ture programming the 4-foot column. However, results were too variable and, probably because the equipment did not incorporate a programming control unit, the heating rate was not linear. Temperature programming was therefore discontinued, results obtained from a 10-foot column operated isothermally being well within the expected experimental error. Analysis of standard test mixtures of 0.01N aqueous fatty acids are shown in Table IV; the results expressed as percentage composition are escellent whereas the absolute values are slightly variable but again well within experimental error. The method has been operated with a minimum of 0.5 mg. of acid present but there is no reason why lower levels of more complex mixtures of acids should not be analyzed, particularly if the gas chromatography unit can be reproducibly temperature-programmed. It was found that with prolonged use of the column for fatty acid analysis the detector became noisy under the usual operating conditions (1250 V. attenua-
tion X10, X3, or X l ) , but this noise could be periodically reduced to negligible amounts by removal of the column, insertion of an empty column, and raising the temperature to 200’ C. overnight, with argon passing through the system. The method is now used routinely in this laboratory for the analysis of the lower fatty acids produced by certain of the obligate Gram negative nonsporing anaerobes (Bacteroidaceae) . ACKNOWLEDGMENT
The authors are indebted to D. G. Land for helpful advice and to Miss R. Haynes for technics1 assistance. LITERATURE CITED
(1) Annison, E. F., Biochem. J . 58, 670
(1954). (2) Bartlett, J. C., Smith, D. M., Can. J . Chem. 38, 2057 (1960). (3) Bvars* B.* Jordan. G.. J . Gas Chro-
(4) Chirl&, A. ~B., Barrett, F. C., J .
Med. Lab. l’echnol. 20, 266 (1063). ( 5 ) Crnig, B. M., Tulloch, A. P., Murty,
N. L., J . Ana. Oil Chemists’ SOC.40, 61 (1963). (6) Gehrke, C. W., Goerlitz, D. F., ANAL. CHEM.35, 78 (1963). (7) Hankinson, C. L., Harper, W. J., Mikolajcik, E., J . Dairy Sci. 41, 1502 (1958). (8) Hawke, J. C., J . Dairy Res. 24, 366 (1957). (9) Hughes, R. B., J . Sci. Food Agr. 11, 47 (1960). (10) Isherwood, F. A , Hanes, C. S., Biochem. J . 55, 824 (1953). (11) Markham, R., Ibid., 36, 790 (1942). (12) Kixelly, J. G., ANAL. CHEM. 36, 2244 (1964). (13) Robb, E. W., Westbrook, J. J., 111, Ibid., 35, 1644 (1963). (14) Schlenk, H., Gellerman, J. L., Ibid., 32, 1412 (1960). (15) Shelley, R. N., Salwin, H., Horwitz, W. J., J . Assoc. Ofic. Agr. Ch,emists 46, 486 (1963). (16) Shrimpton, D. H., Stevens, B. J., In preparation. (17) S‘andenheuvel, F. A., ANAL. CHEW 36. 1930 (1964). (18) ‘Vorbeck, li. L., Mattick, L. R., Lee, F. A., Pederson, C. S.,Ibid., 33, 1512 (1961). (19) Weenink, R. O., N e w Zealand J . Sci., 1, 18 (1958). RECEIVED for review Sovember 2, 1965. Accepted January 31, 1966.
The Preparation and Characterization of Lyophilized Polyacrylamide Enzyme Gels for Chemical Analysis G. P. HICKS and S. J. UPDIKE Deparfment of Medicine, University of Wisconsin, Madison, Wis.
b
The preparation of a stable, lyophilized polyacrylamide enzyme gel of uniform particle size is described. The enzyme gel particles can be accurately weighed and used in packed columns of a standard size to give reproducible activity measurements. When used in this manner, the properties of the immobilized enzyme activity are similar in principle to those for soluble enzymes and are similarly applicable to analysis. The immobilized enzymes have the advantages of convenience and economy when applied to established enzymic methods of analysis and should extend the application of enzymes to analysis by simplifying the development and automation of new, more complex methods based on enzymic reactions.
