chromatographed on the isophthalic acid-Carbowax columns. With most of the compounds tested, this column behaved as an ordinary Carbowax column, the isophthalic acid having no apparent effect on the elution of such compounds. There was, however, one notable exception involving the nitrophenols which may possibly apply to other compound types as well: both ortho- and para-nitrophenol could not be eluted at any temperature on commonly used columns such as Apiezon or Carbowax on glass microbeads. But on the isophthalic acid columns, it was possible to elute only o-nitrophenol. Apparently, intramolecular hydrogen bonding allows the ortho isomer to be eluted on an acidic column.
LITERATURE CITED
(1) Ackman, R. D., Burgher, R. D., ANAL. CHEM.3 5 , 647 (1963). (2) Emery, E. M., Koerner, W. E., Zbid., 3 3 , 147 (1961). (3) Frederick, D. H., Miranda, B. T., Cooke. W. D.. Zbid.. 3 4 . 1521 11962). (4) Giddings, J. C., Zbid.,'35, 439 (1963). ( 5 ) Hardy, C. J., Pollard, F. H., J . Chromatog. 2 , 22 (1959). (6) Hunter, I. R., Ortegren, V. H., Pence, J. W., ANAL.CHEM.3 2 , 682-4 (1960). (7) James, A. T., Martin, A. J. P., Biochem. J . 5 0 . 679 11952). (8) Jowett, P., Horrocks, B. J., Nature 192, 966 (1961).
'R.A., Bate, R., Costa, B., Forman, P., J . Chromatog. 8, 157 (1962). (10) Keller, R. A., Stewart, G. H., ANAL. CHEM.3 4 , 1834 (1962). i l l ) Kirkland. J. J.. ANAL. CHEM.3 5 . 2W3 (1963): (9) Keller,
~~
(12) Macdonell, H. L., Xoonan, J. AI., Williams, J. P., Zbid., 3 5 , 1253 (1963). (13) McKinney, R. W., J . Gas Chromatog. 2 , 108 (1964). (14) Metcalfe, L. D., Suture 188, 142
(1960). (15) Nikelly, J. G., A N A L . CHEY. 3 4 , 472 11962). (16) ?;'ikelly, J. G., I b i d . , 3 6 , 2248 (1964). (17) Sawyer, D. T., Barr, J. K., Ibid., 3 4 , 1518 (1962). (18) Schmeltz, I., Miller, R . L., Stedman, R. L., J . Gas Chromatog. 1, 27 (1963). 119) Smith. Bennth. Acta Chena. Scand. 13, 480 ('1959).- ' (20) Vorbeck, M. L., Mattick, L. R., Lee, F. A., Pederson, C. S., Ibad., 3 3 , 1512 (1961). ~
RECEIVEDfor review June 27, 1963. Reeubmitted July 15, 1964. Accepted September 10, 1964. Division of Analytical Chemistry, 142nd Meeting, ACS, Atlantic City, N . J., September 1962.
Gas Chromatographic Determination of AcetyIsaIicyIic Acid J. G. NIKELLY Deparfment o f Chemistry, Philadelphia College o f Pharmacy and Science, Philadelphia, Pa.
b Acetylsalicylic acid can b e separated quantitatively on a l to 2-meter column of 0.2570 Carbowax 2 0 M and 0.4% isophthalic acid coated on acidwashed glass microbeads of 200micron particle size. The column is operated at 125' C. A calibration curve is necessary because of detector nonlinearity but the analysis requires only about 10 minutes and has a precision of 1% or better. The limit of detectability is about 50 p.p.rn. or 1 O-" gram,
-
A
ACID is usually determined by volumetric titration methods ( 1 , 6, 8 ) or by spectrophotometric methods ( 7 ) . I n all cases the analysis is time-consuming because some preliminary separation steps are usually required even when relatively pure acetylsalicylic acid is being analyzed. For example in the official C S P method (8) for the assay of aspirin tablets, three acidimetric titrations are required because of the interference of acetic acid which is usually present with acetylsalicylic acid. Furthermore, the above methods are not sensitive for low concentrations or small samples. The separation of acetylsalicylic acid by gas chromatography is difficult because of its low vapor pressure and its carboxylic acid functional group which interacts with the solid support causing adsorption and tailing. However, two gas chromatographic procedures have
CETYLSALICYLIC
2248
ANALYTICAL CHEMISTRY
been reported recently. I n the procedure by Crippen and Freimuth (2), the acid is first converted to the methyl ester by refluxing with boron trifluoridk and then chromatographed on a column of 3oyOCarbowax 20M a t a temperature of 175" C. I n the other procedure (S), Hoffman and Mitchell have demonstrated the separation and determination of acetophenetidin, acetylsalicylic acid, and caffeine in APC tablets by using a column of 2% Dow Corning 200 Fluid on tetrafluoroethylene polymer (Haloport F) with temperature programming. I n the present method, acetylsalicylic acid in the presence of other compounds dissolved in an anhydrous volatile solvent such as chloroform can be separated in a few minutes on a column which was developed for the separation of unesterified fatty acids (6). The column, operated at 125' C., is 1 to 2 meters long packed with 0.25% Carbowax 20M and 0.470 isophthalic acid coated on acid-washed glass microbeads of 200-micron particle size. Also, in the same run, the amount of acetic and salicylic acids which are usually present because of the hydrolysis of aspirin can be estimated. EXPERIMENTAL
Apparatus and Materials. A flame ionization gas chromatograph was used ( F & M Scientific Co., ,Model 609) with a recorder of 1-mv. full scale sensitivity.
