Determinations of Molecular Weight of Lignin Degradation Products by Three Methods SONJA K. GROSS, KYOSTI SARKANEN, and CONRAD SCHUERCH Department of Chemistry, State University College of Forestry at Syracuse University, Syracuse 7 0, N. Y.
b Molecular weight determinations on ethanol, hydrol, and kraft lignins have been made in dioxane and ethylene carbonate by cryoscopy and in tetrahydrofuran by an isopiestic method. Special techniques have been used to eliminate errors due to ash content and adsorbed solvents. The concentration dependence of molecular weights in dioxane indicates excessive association. Erratic results observed in tetrahydrofuran in this work and in other solvents by previous workers are probably also due to association. Cryoscopy in ethylene carbonate is the most convenient method of the three. Molecular weights of lignins in this solvent are most reproducible. Association is apparently not a prob'lem with ,this method, for the values are practically concentration independent and are less than by other methods. Its application to other complex products i s indicated, weight determinations on isolated lignins have been reported many times and a variety of results have been obtained ( 2 , 6, 8, 10-16, 18-21, 26, 30, 31, 34). Much of this work is difficult to evaluate. The products are mixtures of polymer fragments of considerable polydispersity, not always adequately defined, and are isolated by a variety of degradative reactions in varying yields. Products would not be expected to be of the same molecular size or size distribution and can therefore not usually be compared with one another. The values obtained usually fall into the difficult range of 500 to 5000,n-here the methods used for small molecules-eg., freezing point depression, boiling point elevation, isopiestic methods-begin to require unusual care to obtain moderate precision, and determinations useful for macromolecules, osmometry, light scattering, and other methods are not yet fully applicable. Isolated lignin samples hold adsorbed water and other solvents tenaciously even after extensive drying ( 2 4 >and unless care is taken, they can also retain ash. Such impurities cause large errors in number average molecular weights of this size. Lignin has a rather restricted solubility in organic solvents, requiring solvents OLECULAR
5 18
ANALYTICAL CHEMISTRY
not only of proper cohesive energy density but more especially of rather high hydrogen-bonding capacity (29). I t s tendency to form hydrogen bonds would suggest the possibility of association in solution. Some lignin samples would be expected to decompose a t the elevated temperatures used in common cryoscopic determinatioiis. These coniplexities suggest the need for the comparison of several methods on the same lignin samples, such as has only occasionally been made in the past, and for more definition of experimental procedures. I n this paper are examined the application of three methods of molecular weight determination to isolated lignins, an isopiestic determination with tetrahydrofuran, and cryoscopic determinations with dioxane and ethylene carbonate as solvents. The experiments were carried out n.ith the intent of avoiding recognizable sources of error. The methods permit the nieasurenients to be made a t temperatures below 40" C. to avoid decomposition. The mineral contents of the lignin samples were tested by ash determinations as well as by comparative measurements of conductivity in an acetonen-ater mixture. The ethanol lignins were usually ash-free by analysis and had conductivities in acetone-mater mixtures of 1 x 10-1 cm.-l ohm-l/gram per cc. The conductivity of ren-ashed pine kraft lignin was about three times this value. Its measured molecular weight may therefore be lower than the true value. Hydro1 lignin (V) had a conductivity as low as that of the ethanol lignins. The concentration dependence of molecular weight was investigated to assess the extent of molecular association, and absorbed solvent was eliminated by careful drying, by freeze-drying from the solvent used in the determination, and especially by a distillation drying technique. EXPERIMENTAL
Lignin Preparations. Ether-insoluble ethanol spruce lignin was prepared in chloroform ethanol solution (1) and isolated finally b y freeze-drying from dioxane. The ethanolysis resulted in 84% delignification, and t h e yields of SI,S2, a n d 5 3 TT-ere 28, 14, and 15%, respectively. The num-