T
HE USEFULNESS OF ENZYMES in analysis is
SOLUBLE
well established (6). The use of immobilized enzymes promises to overcome some of the disadvantages frequently associated with established enzyme methods and to extend their applications in analysis. Several methods for immobilizing enzyme activity have been reported. Enzymes have been diazotized to cellu726
0
ANALYTICAL CHEMISTRY
lose particles (16) and to polyaminostyrene beads (IO). Enzymes have also been immobilized on ion exchange resin ( I ) , polytyrosyl polypeptides ( 7 ) , on a collodion matrix ( g ) , and encapsulated in semipermeable micro capsules made of synthetic polymers (6). Entrapped enzyme activity has also been demonstrated in starch gel (2, 17) and polyacrylamide gel (3). The initial requirement for the analytical application of immobilized enzyme activity is that the insoluble enzyme must be prepared in a stahle, reproducible reagent form, which can be used in a well-defined configuration for activity measurements. One approach to this problem has been to immobilize enzyme activity (cholinesterase) in starch gel impregnated into polyurethane foam pads (2, 11). As another approach, the preparation of a stable, lyophilized enzyme gel of uniform particle size is described and characterized in this report. EXPERIMENTAL
Dehydrogenase color reagent was prepared by dissolving 3.4 mg. of 2,6-dichlorophenolindophenol and 3 mg. of phenazine methoReagents :
sulfate(PMS) in 100 ml. of 0 , l M phosphate buffer, p H 7.4. Oxidase color reagent was prepared by dissolving 20 mg. of o-tolidine hydrochloride and 0.8 mg. of peroxidase (Type 11, Sigma Chemical Co., St. Louis, Mo.) in 100 ml. of 0.2M acetate buffer, p H 4.15. Standard Solutions. Glucose solutions were prepared from wglucose (E. S. P. Grade, Cutter Laboratories, Berkeley, Calif., 50 grams per 100 ml. of solution) by dilution in 0.2M acetate buffer. Glucose solutions were made 1.23 x 10-2X with respect to benzoic acid and stored at 5’ C. H2O2standard solutions were prepared every two weeks from 3% U. S. P. H202by dilution with 0.266 acetate buffer and stored at 5’ C. Lactic acid solutions were prepared fresh for each run by dilution of 59% w/w L-lactic acid solution (Sigma Chemical Co., St. Louis, Mo.) in 0.1M phosphate buffer. NAD and NADH2 (Sigma Chemical Co., St. Louis, Mo.) were made fresh daily in phosphate buffer as required. Enzymes. Glucose oxidase (130,000 units/gram Fermco Laboratories, Inc., Chicago, Ill.). Lactic dehydrogenase (400 micromoles pyruvate to lactate per minute, 37’ C., p H 7.5, Sigma, Type I1 from rabbit muscle). Immobilized Enzyme Gel. PREPARATION. A stock solution of monomer
RESULTS AND DISCUSSION
PULSER
0 2 ml/min. BUFFER rnwrmn, CONTROL SOLUTION
SAMPLE SAMPLE TURNTABLE
1.6 m q m i n .
4
DELAY
r
LINES I mi. SYRINGE FACKED WITH ENZYME-GEL
PHOTOMETER CELLS
LON
EXCESS COLUMN EFFLUEN
1",ZZ Figure 1 .
