The intensities of the copper and iron lines are significantly increased for the reagent blank over those scattered by the Mylar alone. This increase is caused in part by trace elements in the reagents but also is caused by the greater scattering of the x-ray tube radiation by the magnesium sulfate ash. The copper and iron blank intensities were dependent upon the arrangement and amount of the magnesium ash on the Nylar film. l h u s the corrections for the blanks can be made by means of the cobalt internal standard. The xylene sulfonic acid makes the primary contribution to the iron blank, and therefore this blank remains reasonably constant for the same acid lot. The variation in the iron blank contribution is much less than the equivalent iron content in most FCC feed samples. Therefore a varying iron blank correction is not needed, but only a constant one (equivalent to 0.3 in the ratio R F of ~ the iron line to cobalt). Horrever, the copper acid blank contribution is comparable t o the contribution by the copper content of the samples, and therefore correction must be made for the copper blank for each sample. The correction for the copper blank contribution for samples is obtained with relation (6). This relation
is the equation for the correction curve made from copper and cobalt line intensity measurements on a series of reagent blanks with a suitable range of ash contents (magnesium sulfate). The copper blank correction computed with (6) is effectively subtracted from the total copper line intensity for an FCC feed sample in ( 5 ) , and the difference is the measure of the copper in the sample. This procedure yields the desirable detection limit for copper of about 0.01 12.p.m. and also improyes the preciion of the copper determination. A low cobalt content and the cobalt K alpha line are used for internal standardization in this trace metals method because a higher cobalt content map cause the cobalt K beta line to interfere somewhat with the nickel K alpha line measurement a t the trace nickel levels. The extent of radiation absorption or enhancement for the element-3 was determined at the trace levels to ascertain corrections needed in the procedure: The wavelength positions of the copper and nickel K alpha lines occur in the strong absorption region of iron whereas the internal standard cobalt K alpha line falls in the lo^ absorption region of iron. Thus the cobalt K alpha would h a r e some limita-
tions as a reference for the copper and nickel measurements, if no corrections were made for the iron absorption effect. The effect of the iron on the copper and nickel were sufficiently proportional to the iron content to be calculable for the method precision desired. The tests confirmed that the effect of iron on the vanadium trace determinations was not significant within the method precision, and no other interelement effects were significant. Consequently, the calibration data for copper and nickel were obtained each lvith iron a t the concentration levels of 0.1, 1.0, and 5.0 p.p.m. Sickel was suppressed significantly (in regard to the method repeatability) only a t about the 5-p.p.m. iron level, and copper was suppressed starting a t the 1-p.p.m. iron level. These effects are included in thc computation relations given under Procedure. LITERATURE CITED
(1) Hansen, J., Hodgkins, C. R., BXAL. CHEM.30,368 (1958). (2) Shott, J. E.,, Jr., Garland, T. J., Clark, R. O., Zbzd., 33,506 (1961). RECEIVEDfor review Map 7 , 1962. Accepted January 7 , 1963. Presented a t the 1962 Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, March 7 , 1962.
Microdetermination of Long-chain Carboxylic Acids Transesterification with Boron Trifluoride OLGA SALAME DURON and ALOE NOWOTNY' Department of Experimental Pathology, City of Hope Medical Center, Duarte, Calif.
A quantitative micromethod has been developed which is specific for the determination of long-chain carboxylic acids in materials containing acetyl groups. The method is based on quantitative transesterification with boron trifluoride dissolved in methanol. From the hexane extract of the methylesters the methyl acetate can b e removed at 67' C., a treatment which does not influence the amount of CS or higher fatty acid esters. For the quantitative determination of the methylesters the hydroxylamine-ferric perchlorate method of Snyder and Stephens was used. The lowest amount of long-chain carboxylic acids that can be determined accurately by this method is 0.1 pg.