bers correspond t o those of ( 1 ) . Maple hydrol lignin was prepared by Hibbert's method (3) and precipitated into ether. The reaction gave 80% delignification. The ether-insoluble fraction was 32%. fraction I11 was 2%, and fraction 1- was 7% of the total lignin. The isolation of the numbered fractions has been described (28). Ren ashed pine kraft lignin, presented by the Mead Corp., mas also tested. All the samples were dried for 3 days in a vacuum oven in the presence of phosphorus pentoxide a t 25" C. and stored over phosphorus pentoxide until used. Cryoscopic Determination Using Ethylene Carbonate (Cyclic Carbonate of Ethylene Glycol). Ethylene carbonate (Jefferson Chemical Co., Inc., S e w York) n a s purified (9) by partial crystallization and removal of the supernatant and then by vacuum distillation (10 mm. of mercury) of the higher melting fraction through a Todd colunin packed n ith single-turn glass helices. Fractions of 50 to 100 ml. were taken and those with melting points within 0.02" were combined as liquids. Each combined fraction was allon ed to crystallize n-ith stirring to form small crystals, and the crystals were vie11 mixed. K h e n purification is not thorough, freezing points are sometimes not stable. A standard Beckman freezing point apparatus was used with air and water bath. The water ba)h temperature v a s kept a t approximately 33" C. in a Den-ar flask. The freezing point of 20 grams of solvent was determined. The lignin sample mas then introduced and the freezing point of the solution determined. The freezing point was taken as the temperature of the plateau reached after slight supercooling of 0.1' or less. p-Kitroaniline nas used to determine the exact cryoscopic constant for each fresh bottle of ethylene carbonate. Nine to tnelve deterniinations were obtained easily in a day n-ith no special routine, when each batch of ethylene carbonate was used for three or four deterniinations (Table I). Cryoscopic Determination Using Dioxane. Purified dioxane (Eastman Chemical Co., n h i t e label grade) was refluxed over sodium for 3 hours, distilled, and stored over calcium hydride. The apparatus is shown in Figure 1. The weighed sample is introduced bv means of a glass spoon into bulb A . Exactly 15 nil. of dioxane was pipetted onto calcium hydride in flask E . B was cooled in liquid nitrogen and the system
evacuated. After the stopcock was closed, B was warmed and bulb A cooled in ice so that some of the dioxane distilled over and dissolved the sample. The dioxane was distilled back by warming bulb A and cooling flask B , and \vas dried by the calciuni hydride. Solvent that distilled into tube C lvas removed by tipping, so that it flowed back onto the sample. After this operation \vas repeated three times, C was chilled in a n ice bath and all the solvent was distilled into C, over a period of 1 hour. Air which had been dried by passing through two U-tubes containing barium perchlorate was now passed into the system. This was necessary because determinations in a vacuuni yielded erratic results. (Variations in pressure on the Beckman mercury bulb changed temperature readings niarkedly.) The apparatus \vas provided n-ith an air jacket and a cooling bath a t approximately 8" C., and the freezing point of the dioxane ivas determined. The stirrer ivas moved u p and dorm by magnet (Id). The ap11aratus was tipped so that the solvent ran into bulb -4.and dissolved the lignin sample. I t was then tipped back and the freezing point of tlie solution was determined. This experiment was carried out in 3, constant temperature room to niinimize the variation in stern temperature. A standard, azobenzene or p-iiitroaniline. to determine t'he freezing point depression constant, ivas run from tinie to tinie ( K = 5.3 i 0.5). Isopiestic Method Using Tetrahydrofuran. Isopiestic cells of t h e t y p e described by Clark (4)and by Parrette (17) were used. The total volume of t h e graduated measuring tubes was 2 ml. For molecular n-eight determinations of known compounds, Parrette's procedure was essent'ially followed. The equilibration of the tubes was carried out in a constant temperature water bath a t 30" & @ . @C., l othe t,op and sides of which xvere carefully insulated. The tubes were rocked with a gentle pendulum motion 60 times per minute. Agitation was necessary to avoid false equilibria. The procedure was identical for lignin samples, except that both lignin and standard were predried by distillation in a manner similar to that used for tlie cryoscopic method Ti-ith dioxane. IYeighed samples of lignin and reference substance axobenzene were placed in bulbs A and A' (Figure 2 ) . Tube C n-as sealed off and C' sealed to distillation tube E. Flask D contained t,etrahydrofuran somewhat, in excess of the amount needed for the determination and, in addition, a feiv grams of calcium hydride. Flask D n-as chilled in dry ice-acetone cooling niixture and the whole system was eracuated through stopcock F and thoroughly degassed. Half of the solvent was subsequently distilled int,o il and A' and the solids were dissolved. The solwnt was again distilled back into D and the process repeated twice. Finally, the correct amount of solvent was distilled back into -4 and 8' for the deterniination. Tube C' was sealed off and equilibration a t constant temperature
Table 1.