I91 04
The glass microbeads used as the solid support were supplied through the courtesy of Microbeads, Inc., Jackson, Miss. The microbeads were pretreated by washing for a few minutes with 1% H N 0 3 followed by several rinsings, first with water and then with acetone. The choice of acetone for the final rinsings permits the rapid removal of solvent in the next step. Hot methanol was used to dissolve both the 0.4 wt. yo isophthalic acid (Fisher Scientific Co., Pittsburgh, Pa. 15219) and the 0.25 wt. % Carbowax 20M which were then added together to the glass microbeads. The packing was then heated and stirred until the solvent was completely volatilized. The dry and free-flowing packing was packed into a 2-meter column of '/(-inch aluminum tubing with vigorous tapping along the side of the tube until no more packing was accepted. About 52 grams of packing are required for a 2-meter column. The ends were plugged with glass wool. Procedure. A stock solution of acetylsalicylic acid in anhydrous chloroform was prepared from commercial 5-grain aspirin tablets which were pulverized in a mortar. Anhydrous chloroform was a suitable solvent because of the favorable solubility of aspirin (1 gram in 17 ml.). Aspirin is less soluble in other solvents such as acetone and methanol; furthermore, these cannot easily be kept anhydrous so that some hydrolysis of aspirin takes place, particularly in the heated injection port. This was evident by the appearance of the acetic and salicylic acid peaks when these solutions were chromatographed.
Table 1.
Peak Height Measurements of Standard Acetylsalicylic Acid Solutions
Concentration, mg./ml. Attenuation factor Peak height,o mm. 1. 2. 3. 4.
33.86 4
13.55 2
1216 1242 1232 1238 1232 20.3 1.6
596 588 604 610 600 6.9 1.2
5.424 1
2.210 1
0.900 1
254 107 44 __ 253 106 43 257 107 43 255 108 44 Mean 255 107 43.5 Std. error 1.6 1.2 0.5 % Std. error 0.6 1.1 1.1 0 Computed from the actual peak height multiplied by the attenuation factor. A C E T I C ACID
SALICYLIC
0
2 RETENTiON
4
6
a
Prepare a calibration curve by plotting per cent area or per cent height of the peaks vs. the concentration. Inject a n equal volume of the unknown solution and determine the concentration from the calibration curve.
TiME, MiN. RESULTS A N D DISCUSSION
Figure 1 . Chrornatcigrarns of aspirin in different solvents 2 - M e t e r column packed with acid-washed 200micron glass microbeads coated with 0.2570 C a r b o w a x 20M and 0..470 isophthalic acid. Column temp., 125' C. Injection port temp., 250' C. H e flow rate, 60 ml. p e r minute. Sensitivity, 8 X lo-" ampere full scale. Sample, 1 PI. o f 2 mg. p e r ml. The peaks labelled ospirin a r e acetylsalicylic acid
The above stock solution was standardized with the official USP method (8). A series of standard solutions was then made by successive volumetric dilutions of the stock solution, using anhydrous chloroform. One-microliter volumes of the standard solutions were injected with a Hamilton 10-fil. syringe (without a Chaney adapter). I n order to obtain reproducibility data, the runs were made over a period of six days, the instrument being turned off overnight and reset with the same conditions the next day. The height of the acetylsalicylic acid peaks was measured! to the nearest millimeter and normalized by using the height of the chloroform peak as an internal standard. The use of chloroform solvent as an internal standard was an acceptable alternative which eliminated the quantitative steps of adding an internal standard having a concentration and retention time similar to acetylsalicylic acid. The above procedure may be summarized as follows. Prepare and standardize volumetrically a stock solution containing about 30 mg. of acetylsalicylic acid per milliliter of anhydrous solvent. Prepare a series of standard solutions by volumetric dilution of the standard stock solution. Inject equal volumes of each of the st,andard solutions usi:ng the instrument conditions described in Figure 1 and determine the height or area of the solvent and acetylsalicylic acid peaks (retention time of acetylsalicylic acid, 4 to 5 minutes).
