Investigations of Adsorption of Unsaturated Fatty Acid Methyl Esters on Silicic Acid-Silver Nitrate Robert A. Stein and Vida Slawson Department of Biological Chemistry, School of Medicine, University of California Los Angeles, Los Angeles, Calif. 90024 Silicic acid-silver nitrate adsorbent was prepared by the addition of ethyl ether to an acetonitrile solution of silver nitrate on a silicic acid chromatographic column. Partition isotherms were measured for methyl esters of cis-unsaturated fatty acids between the adsorbent and cyclohexene-ether-pentane mixtures, and solvents that gave 4040% of a solute in solution eluted the solute satisfactorily from a chromatographic column. A scheme was developed that gave good separations by elution chromatography of a mixture of methyl esters of fatty acids containing from 0 to 6 methylene-inter- rupted ck-double bonds. Competition for adsorption sites between methyl oleate and methyl docosahexaeno- ate was dependent upon the concentration of each ester. Electron micrographs of the support suggest that the silver nitrate is distributed as small crystals within the silicic acid granules. MEASUREMENT OF PARTITION coefficients for a variety of olefins with silver ion (1) give a clear indication that molecules differing in number, configuration, and degree of substitution of olefinic centers may be separated by the use of these com- plexes. The subsequent development of partitioning (2) and chromatographic (3, 4) methods that use silver complexes for separations has been reviewed recently (5). In the present paper, a more detailed investigation has been made of the physical nature of the support and some of the problems encountered in separations of methyl esters of naturally- occurring fatty acids on silicic acid-silver nitrate adsorbent. A modified preparation of a uniform silicic acid-silver nitrate mixture has been used to assess the partitioning of the methyl esters of common naturally-occurring fatty acids. With these data, an elution scheme was developed that gives good separation of a mixture of fatty acid esters which contain from 0 to 6 methylene-interrupted cis-double bonds. Deter- mination of adsorption isotherms for mixtures of methyl oleate and methyl docosahexaenoate showed that the com- petition between monoene and polyene for adsorption sites was dependent upon the concentration of each fatty ester. EXPERIMENTAL Reagents. Silicic acid powder (J. T. Baker 0324) was used as received. All solvents were redistilled in glass, except for diethyl ether (J. T. Baker 9244, anhydrous) which was used directly from the can. Cyclohexene was distilled from metallic sodium. Methyl stearate (18 :O), oleate (18: l), linoleate (18:2), and linolenate (18:3) were >99% pure; methyl arachidonate (20 :4), eicosapentaenoate (20 : 5), and docosahexaenoate (22 :6) were >90% pure (Hormel Insti- tute, Austin, Minn). Tetracosane, recrystallized to a mp of 50 "C [lit. (6) mp 51.1 "C], was >99% pure by GLC. (1) S. Winstein and H. J. Lucas, J. Amer. Chem. SOC., 60, 836 (2) H. J. Dutton, C. R. Scholfield, and E. P. Jones, Chem. Znd. (3) B. de Vries, J. Amer. Oil Chem. SOC., 40, 184 (1963). (4) 0. S. Privett, M. L. Blank. and 0. Romanus. J. Lioid Res.. 4. (1938). (London), 1961, 1874. I , 260 (1963). (5) L. J. Morris. ibid.. 7. 717 (1966). Apparatus. A glass chromatographic column, 2 X 33 cm, equipped with ground glass joints, was fitted with a 250-ml pressure-equalizing separatory funnel. The equalizing arm was fitted with a gas inlet tube so that the solvent in the funnel and column could be kept under a nitrogen atmosphere. Solvent flow was regulated by vacuum from a water aspirator, Gas-liquid chromatography (GLC) occurred on a Barber- Colman Model 10 gas chromatograph with a flame ionization detector, which gave peak areas proportional to weight (with i 3 for each peak) for a standard mixture containing equal weights of methyl palmitate, stearate, arachidate, and behen- ate. Polar columns used were 12-14% ethylene glycol succinate on acid-washed, siliconized Chromosorb G (Johns- Manville) or acid-washed, siliconized Chromosorb P (Johns- Manville). The GLC columns were 6 rnm (i.d.), 3 to 5 feet in length, and were used at 186 i 10 "C with carrier gas flow regulated to elute methyl stearate at about 5 minutes. The nonpolar column was 0.29x SE-30 (General Electric) on glass beads (80-120 mesh). Standard Mixtures. Aliquots