Experimental Molecular Weight Values on Lignin Preparations b y Different Methods
Cryoscopy in Cryoscopy Isopiestic Detn. in Ethylene Carbonate in Dioxane Tetrahydrofuran Concn., hiol. Concn., hiol. Concn., Mol. Type of Lignin grams/liter wt. grams/litei wt. grams/liter" v-t . , . . Ethyl alcohol lignin SI ( 1 ) .. 7.0 908 ... ... 910 spruce i . 1 .. . , , . . .. ... 14.5 852 2710 Ethyl alcohol lignin SI1 ( 1 ) 8.2 2420 20.3 1842 9.7 3150 16.3 3140 43.6 1560 13.5 b spruce 45.6 li20 24.2 3380 21.5b 1615 49.1 2495 3100 21.7 2000 4150 30.2 54,8 2180 79.1 4690 Ethyl alcohol lignin SI11 ( 1 ) 7.6 2150 , . , . . 35.4 3610 43.4 1910 .. ... 15.3 spruce 3000 .. ... 90.5 23.6 1900 1735 , . ... Rexvashed pine kraft lignin 7.0 1085 10.4 21.0 1810 , . ... 1065 13.7 1940 .. ... 1150 27.1 21.4 ... 1930 , . 1165 31.2 27.8 ... .. Maple hydrol lignin I11 (86) 10.0 1915 , . ... ... 17.7 , . ... .. 1805 ... .. ... .. Maple hydrol lignin T (26) 6.1 905 ... ... .. 11.4 1005 .. 2980 .. ... Ether-insoluble 7.7 1120 19.7 .. , . . 3300 15.4 Maple hydrol lignin ( 3 ) 1000 24.2 3520 28.1 23.4 975 a At equilibrium. In dioxane not distilled in apparatus. "
?
Figure 2. Apparatus for determinations
Figure 1. Apparatus for cryoscopic determinations in dioxane
begun. Readings were taken a t convenient intervals until equilibration. RESULTS AND DISCUSSION
These three methods mere found to be accurate and satisfactory with known compounds of ordinary molecular weight. By far, the most convenient is the cryoscopic determination in ethylene carbonate. It is a n excellent solvent for a variety of substances and for practical purposes it is nonhygroscopic. KO special precautions need t o be taken and several determinations can be completed in a working day. Presumably, by the use of thermistor or thermocouple circuits, the determinations could be run equally well on smaller samples.
isopiestic
On compounds of lon moleculai weight the most precise method is the isopiestic technique. Hoir ever, preparation of the tubes is sonierrhat tedious and 4 days or more arc required for equilibration. Cryoscopic determinations of molecular weight in the solvent dioxane have been used frequently but it is a troublesonic method, even for small molecules. Deterniinations TT ere repeated and duplicate ~ a l u e svere obtained consistently only when tlie 012eration was carried out in a closed system or prcferably in one open t o the atmosphere through dr\ ing tubes. L-nder other conditions moisture 1% as absorbed from the atmosphere and the results were completely unreliable. Klien these three niethods mere applied t o various isolated lignin samples, the cryoscopic determination n ith ethylene carbonate as solvent gave satisfactory results (Table I). The technique v a s simple, the values n ere invariably VOL. 30, NO. 4, APRIL 1958
519
consistent, and the molecular weights were almost concentration-independent, indicating little association. There is no reason t o doubt the accuracy of these determinations. However, on long standing both ethanol spruce lignin fractions and pine kraft lignin fractions increased in molecular weight as though condensations were occurring during storage. After several months Vaino Era in this laboratory obtained niolecular weights about IO t o 20% higher than those obtained by S. K. Gross. The same phenomenon had been previously observed by Gross and has been reported by Enkvist and Hougberg (7). Cryoscopic determinations were repeated on some of the samples using dioxane as solvent. It was possible to remove a n y adsorbed extraneous solvent which would lower the molecular weight by freeze-drying the lignin from dioxane before determination, or by the distillation drying technique described. A duplicate determination using the two drying methods indicated they mere probably equally effective (Table I), though transfer into the apparatus after freeze-drying introduces a n element of risk. Several conclusions can be d r a m from the comparison between determinations in dioxane and ethylene carbonate. First, all three lignin samples whose molecular weights were measured in both solvents were more associated in dioxane than in ethylene carbonate, because their molecular weights showed a higher concentration dependence in dioxane. Second, extrapolation of the molecular weight values in dioxane to zero concentration gave roughly the same molecular weight in one case, and on the other two samples, higher molecular weight values than those obtained in ethylene carbonate. While extrapolation to zero concentration will generally correct for nonideal behavior of the system, this is not always true. Highly associated compounds like phenol in benzene solution give on extrapolation molecular weights substantially higher than the correct value (32). However, there is some possibility that tightly adsorbed dioxane is present on the lignin and depresses the molecular weight values obtained in ethylene carbonate though not appreciably in dioxane. At present, it is not possible t o assert that the lower values are completely accurate, even though ethylene carbonate is clearly a superior solvent. Third, the concentration dependence of molecular weight of the different lignins in dioxane is not the same and is least for kraft lignin. The high concentration dependence of molecular weight of the other two lignins in dioxane, and the poorer precision obtained with ethanol lignin also place limitations on the accuracy of the extrapolation t o