Diagram of instrumentation
was prepared by dissolving 40 grams of acrylamide (Eastman Distillation Products, Practical Grade) in 100 ml. of 0.1JI phosphate buffer, p H 7.4. Crosslinking reagent was prepared by dissolving 2.3 grams of N,X-methylenebis (scrylamide) in 100 ml. of 0.1.11 phosphate, p H 7.4. Both solutions were stored a t 5' C. Gels were prepared by mixing 1 ml. of the acrylamide solution with 4 ml. of the N,N-methylene-bis (acrylamide) solution and adding 1 ml. of an enzyme solution (containing from 0.1 to 20 nig. of enzyme). To catalyze the photopolynierization, approximately 0.03 mg. of riboflavin and 0.03 mg. of potassium persulfate were added. The concentration of enzyme in the gel is expressed as mg. of enzyme per 100 ml. of gel. Since oxygen inhibits the copolymerization that occurs in this system, the reagent mixture was deoxygenated by bubbling with nitrogen before the addition of the enzyme. Upon photocatalysis with a No. 2 photo floodlamp the copolymerization reaction was completed in two to fifteen minutes, with the end point defined as the time taken for the gel to reach maximum opacity. Copolymerization proceeded rapidly when care was taken t'o thoroughly remove oxygen from the solutions composing the reaction mixture. To reduce the effect of heat denaturation of the enzyme during the exothermic reaction, the reaction vials were placed in an ice bath during polymerization. MECHASICAL DISPERSIOS. The resulting block of polymerized enzyme-gel was mechanically dispersed into particles by passing it first through a #I3 syringe needle, and secondly, through a #lS syringe needle to break the gel into even smaller part'icles. The suspension of gel particles was washed 10 times by decantation with 0.1-11 phosphate buffer a t room temperature, allowing it to settle about 2 minutes between each decantation. The smaller particles and any soluble enzyme were discarded in this process. LYOPHILIZATIOS AND STORAGE. The suspension of washed particles was finally suspended in distilled water and lyophilized. The dry part'icles were seived to a size between 20 and 40 mesh and stored in a desiccator a t 5' C.
This enzymc gel reagent will be referred to hereafter ais "E-G 20-40." Preparation of Enzyme Gel Columns. Glucose oxidase (GO) and lactic dehydrogenase (LDH) columns were prepared by equilibrating a dry weight of 25 mg. of E-G 20-40 for one hour in 2 ml. of buffer and quantitatively transferring the hydrated gel to a chromatographic column made from a 1.0 ml. disposable syringe as shown in Figure 1. The GO E-G 20-40 was hydrated with 0.2M acetate buffer p H 4.15 and the LDH E-G 20-40 was hydrated with O.lil! phosphate buffer p H 7.4, giving a final bed volume of 0.25 ml. (5-mm. i.d. x 10-mm. height). All columns prepared with E-G 20-40 gel tolerated a flow rate of 0.8 ml. per minute without plugging. Apparatus. The instrumentation used to study the immobilized enzyme systems is diagrammed in Figure 1. An enzyme gel column packed with E-G 20-40 is perfused with substrate solution a t a constant rate of 0.8 ml. per minute. For temperature control, the column and tubing preceding the column were placed in a regulated temperature bath. A portion of the column effluent is metered a t 0.2 ml. per minute in stream SI and mixed with a stream of either dehydrogenase or oxidase color reagent to detect the reaction products in the effluent stream. After passing through a short delay line to permit the color reaction to proceed, the reaction stream passes through a photometer cell. The color reagent is also mixed with a buffer or control solution and passed through a second delay line and photometer cell to serve as a reagent blank. The steady state absorbance difference between the two photometer cells is a measure of the product concentration in the column effluent stream. The delay lines are kept at room temperature, since, in contrast to previous applications of this photometer system (6, 12, 1 3 ) , the flowing stream is being used to monitor the products in the effluent stream and not to measure a rate of reaction. Standard solutions are introduced in stream S I to permit calibration of the photometer. A sample turntable ( I S ) was used to change the substrate solutions in the enzyme gel column stream at the rate of 20 per hour.
Polyacrylamide enzyme gels have been prepared in this laboratory in the forms of particles, blocks, strings, rods, coatings, and tubes, all containing the same amount of enzyme per unit volume of gel. I t is apparent that the specification of immobilized enzyme activity, in contrast to soluble enzyme activity, is dependent upon the physical form of the immobilized enzyme reagent and configuration of the immobilized system. E-G 20-40 particles can be accurately weighed and packed into a column of standard dimensions permitting the determination of immobilized enzyme activity with good precision ( 3 ~ 5 % ) . The technique of preparing E-G 20-40 enzyme gel particles has been applied in this laboratory to glucose oxidase, catalase, LDH, amino acid oxidase, glutamic dehydrogenase, and t o enzyme activity in human serum. The development of E-G 20-40 included studies t o determine the optimum acrylamide gel composition, the effect of enzyme and substrate concentration on the immobilized enzyme activity, and the stability of the immobilized enzymes to compare the uses of immobilized and soluble enzymes in analysis. Both a dehydrogenase (LDH) and an oxidase (GO) were used in these studies. Principles of Activity Measurements. The reactions used to detect dehydrogenase activity are given below :
NADH,
+ Dye (blue?