of a mixture of methyl esters (Table I) were sealed in vacuo after first being saturated with nitrogen, and were stored at -20 "C. GLC showed that no significant changes occurred during storage for six months. Deviation between weighed composition and composition determined by GLC is due to impurities in some of the esters used to make the mixture, but because the following data are from comparative GLC analyses, the differences between weight and GLC area are of significance only when a material balance is obtained. Silicic acid (14 grams) was slurried with acetone, allowed to settle, and the suspended fine particles were removed by decantation. The thick slurry was poured into the chromatographic tube, which was connected to a slight vacuum, and the top of the adsorbent was covered with a porous disk to keep it level during sub- sequent additions of solvent. The column was impregnated with silver nitrate and activated in the following sequence: (1) 50 ml of acetone, (2) 50 ml of acetonitrile, (3) 5.6 -10.1 grams of AgN03 in 20 ml of acetonitrile, (4) 100 ml of diethyl ether, (5) 500 ml of 2 5 x (v/v) cyclohexene in diethyl ether, (6) 50 ml of pentane. As precautions against the effects of oxygen and light, nitrogen was bubbled through the solvents for 1-2 min before use, and the column was protected from light by a paper shield. Fractions (10 or 15 ml) were freed of solvent in a stream of nitrogen on a steam bath, and were stoppered and stored in a freezer at -20 "C. The column Column Preparation and Procedure. Table I. Composition of Methyl Ester Mixture Used to Test Separations on Silicic Acid-Silver Nitrate Columns Methyl ester Wt z GLC Area p 18:O 13.7 14.8 (14.3-15.4) 18:l 16.1 17.8 (17.3-18.4) 18:2 13.6 14.7 (14.5-15.3) 18:3 13.8 14.9 (14.8-15.3) 20.4 12.2 10.9(10.7-11.3) 22:6 16.1 15.2 (14.6-15.8) 20:5 14.5 11.7 (11.6-11.7) a Average of 3 values and range. . ,, . , (6) W. M. Mazee, Rec. Truc. Chim. Pays-Bus, 67, 197 (1948). VOL. 40, NO. 13, NOVEMBER 1968 2017
Transcript
Investigations of Adsorption of Unsaturated Fatty Acid Methyl Esters on Silicic Acid-Silver Nitrate
Robert A. Stein and Vida Slawson Department of Biological Chemistry, School of Medicine, University of California Los Angeles, Los Angeles, Calif. 90024
Silicic acid-silver nitrate adsorbent was prepared by the addition of ethyl ether to an acetonitrile solution of silver nitrate on a silicic acid chromatographic column. Partition isotherms were measured for methyl esters of cis-unsaturated fatty acids between the adsorbent and cyclohexene-ether-pentane mixtures, and solvents that gave 4040% of a solute in solution eluted the solute satisfactorily from a chromatographic column. A scheme was developed that gave good separations by elution chromatography of a mixture of methyl esters of fatty acids containing from 0 to 6 methylene-inter- rupted ck-double bonds. Competition for adsorption sites between methyl oleate and methyl docosahexaeno- ate was dependent upon the concentration of each ester. Electron micrographs of the support suggest that the silver nitrate is distributed as small crystals within the silicic acid granules.
MEASUREMENT OF PARTITION coefficients for a variety of olefins with silver ion ( 1 ) give a clear indication that molecules differing in number, configuration, and degree of substitution of olefinic centers may be separated by the use of these com- plexes. The subsequent development of partitioning ( 2 ) and chromatographic (3, 4 ) methods that use silver complexes for separations has been reviewed recently (5). In the present paper, a more detailed investigation has been made of the physical nature of the support and some of the problems encountered in separations of methyl esters of naturally- occurring fatty acids on silicic acid-silver nitrate adsorbent.
A modified preparation of a uniform silicic acid-silver nitrate mixture has been used to assess the partitioning of the methyl esters of common naturally-occurring fatty acids. With these data, an elution scheme was developed that gives good separation of a mixture of fatty acid esters which contain from 0 to 6 methylene-interrupted cis-double bonds. Deter- mination of adsorption isotherms for mixtures of methyl oleate and methyl docosahexaenoate showed that the com- petition between monoene and polyene for adsorption sites was dependent upon the concentration of each fatty ester.
