INDUSTRIAL AND ENGINEERING CHEMISTRY
918
from other interfering constituents, such as silica and phosphorus, by the in a apparatus designed to ensure ease of cleaning and charging. The arsenic is oxidized to arsenic acid and the density of the blue color obtained by formation of the stable arsenicmolybdenum compound is measured photometrically. Three ranges are used for the quantitative estimation of the arsenic-namely, 0 t o 10, 0 t o 50, and 0 to 100 m i c r o g r a m s the accuracies for the above ranges being, respectively, * O , l , +0.5, and *l.O micrograms. A reagent blank which varies from 0.5 t o 2.0 micrograms is run simultaneously with each set of analyses and i t is determined with a n accuracy of *0.1 microgram, thus securing an accuracy Of *OS2 microgram for the lowest range.
Vol. 13, No. 12
Literature Cited (1) Chancy, A. L.,and Magnuson, H. J,, IN=. E X G . CHEM.,ANAL. E D . , 12, 691-3 (1940). (2) Hinsberg, K., and Riese, M., Biochem. Z.,290, 39-43 (1937). ANAL.ED.,12, 768-71 (1940). (3) Hubbard, D. M., IND. ENG.CHEM., (4) Kahane, E., and Pourtoy, M., J . pharm. chim., Ser. 8 , 23, 5 (1936). ( 5 ) Maechling, E. H.,and Flinn, F. B,,J . Lab. Clin. &fed., 15, 779 (1930). (6) Morris, H. J., and Calvew, H. O.,IND.ENQ.CHEW,ANAL.E D . , 9, 447 (1937). (7) Rodden, C. J., J . Research Natl. Bur. Standards, 24, 7 (1940). (Research Paper 1267.) (8) Schemer, J. A., Ibid., 16 (1936). (Research P a p e 871.) P R ~ S E N Tbefore E D the Division of Analytical and Micro Chemistry at the 102nd hleeting of the American Chemical Society, Atlantic City, N. J.
Apparatus for Extraction of Lipoids from Wet Tissues FREDERIC E. HOLMES Children’s Hospital Research Foundation and Department of Pediatrics, College of Medicine, University of Cincinnati, Cincinnati, Ohio
T
HE small apparatus shown in Figure 1 has been used in
this laboratory since 1932 for the extraction of lipoids from blood in the determination of cholesterol. The method used has been published only in the abstract (8) without a sketch of the apparatus. The essential modification in this extractor from previous ones of similar type, such as those of Leiboff (19, IS), Ling ( 1 4 , and numerous ot,hers. is the trap fixed to the reflux condenser at its lower end. Moisture from the sample distills with the solvent and is retained in the trap. A notable improvement in consistency of results has been obtained, and there are reasons for concluding that direct extraction with chloroform under these conditions results in complete, or nearly complete, removal of cholesterol from the sample. However, although most of the author’s work has been done with chloroform as the solvent, this extractor, and the larger ones shown in Figures 2 and 3, are adaptable to use with other solvents.
