(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 added 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
Table I.
Separation of n- and ;-Butane in Natural Gas
Surface area, Col no.
wt.,
1
Column packing ?-Alumina, Alcoa F-20
2
Fibrillar boehmite with acetate ions
7.4
3
Fibrillar boehmite, without acetate ions
5.3
4
-,-Alumina from fibrillar boehmite
5.4
b
grams 12.4
Treatment Elutriated in water, dried at 115" C. in vacuo for 16 hr . Baymal elutriated in methanol, dried at 105" C. in vacuo for 16 hr. Baymal heated at 300" C. for 64 hr., soaked in distilled water for 1 hr., dried for 3 hr. at 115' C. in vacuo Baymal heated at 600" C. for 16 hr., soaked in distilled water for 1 hr., dried for 2 hr., at 115" C. in vacuo.
Retention mz/ times,b minutes grama in161 14.5 17.5
Theoretical plates, 2-
790
Reten tion
Separa-
packing in56.5 68.6
factor, i-/n1.21
vol., cc./gram
tion
271
5.3
6.4
1060
34.6
40.2
1.21
256
6 4
8.6
660
56.6
79.3
1.3s
259
3.2
3.6
790
2 5 , %5
29.2
1.12
Nitrogen flow technique (8). Air retention time, 0.45 minute.
pletely understood, since there is little change in the physical shape of the gel particles or individual fibrils brought about by the heat treatments employed (4, 7 ) . Presumably, the acetate-modified boehmite surface in column 2 has a much narrower range of active adsorption sites, energy-wise, ~. than the other materials. The acetate groups are probably distributed on the surface of the boehmite as a monomolecular layer with the methyl groups oriented outward. This would result in a more homogeneous adsorption-desorption process with attendant higher column efficiency, The significantly lower efficiency of column 3, fibrillar boehmite without acetate, probably represents the opposite situation in which a wide range of sites of varying energies contributes to a more heterogeneous adsorption-desorption process. The intermediate efficiency of column 4, y-alumina prepared from fibrillar boehmite, might reflect the narrowing of range of active sites of the parent material, through elimination of chemicallybound water from the surface. The ease with which fibrillar boehmite can be deposited onto a variety of surfaces (4, 6 ) , makes i t a n attractive adsorbent for preparing custom surfaces for gas solid and modified gas solid chromatography. Figure l a shows the separation of a prepared mixture of Freon fluorocarbons 12, 114, and 11 (registered trademarks of E. I. du Pont de Nemours & Co., Wilmington, Del.) carried out on a 1-meter column of Baymal-treated GC-22 firebrick Super Support (The Coast Engineering Laboratory, Hermosa Beach, Calif.), operated at 100' C. 30separation of the fluorocarbons was found with a n untreated column of GC-22 under the same conditions. The support was treated by slurrying 50 grams of the firebrick with an excess 1296
ANALYTICAL CHEMISTRY
of 27, Baymal sol (6) for about 15 minutes. After filtering off the excesol, the wet cake was dried for 2 hour. at 110" C.; it was then ~ a s h e c lwith
UNMODIFIED
about 4 liters of dihlled water, and dried a t 110" (', for 3 hour.;. Duplicate -urface area meaqurements (B.E.T. method) carried (Jut on the untreated
STEARIC ACID MODIFIED
COLUMN TEMP-1009:
COLUMN TEMP-SVC
F-12 F- 12
W
'-114
v)
z 0
I I v)
114
W
a a W
L3
e 0 0
w
a F-ll
1 0
i 60 90
0
60 90
RETENTION TIME, seconds
la
b
1 .