PMS
NAD f Dyered (2) (colorless)
I n Reaction 1, when a column of immobilized dehydrogenase enzyme (DH,) is perfused with a mixture of substrates (S) and nicotinamide adenine dinucleotide (NAD), reduced KJAD (NA4DH9) and products (P) are produced. The concentration of NADHz in the column effluent stream is measured in Reaction 2 a? the decrease in the absorbance of an oxidized blue dye (Dye,,, 2,6-dichlorophenolindophenol) a t 620 mp in the presence of a catalyst, phenazine methosulfate (P?VIS). The detection of immobilized oxidase activity was based on the following reactions:
s + O2 Hi02
P
+ H202
+(colorless) Dyered Peroxidase
+ Dye,.
(3)
_ _ _ _ f
HzO
(blue)
(4)
I n Reaction 3, when a column of immobilized oxidase enzyme (OX,) is perfused with a substrate (S) and Oz, products (P) and H z O ~are produced. VOL 38, NO. 6, MAY 1966
727
mp. GO per
100 ml.of GEL
.I0
I'
/
420
I
The H202 in the column effluent stream is detected in Reaction 4 by measuring the increase in absorbance of an oxidized blue dye (Dyeox,o-tolidine) at 620 mp in the presence of a second enzyme, peroxidase. Study of Gel Composition. A series of gels with t h e same L D H concentration (10 mg. L D H per 100 ml. of gel) but with different compositions were prepared to determine empirically the conditions for optimizing t h e two properties which make the gel particles most uqeful in flowing stream systems, that is, mechanical rigidity and activity. Other potentially useful properties such as optical clarity and ability to adhere to surfaces were also observed. A11 gels were prepared as previously described except that the ratios of the monomer and crosslinking reagents were varied. More dilute gels for a particular ratio were obtained by diluting the mixture of monomer and crosslinking agents with 0.l111phosphate buffer, p H 7.4, before adding the enzyme and catalyst. A list of gel compositions and s o m of the properties are shown in Tsble I. Best mechanical rigidity was obtained a t higher gel concentrations over the concentration range studied. At any one concentration, increasing the percent of crosslinking agent decreased mechanical rigidity, but favored a higher
yield of immobilized enzyme activity per unit of soluble enzyme activity introduced before polymerization. Proteolytic enzymes have been trapped by polymerizing only the crosslinking agent (3). Such a gel, however, is very soft, sediments slowly, and is unsuitable for use in flowing system applications. On the other hand, high gel concentrations tend to reduce the activity of the gel a t a given percentage of crosslinking agent. The most suitable gel material requires both a relatively high concentration of monomer to lend mechanical rigidity and a high concentration of crosslinking agent to achieve the highest possible yield of immobilized enzyme activity. The concentration of crosslinking agent is limited by its solubility in aqueous solutions, which is less than 3 grams per 100 ml. of gel. The first gel listed in the table (8.2 grams per 100 ml.) was selected for routine use. The catalyst best suited for polymerization depends upon the gel composition. I n general, mixtures with a high percentage of monomer polymerize more effectively with persulfate as a catalyst, while solutions with a higher percentage of crosslinking agent polymerize better with riboflavin and a photocatalyst. I n the composition selected for use the inclusion of both
8.2
5.0"
5.0"
11.1
14.7
5.0"
5.8
5.0"
4.1
3.2
81
90
95
90
95
81
32
32
49
68
19
10
5
10
5
19
68
68
51
32
100
66
60
32
8
White, opaque, minimally active, unsatisfactory for use due to poor rigidity which caused columns to plug.
Excellent Excellent Excellent Fair White Clear Clear White slightly slightly opaque opaque Diluted with 0.1M phosphate buffer, pH 7.4, before adding enzyme and catalysts.