B
long hydrocarbon chains are the only components characteristic of materials of a lipide nature, early lipide determination methods were ECAUSE
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ANALYTICAL CHEMISTRY
EXPERIMENTAL
tubes. Efficient reflux condensers with well fitting T 12/30 joints were obtained. All the glassware used in this determination had to be cleaned with chrom-sulfuric acid. The use of detergents occasionally led to errors. The boron trifluoride (BF3)-methanol reagent can be obtained from Applied Science Laboratories, Inc. (State College, Pa.), or prepared in t h e laboratory by dissolving BF3 gas in methanol to obtain a 14% wt./ivt. concentration. Reagent grade hexane n a s used to dissolve the methylesters Procedure. Transesterification of Long-chain Carboxylic Acids into their Methylesters. One milliliter of B F 3 methanol reagent was added to a 2- to IO-mg. sample of the d r y preparation in a 16- X 15O-mm. test tube with T 12/30 joint. The suspension
and Reagents. Test tubes used for t h e transesterification 12/30 were 16- X 150-mm. with joints, and for t h e ester determination, accurately graduated 10- X 120-mm.
1 Present address, Department of Microbiologv, Temple University School of Medic'ine, Philadelphia, Pa.
based on titrimetric ( 2 , ?) or oxydimetric (1, 5 ) measurements of the longchain carboxylic acid content. Recently, more sensitive hydroxylaminolytic methods (4, 8 ) have come into favor which are specific for esters regardless of their chain length. Many biological materials, especially bacterial antigens, contain 0-acetyl and N-acetyl groups which interfere viith the determination of true lipides. V o r k with gram negative bacterial lipopolysaccharides necessitated the development of a niicromethod which included the reliability and sensitivity of the hydroxylaminolytical methods while excluding the interference of short-chain carboxylic acids.
Apparatus
4.0 C u
z
I
2
3.0
hl
-
.-C
In C
0
.-a
2.0
V
u
:
-
.-
L
e
p
YI
W
1.0
L
0
c
0'
I
2
4
6
8
IO
Hours o f B F 3 - M e O H Treatment
0
Figure 1 . Transesterification of long-chain carboxylic acids present in a bacterial 0-antigen perparation with use of BFa-methanol reagent
was refluxed for 6 hours using a T 12/30 joint condenser on a hot mater bath. Before the tube was disconnected, the whole unit was immersed in ice water. X h e n the suspension was completely cooled, 4 ml. of reagent grade hexane was added and the tube \vas closed tightly and shaken for 30 seconds. After 15 minutes a t room temperature, 2 ml. of the clear supernatant was transferred into a 10- x 120-min. graduated test tube (Kimble 46350) to be used for ester determination. Determination of Total Methylesters in Hexane Extract. The reagents used were prepared according to Snyder and Stephens (8). Two milliliters of hydroxylamine reagent m r e added to the 2-1111. hexane extract, carefully mixed, and let stand at room temperature for 15 minutes. this the tubes were carefully immersed into a 67" C. mater bath. Because of the vigorous boiling of the hexane-hydroxylamine mixture during t h e first minute, the rack with the tubes had t o be lifted out briefly to prevent loss of the material from boiling over. Jl'hen the mixture simmered down, the rack was left in the bath for 15 minutes. During this time, the hexane evaporated completely, without any loss of the methylesters, which reacted very rapidly with the hydroi! laniine reagent. The final volume after 15 minutes a t 67" C. was 2 ml. Ferric perchlorate reagent was added to the cooled tubes until they were filled precisely to 7 nil. The purple color developed in 30 minutes at room temperature and mas read in the spectrophotometer a t 520 mp against a reagent blank treated exactly as the unknonn sample, using 2 ml. of reagent grade hexane instead of the 2-ml. hexane extract. Specific Determination of Long-chain .kid's Methylesters. This n as done after the removal of methyl acetate. Since hexane does not boil without the hydroxylamine reagent a t 67" C., hexane extracts in the open graduated test tubes could be placed in a water bath a t 67' C. and let stand for 60
D 20
40
60
120
1 eo
M i n u t e s i n 6 7 O C water bath
Figure 2.