Ib
Figure 1. Separation of Freon fluorocarbon mixture with packed columns of fibrillar boehmite-coated firebrick Sample, 10 pl. of gas mixture; carrier, 50 cc. per minute of H e
siipj)ort gave 2.22 and 2.46 sq. meters per gmi. The treated support had a specitic surface area of 12.84 and 1:3.0.7 q. meters per gram, demonstr:itiiiy thr 4gnificant increaAe in siirfsrr cuustd by the addition of the fi I)rillar borhriiit,t.. The surfact. of fibrillar boehmite can tic, rq)idly and conwniently modified with a vciritty of organic and inorganic 4 ) s t a i i c r s ( 2 , 6 ) . The curve in Figure l b illustrates the separation of the Freon fluorocarlmi mixture with a ~ ~ : ~ ~ , i i ~ ~ GC-22 l - ~ , ~ )column a t , ~ , ~which has heeri c*heruically modified with stearic acid. The dractivating effect ot' the stearic acid permitted the colrinin to accomplish n separation at 5 0 3 C., siniilar to th:it found for the uiiincitlitirti ~ d u i u nopwated at 100' C. (Figure l u j . Sorile effect on the relative retentioil times for the vari(xis roiul)oiwrits is also ;een, no doubt rcprescwting the influence of the organic iimiificr. Thc~ 12ayinai -trea :ed GC-22 fire1)rit.k tltwril)rd a h v c i ~ a 5modified by refluxing 10 grains of this material in 1 ,> nil. oi iiiethanoi. cmcmtaining 1 gram of straric w i d , for 4.5 minutes. The solid was filtered off and heated to Imilinq in 135 mi. or' methanol three t i t i w ~ ,rat41 time filtering off the solids :iftc>v thr treatiiient. The final mate. . rxi! w n . ~ xir-dried, then heat'ed a t .iO" C. !'or 1 ti holm in vacuo. The fin:]! > r c w i c a ~~,~id-ninc-lified material had n >urf:iw :iiwi of 10.3 q.meters per g ~ ~ i nTiit, . .;traric i: - i i d i i probably c1irwiwrt)t.d us a11 ~ i r m t e dnionomoleriilur !a~.ero n the surracc of the boeh-
F-I2
--
F-ll
I_ 0
h 60
RETENTION T I ME,
I20
ACKNOWLEDGMENT
The author is indebted to John Bugoih, R . Iiirfatainedwith a blank capillary cleaned identically, but containing no Baymal. Similar preparations were successfully carried out using 0.01-inch i.d. glass capillaries which were likewise coatcd with 5 5 Baymal sol.
Figure 2. Separation of Freon fluorocarbon mixture with fibrillar boehmitecoated gas solid chromatography capillary column Sample, 0 . 6 pl. of gas mixture; column temp., 2 2 ' C.; flow rate, 3.6 cc. per minute of He; splitter ratio, 27: 1 ; flame ionization detector, full sensitivity
=1 25-ft. length of 1 16-inch o.d . 0,020-inch i d . , stainless steel capillary tubing was cleaned ( 5 ) . and completely filled with a i%aqueous sol of Baymal from a reservoir using nitrogen preswre. in the usual manner for preparing capillary column*. (Yote: When preparing longer or kmaller i.d capillaries, a 4 t o 57c sol of lower visco-it? may be required ) The capillary inlet pressure was then adjuqted t o elute the sol at a rate of about 1 drop every 3 minutes. After about 1.5 hours. all excess sol nas removed. The n e t capillary was then dried OT ernight a t room temperature nith a current of 7 to 8 cc. per minute of nitrogen, folloned by heating a t 120' C. for 2.5 hours, also with nitrogcn floning The resulting capillary had an internal
LITERATURE CITED
1 Ihigosli, J . (to E. 1. du Pont de Semiiiire k C o . 1, T-. S. Patent 2,915,475 Dec. 1. 1~1.50I . 2 I h i d . . 3,013,901 ( I l r c . 19, 1961). 3 , Hugiisli, J., J . P h p . Cheiri. 65, 1 i 8 9 I1061 I . 4 1 Bugoeh, J.. Brown, R . L., SIcJThorter, ,J. It., Sears, (;. JV.j Pippel, R. J., l i i d . Eiig. ( ' h c i > i . Prod. Res. Develop. 1, 1.5; t l$I61i . .jI Aurc~hfield. H. P., Storrs, E. E., ~
t
6 1 E. I. di! Pant de Seniours S- Co., ll.ilniingtim l k l . , Buil. -1-18211,"Bayriial Colloidal .~luniina,"1961. 7 ! Iler. 11. K., J . .Ani. L'crai7i. SOC.44, 61% (]!I61
1.
11.. Ilggertsen, F . T., 30, 138; (19%). ! ) ' Patton, H. IV Lewis, J. S.,Kaye, I!., I.. I M . , 27, ifo (iojj). t 1 0 1 Petitjean, 1). L., Leftault, C. J., rh
I
Selsen. 1:.
.\S.\I..
C'AEV.
.Jr.. Pittsburgh Conference on Analytical
Cheriiistry and Applied Spectroscopy, SIarch, 1962,
J. J. KIRKLAND
Industrial a n d Biochemicals Department E. 1. du Pont de ?;emours & Co. Esperinient a1 Bta tion \\-ilniinyton, Del. I?ECEITBI) for review May 16, 1963. -4ccepted .June 5 , 1963. VOL. 35, NO 9 . AUGUST 1 9 6 3
1297