Typical Chromatograms. Typical chromatograms appear in Figure 1 in which t h e acetylsalicylic acid peaks are labelled aspirin. Although the column used is undoubtedly overloaded with respect t o t h e solvent, the acetylsalicylic acid peaks are symmetrical and well resolved. I n fact, it is apparent that a shorter column may be used to give adequate separation with a shorter analysis time, particularly if it is not necessary to estimate the acetic acid present in the sample; a shorter column would be even more susceptible to overloading by the solvent so that the solvent peak would obscure the acetic acid peak. The lower recording in Figure 1 shows the formation and separation of the acetylsalicylic acid hydrolysis products, acetic and salicylic acids. These are formed from the presence of water
10
20
30
CONCENTRATION, rnq./ml.
Figure 2. Calibration curve for acetylsalicylic acid The detector response i s deflned here as p e a k height in millimeters divided by concentration in milligrams p e r milliliter. In the concentration r a n g e o f interest, the response decreases with increasing concentration-Le., the detector i s nonlinear. The instrument conditions a r e as in Figure 1 , with chloroform as the solvent
in the ethanol solvent. Both peaks show tailing; typically, acetic acid tails more than other fatty acids (6). while salicylic acid tails presumably because of the interaction of the hydroxy group with the column packing. As a result of tailing, it was difficult to determine quantitahively the amount of acetic and salicylic acids in these solutions, especially a t the low concentration levels a t which they are normally expected. It is possible however to estimate the relative concentrations of these acids in different samples by comparing their relative areas as shown in Figure 1. Precision and Accuracy. The precision in the determination of acetylsalicylic acid was about 1%, based either on peak height or peak area measurements. The results for peak height measurements shown in Table I were obtained over a period of 6 days SO t h a t they may reflect the repeatability of instrument conditions. For accurate results, a calibration procedure is required because of nonlinearity in the detector signal for the concentration range used, 0 to 34 mg. per milliliter. This is shown in Figure 2 . For l-fil. samples in the concentration range 2 to 34 mg. per milliliterLe., up to the solubility limit of aspirin in chloroform, the detector response, peak height per mass, is not independent of the mass injected. Below 2 mg. per milliliter the response is apparently independent of the injected mass,-i.e., the signal is linear. However, this concentration range is near the lower concentration limit of detector sensitivity for 1-fil. samples. Larger sample volumes were not tested. Under the conditions used in this work the point a t which the detector departs from linearity corresponds to about 10-8 gram. This amount is considerably lower than the corresponding amount reported for propane in propane-nitrogen mixtures for which the detector is linear up to 10-4 or 10-3 gram (4). However, this difference may be caused by column overloading as well as by differences in molecular weight and ionization efficiency. VOL. 36,
NO. 12, NOVEMBER 1964
a
2249
LITERATURE CITED
(1) ~ssociat,ion of
Official A4gricultural Chemists, "Official Methods," 9th ed., pp, 4R1-g3, 502, Washington, D, C,, 1960. ( 2 ) Crippen, R. D., Freimuth, H. D., ANAL. CHEN. 3 6 , 273-5 (1964).
(3) Hoffman, A. J., Mitchell, H. I., J . Pharm. Sci. 52, 305-6 (1963). ( 4 ) Instruction Manual, Model 609, F & AT Scientific Corp., Avondale, Pa. ( 5 ) National Formulary, 11th ed., PP. 19-20, American Pharmaceutical Association, Washington, D. C., 1960. (6) Xikelly, J. G., ANAL.CHEM.3 6 , 2244 (1964).
