Table 111.
Tests with Sea W a t e r
English Channel, 9/24/62 Kitrate Found, pg. atom S,' liter Hydrazine Sample reduction T.V. no. Added (6) method 2 5 1 9 2 0 11 5 11 7 10 4 0 22 2 6 10
6 8
0 10 0 10
13 3 14 3 14
3 5 4
1 0
12 4 14 3 13
8 3 3 9
6
SEA ~I'ATER. Results for samples from the English Channel with salinity approximately 35°/00 are given in Table 111. S o completely satisfactory alter-
native method for nitrat'e in sea water is a t present available; the Tvidely used reduction m-ith hydrazine to nitrite (6) !vas employed. Since it gave less consistent results than the ultraviolet method, the agreement between the methods can hardly be expected to be very close. Reproducibility. I n the range 0.03 to 3 p.p.m. of SOa-S, (1-cm. cuvette) the standard deviation was =0.05. I n the range 0.25 to 25 pg. atom of K03-IK per liter (9-em. curette), the standard deviation was 0.4.
*
Kote: The sulfuric acid solutions on which absorbance meawwiients are made are viscous: highly refractil-e. and saturated with hydrochloric acid. Care must be taken. in transfer to cur-ett'es. to avoid "schlieren" clue to imperfect mixing, and bime may be niwled to allonsmall bubbles to clear.
LITERATURE CITED
(1) .%rmstrong, F. -4.J., Boalch, G. T., J . Slorine Bzol. dssoc. U.K. 41, 591-7
(1961).
( 2 ) Bastin, R., Keberling, T., Palilla, F., AN.41.. CHEM. 29, 1795-7 (1957). ( 3 ) Goldman, E., Jacobs, R . J , J . - 4 ~ 1 . W a t e i W o r k s Assoc. 53,187-91 (1961). 1 3 ) HoathPr. R. C . Rackham. R . F.. d n ~ ( u s84, t 548-51 (1959)
( 5 ) Scott, IT I T , "Standard Method8 of Chemical .%nalvsis,'' 5th ed., Vol. 2 , I) 2076, Tan Sostrand, Nen- Yorli, 1944.
(6) Strickland, J. D. H., Parsons. T. R., Bull. Fisheries Res. Board Can. 125, 61 11960).
F. A . J. ARMSTROXG
3Iarine Biologicla1 ;Issociation of the United Kingdom Citadel Hill Plymouth, England RECEIT E D fc!r revim December 26, 1962. .-lccepted IIal-13. 1963
2-Methylalkanoic Acids as Internal Standards in Gas Liquid Chromatographic Assay of Fatty Acids SIR: Gas liquid chromatography (GLC) has proved to be a rapid technique to determine the relative composition of fatty acid components. as well as their radioisotope levels, in lipid mixtures often weighing lesq than 100 pg. It is frequently of interest. however, to calculate absolute levels of fatty acids in mixtures, but standard titrimetric or gravimetric procedures are difficult Then assaying microgram quantities. Through the addition of a known amount of an internal standard to mixtures of fatty acid e*ters prior to GLC, we have found it relatively simple to determine microgram levels of fatty acid components. The advantages of an internal standard obviate volatilization of solvent or loss of sample a t the injection ports, both of nhich are frequent critical events when using microsyringes to inject samples quantitatively. Thus. calibration of recorder response to known amounts of fatty acid esters iniected is not necessary. I n the course of studies of the properties and metabolism of 2-alkylalkanoic esters synthesized in our laboratory (f), we have found that the nonnaturally occurring 2-methyl homologs of dc decanoic, tetradecanoic, and hexadecanoic acid have retention times that are distinct from common, naturally occurring fatty acids when assayed by GLC with 17% polyethylene glycol succinate (EGS) columns. Methyl substitution a t the a- or 2-position results in a relative retention time (0.879) t h a t is distinctly less than that of the unsub1294
ANALYTICAL CHEMISTRY
stituted straight chain acid (Figure 1). This would be in contrast t o naturally occurring fatty acids having methyl substitution at the ultimate (iso) or penultimate (anteiso) positiontraight chain acids (2, 5 ) . The decreavd relative retention times on polar polyester liquid phases resulting from subditution of alkyl groups a t the 2-position wggebt that diminished polarit\- j-teric hindrance) of the ester grouping outn eigli. the increase in molecular n eight or number of carbon atom< 1. elpected. honever, a plot of the logarithm of the retention time 2's. the number of carbon atoms for the 2-methyl fatty acid esters is linear. K h e n EGS columns are employed under conditions noted in Figure 1, the separation factor (5) hetneen the
2-iiiethj-1 substituted acids and their uii.;ub.tituted analogs-e.g., retent'ion time of laurate to retention time of dl2-methyllaurate-was found to be 1.14 for the three derivatives studied, as compared. for example, to a separation factor of 1.20 betiveen oleabe and Aearat'e. Figure 2 illustrates GLC of microgram level* of fatty acids obtained from 34 mg. (wet weight) of a human intestinal biopsy (courtesy of Dr. Robert J. Bolt,). The lipids were extracted (3) and the fatty acid methyl esters prepared for GLC by tranqesterification with methanolic-HC1 ( 6 ) . The dried (in va.cwo) fatty acid esteri were dissolved in 50 pl. of hexane containing dl-2-methylmyristate (100 mg. per 100 ml.), t'hen injected into the GLC apparatus. The relative composition of the fatty acids was then
Figure 1 . GLC of the methyl esters of dl-2-methyl substituted laurate, myristate, and palmitate as compared to the unsubstituted normal homologs. in., 1 7y0 EGS on 80-1 00 mesh Chromosorb-W; tritium ionization Column conditions: 185', 8 ft. X detector (220'C.) and argon inlet pressure 26 p.s.i.
