in contact with the titrant. Also, in the 0.005M solutions, a precipitate begins to form after a few days nhich tends to interfere with proper drainage of the buret. For these reasons a fresh dilution from the 0.231 stock solution was made and restandardized each day. S.13.S. standard sample 166B has been run using the S a H C 0 3standardization procedure and crucibles which have been prefired in the induction furnace. Table I gives the results on this sample for several runs of different sample sizes. The sensitivity of the dilute titrant is about + 1 p g . of carbon. The 0.02Jf titrant is used a h e n total carbon was expected to exceed 200 pg. I t is comparable to the more dilute t i t r a n t and no unusual problems were
encountered in its use. The sensitivity was about +4 pg. of carbon. The general procedure works well for other samples and has been used for determining trace carbon in such metals as uranium, beryllium, and boron, as well as various alloys and ceramics. Sometimes extra flux or accelerator must be added to a prefired crucible to obtain proper burning characteristics and ensure complete combustion. I n this case the blank on the added material must be determined. The high oxygen flow rate, the continuous visual observation of the CO, as it is titrater', and the simplicity and sensitivity of the detection system afford a procedure well suited for trace carbon determinations in diverse ma-
terials and for investigational procedures for determining the proper conditions for the induction furnace technique. LITERATURE CITED
(1) Blom, L., Edelhausen, L., Anal. Chzm. Acta 13, 120 (1955). (2) Grant, J. A,, Hunter, J. A,, Mamie, W. H. S , Analyst 88, 134 (1963). (3) Patchornik, A,, Shalitin, Y., ANAL.
CHEM. 33, 1887 (1961). (4) White, D. C., Talanta 10, 727 (1963).
WALTER G. BOYLE,JR. FREDERICK B. STEPHENS WILLICVI SUNDERLAND Lawrence Radiation Laboratory University of California Livermore, Calif. WORKperformed under the auspices of the U. S. Atomic Energy Commission.
Flash Exchange Method for Quantitative Gas Chromatographic Analysis of Aliphatic Carbonyls from Their 2,4Dinitrophenylhydrazones SIR: Separating, identifying, and quantitatively determining the amounts of 2,4-dinitrophenylhydrazones ( D S P ' s ) in complex mixtures have been the subject of many techniques and investigations. I n one of the more promising methods, described by Ralls (8),the D S P ' s of volatile carbonyl compounds were pyrolyzed with CYketoglutaric acid, and the liberated carbonyl compounds were flashed into a gas chromatograph and separated on 307, Carbowax 2011 or 30Y0 LAC-446 columns. Ralls suggested that the method could be made quantitative, and, subsequently, Stephens and Teszler described a modified procedure in which formaldehyde D N P mas added to the mixture of derivatives in an effort to aid the "flashing" of liberated carbonyls into the gas chromatograph (10) equipped with a thermal conductivity detector. Standard deviations were reported for acetaldehyde D S P (*7 pg.) and propionaldehyde D S P ( k 5 pg.) over a range of 30 to 250 pg. of derivative with a recoverability of 104.3 f 0.57, of acetaldehyde. This procedure was extended to include acetone, isobutyraldehyde, 2-butanone, isovaleraldehyde, and valeraldehyde DXP's (1s) and applied to their quantitative determination in the steam distillates of various tobaccos. Further work in this area (f 2 ) was subsequently reported; honever, large variations in peak area were noted in addition to orcasional new unexplained peaks and it was felt that the method as described ( I d , I S ) did not provide the quantitative data
required. T o that end, a rigorous investigation of the various parameters involved in the analysis was initiated, using flame ionization detection. EXPERIMENTAL
Apparatus. An F and 11 Model 300 gas chromatograph equipped with a Model 1609 flame ionization detector a n d a l-mv. recorder was used. Helium, hydrogen, and oxygen flow rates were precisely controlled by Kupro Model 2SA very fine metering valves (Suclear Products Co., 15635 Saranac St., Cleveland, Ohio). Oven temperature was maintained a t 70" C., with the detector block at 175" C. a n d injector port at 165" C . A helium flow rate of 47.5 ml. per minute a t a column pressure of 15 p.s.i. was employed and hydrogen and oxygen flow rates were 80 and 350 ml. per minute, respectively. Of the large number of liquid substrates examined, a 16-foot X '/*-inch column of 8y0XE-60 and 127, QF-1 on Chromosorb P gave superior separation of the carbonyls of interest. However, when this column was heated above 125" C. and then returned to the operating temperature of 70" C., the retention times of the carbonyls decreased. Equilibrating a t 70' C. resulted in a gradual return to the original retention times (approximately 48 hours). This effect has been previously attributed to conformational changes occurring in liquid substrates, particularly XE-60 ( 2 ) . Materials. All carbonyl compounds were obtained from commercial sources and purified by repeated fractional distillation until gas chromatography indicated better t h a n 99% purity.