728
catalysts gave the best final gel material. Gels can also be polymerized with no persulfate or riboflavin with highly purified glucose oxidase, and exposed to room light. The glucose oxidase reaction consumes O2 and generates H202which apparently is sufficient for catalysis. Other properties of the gels should be potentially useful in analysis. The optical clarity of some of the gel compositions would permit their use with a photosensitive device, such as a multiplier phototube, and is under investigation. The tendency for some gels to adhere to surfaces suggests the immobilization of enzymes on electrodes or other sensing devices. An investigation of this application is in progress (4). Effect of Enzyme Concentration. T o study the effect of enzyme concentration on the enzyme gel activity, gels were prepared with enzyme concentrations ranging from 5 to 420 mg. glucose oxidase per 100 ml. of gel. Identical enzyme gel columns were prepared from each gel and the steady state responses of each column when perfused with glucose solutions from 1 to 10 mg. per 100 ml. at 30' C. were measured. The results are shown in Figure 2. The concentration of H202 produced in the effluent stream was proportional
Characteristics and Activity of Enzyme-Gel Particles Prepared with Different Gel Recipes
Excellent White opaque
~~
a
200
Figure 3. Dependence of gel activity on enzyme concentration
Figure 2. Effect of enzyme concentration on column responses
+
'
IW 400
I
mg. GLUCOSE OXIDASE per 100ml. of GEL
rng. GLUCOSE per 100 ml.
Table I. Total grams (monomer crosslinking) of polymer per 100 ml. of gel % mono. _ Acrylamide mer yoN,N-methylene-bis (acrvlamide) crosslinkhg agent Relative activity of immobilized enzyme gel particles Mechanical rigidity Appearance
I
too
0
ANALYTICAL CHEMISTRY
mg.GO per
100m1.d GEL
0
I
I
4
a
Gm. GLUCOSE, per 100 ml.
Figure 4. Substrate curves for glucose oxidase gel
to the glucose concentration for the activity in each gel. The data in Figure 2 can be considered as a series of calibration curves for the determination of glucose with different levels of enzyme activity. The slope of each curve increases with the amount of enzyme in the gel. Figure 3 is a plot of the initial slope of each calibration curve against the amount of enzyme in the gel. As shown, the slope of gel activity is proportional to the enzyme concentration up to 50 mg. per 100 ml. Above 50 mg. per 100 ml. a large increase in the amount of enzyme gives only a small increase in activity of the gel. Optimum activity of the gel for analytical purposes, considering the economy of enzyme, is obtained a t about 25 mg. per 100 ml. for glucose oxidase. Similar results were obtained with lactic dehydrogenase, where only a small increase in gel activity was obtained above 100 mg. rabbit muscle LDH per 100 ml. of gel. Effect of Substrate Concentration. Several experiments were run t o study the effect of substrate concentrations for GO and LDH E-G 20-40 t o determine the similarity between the analytical use of enzymes in immobilized and solution systems.
gel may reflect either a process limited by diffusion into the gel particles or the preferential entrappment of specific isoenzyme activiby which represents only a small fraction of the original purified enzyme in solution, as will be discussed further. The interpretation of the gel kinetics must, therefore, be made very cautiously. Stability and Effect of Temperature. Lyophilized GO E-G 20-40, when kept a t 0" to 4" C., showed no loss of activity after 3 months, while lyophilized LDH E-G 20-40 lost about 10% of its activity per month over a period of three months. A hydrated column of glucose oxidase gel lost less than 5% of its activity in 6 weeks when kept in a refrigerator. A hydrated LDH column was less stable, losing most of its activity in about 3 months a t 4" C. Some attempts were made to determine whether or not the enzyme activity in the gel was actually more stable than in free solution. Data from one such experiment is shown in Figure 6. When an LDH column (150 mg. of LDH per 100 ml. of gel) was perfused continuously with lactate a t 37" C., no loss of activity was noticed for a t least 10 hours. After 10 hours, the activity began to decrease, losing 50% of its activity after about 20 hours. The stability was a function of time, but not flow rate, suggesting an actual denaturation process as opposed to the "wash-out" of the enzyme. I n starch gels, it has been reported that cholinesterase activity could be "washed-out" of the gel more rapidly with high flow rates (2). The same purified LDH enzyme used in the preparation of the gel was very unstable in solution when maintained a t 37O C., losing 90% of its activity in only 2 hours. After two hours, the activity remained constant at
