Two Improved and Simplified Methods for the Spectrophotometric Determination of Hydroxyproline MANUEL BERGMAN crnd ROY LOXLEY Safety in Mines Research Establishment, Ministry o f Power, Sheffield, England
b A method, based cm that of Stegemann but with a simplified procedure, has been developed for the spectrophotometric determination of hydroxyproline. Radical improvements have been achieved in the stability of the reagents and of the Final color. The variation of the color yield with the conditions a t various stages in the analytical procedure has been minimized, and been made independent of hydroxyproline conceitration over a wider range than hithel'to reported. In addition, a procedure has been developed which involves no thermostat bath, as the color development is carried out overnighi a t room temperature instead of in an accurately timed period a t 60" C.
A
NUMBER of analytical methods for tEe spectrophotometric estimation of hydroxyproline have been described. Since 1950 at least 18 have been based on the oxidation of hydroxyproline to a compound related to pyrrole, and the subsequent condensation of this intermediate compound with Ehrlich'si reagent (p-dimethylamino benzaldehyde) to give a red dyestuff. The finad color yield and the reproducibility and stability of the color depend on many factors. Some authors have investigated a number of these factors and have produced methods which, if used with care, would produce good results for the particular system in question. ?he frequency of new papers dealing with further modifications to the method, however, .points to the lack of a publication in which the variables of the method have been comprehensively studied, and their effect on analytical results m nimized. The two basic methods of Stegemann ( 9 ) and of Neuman and Logan (6) have been studied in this laboratory. The method of Stegemann has been used as the basis of the procedures suggested in the present work. Isorropanol has been substituted for methoxy-ethanol (9) and n-propanol ( 6 ) , and the variables studied have includelj the optimum time and temperature for oxidation and color development, 5,s well as the optimum concentrations of organic solvent and of the rsagents a t each stage of the procedure
LARGE
(3) Hydroxyproline Standard Solution. This was normally 400 p.p.m. 1-hydroxyproline (British Drug Houses, Laboratory Reagent) made up in 0.001M HC1 to minimize bacterial growth. The solution was stable for several months if kept a t 4 ' C., and only the portions taken for dilution were allowed to reach room temperature. RAPID PROCEDURE A. Clean, dry, 30-ml. test tubes were used in this procedure. (1) A 1-ml. portion of the neutral or faintly acid solution to be analyzed was pipetted into each test tube, 2 ml. of isopropanol were added from a pipet EXPERIMENTAL with mixing, 1 ml. of the oxidant solution was added from a pipet, and the All the chemicals used were of solutions were well mixed and allowed 'AnalaR' grade where available. Abto stand for 4 ( + I ) minutes a t room sorbances were measured on a Hilger temperature (17" to 21" C. in the presUvispek spectrophotometer and, where ent work). the shape of the absorption curve was of (2) Thirteen milliliters of the interest, on a Gary recording spectroEhrlich's reagent solution were added, photometer. Analytical Method. REAGENT the solutions were well mixed, and the tubes heated for 25 minutes (*15 SOLUTIONS. (1) Oxidant Solution. seconds) a t 60" ( i 0 . 2 " ) C. in a water (a) A 7% w./v. aqueous solution of bath. The tubes were cooled for 2 to 3 Chloramine T (the sodium salt of p minutes in running tap water, and then toluene sulfon-chloramide) was made the solution was diluted to 50 ml. in a up daily in the present work (about standard flask with isopropanol. The 0.25 ml. per analytical determinaabsorbance against water was measured tion, allowing for manipulation), but 4 hours in a 1-em. cuvette a t within was found t o be active for several 558 mp. weeks. A reagent blank was included in the (b) An acetate/citrate buffer of p H procedure by substituting water for the value 6.0 was made up by dissolving 57 unknown solution and the absorbances grams of sodium acetate (3Hz0), 37.5 were corrected accordingly. A hygrams of trisodium citrate (2Hz0), droxyproline standard solution was also 5.5 grams of citric acid (HzO) and 385 included as a precaution. Whenever a ml. of isopropanol in water and