520
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ANALYTICAL CHEMISTRY
zero concentration. I n conclusion, the convenience and precision of the ethylene carbonate method are much greater than with dioxane but its accuracy can not yet be considered proved. However, another series of determinations in ethylene carbonate has been carried out in the laboratory in addition to those reported here (27) and the results have been completely consistent. I n the authors’ opinion, this method is superior t o any other published method for application to isolated lignins. Interest in the isopiestic technique arose in part from the assumption that volatile solvents adsorbed on the lignin might equilibrate and would not interfere with the molecular weight determination. An experiment using water as the volatile impurity showed however that equilibration can be incomplete even after 70 hours. -4 slowly changing reading could therefore be mistaken for a constant value which was substantially different. To avoid this source of error any extraneous solvent adsorbed on the lignin was removed by the distillation drying technique described. Furthermore, several of the values were obtained by equilibration in both directions, so that false equilibria were eliminated. Kevertheless, the scatter of points obtained from individual determinations a t different concentrations was too great to permit a close estimate of molecular weight. Similar erratic results have previously been reported on lignin molecular weight determinations (16). Because the accuracy obtained on simple compounds was to better than 2%, these erratic results can only be ascribed to a strong association of lignin in the solvent tetrahydrofuran. Apparently the degree of association is dependent on small uncontrollable variations in the experimental procedure. The fact that ethylene carbonate proved to be a n excellent lignin solvent is surprising, for esters are generally very poor hydrogen bond acceptors. However, the melting point, boiling point ( I S ) , and dielectric constant (33) of ethylene carbonate are all markedly different from the same physical properties of common esters, and there seems to be no reason to doubt that it may have a reasonably strong hydrogenbonding capacity. The association of lignin in dioxane might be expected, for the solvent power of dioxane is substantially increased by the addition of small amounts of methanol, and dioxane has one of the lowest hydrogen-bonding capacities of known lignin solvents. Polyamides, which also require hydrogenbonding solvents, are known to be associated in dioxane ( 5 ) . Furthermore, acetone has almost the same cohesive energy density and hydrogen-bonding
capacity as dioxane and should therefore have similar solvent power for lignin. Puddington (92) has observed a substantial concentration dependence of the molecular weights of maple hydro1 lignin fractions dissolved in acetone. CONCLUSION
In the past, liquids have been used as solvents for lignin molecular weight determinations which are not good enough lignin solvents to give correct molecular weight values. Molecular weights in such solvents show a substantial concentration dependence and a tendency to give erratic results in successive determinations. Extrapolation to zero concentration may not correct for association (32). Apparently the same difficulty has been observed in the determination of molecular weights of tannins (93). Ethylene carbonate is a n excellent and convenient solvent for the cryoscopic determination of lignin molecular weights. Successive determinations give consistent results with little concentration dependence. It can probably be recommended for other complex natural products such as sterols, lignans, and tannins. It suffers from the sole disadvantage that the absence of adsorbed solvent is difficult to establish. An ideal solvent for the determination of molecular weights for amorphous substances such as lignin should be sufficiently volatile to permit the isolation of the sample from the same solvent as is used in the determination. Previous reports that some lignin samples increase in molecular weight on storage have been confirmed. ACKNOWLEDGMENT
The authors gratefully acknowledge fellonship support for Kyosti Sarkanen from the Research Corp. and the Technical Association of the Pulp and Paper Industry. The distillation-drying technique described was kindly suggested by E. W, Abrahamson of this college. The authors wish to acknowledge the work of Vaino Era in confirming the low concentration dependence of lignin molecular weights in ethylene carbonate and the increase in lignin molecular weight on long standing. LITERATURE CITED
(1) Arlt,
H., Sarkanen, K., Schuerch,
C.. J . Am. Chena. Soc. 78. 1904 ).