The 2,4 - dinitrophenylhydrazones (DNP's) were prepared by the method of Johnson (4) and each derivative was recrystallized several times from at least three different solvents. Melting points agreed with previously published constants (6). Standard mixtures of the D S P ' s were prepared by accurately weighing the derivatives of acetaldehyde (0.10000 gram), propionaldehyde (0.07030 gram), acetone (0.07030 grain), isobutyraldehyde (0.05620 gram), 2 - butanone (0.05620 gram), isovaleraldehyde (0.04750 gram), and valeraldehyde (0.04750 gram). These were then dissolved in reagent grade carbon tetrachloride and the solution was added to 2.0000 grams of Celite. Evaporation of the carbon tetrachloride with stirring, followed by oven drying (110" C,), gave the master analytical sample. Arbitrarily, seven replicate analyses were performed on several levels of sample prepared by mixing 1, 2, 4, or 6 mg. of the master D N P standard with sufficient Celite to give 8 mg. of sample. This provided five samples with the following representative ranges of carbonyl weight (micrograms) : acetaldehyde 8.42 to 64.32; propionaldehyde 7.36 to 56.22, acetone 7.35 to 56.14; isobutyraldehyde 6.89 to 52.61 ; 2butanone 6.89 to 52.61 ; isovaleraldehyde 6.59 to 50.37, valeraldehyde 6.59 to 50.37. The butyraldehyde D S P was separately weighed (5.25 mg.) and made up to 5.00 ml. with reagent grade carbon tetrachloride. This solution was freshly prepared every third day. Procedure. I n general, 8 mg. of t h e DX'P mixture was accurately weighed, 50 p l . of the butyraldehyde D X P VOL. 37, NO. 7, JUNE 1965
935
solution added as an internal standard, and the mixture oven-dried. To this was added the required amount of a-ketoglutaric acid and formaldehyde D N P (12) or 6.0 mg. of purified p dimethylaminobenzaldehyde mixed with 5.3 mg. of oxalic acid dihydrate. The latter reagents were found to effect maximum regeneration of the carbonyls from their DNP’s. After thorough mixing, the sample was flashed into the chromatograph as described by Stephens and Teszler (10). Detector response was measured in terms of peak height or peak area (peak height times width a t half peak height). RESULTS AND DISCUSSION
Under the conditions of the exchange reaction (IO), 3.9 mg. of a-ketoglutaric acid alone produced peaks with
compounds could be erroneously identified using this exchange mixture, and efforts were made to determine some new method for the regeneration of carbonyls from their DNP’s. A literature search revealed that several types of exchange reagents have been proposed or employed for such a regeneration : diacetyl in different acids (11); excess pyruvic acid ( 1 ) ; pyruvic acid in hydrobromic and glacial acetic acids ( 7 ) ; refluxing acetone-hydrochloric acid mixtures (3) ; formic acid and copper acetate (9); and levulinic acid-lOyo mineral acid (6). Whether the mechanism is one of hydrolysis and/or actual carbonyl exchange has not been established, although there seems to be general agreement that carbonyl groups, acid, and
retention times identical with those obtained for formaldehyde (1.84 minutes), acetaldehyde (4.17 minutes), and propionaldehyde (5.50 minutes). A large peak appeared a t 28.61 minutes followed by a smaller one a t 34.50 minutes, but the compounds responsible for these have not been identified. The presence of propionaldehyde could conceivably be attributed to the double decarboxylation of a-ketoglutaric acid. When the reaction was repeated with 0.5 mg. of formaldehyde D K P and 5 mg. of a-ketoglutaric acid, a marked increase in acetaldehyde and propionaldehyde was apparent, with the appearance of a new peak a t 5.66 minutes. The two peaks with the longer retention times remained unchanged (Figure 1). I t was apparent that with unknown mixtures of DPiP’s,
’OAMALMHYDE IX
ACETALOOCIDE
I
I X
1
1
I
I
1
1
1
I
\
1
1
1
1
1
1
1
1
PROPI)NAL#HY# I X
SIURLE: 39 m3 AlLRU-KETOQUT&C
0.5 Yo f O W c o ( y #
ACO
DNP.