EXPERIMENTAL
Reagents. Silicic acid powder (J . T. Baker 0324) was used as received. All solvents were redistilled in glass, except for diethyl ether (J. T. Baker 9244, anhydrous) which was used directly from the can. Cyclohexene was distilled from metallic sodium. Methyl stearate (18 : O ) , oleate (18: l), linoleate (18:2), and linolenate (18:3) were >99% pure; methyl arachidonate (20 :4), eicosapentaenoate (20 : 5 ) , and docosahexaenoate (22 :6) were >90% pure (Hormel Insti- tute, Austin, Minn). Tetracosane, recrystallized to a mp of 50 "C [lit. (6 ) mp 51.1 "C], was >99% pure by GLC.
(1) S . Winstein and H. J. Lucas, J. Amer. Chem. SOC., 60, 836
(2) H. J. Dutton, C . R. Scholfield, and E. P. Jones, Chem. Znd.
(3) B. de Vries, J . Amer. Oil Chem. SOC., 40, 184 (1963). (4) 0. S . Privett, M. L. Blank. and 0. Romanus. J . Lioid Res.. 4.
(1938).
(London), 1961, 1874.
I ,
260 (1963). (5) L. J. Morris. ibid.. 7. 717 (1966).
Apparatus. A glass chromatographic column, 2 X 33 cm, equipped with ground glass joints, was fitted with a 250-ml pressure-equalizing separatory funnel. The equalizing arm was fitted with a gas inlet tube so that the solvent in the funnel and column could be kept under a nitrogen atmosphere. Solvent flow was regulated by vacuum from a water aspirator, Gas-liquid chromatography (GLC) occurred on a Barber- Colman Model 10 gas chromatograph with a flame ionization detector, which gave peak areas proportional to weight (with i 3 for each peak) for a standard mixture containing equal weights of methyl palmitate, stearate, arachidate, and behen- ate. Polar columns used were 12-14% ethylene glycol succinate on acid-washed, siliconized Chromosorb G (Johns- Manville) or acid-washed, siliconized Chromosorb P (Johns- Manville). The GLC columns were 6 rnm (i.d.), 3 to 5 feet in length, and were used at 186 i 10 "C with carrier gas flow regulated to elute methyl stearate at about 5 minutes. The nonpolar column was 0.29x SE-30 (General Electric) on glass beads (80-120 mesh).
Standard Mixtures. Aliquots of a mixture of methyl esters (Table I) were sealed in vacuo after first being saturated with nitrogen, and were stored at -20 "C. GLC showed that no significant changes occurred during storage for six months. Deviation between weighed composition and composition determined by GLC is due to impurities in some of the esters used to make the mixture, but because the following data are from comparative GLC analyses, the differences between weight and GLC area are of significance only when a material balance is obtained.
Silicic acid (14 grams) was slurried with acetone, allowed to settle, and the suspended fine particles were removed by decantation. The thick slurry was poured into the chromatographic tube, which was connected to a slight vacuum, and the top of the adsorbent was covered with a porous disk to keep it level during sub- sequent additions of solvent. The column was impregnated with silver nitrate and activated in the following sequence: (1) 50 ml of acetone, (2) 50 ml of acetonitrile, (3) 5.6 -10.1 grams of AgN03 in 20 ml of acetonitrile, (4) 100 ml of diethyl ether, (5) 500 ml of 2 5 x (v/v) cyclohexene in diethyl ether, (6) 50 ml of pentane. As precautions against the effects of oxygen and light, nitrogen was bubbled through the solvents for 1-2 min before use, and the column was protected from light by a paper shield. Fractions (10 or 15 ml) were freed of solvent in a stream of nitrogen on a steam bath, and were stoppered and stored in a freezer at -20 "C. The column
Column Preparation and Procedure.
Table I. Composition of Methyl Ester Mixture Used to Test Separations on Silicic Acid-Silver Nitrate Columns
Methyl ester Wt z GLC Area p 18:O 13.7 14.8 (14.3-15.4) 18:l 16.1 17.8 (17.3-18.4) 18:2 13.6 14.7 (14.5-15.3) 18:3 13.8 14.9 (14.8-15.3) 20.4 12.2 10.9(10.7-11.3)
102-115 14.4 81.4 (22:6) 100-125 20.9 77.6 (22:6) Q Fractions (10 ml) 1-16 eluted by 1 % ether-pentane; 17-32 by 4% ether-pentane; 33-48 by 10% ether-pentane; 49-65 by 20% ether-
pentane; 66-79 by 50% ether-pentane; 80-96 by 75% ether-pentane; 97-112 by 5 % cyclohexene-75z ether-20Z pentane; 113-125 by 10% cyclohexene-75 % ether-I5 % pentane.
b Fraction 65 (0.8 mg) not included.
volume (12 ml) was determined by measuring the amount of ether necessary to carry an unadsorbed solute (tetracosane) through the column.