A. J., Paper Trade J . 111,
(1941).
Doty, P. X.,Bradbury, J. H., Holtzer, A. M., J . A m . Chem. SOC.78,947 (1956). Enkvist, T., Svensk Papperstidn. 51, 225 (1948).
Enkvist, T., Hougberg, B., Paper and Timber (Finland) 37, No. 5, (17)
201 (1955).
Gralen, iX.,J . Colloid Sci. 1, 453 (1946).
Gross, S. K., Schuerch, C., AXAL. CHEM.28, 277 (1956). Haaalund, E., Urban, H.. Cellulose-
(18) (19) (20)
Zbid., 9, 49 (1928).
’
Hess. K.. Heumann., K.., Ber. 75. 1802 (1942).
Jefferson Chemical Co., Kew York, “Ethvlene Carbonate.]’ Klages,“F., Ann. 541, 25 (1939). Loughborough, D. L., Stamm, h., J . Phys. Chem. 40, 1113 (1936). Mikam-a, H., Okada, S . , J . Chem
(21) (22) (23) (24) (25)
SOC.Japan, Ind. Chem. Sect. 54, 239 (1951). Parrette, R. L., J . Polymer Sci. 15, 447 (1955). Pauly, H., Ber. 67, 1177 (1934). Payne, J. H., Fukunaga, E., Kojima, R., J . Am. Chem. SOC.59, 1210 (1937). Pennington, D., Ritter, D. M., Zbid., 69, 665 (1947). Phillips, M., Zbid., 49, 2037 (1927). Puddington, I. E., Ibid., 72, 3840, Table I1 (1950). Roux, R. D., J . SOC.Leather Trades’ Chemists 37, 259 (1953). Sachtling, H., Zocher, H., KolloidBeih. 40, 413 (1934). Samec, M., Kolloid 2. 51, 96 (1930).
Sarkanen, K., Schuerch, C., ANAL CHEM.27, 1245 (1955). Sarkanen, K., Schuerch, C., J. Am. Chem. SOC.79, 4203 (1957).
Schuerch, C., Zbid., 72, 3838 (1950).
Ibid., 74, 5061 (1952).
Schwabe, K., Hasner, L., Cellulosechemie 20, 61 (1942).
Staudinger, H., Dreher, E., Ber. 69, 1729 (1936).
Turner, W. E. S., English, S., J. Chem. Soc. 1914, 1786.
Watanabe, J., FUOSS, R. M., J . Am. Chem. SOC.78, 527 (1956).
Wedekind, E., Katz, J. R., Ber. 62, 1172 (1929).
RECEIVEDfor review May 31, 1956. Accepted December 19, 1957.
Photometric Determination of Beryllium UNO T. HILL, Inland Steel Co., lndiana Harbor, Ind. b Existing methods for the determination of beryllium require chemical separations from interfering elements. Direct photometric methods for the determination of beryllium in aluminum, steel, copper, titanium, and mixed oxides are described. Beryllium reacts with Eriochrome Cyanine R to form a red complex having a maximum absorbance a t 512 mp a t pH 9.8. Addition of Versenate and cyanide eliminates interfering ions. The methods are accurate and rapid.