(7) Snell, F. D., Snell, C. T., "Colorimetric Methods of Analysis," Vol. 11, p. 385, Val. 111, pp. 412-13, Vol. IIIA, pp. 387-90, Van Nostrand, Xew York, 1946. (8) r.s. PharmacoPea, XVI ed. PP. 20-1, USP, Washington, I). C., 1960. RECEIVEDfor review July 15, 1964. Accepted August 26, 1964.
Sulfonation and Dehydrogenation as an Aid to Gas Chromatography in Evaluating 90" to 150" C. Petroleum Fractions W. J. HINES and D. E. SMITH Phillips Petroleum
Co .,
Bartlesville, Okla.
b A combination of sulfonation, catalytic dehydrogenation, and gas chromatography is used to analyze petroleum fractions through about Clo. For a 90" to 150" C. fraction, procedures are described to determine most of the individual components through n-heptane, individual aromatics through CScompounds, ethylcyclohexane and the four isomers of dimethylcyclohexane (cis and trans structure combined), combined total of CS and Clo aromatics, individual n-paraffins, and an approximate carbon number distribution of the remaining paraffins and naphthenes above C7 compounds. A portion of the original sample is sulfonated to remove aromatics, dehydrogenated to convert ethylcyclohexane and the disubstituted cyclohexanes to aromatics, and again sulfonated to determine the amount of ethylcyclohexane. Chromatograms are obtained at each step using a capillary gas chromatography column. Calculations are made by comparing peak areas on these chromatograms to the peak areas on a chromatogram of the original sample. This combination of techniques is used to obtain both qualitative and quantitative analyses of the samples of this type.
Several techniques for the analysis of petroleum fractions were investigated. Sulfonation has long been a standard procedure in determining aromatics in petroleum fractions. Rampton (11j and Polishuk, Donnel, and Wood (10) have described the selective dehydrogenation of cyclohexyl rings to aromatics. This was successfully used by Howard and Ferguson (6) and Cousins and Chaney ( 1 ) in developing mass spectroscopic methods to differentiate between cyclopentyl and cyclohexyl naphthenes. Polgar, Holst, and Groennings (9)e mployed long capillary gas chromatography columns to resolve the cyclohexanes in the Cs range.
UARTZ PELLETS
H
TALYST TUBE
FURNACE
T
HE VALUE of gas chromatography as a means of analyzing petroleum fractions has been adequately demonstrated in the literature. Examples of this application of gas chromatography are given in the reviews of Le Tourneau (7j, and Dal Nogare and Juvet ( 2 ) . However, the large number of components present in even relatively narrow boiling ranges of petroleum has caused difficulties in obtaining quantitative data for specific components in the C7 and Cg boiling range.
2250
ANALYTICAL CHEMISTRY
COLD WATER
r
i4/35 JOINT
Figure 1
.
Dehydrogenation apparatus
This paper reports the results of combining sulfonation and dehydrogenation with gas chromatography to increase the utility of the latter technique in the analysis of petroleum fractions boiling in the range of 90" to 150" C. EXPERIMENTAL
Apparatus. A Perkin-Elmer Model 154B equipped with a flame ionization kit and a 150-foot X 0.01-inch i.d. stainless steel column coated with squalane was used for this study. The sample splitter was of concentric tube design and was controlled by an Ideal valve with a micrometer handle. T h e injection port and inlet line to the splitting chamber were heated to 200" C. and the column was operated a t 35" C. The hydrogen flame ionization detector had a polarizing voltage of 300 volts across the electrodes. The detector response was coupled to an ionization detector amplifier. The output of the amplifier was fed in parallel to a Brown 5-mv. recorder with a 1second full scale pen speed and a digital chromatograph readout system, Model CRS-1, Infotronics Corporation. Figure 1 is a block diagram of the dehydrogenation apparatus. I t was equipped with a motor driven syringetype charging device and a cold collection trap. A 500-watt single-wound furnace drawing 5.6 amperes was used. A borosilicate tube Rith a 10-cc. catalyst chamber was modified so the effluent vapors were carried directly into the collection trap which was cooled in an ice bath. This modification minimized entrainment of hydrocarbon vapors in thp vented hydrogen stream. Reagents. The sulfonation acid wa? prepared as described in test method ;\ST11(TI 1019). Platinum(ic) chloride was obtained from Fisher Scientific Co. The catalyst support was Columbia activated carbon, Grade L, 20 to 30 mesh. Products of sulfonation were removed from the samples by