Figure 4 shows substrate curves for two glucose oxidase columns. At high glucose concentrations, the amount of H202generated by the columns a t 30" C. far exceeded the analytical range of the photometer system, and it was necessary to cool the columns to 0" C. in an ice bath to make the determinations. It is interesting to note that the sensitivity of the photometer system and the columns used to obtain the data in Figure 4 were identical to those used for the 5 and 10 mg. GO E-G 20-40 curves in Figure 2, but with a thousand-fold higher glucose concentration. These data demonstrate the wide analytical range which can be achieved by varying the activity in the enzyme gel and the temperature of the columns. It also suggests the possibility of increasing the stability of the gel activity by operating a t low temperatures. Non-rate-limiting concentrations of lactate and NAD were determined for LDH E-G 20-40 a t two temperatures. The results are shown in Figure 5. Lineweaver-Burk plots were made with the photometer responses obtained to determine an apparent Michaelis Constant (K,) for each substrate. The plots obtained demonstrate the great similarity between the behavior of immobilized enzymes and enzymes in solution. Just as knowing the K , for enzymes in free solution helps to define the analytical range for substrate analysis ( 5 ) , it also appears that the K , value in immobilized systems might be similarly useful. While the applications of immobilized enzymes appear to be similar in principle to those of soluble enzymes, the K , values obtained for the gels were generally higher than the values obtained with the same enzyme preparations in free solution. The K , and other characteristics of the enzyme
I
m G i E
Km
-
00
0 M
LACTATE
.
0056 M
20
40
0
Substrate curves for LDH gel
4
6
0
10
60
HOURS AT
I
M LACTATE
Figure 5.
2
Figure 6.
37°C
Stability of LOH in gel and solution VOL 38, NO. 6, MAY 1966
729
10% of its original value, as shown in Figure 6. These data seem to demonstrate that the L D H activity is much more stable when entrapped in the acrylamide gel than in free solution. Other interpretations, however, are possible. The rabbit muscle enzyme used in the preparation of the enzyme gel was composed of more than a single isoenzyme of LDH. On an electrophoretic separation as previously described (fa), i t was demonstrated that the preparation was primarily the slowest moving, less stable M-type isoenzyme (8), but a small amount of a faster migrating, more stable H-type isoenzyme (8) was also present. An equally probable interpretation of the data in Figure 6 is t h a t the labile L D H isoenzyme was inactivated during the gel preparation, and the entrapped enzyme activity reflected only the more stable LDH fraction which was able to survive the polymerization and lyophilization procedures. I n a more severe test of stability, the activities of a series of identical glucose oxidase columns, after heating for 10 minutes at room temperature from 37’ to 70” C., were compared with activities of a series of glucose oxidase solutions treated in the same manner. The stability of columns containing gels with 100 mg. and 10 mg. of GO per 100 ml. were identical, and when compared with glucose oxidase solutions (200 mg. GO per 100 ml.), demonstrated no significant increase in stability. About half of the activity was destroyed in 10 minutes a t 60” C. and all of the activity at 70” C. in both the gel and solutions. Analytical Implications. The use of immobilized activity should extend t h e applications of enzymes in analy-
sis. Aside from t h e advantage of economy, another major advantage of the immobilized system, which should permit the development of many new analytical applications, is t h e fact t h a t the products of the enzyme reaction are easily separated from enzyme catalyst with high efficiency. Thus, an analytical method could be based on a series of enzymic reactions by utilizing a series of enzyme gel columns, even though the individual enzymes were not mutually compatible in a single solution. This “automatic” separation of enzyme and products should also greatly facilitate the automation of more complex analytical procedures such as enzymic amplification with cyclic reactions (16). Furthermore, the exclusion of molecules from the enzyme reaction system trapped in the gels on the basis of molecular size in a manner similar to that for “gel filtration” techniques may help to reduce the number of interferences, which is a major problem with many enzymic methods of analysis. It is possible, in principle, to develop a compact “reagentless” continuous analyzer for determinations where the enzyme is the only “reagent” required in addition to the sample for the analysis. For example, glucose oxidase has been used with an oxygen electrode to continuously monitor blood glucose (14). An immobilized glucose oxidase system coupled to an oxygen electrode should provide a “reagentless” method of continuous glucose analysis and is currently being developed in this laboratory. ACKNOWLEDGMENT
The encouragement and informative discussions with Dr. C. E. Reed are gratefully acknowledged. The assistance of Mrs. Martha White in
making measurements on the enzymes in free solution and of Mr. Pankonin in modifying the photometer system were greatly appreciated. LITERATURE CITED
(1) Barnett, L. B., Bull, H. B., Biochim. Biophys. Acta 36, 244 (1959). (2) Baunian, E. K., Goodson, L. H.,
Guilbault, G. G., Kramer, D. N., ANAL.CHEM.37, 1378 (1965). (3) Bernfeld, P., Wan, J., Science 142, 678 (1964). (4) Blaedel, W. J., Haas, R., Department of Chemistry, University of Wisconsin, personal communication, November, 1965. (5) Blaedel, W. J., Hicks, G. P., Advan. Anal. Chem. Instr. 3 , 105 (1964). (6) Chang, T. 31. S.,Science 146, 524 I 1- 864). - - -,.