Removal of methyl acetate from open tubes a t
67" C.
minutes. During this period the methyl acetate evaporated. Two milliliters of hydroxylamine were added to the cooled tube and the procedure was completed as described in the previous paragraph. Calibration Curve. Between 100 to 1000 pg. of known amounts of chromatographically pure palmitic acid were weighed into test tubes from solid material or from a standard stock solution made in reagent grade hexane. The dried samples were treated with BFs-methanol and hydroxylamine as previously described. The calibration curve obtained ga\-e a straight line. The amount of the fatty acids expressed as palmitic acid in the unknown sample can be determined by reading the absorbance from this calibration curve. The curve may be checked n i t h every determination of unknown material by carrying simultaneously a known concentration of palmitic acid. Calculation of the micro ester equivalents per milligram of material gives more exact information on a n unknown preparation than the carboxylic acid content expressed as percentage of palmitic acid, since the latter is merely a more or less correct average value. RESULTS AND DISCUSSION
The crucial point of this determination method is the quantitative transesterification of all carboxylic acids present in the highly intricate bacterial 0-antigen structure into their methylester derivatives. K i t h the method described herein, commercially available, chromatographically pure fatty acid preparations gave 98 to 1 0 0 ~ orecovery as niethylesters. It was more difficult t o follow quantitatively the transesterification in bacterial lipopolysaccharides, because no reliable data are available in the literature concerning the long-chain carboxylic acid content of complex natural products such as bacterial 0-antigens, mainly
because they could not be isolated in sufficient quantities for well established quantitative macro procedures. Micromethods such as hydroxylaminolysis, nhich give reproducilbe data when working with glycerophosphatides or brain glycolipides, TT ere unreliable n hen allplied to more complex materials. In this 11ork, therefore, the time necessary to obtain a maximal transesterification \\as followed. The results are shonn in Figure 1 where the time of transesterification was plotted again.t the amount of carboxylic acid niethylesters obtained. The maximum was reached in 6 hours. It is interesting to note t h a t after only a few minutes, two thirds of the total carbovylic acid content n as transesterified into methylesters. The transesterification of the laqt one third required approximately 6 hours, shoi\ ing a much stronger linkage b e h e e n these fatty acids and the rest of the lil,opolysaccharides. This ir in accordance with earlier observations (6). The total carboxylic acid content of the same 0-antigen preparation iva5 also determined by titration. Hydrolysis of approximately 5O-mg. lipopolvraccharide samples Ivaq carried out with 6N HC1 on boiling water bath for 8 hours followed by extraction of the liberated fatty acids with ethyl ether. The amount of extracted fatty acids was determined by acidimetric titration with 0.02N potassium ethylate in anhydrous ethanol, with phenolphthalein used as indicator. I n another series of experiments, the procedure of Haskins (3)was used in which liydroxylaminolysis was applied for the quantitative determination of carboxylic acid content in the same 0-antigen 1)reparation. Carboxylic acid content of a SerVOL. 35, NO. 3, MARCH 1963
371
Table 1. Carboxylic Acid Content of a Serratia Marcescens 0-Antigen Preparation Determined by Different Methods
Ester micro equivaMax. lent in deviation Method of 1 mg. of in 10 detn. material detns., % Titrimetric 0.80 =I=8 . 4 Hydroxylaminolytic 0.93 221.2 BFa, total acids 1.19 f 3.8 BFs, long-chain acids 0.86 =k 5 . 0
ratia marcescens 0-antigen preparation determined by different procedures is shown in Table I. The transesterification described converts the carboxylic acids into their methylesters, without any respect to their chain length. The differentiation between acetyl and long-chain (C, and higher) carboxylic acids is based simply on the different volatility of their methylester derivatives a t elevated temperature. In preliminary experiments, sam-
ples of pure methyl acetate and methyl caprylate and their equimolar mixtures were dissolved in hexane and heated in open test tubes in a 67’ C. water bath. After different time intervals, the ester content of the tubes was quantitatively measured by the Snyder and Stephens procedure (8). The results (Figure 2) show that methyl acetate completely escaped from the open tubes in 60 minutes, but the amount of methyl caprylate was unchanged after 3 hours. This method makes possible simultaneous determination of volatile and nonvolatile carboxylic acids. When the hexane extract of the transesterification mixture is divided into 2 aliquots, one may serve for the determination of the total carboxylic acids. The other aliquot after 60 minutes a t 67’ C. will contain only the methylesters of the higher carboxylic acids. Naturally other acids between Cz and Cs will not be removed completely during 60 minutes at 67’ C. Their residual amount can be measured as long-chain
methyl esters. However, the amount of these acids in bacterial lipopolysaccharides is very low. Whether the method described herein can be applied for the determination of fatty acid content of other lipides and lipide complexes is under further inyestigation. LITERATURE CITED
R.,J . Biol. Chem. 170, 671 (1947). ( 2 ) Bloor, W. R., Pelkan, K. F., iillen, D. M., Ibid., 5 2 , 191 (1922). (3) Haskins, W. T., AXAL. CHEW. 33, 1445 (1961). (4) Hill, U. T., IND.ENG.CHEM.,ANAL. ED. 18, 317 (1946). (5) Kibrick, .4. C., Skupp, S. J., Arch. Biochem. Biophys. 44, 134 (1953). ( 6 ) Nowotny, A., J . A m . Chem. SOC. (1) Bloor, W.