i
X
-
2x
8 >-
1
i
I
I
I
1
5
I
I
Figure 1.
1
1
ANALYTICAL CHEMISTRY
*
1
I
I
I
I
Flash pyrolysis of 3.9 mg. of a-ketoglutaric acid Bottom.
936
I
10 25 RETENTION TIME IN MINUTES
With 0.5 rng. of f o r m a l d e h y d e DNP
1
30
I
I
I
I
I
35
I I
2-BUlANONE 3*2 X
ACETALDEHYDE
RETEMION TIME IN MINUTES
Figure 2. Chromatogram with DMAB-oxalic acid (6.3 to 5.0 mg.) as exchange reagents in flash exchange of 6.0mg. of DNP standard mixture and 2 mg. of Celite
water are required. A number of reagents were evaluated and it was found that a mixture of oxalic acid dihydrate and p-dimethylaminobenzaldehyde (DNAB) regenerated carbonyls from their DNP's efficiently and a blank run of the aldehyde-acid mixture showed only one small peak with a retention time similar to that of acetaldehyde. Since the area was small relative to the signal of the analytical sample, it was neglected. A chromatogram of the carbonyls liberated by this mixture is shown in Figure 2.
Table I.
Method Weight us. peak height, Yb
Weight us. area, Ye
Weight ratiod us. area ratio, Y
of known carbonyl area per area of butyraldehyde. I n computing calibration curves, it is usual to relate the response variable, Y (in this case, detector response), and the amount of known compound X. The regression line then determined by the method of least squares (14) takes the form
Reasoning that pyrolytic reactions are subject to certain inherent sources of error (differences in capillary packing, total sample size, etc.), an internal standard was employed-Le., butyraldehyde DXP-in an attempt to minimize such variations. The quantitative relationships were investigated as a function of the weight of carbonyl and (1)peak height, (2) peak area determined by the peak height multiplied by the width at half height, and (3) the ratio of known weight of carbonyl per known weight of butyraldehyde and the ratio
where g is the mean detector response, z is the mean weight of carbonyl, and b is
Statistical Relationships" among Carbonyl Weights and Peak Height, Area, and Area Ratio
Carbonyl, pg. hcetaldehyde Propionaldehyde Acetone Butyraldehyde %Butanone Isovaleralde hyde Valeraldehyde Acetaldeh de Propionadeh y de Acetone Isobutyraldehyde 2-Butanone Isovaleraldehyde Valeraldehyde Acetaldehyde Propionaldehyde Acetone Isobutyraldehyde 2-B utano ne Isovaleraldehyde Valeraldehyde
a
2
b
r1
S
S'
a
704.31 620.45 451.22 397.71 290,50 262.46 210.26 310.04 405.15 374.74 419.86 391.01 408.00 412.26 1.332 1.737 1.605 1 ,806 1,670 1.746 1.759
34,47 30.13 30.04 28.15 28.28 27.03 27,08 34.08 29,79 29,75 29.88 27,88 26.69 26.69 2.256 1.973 1.970 1.846 1,846 1.767 1.767
18.72 18.25 13.61 12,62 9.14 8.41 6.72 8.54 12,55 11.79 13.75 12.89 13.98 13.79 0.615 0,903 0.843 0.983 0.923 1,001 0.983
0.993 0,990 0.994 0.994 0.994 0,989 0.994 0.998 0,998 0.995 0.997 0,999 0,998 0.995 0.999 0.999 0.995 0,998 0,999 0,999 0,999
77.52 74.19 68.36 55.18 49 60 43.93 36 25 21.82 33.56 45 18 40 84 57 16 53 04 61 18 0.1129 0.1223 0 0945 0 1281 0 1435 0 1036 0 1693
4.14 4.06 5.02 4.37 5 43 5 22 5 39 2.56 2 67 3 83 2 97 4 44 3 79 4 46 4.87 3 59 2 97 3 45 4 12 4 56 2 76
1.98 2.63 4.66 3.79 7.18 7.32 8.16 0.70 1.03 2.36 1.51 4.09 3.02 4.70 0.84 0.58 0.39 0.61 0.90 1.23 0.42
a+