Equilibrium Experiments. Glass stoppered flasks contain- ing the adsorbent and solutions of esters were shaken gently for 30 minutes at 25 "C. Tetracosane, which was not ad- sorbed under conditions used in these experiments, was in- cluded as an internal standard for GLC.
The air-dried weight of the adsorbent was determined at the conclusion of the experiment after removing the esters with 1 : 3 cyclohexene-ether.
RESULTS AND DISCUSSION
Determination of Adsorption Isotherms. Our initial ap- proach in separating a wide range of unsaturated esters on silicic acid-silver nitrate columns was to elute the esters with increasing amounts of diethyl ether in pentane. This system was moderately successful, in that good separation of satu- rated, triene, and tetraene esters could be obtained, but the upper limit of such a system seemed to be four double bonds because of the very slow elution of the tetraene. Distribu- tion ratios of various olefins between carbon tetrachloride and aqueous silver nitrate solutions ( I ) suggested that cyclo-
I I I 1
Me 184
Me IB I
Mel8.2 A I 2 3 4 5 6 7 8 9 1 0 1 1 1 2 Me I82
60 50 40 30 20
10 20 30 40 50 60 70 80 90 100 .- c % E t h e r in Pentane % Cyclohexene in Pentane
80- 70!
30 20
0 5 I O 15 20 25 0 10 20 30 40 50 60 70 80 90 100
50 6ol
0 10 20 30 40 50 60 70 % Cyclohexene in 8.1% Ether -Pentane %Cyclohexenc in 75% Ether-Pentone
Figure 1. Distribution (wt %) of methyl esters between silicic acid-silver nitrate and various solvent mixtures (v/v)
hexene included in the solvent mixture would facilitate the displacement of the unsaturated fatty esters from the silver complexes. Because the specific elution characteristics of ternary solvent systems are complex, assessment of the efficiency of the vast array of possible mixtures by the (trial and error) column technique would require considerable time. An approximate idea of the effects of solvent composition was therefore obtained from a simple equilibrium system, analogous to those used for determining optimal composition of solutions used for elution of fatty acids (7) or fatty esters (8) from other types of column. Quantitative differences in a mixture of esters before and after equilibration between various solvent mixtures and adsorbent were measured by GLC. These data are shown as isotherms in Figure 1. Some factors that are important to the correlation of iso- thermal data and column performance have not been evalu- ated. Among these are the cumulative effect of solvents in spreading the bands of solutes on the column. As this spreading occurs, elution from the column will be earlier than expected from the isothermal data and separations will be poorer. A subjective correlation of column elutions with batch equilibria suggests that for successful elution of an ester from a column, the amount of ester in solution at equilibrium should be between 40-80%, and compounds that are a t the extremes of this range could be separated from each other. If the amount in solution was more than SO%, the solute moved with the solvent front, and if less than 40 %, the compound was spread over a large number of fractions.
Column Separation of Fatty Ester Mixture. The results of a typical column separation of a sample of the standard mixture are given in Table 11. The arbitrary distinction between heart and end fractions was made to give the largest heart fractions consistent with high purity. Where there was considerable overlap between components, the heart fraction was a smaller proportion of the total fraction than when the overlap was small.
A total of 135.4 mg was recovered from fractions 1-125, and in addition 7.9 mg was eluted by 200 ml 25% (v/v) cyclohexene-ether from the 153.7 mg applied to the column. To assess the losses that occurred during chromatography and related handling, GLC peak areas were compared to the areas from the original mixture. Normalizing the com- ponents with respect to methyl stearate made it possible to calculate the relative recovery of each compound (Table 111). Not unexpectedly, the greatest losses occurred with the most
(7) F. D. Gunstone and P. J. Sykes, J. Chem. Soc., 1960, 5050. (8) J. Hirsch and E. H. Ahrens, Jr., J. Biol. Chem., 233, 311 (1958).