(4). Thus, by the addition of a known amount of the methyl ester of the standard to known amounts of lipid mixtures, the effectiveness of methylation (or transesterification) techniques and recovery of components through GLC may be determined. ACKNOWLEDGMENT
Figure 2. GLC of the fatty acid esters prepared from a human intestinal mucosal biopsy with a d d e d dl-2-methylmyristate as an internal standard
The author is indebted to Kathryn Howell for technical assistance and t o H. Marvin Pollard for encouragement and support.
GLC conditions same as Figure 1 LITERATURE CITED
computed by planiinetiy, and the microgram levels of each fatty acid were t h u i obtained b y comparison of the area of each component t o the area of the added 2-methylmyristate of known concentration. I n Figure 2, for example, palmitic acid was found to represent 40.2070 of the total fatty acids and to have an area 1.81 greater than the 50 pg. of added internal standard. Thus, 90.4 pg. of palmitic acid and 224.0 pg. of total fatty acids (0.66%) were ontained from the biopsy. Similarly. spwific activities of fatty acids may also be readily determined n hen approprilite collection devices are used for radioassay of GLC fatty acid fraction.. . Injection of k n o w mixtures of 2inethyl substituted arid straight chain homologs demonstrated equivalent dotcctor rwponcc, \\liic I permit- direct
comparison of areas of the 2-methylmyristate with those of the normal qeries of fatty acids. Although the separation factor of the 2-methyl substituted acid us. the normal acid is not as high as one observes for stearate us. oleate, for example, the unsubstituted acid-e.g., myristate, Figure 2-is readily discernible if preaent in any significant amount i>2%). The 2-methyl substituted acid to be used as an internal standard may b e d he evaluated in terms of GLC conditions and the fatty acids to be assayed. Although our principal interest in the use of a n internal standard has been to determine the level of fatty acid components in small tissue samples (10 to .50 mg. wet weight), we also found i t r onvenient to employ such standards a- a routine control of GLC procedures
(1) Cason, J., Allinger, K. L., Williams, D. E., J . Org. Chem. 18,842 (1953). ( 2 ) Farquhar, J. W., Insull, W., Rosen, P., Stoffel, W., Ahrens, E. H., Nutr. Bee. 17, No. 8, Part 11, 1 (1959).
(3) Folch, J., Lees, M., Stanley, G. J . Biol. Chem. 2 2 6 , 497 (19c7). (4) Gehrke, C. W., Goerlitz, D. ANAL.CHEM.35, 76 (1963). (5) James, A. T., Martin, A. J. Biochem. J . 6 3 , 144 (1956). ( 6 ) Stoffel, W.,Chu, I., Ahrens, E. ANAL. CHEM.31, 307 (1959).
H.,
F.,
P., H.,
EDWARD A. NAPIER,JR. Gastroenterology Research Laboratory Lniversity of Michigan School of Medicine Ann Arbor, Mich. RECEIYED for review February 25, 1963. Accepted May 27, 1963. Work supported in part by the American Cancer Society (VM CRI-39) and the USPHS (C-3312).
Fibrillar Boehmite-A New Adsorbent for Gas Solid Chromatography SIR: -1 ncw fine alumina, fibrillar colloidal boehmite ( f - 4 j 6 ) , has been found to lie a usef;d adsorbent for carrying out) certain scparations by gas solid and modified gas solid chromatography, using either pecked or capillary columns. This t'ype of alumina (Baymal Colloidal .ilumintt, trademark regiptercd by E. I. d u Font de Semours & Co.. Klmington, Del.) consi sinall aggregates which arc composed of a m a s of intei.locked, alumina fibrils, each of which is about 50 A. in diameter and over 1000 &I.long; each fibril is a crystal of boehmite, AIIO(OH). Thc surfaces of the fibrils arc modified by acetate ions which, tiowever, may be removed or replaced by other types of inaterials. The unique gas chromatographic properties of this material apparently are primarily due to the high specific surface area of AlO(0H) (about 2T5 sq. meters per gram), the crystal-
line nature of the alumina, and the relatively large pores between the fibrils. This type of fibrillar boehmite, in the form of granules, was used as an adsorbent medium in packed columns for separating low-boiling materials, w c h as hydrocarbons, fluorocarbons, and fixed gases. Table I lists partial results of the gas solid chromatographic ,eparation of 0.25 cc. of natural gas using a Perkin-Elmer Model 154-B gas chromatograph with I-meter, 31'16-inch i d . , stainless steel columns packed with 80- to 100-mesh fractions of the various materials indicated. All runs were made n i t h a column temperature of 60' C. and a carrier gas flow rate of 50 cc. per minute of helium. Kornial and isobutane data were selected for presentation because it is possible to calculate meaningful separation factors for these close-boiling compounds. Data from separations carried out on Alcoa F-20, a
typical y-alumina, are included for comparison (9). The data in Table I show that the Baymal .410(OH) surface, particularly when the acetate ion has been removed, exhibits some unique adsorption properties. Xormal butane is more -trongly retarded by this surface (colunin 2), a i judged by retention volumes, cc. per gram of packing, than by F-20 y-alumina (column 1) or yalumina prepared from fibrillar boehmite (column 4). This is albo demonstrated in the higher separation factor shown by the fibrillar boehmite for the i-,'n-C4Hlo pair. Xlthough the y-ahmina packing in column 4 demonstrates a separation factor nhich is identical to that of conventional y-alumina (column l), only half the time is required for an equivalent analysis. The reasons for the variation of the efficiency of Columns 2-4 are not comVOL. 35, NO. 9, AUGUST 1963
* 1295