Microextractor The apparatus (Figure 1) consists of an extraction tube having bulb at the bottom calibrated to contain 5 ml. of extract and four lugs to support the sample, and a separate one-piece assembly of condenser and tra which fits into the top part of the tube. The body of the t u i e above the constriction is of 2 4 to 25-mm. tubing and has an approximate length of 230 mm. The four lugs, shown laterally and in section, are approximately 50 mm. above the top of the constriction. The constricted part is of 11-mm. tubing and has a length of 13 to 15 mm., these dimensions having been found best to give sufficient accuracy in making up to volume and still to permit rapid and thorough mixing of the extract with the reagents in the determination of cholesterol. The condenser has a long skirt which serves to hold it and the trap in the center of the extraction tube and also to deflect any moisture from the air down the outside of the a p paratus. The extraction tubes and condenser skirts are kept within close enough tolerances so that a large number of tubes and eeveral condensers are interchangeable. The outer shell of the trap and the part of the condenser hanging in the extraction tube are of 16-mm.tubing. The inner tube of the trap is of 11-mm. tubing extending a t least 12 mm. below the ring seal joining it to the shell, and the shell extends like a collar at least 12 mm. above the seal. The openings through the sup orting member connecting the trap to the condenser have a Zameter of not less than 5 mm. The holes to the outside through the shell below the ring B
seal have approximately 3 mm. diameter. The bottom of the trap should be between 15 and 30 mm. above the lugs. For operation, 5 to 6 ml. of solvent are placed in the bulb, a piece of fat-free filter paper 20 X 20 X 1 mm. with its corners bent at right angles is dropped ‘‘ears up” onto the lugs, 0.25 ml. of the specimen (serum, h,ep a r i n plasm a , whole blood, “saline” suspension of cells, o i other fluid) is delivered onto the paper from a graduated 1-ml. pipet or from a special pipet ( 7 ) , and the extraction tube with the condenser in p l a c e is i m mersed to the level of the sample in a boiling water bath. (Atall-form 400-ml. Detail beaker makes a f Trap satisfactory bath for two tubes.) The extraction is continued for 1 hour. Vapor of the boiling solvent around and t % ? ~ ~ ~ the sample, carrying moisture from J J the sample to the - S e c t i o n Ir, condenser. T h e condensate flows into the trap through the openings in the sup-
6 8
8 T : : : ;
separate in the inner tube of the trap, the water beinn
vntn;nnA
anrl
th; solvent flowing around the bottom of the inner tube
FIG^ 1. MICROEXTRACTOR FOR EXT~ACTION OF LIPOIDS FROM B L O O D Condenser and trap in position in tube
December 15, 1941
ANALYTICAL EDITION
and out the small holes in the outer shell of the trap and dripping back onto the sample (heavy solvents). For solvents lighter than water, a simple $;; supported by a extending to the bottom of the cup seems to be adequate. Such a trap has been tested with ethyl ether in two or three determinations. A trap suitable for both heavier a n d lighter solvents could be made by extending the outer shell of the trap shown in l?i about 15 mm. the bottom of the inner tube. Such a trap has not been tested.
E;
Large Extractors
with Water Traps The
performance
of t h e microextractor (discussed below) suggested the u ~ of e similar apparatus for extractions on a larger scale. I n 1940, K a e and co-workers described an a paratus FIGVRE 2. TYPEA LARQEExfor drying anzextractTRACTOR FOR Usm WITH THIMBLE ing specimens in a practically continuous Left half of pressure trap in detail a t right; arrow ahows path of vapor operation without transfer from the one apparatus. The water trap in Kaye’s apparatus could easily have been redesigned t o retain water above a heavier solvent. Instead, the entire a p paratus waa redesigned to accomplish several objectives: (1) to eliminate the large ground joints a t the top of the extraction chamber and below the reflux condenser, (2) to eliminate fragile outside tubes, and (3) to make the parts of the apparatus coaxial in order to save space and to eliminate the extra water bath. Whether or not these objectives are also advantages depends upon the personal preference of the person-using the apparatus.
6)
The first extractor tested is shown in Figure 2. This was again redesigned with the object of forcing vapor through the sample to produce vigorous agitation of the sample in the solvent, and the extractor shown in Figure 3 was built. These two extractors have been tested only t o the point of performing extractions in them. It was intended to compare extracts of wet material with those of dried material in respect t o total lipoid extracted, volatile constituents, and possibly the changes in labile constituents such as unsaturated f a t t y acids, vitamins, etc., but this work has been indefinitely postponed. It seems advisable t o make the apparatus available now, rather than t o wait until work can be resumed. LARGEEXTRACTOR FOR UBE WITH A THIMBLE.I n the extractor shown in Figure 2, Type A, solvent vapor from the boiling flask passes upward through the large center tube (containing the siphon) into the drainage chamber, through the vapor
919