a General equation. Y = b (y - a ) , T * = square of correlation coefficient, s = standard error per determination in Y , S' s / b , and PZ = number of determinations required for true carbonyl weight d ~ l O 7 95% ~ , of time (cf. 14).
Peak height in cm. Area = peak height X width a t half peak height. d Weight ratio = weight of unknown per known weight of butyraldehyde, area ratio
=
b
c
~
=
area of unknown per area of butyraldehyde, ~~
VOL. 37, NO. 7 , JUNE 1965
937
the slope of the line. Measures of precision can then be determined in terms of s, the standard error per determination in the detector response, Y , at mean values of X and Y , and T * , the measure of goodness of fit around the line. The results are shown in Table I. In the weight us. peak height correlation, no isobutyraldehyde was present as in the other two methods. All of the r2 values show that the fit about the calculated line is satisfactory and in all three methods the standard error is approximately 10%. Without further calculations, it might be presumed that the three methods are equally precise, and, because the measurement of peak height is more convenient, that this technique would be satisfactory. However, the calibration curve given in Equation 1 is used for predicting the unknown weight of carbonyl, X’, as a function of the detector response, Y , resulting in the inverted equation
I n this form the standard error is now dependent on the carbonyl weight as well as random errors associated with the detector response, Y . Under these conditions, the standard error in Y does not apply and the standard error per determination in weight, s’, is determined as s’ = s / b , which in itself is a measure of the relative precision. The s’ values show that the peak height us. carbonyl weight is the least precise of
the three methods, the other two being apparently equal in precision. Another method of determining the relative precision of several methods is to compare the number of determinations, fii, necessary to obtain the weight of carbonyl within certain limits (14). In this case the limits were set such that the true weight of carbonyl +lo% would be determined 95% of the time. A comparison of methods then consists of comparing f i and s‘ and, as shown in Table I, the weight ratio-area ratio method is decidedly more precise than either the peak height or peak area relationship, the peak area being the next most precise. Thus one determination using the weight ratio-area ratio method will yield results which will include the true weight of carbonyl 95% of the time within +IO%. fi was calculated from data having the largest error. Presumably, a higher degree of precision could be calculated using carbonyl weight values close to the mean value. The use of the internal standard, butyraldehyde D N P , and the exchange mixture of oxalic acid-DMAB permits the determination of aliphatic carbonyls with satisfactory precision and it appears likely that the procedure could be applied to other analyses involving pyrolytic conditions for compound liberation. The statistical analysis described indicates that methods presented with nearly identical correlation coefficients do not necessarily reflect equal precision in the analytical procedures.