(7) Eli, A. B., Katchalski, E. J., J. Biol. Chem. 238, 1690 (1963). (8) Fondy, T. P., Kaplan, ?J. O., Trans. N . Y . Acad. Sci. 119. 888 11965).
wissenschajten 40, 508 (1953). (11) Guilbault, G. G., Kramer, D. N., ANAL.CHEY.37. 1675 (1965). (12) Hicks, G. P.,’Nalevac, G’. N., Anal. Biochem. 13, 199 (1965). (13) Hicks, G. P., Updike, S. J., Zbid., 10, 290 (1965). (14) Kadish, -4.H., Hall, D. A., CZin. Chem. 11, 869 (1965). (15) Lowry, 0. H., Passonneau, J. V., Schulz, D. W., and Rock, 31. K., J. Biol. Chem. 236, 2746 (1961). (16) Mitz, M. A., Summaria, L. J., .Vatwe 189, 576 (1961). (17) Vasta, B., Usdin, T., Aldrich, F., U. S. Army Rept. DA-18-108-405CML-828, (1963).
RECEIVED for review November 22, 1965. Accepted March 9, 1966. One of the authors (SJU) was supported as a postdoctoral fellow by the Badger State Civics Fund. I n part, Division of Analytical Chemistry, 150th meeting, ACS, Atlantic City, N. J., September 1965.
Solution Technique for Analysis of Silicates N. H. SUHR
and C.
0.INGAMELLS
Mineral Constitution Laboratories, The Pennsylvania State University, University Park, Pa,
A new solution technique makes possible the rapid and precise determination of most major and minor constituents of silicates in a single sample. Solution is effected by adding the melt of sample fused with lithium metaborate directly to cold dilute nitric acid that contains the internal standard cobalt. The solution is analyzed with an emission spectrometer using a rotating disk technique. Data for precision are given for the oxides of Si, AI, Mg, Ca, Sr, Ba, Ti, Mn, Fe, Cr, Cu, Zn, Zr, and Ni. Accuracy of the method is evaluated by comparison with chemical analyses of four rock samples. N a and K are de-
730
ANALYTICAL CHEMISTRY
termined on the same sample solution by flame photometry. The same solution can also be used to determine various elements colorimetrically, spectrographically, and by x-ray fluorescence spectrometry.
T
growing need for reasonably accurate analyses of large numbers of geologic samples has led during recent years to numerous developments, both chemical and instrumental. Rapid chemical methods have proved very useful, but they are intrinsically slow, and even when simplified to the utmost, demand skills which are not always available. X-ray fluorescence is widely HE
used, but requires special equipment for the determination of sodium and magnesium. I n the method here described, in which the sample is treated with dilute nitric acid after lithium metaborate (LiB02) fusion, almost all rock-forming minerals yield a clear solution which can be examined spectroscopically, flame photometrically, and chemically. A wide range of sample compositions can be tolerated. If a constituent falls outside the range of one method, another can be used without preparing a new sample. With a direct-reading spectrometer, very rapid analyses are possible. Precision compares favorably