tube (side tube, not labeled) to the extraction chamber, and upward to the condenser. The reflux & the water trap and flows down the walls of the extraction chamber and back into the drainage chamber. When the drainage chamber becomes full enough to cover the lower end of the vapor tube, solvent is forced up into this tube, and the pressure of the va or is increased, forcing it through the small amount of reflux colfected in the pressure trap. The purpose of the pressure trap is to prevent drainage of all but a small portion of the liquid solvent from the extraction chamber. Small increments of refluxed solvent draining into the lower chamber raise the level in the vapor tube and hence the pressure of the va or below the trap, so that the vapor can su port, and is forcefthrough, an increasing depth of liquid in extraction chamber. When solvent in the extraction chamber reaches sufficient de t h to fill the thimble to within about 8 mm. of the top edge, it k g i n s to splash over into the vapor tube. The bulblike expansion in the top of the extraction chamber allows this splash to be directed outward toward the wall instead of into the top of the thimble. Continued increase of solvent in the extraction chamber results in overflow into the vapor tube, and this excess flows into the drainage chamber until it overflows through the siphon. D r a i n a g e of solvent from the thimble can then continue during a considerable period of time until the level of li uid in the lower cham%er again reachea the bottom of the va rtube. %e pressure of the vapor against the liquid in the drainage chamber is practically equal to that exerted through the siphon from the boiling flask, and consequently the siphon operates like an ordinary siphon at a t mos p h e r i c pressure. It is essential that the top of the siphon be correctly designed to prevent dripping and failure to trip. The design shown here has worked well. The arm of the siphon in the drainage chamber should have a slightly larger inside diameter than that of the arm descending into the boiling h k (inside the large center tube), and should be of a size that will not entirely dam the small surges of Piquid which aid in tripping t h e siphon. Inside diameters of 4 and 3 mm., res ectively, were found toe! satisfactory. To prevent its tripping too soon, the top of the descending limb of the siphon should be between 15 and 20 mm. above the level of the top of the o ning into the vapor t u g . The extractor may be built in two parts, the upper diaphragm of the trap being formed on the lower end of the extraction chamber and the lower half of the trap on the top of the drainage chamber. The upper and lower halves of the vapor tube may then be joined, and FIGURE 3. TYPEB LARGIU Exwhile the glass is still TRACTOR PRODUCING AGITATION soft, the halves of the O F SAMPLE
tg
n
I KI
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INDUSTRIAL A N D ENGINEERING CHEMISTRY
trap may be brought together and fused, a fine glass rod being used to encircle the junction, filling any cracks and forming a smooth ring seal. Immediate thorough annealin is necessary. LARGE EXTRACTOR PRODUCING AGITATIONOF #AMPLE. I n the extractor shown in Figure 3, Type B, the operation of the siphon and solvent vapor ducts is the same as in the Type A apparatus. The solvent backs up in the vapor tube and produces pressure on the vapor in the chamber below the filter disk in the same manner. However the reflux, instead of flowing down the walls of the extraction chamber, is caught in the annular channel at the top of the chamber and directed down the solvent tube. It asses down this tube, up &rough the outer ortion of the filter, over the hter, and down through the center part of the disk (Figure 4), flushing any residual extract from the preceding cycle out of the bottom of the mass being extracted. When the pressure of the vapor below the disk becomes suf6ciently reat to stop this flow, ubbles of vapor begin to pass upward into the extraction chamber. As soon ~ t 9 the hot vapor has warmed the solvent in the extraction chamber to its boiling point, the bubbles pass through the chamber, producing an almost violent agitation of the F I Q W 4. DIDTAJL OF Frrc solvent and of the solid maTER DISK IN TYPE)B terial suspended in it. EXTRACTOR The ressure required to Plan below and longitudinal #eoforce Piquid solvent up tion of column above through the outer part of the filter is so low that the level of the top of the liquid in the solvent tube remains within a few millimeters of that of the liquid in the extraction chamber. When the liquid in the extraction chamber has been sufficiently augmented by reflux from the condenser and water trap, solvent flows over the top of the vapor tube and down into the drainage chamber, raising the level in this chamber gradually until it trips the siphon. The drop in pressure resulting from the emptying of the vapor tube permits solvent to drain from the extraction chamber through the filter. After flow stops in the siphon, all the solvent in the extraction chamber can be accommodated in the drainage chamber. Extract and the continuing reflux fill the chamber to the bottom of the vapor tube, and the cycle of operations begins to repeat itself. The relationship between the height of column in the va or tube and the porosity of the filter disk in the extractor teste! is satisfactory for a disk of the porosity of the one described. The disk used was rather coarse. A greater head, to force vapor through a finer filter, could be obtained by making the vapor tube and drainage chamber longer. I n the apparatus used, a maximum elevation of solvent (chloroform) of 370 mm. in the vapor tube could be reached, but it actually operated most of the time at between 230 and 300 mm. The filter disk was made by sintering powdered Pyrex in a 50ml. beaker, with the center tube in place which divides the filter into central and peripheral parts. A layer 1 mm. deep of 60to 80-mesh grains was spread on the bottom of the beaker and over it was laid a layer 2 mm. deep of 40- to 60-mesh grains. The center tube, cut to final length, was pushed into the ground Pyrex to the floor of the beaker, and the beaker and contents were heated in a furnace a t about 800' C. until the walls of the beaker began to thicken and bend slightly. After annealing, the glass of the beaker was ground away against a grindstone (Norton, No. 37150-1).