201 8 0 ANALYTICAL CHEMISTRY
50 x 10-31
40 - - E \ 30- 0,
Methyl 22:6 ,um/g Adsorbent
Figure 2. Effect of adsorbed methyl docosahexaenoate on distribution ratio of methyl oleate partitioned between 1 (v/v) ether-pentane and silicic acid-silver nitrate.
highly unsaturated esters, since these are probably the most susceptible t o autoxidation. Autoxidation of the polyun- saturated esters is accompanied by conjugated diene forma- tion. The UV spectrum of a 2,2,4-trimethylpentane solution of the final recomposite gave no evidence for the presence of conjugated diene with a concentration that would have demonstrated a minimum of 0.5z. If losses of the poly- unsaturated components of the mixture occurred by autox- idation prior to or during chromatography, the initial products were not eluted from the silicic acid column in any of the fractions. The individual GLC peak areas of the com- posite sample in Table 111 amount to a total recovery of 93 %. On a weight basis, the same sample amounts to an 88 z recovery. The larger loss of weight compared to GLC area probably represents a uniform loss in handling the numerous fractions and removal of material by elution chromatography that did not appear in GLC chromatograms. A final column elution by 200 ml 25 z (v/v) cyclohexene-ether brought out 7.9 mg of brown material which may be from degradation of the polyunsaturated compounds or an accumulation of material that arose from the solvents.
Effect of Sample Size and Competition on Adsorption. Equilibria of methyl 18 : 1 and 22 : 6 between silicic acid-silver nitrate and solvent [ l z (v/v) ether-pentane] were used to determine the effect of ester load and competition between types of esters upon adsorption. When only methyl l 8 : l was present, the distribution ratio (D, = methyl 18: l in solution,/methyl 18 : l / g silicic acid-silver nitrate) is constant below a solution concentration of 0.1 pm/ml. With higher concentrations D, increases because the amount adsorbed becomes a smaller proportion of that in solution.
Because methyl 22:6 is completely adsorbed from the solvent used in these experiments, its inclusion with methyl 18 : 1 in the partitioning system gives an indication of equiv- alency of adsorbed monoene and polyene. In theory, the distribution ratio of the system containing both methyl 18: l and 22:6 [D, = methyl 18:l in solution/(methyl 18 : l + methyl 22 :6) adsorbed] compared to D, should be a measure of any equivalency of the two esters. However, D, is not constant over a range of concentrations, because ratio D, is a function of the amount of polyene present in the system as shown in Figure 2.
By using the data of Figure 2 and making the assumption that conditions can be met such that D, = D,, it is possible to calculate values that relate adsorbed polyene in terms of adsorbed monoene. The calculated curves showing the
Table 111. Comparison of GLC Peak Areas from Composite Sample before and after Silicic Acid-Silver
Nitrate Chromatography
Relative peak area Recovered, Methyl ester Before After. z
equivalency of adsorbed monoene and polyene, Figure 3, were obtained by using D, = 4.3 X gram/ml (which is constant at low concentrations) and setting the denominators equivalent to one another in the equation D, = D, when concentration of monoene in solution is arbitrarily selected. For example, when [monoene in solution] = 0.05 km/ml we can write
0.5pm/ml - 4.3 X 10-3 gram/ml = -
[monoene adsorbed]
0.05 um/ml [monoene adsorbed + polyene adsorbed]
The adsorbed monoene on the left side of the equation is calculated directly to be 12 pm/gram. The adsorbed monoene on the right side is calculated from D, in Figure 2 for any value of adsorbed polyene. When adsorbed polyene is 20 km/gram, D, = 12 X gramiml and the corresponding adsorbed monoene is 4.2 pm/gram. Thus 12 pm/gram monoene is equivalent to (4.2 pmigram monoene + 20 pm/gram polyene) or 20 pm/gram polyene is equivalent to 7.8 pm/gram monoene when 0.05 pm/ml monoene is in solution.