A
RECEKT paper
from this laboratory (1) indicated Eriochrome Cyanine R as a specific reagent, for beryllium when sodium Versenate [(ethylenedinitrilo)tetraacetate] was used to mask interfering elements. The present study was made to establish more definitely the accuracy and limits of applicability of the Eriochrome Cyanine R method. Efforts were directed in particular to the development of a rapid and specific method for trace amounts of beryllium in aluminum, iron, copper, lead, titanium, and their alloys without chemical separations. ’ APPARATUS AND REAGENTS
Beckman DU spectrophotometer. Reagents. Eriochrome Cyanine R, 0.09%, General Dyestuff Corp. Dissolve 0.90 gram of Eriochrome Cyanine R in about 250 ml. of water, add 25 grams each of sodium chloride and ammonium nitrate, add 2 ml. of nitric acid (specific gravity 1.42) and 100 ml. of 95% ethyl alcohol, dilute to 1 liter, and mix. A 0.009yo solution a t p H 6.0 should have an absorbance of 0.9 in a 1-em. cell a t 438 mp (1). Sodium Acetate buffer. Dissolve 50
grams of sodium acetate in water and dilute to 1 liter. Sodium Versenate Solution. 5%. Hach Chemical Co. Dissolve 50’ g g m s of disodium dihydrogen Versenate [(ethylenedinitri1o)tetraacetatel in water and dilute to 1 liter. Standard Beryllium Solution (1ml. = 2.0 y of beryllium). Dissolve 0.1965 gram of beryllium sulfate tetrahydrate in 25 ml. of hydrochloric acid, dilute to 1 liter, and mix. Transfer 100 ml. of this solution to a 500-ml. volumetric flask, dilute to the mark with O.lyo hydrochloric acid, and mix. I
PROCEDURES
Aluminum. T o a 1.OOOO-gram sample in a 250-ml. beaker add 20 ml. of 1 to 1 hydrochloric acid, warm on a hot plate until in solution, cool, and dilute to 100 ml. in a volumetric flask. Transfer a 1.00-ml. aliquot into a 50ml. volumetric flask and add 5 ml. of 5% sodium Versenate, 5 ml. of 5% sodium acetate, and 5 ml. of 0.09’% Eriochrome Cyanine R. Adjust the p H to 9.7 to 9.8 with lOY0 sodium hydroxide by titration from a buret. The color change of Eriochrome Cyanine R is through red, yellow, and finally dark purple. When this last end point is reached, add 5 drops of 10% hydrochloric acid to obtain a p H of 9.7 to 9.8. Dilute t o the mark and mix. Alternatively, employ a p H meter to adjust the pH. Obtain the absorbance of the sample against a blank of beryllium-free aluminum carried through all the steps of the procedure. The measurement may be carried out in a 2-em. cell a t 512-mp wave length. Obtain per cent of beryllium from a previously prepared calibration curve. Steel. Process a 1.OOOO-gram sample as for aluminum b u t adjust the p H with 10% potassium hydroxide. T h e iron must be in the reduced state.
Measure the absorbance of the sample against a blank of beryllium-free steel carried through all the steps of the procedure. Obtain per cent beryllium as for aluminum. Copper. T o a 1.0000-gram sample in a 250-ml. beaker add 20 ml. of 1 to 1 nitric acid. Warm, boil off the oxides of nitrogen, cool, and dilute t o 100 ml. in a volumetric flask. Transfer a 1.00-ml. aliquot into a 50ml. volumetric flask, and add 5 ml. of 5% sodium Versenate, 5 ml. of 5% sodium acetate, and 5 ml. of 0.09% Eriochrome Cyanine R. Add sufficient 10% potassium cyanide (2 ml. is usually sufficient) to discharge the blue coloration due to cupric complexes, and adjust the p H as for aluminum with sodium hydroxide. Dilute to the mark and mix. Obtain the absorbance against a blank of beryllium-free copper in a 2em. cell a t 512 mp. From a calibration curve obtain per cent beryllium. Titanium. T o a 1.0000-gram Sample in a 250-ml. beaker add 25 ml. of concentrated hydrochloric acid, cover, and heat on a water bath until in solution. Replenish the acid if necessary. When in solution cool and dilute to 100 ml. in a volumetric flask. To a 1-ml. aliquot in a 50-ml. flask add 4 drops of 30% hydrogen peroxide, 2.5 ml. of 5% sodium Versenate, 5 ml. of 57, sodium acetate, and 5 ml. of 0.09% Eriochrome Cyanine R, and adjust the p H to 9.7 to 9.8 as for aluminum, using 10% potassium hydroxide. Dilute to the mark and mix. Obtain the absorbance against a blank of beryllium-free titanium carried through all the steps of the procedure. From a calibration curve obtain the per cent beryllium. Oxides. T o a 1.0000-gram sample in a 250-ml. beaker add 25 ml. of concentrated hydrochloric acid, cover, and digest a t low heat. Remove cover, take to dryness, and bake to dehydrate silica. Add 10 ml. of VOL. 30, NO. 4, APRIL 1958
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