%
~
Vol. 13, No. 12
A variety of traps could be designed for the large extractors. Of several types tested, that shown, for solvents heavier than water, retains the least amount of moisture when its contents are poured into a cylinder or buret for measurement. The Type B extractor would accommodate a deeper trap having an inner tube, similar to the one in the trap of the microextractor, graduated for direct reading of small amounts of moisture. However, from large samples having the water content of fresh tissues, the amount of water recovered is so large that the loss of one or two drops would constitute a small error. For the micro- and Type A extractors, the method of weighing the dry residue is always available. Traps for solvents lighter than water were not tested. A lain cup similar to the trap shown but without the overflow tuge is suggested. A small powder funnel in the top would direct water below the surface. Small amounts of moisture can be recovered with a pipet with capillary tip, and can be measured by drawing up chloroform below the water until the water is in the graduated part. A satisfactory tool for inserting or removing the tra or extraction thimble can be made of 3-mm. (0.125-inch) dri! rod or 4mm. (0.156-inch) brass rod. A deep V with a short coil at the apex is made by bending the rod one and a half times around a piece of pipe. The ends are then bent outward slight1 to engage the top of the trap or the inside surface of the thimbc.
Performance of Microextractor Since the apparatus is adaptable for use with other solvents
and for the extraction of other lipoids, its performance in the extraction and determination of cholesterol is a rather narrow criterion of its general usefulness. However, the results shown in Table I are satisfactory in significance and implication aa to the possibility of approaching complete extraction, especially when it is considered that, u p t o 6 or 8 per cent, a large part and probably the greater part of the differences can be attributed to small variations in both the rate of development and the reading of the Liebermann-Burchard color. The consistency of values of duplicates, in the first line of the table, suggests a close approach to a limit. The comparison of extraction in the microextractor and colorimetric determination of cholesterol with alcohol-ether extraction and determination by the digitonide method of Kirk, Page, and Van Slyke (11) must be interpreted with caution. Excess color due to esters (Kelsey, 10) could be thought to have compensated for and concealed a deficiency in the extraction, particularly a failure t o extract a definite portion associated with the proteins (6, 6). T h a t the deficiency, if any, in the extraction itself is much smaller than might be inferred, is shown by the small amount of residual cholesterol found after saponification in previously extracted samples (saponified, acidified, and reextracted) . The explanation of the small effect of the esters and of other substances producing excess color (2, 4, 16, 16) has been learned only in part. "Recovery" of added cholesterol, since i t is so easily extracted, has only the negative value of showing that no gross errors occurred in the colorimetry.
TABLE I. PERFORMANCE) OF MICROEXTRACTOR Number Nature of Data.
Tifala
Differences Found Maxi- In96% mum of cases Average
%
Water Traps and Condensers The trap of the microextractor is not suitable for measuring the volume of water collected, and separation of trap and condenser would sacrifice the convenience and rapidity with which they can be handled. The separate construction of trap and condenser in the large extractors facilitates interchange of parts and removal of the water collected. The condenser shown has been adequate.
Difference between duplicate colorimetrio determinations on chloroform extracts Comparison of values in same sample by above method and by Kirk's method Recovery of added cholesterol by above method (per cent of total, t h a t in sample t h a t added) Re-extraction after saponification of extracted sample (24 samples combined, yield o 22 mg. "To. Wrattan No. 71-A red filter in eyepiece, extract, brown)
+
a
%
% 0.7
603
13