The curves of Figure 3 suggest that at low concentrations of monoene, when the adsorption sites of the silicic acid-
Methyl l8:l in Solution A \
O . O l ~ m / m l O.OS/rn/ml O.lO/m/ml 50-
E 2
40- N - =- r t 30- I -0 ; 20-
51 a 10- 0
0 2 4 6 8 10 12 14 16 18 20 Absorbed Methyl 18: I f im/g
Figure 3. Calculated equivalence of methyl docosahexaenoate and methyl oleate adsorbed on silicic acid-silver nitrate in the presence of methyl oleate in 1 (v/v) ether-pentane
VOL. 40, NO. 13, NOVEMBER 1968 2019
silver nitrate are not fully occupied, there is very little com- petition between monoene and polyene, but with increasing amounts of monoene in solution, the effect of adsorbed polyene is more pronounced. When the concentration of monoene in solution is 0.1 rm/ml, the adsorbed monoene is about 20 pm/gram and the adsorption sites are uniformly filled, because a further increase of monoene in the system allows a proportionally smaller amount of adsorption. For the polyene added at this point, there are no available free adsorption sites, and the polyene displaces an equivalent amount of monoene, making this the most favorable con- centration at which to observe the maximum effect of polyene. At a concentration of 0.1 pm/ml monoene in solution, the adsorption of 3 pm/gram polyene is calculated to be equivalent to 8.6 pmigram monoene. This means that when the adsorp- tion sites are uniformly filled, the presence of one molecule of methyl 22:6 prevents the adsorption of nearly three molecules of methyl 18 : 1.
A silicic acid-silver nitrate column of the type used in these experiments was divided into thirds, Each segment was digested with nitric acid, and the amount of silver determined by a standard thiocyanate titration (9). The top, middle, and bottom contained 0.141, 0.144, and 0.135 gram AgNOa/gram adsorbent, respectively. A second column similarly analyzed showed 0.158, 0.132, and 0.125 gram AgNO,/gram adsorbent. The total amount of silver nitrate on the column as determined by the difference between the weight originally added and the amount collected duripg the activation process agreed within 10% of the amount determined by thiocyanate titration. Routine gravimetric determinations showed that the average amount of silver remaining on the silicic acid was 13.1% (w/w) and ranged from 11.9-14.9z for 9 columns.
The amount of silver nitrate in the adsorbent prepared here is in great excess of the amount of olefin that can be adsorbed if the complexing were on a 1 :1 mole ratio. Ap- parently the silver nitrate is collected in areas of the silicic acid which are not approached by the olefin or forms crystals of which only a surface or edge may he used for adsorption of the olefin, In either of these cases, if the collection of silver is large enough, electron microscopy could differentiate between the silicic acid and aggregates of silver nitrate. To investigate these possibilities, particles of silver nitrate- impregnated and plain silicic acid were imbedded in polyester resin and sectioned for miscroscopy. Electron micrographs (Figure 4) showed the presence of dense crystals within the silicic acid particle which had been silver nitrate treated. If these are silver nitrate crystals, as the evidence suggests, they must have formed within the interstitial spaces of the silicic acid particles when ether was added to the acetonitrile solution of silver nitrate on the column. At thi$ time it cannot be established that these crystals are responsible for the silver ion-olefin complexing, and it is possible that the adsorptive silver ions are distributed over the silicic acid in a
(9) 1. th. Kolthoff and E. B. Sandell, “Textbook of Quantitative Inorganic Analysis,” 3rd ed., Macmillan, New York, 1952, p 455.
Distribution of Silver Nitrate.
2020 ANALYTICAL CHEMISTRY
Figure 4. Electron micrographs of slices of silicic acid- silver nitrate (upper picture) and silicic acid (lower pic- ture) granules
Dark structures in upper picture are suspected to be silver nitrate crystals. Uniform grey areas are polyester resin used as embedding medium. White areas in upper picture are pores that are incompletely filled with resin
manner that can not he detected by electron microscopy. Measurements of the supposed silver nitrate crystals show approximate dimensions of 0.35 x 0.09 X 0.09 p. If this were the size of the average crystal, the silver nitrate would he distributed so that it had about 10 mP/gram surface area. By way of comparison, silicic acid commonly used for chro- matography has a surface area that ranges between 340-830 mZ/gram (IO).
ACKNOWLEDGMENT
We are indebted to Frank Galey, Laboratory of Nuclear Medicine and Radiation Biology, University of California Los Angeles, for his efforts in preparing the sections of the silicic acid granules and performing the electron microscopy.
RECEWED for review July 1,1968. Accepted August 19,1968. Work supported by Public Health Service Research Grant H-4120 from the National Heart Institute, National Institutes of Health, U. S. Public Health Service, Bethesda, Md.
