)2 (CH&
I PH2 ”CH3 “‘2
”2
Compound
Figure 1. RF values of substituted trinitrobenzenes with formarnide- [cyclohexane/ benzene (1 / 1 ) ] system