ACKNOWLEDGMENT
The authors are indebted to Sara Griffin and Katherine Harper for technical assistance in the accumulation of analytical data required for this work. LITERATURE CITED
(1) Anchel, AI., Schoenheimer, R., J . Biol. Chem. 114, 539 (1936).
(2) Chen, C., Gacke, D., ANAL. CHEM. 36, 72 (1964). (3) Demaecker, J., Martin, R. H., Sature 173. 266 11954). (4) Jdhnson,~G. D., J . ,4m. Chem. SOC. 73, 5888 (1951). ( 5 ) Jones, L. A , , Hancock, C. K., J . Org. Chem. 25, 226 (1960). (6) Keeney, > f . , ANAL. CHEM.29, 1489 (1957). (7) Mattox, V. R., Kendall, E. C., J . Am. Chem. SOC.70, 882 (1948); 72, 2290 (1950). (8) Ralls, J. W., A N ~ L CHEM. . 32, 332 (1960). (9) Robinson, R., lVature 173, 541 (1954). (10) Stephens, R. L., Teszler, A. P., ANAL.CHEM.32, 1047 (1960). (11) Strain, H. H., J . A m . Chem. SOC. 57, 758 (1935). (12) Weybrew, J. A., Jones, L. A , , Tobacco Sci. 6. 164 11962). 3) ’#&brew, J. ‘A,, Stephens, R. L., Ibid., 6, 53 (1962). 4 ) Williams, E. J., “Regression Analysis,” Chap. 6, Wiley, Sew York, 1959.
L. A. JONES R. J. MONROE martment of Chemistrv Ndrth Carolina State Uriiversity Raleigh, N . C. PUBLISHED with the approval of the Director of Research of the Xorth Carolina Agricultural Experiment Station, Raleigh, X. C., as Paper No. 1902 of the Journal series.
Spectrophotofluorometric Analysis of Nonfluorescent Compounds on Paper and Thin Layer Chromatograms SIR: Recently two articles appeared in the literature describing methods for the direct spectrophotofluorometric analysis of aromatic and aza heterocyclic hydrocarbons on thin layer chromatograms (6 8J. These techniques save many man-hours usually spent with the more conventional procedures involving spot extraction followed by spectral examination of the extracts. Direct spectrophotofluorometric analysis is not only applicable to the analysis of compounds that are naturally fluorescent in neutral, acid, or basic environment but should be usable also for those nonfluorescing compounds that can be made to fluoresce on paper or thin layer chromatograms by reaction with an appropriate reagent(s). Many methods for the fluorometric assay and/or detection of nonfluorescing molecules are available in the literature. 938
ANALYTICAL CHEMISTRY
Methods for formaldehyde and its precursors (5, 9 ) , estrogen steroids (Z), vanillin ( I ) , malonaldehyde ( 7 ) , and tryptophan (4, have been modified and shown in this communication to be amenable to the characterization of nanogram amounts on paper and thin layer chromatograms by direct spectrophotofluorometric techniques. EXPERIMENTAL
Apparatus. -411 spectral d a t a were obtained with a n Aminco-Bowman spectrophotofluorometer from the American Instrument Co., Silver Spring, M d . T h e instrument was equipped with a solid sample accessory and with a special holder for strips of glass, plastic, or paper. Slit arrangement No. 2 as described in the manual gave good resolution with minimum noise levels. Thin layer
chromatography plates were obtained from Custom Service Chemicals, Inc., Wilmington, Del. A11 other materials were obtained from the nearest commercial source. Reagents. Chemicals were commercially available and of known purity, or they were purified through distillation or recrystallization to a constant boiling or melting point. General Procedure. Satisfactory procedures are available for obtaining excitation and emission spectra directly from thin layers of adsorbents and on glass and plastic films ( 8 ) . These procedures were followed without modification in this investigation. Formaldehyde Procedure. T o a spot containing 2 pl. of 0.5% J-Acid (6-amino-1-naphthol-3-sulfonic acid) 1 pl. of an aqueous solution of formaldehyde was added; after the moist area stood for 2 minutes, the fluorescence spectrum was recorded.