83,501 (inn" YUI).
(7) Page, E .. Michaud., L.., Canad. J . M . Sc. 29, 239 (1951). (8) Snyder, F., Stephens, K., Biochem. Biophys. Acta 33, 244 (1959). RECEIVEDfor review June 11, 1962. Accepted January 2, 1963. Work supported in part by Grant E-3849 from the U. S. Public Health Service.
Limited Area Flame Spectrometry Chemiluminescence BRUCE
E. BUELL
Union Research Center, Union Oil Co. of California, Brea, Calif.
Limited area techniques are utilized to investigate the excitation possibilities of the oxyhydrogen flame atomizing organic solvents. Chemiluminescence, or inner cone excitation greater than expected from thermal excitation, is studied by correlating excitation potentials and dissociation energies with height of maximum emission above the burner tip and solvent-enhancing factors. Over 600 atomic lines, with excitation potentials varying from 2.1 to 9.0 e.v., were recorded during this study, many for the first time in flames. Only selected lines are presented in tables and representative spectra. The tables establish conclusively that as excitation potentials increase, solvent-enhancing factors increase and height of maximum emission in the flame decreases until that for CO and CH emission is reached. Chemiluminescence and chemical reduction in the flame are offered as possible explanations for this and as a major contributor for large solvent-enhancing factors (established as exceeding 40,000-fold).
372
ANALYTICAL CHEMISTRY
C
has been established as occurring in hydrocarbon flames and hydrogen flames containing added organic substances and has been discussed and defined by Gaydon (9) as electronic excitation produced directly by chemical reaction. Chemical reduction in the flame followed by thermal excitation will not be strictly differentiated from chemiluminescence in this study and the approach used is to simplify rather than delve into details of complex-multiple reactions. To simplify further, the excitations studied are those occurring in the inner cone, which appear to be greater than expected from the thermal energy provided by the oxyhydrogen flame. Recent reviews by Gilbert ( l a ) on chemiluminescence and Mavrodineanu (17) on flame characteristics and emission discuss the history and development in this field, which appears to date back to 1877 but has been little used and poorly understood. Referring to the inner cone, Mavrodineanu says, “The analytical flame spectroscopist has found little or no interest for this HEMILUMINESCENCE
particular region of the flame, except for the efforts he is making to avoid it.” Perhaps this is partly due to limited knowledge of the inner cone excitations and poor dissemination of such knowledge to analytical spectroscopists. Inner cone excitation or cherniluminescence can be very useful not only for exciting elements such as tin, zinc, and nitrogen (via CN bands) which are not excited adequately otherwise, but also for increasing sensitivity. For example, upon atomization of cleaner’s naphtha into the oxyhydrogen flame, the height of maximum emission for lead a t 4057.8 drcps from 35 mm. to 17 mm. above the burner tip (in the top of the inner cone) and emission is enhanced 18-fold. The purpose of this investigation is to promote the use of and a better understanding of chemiluminescence. Previous work in this laboratory (4) reported a simple modification of a Beckman Model DU flame spectrophotometer for limited area flame spectrometry in conjunction with atomization of organic solvents into an