made new batch of any of the solvents or up to 1 liter with water. This solution reagents was used, three to five hydroxywas stable indefinitely. proline standard solutions of between 5 Just before the start of each series of and 40 p.p.m. were included in the determinations, solutions 1(a) and 1(b) determination. were mixed in the proportion of 1 The procedure as described gives an volume of I(a) to 4 volumes of l ( b ) absorbance of 0.54 in a 1-em. cuvette to give the oxidant solution (about 1.25 for 40 pg. of hydroxyproline, and is ml. per analytical value required). therefore best suited to solutions con(2) Ehrlich's Reagent Solution. (a) taining between 20 and 35 p.p.m. The p-Dimethylamino-benzaldehyde was absorbance of the reagent blank is dissolved in 60% perchloric acid (S.G. normally below 0.01 for a 1-cm. cuvette, 1.54) in the proportions of 2 grams of and therefore solutions containing less aldehyde to 3 ml. of acid. This solution than 20 p.p.m. of hydroxyproline can be was made up fresh in the present work accurately estimated by measuring the (about 3 ml. per analytical value reabsorbance of the colored solution in 2 quired) but was stable for several weeks or 4-em. cuvettes. If the final in a dark bottle. dilution step of the procedure is omitted, (b) AnalaR Isopropanol. Just bethe sensitivity of the method rises fore the start of each series of deterthreefold, but the stability of the color minations, solutions 2(a) and 2(b) were is decreased, and the absorbance should mixed in the proportions of 3 volumes of be measured within half an hour. I n 2(a) to 13 volumes of 2(b) to give a addition, the possibility of error owing to final volume of about 15 ml. per analytical value required, allowing for variable volume changes by evaporation manipulation. during the heating stage is introduced The present methods are convenient, little affected by small variations in most of the parameters involved, and easily adapted to individual requirements. The stability of the reagents and of the final coior has been markedly improved. The color yield is accurately proportional to the hydroxyproline concentration over a wide range of concentrations; calculation shows that the yield is higher for a given concentration than that of any other method described in the literature.
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If the hydroxyproline solution is 30 highly colored-e.g., with pigments resulting from the hydrolysis of tissuethat the absorbance of the final color may be affected, this may be allowed for by the inclusion of another blank. One milliliter of the solution to be analyzed is taken through the procedure, but the heating stage is omitted, and no aldehyde is added. The absorbance obtained, added to that of the reagent blank, is used to correct that of the solution obtained by the normal procedure. OVERKIGHT PROCEDURE B. This procedure does not require dilution to stabilize the final color; i t is suitable for the analysis of solutions containing 2 to 15 p.p.m. of hydroxyproline (ideally 6 to 12 p.1i.m.). Procedure B is conveniently carried out in glass-stoppered tubes, with the color development a t room temperature. If there are likely to be fluctuations of temperature, the samples and standards should be placed in a thermally insulated box. The most suitable acid concentration for color development a t about 20' C. was found to be about 0.6X perchloric acid instead of 0.815M. The Ehrlich's reagent solution was, therefore, made up as follows: the aldehyde was dissolved in 607, perchloric acid in the proportions of 10 grams of aldehyde to 11 ml. of acid (about 3 ml. per analytical value required), and stored in a dark bottle. Just before the start of each series of determinations, this solution was mixed n i t h isopropanol in the proportions of 3 ml. to 16 ml. of isopropanol (15 ml. per analytical value). The absorbance was measured a t 558 mp after about 17 hours. RESULTS AND
DISCUSSION
The Suggested Methods. A calibration curve for one of t'he suggested methods (Procedure A) is shown in Figure 1 and is compared with those of some other authors. The color yield (molar absorptivity relative t o hydroxyproline 90 X l o g liters per em. mole) is higher and independent of hydroxyproline concentration over a wider range than for any other method described in the literature. For convenience in the consideration of variahles likely to affect the color yield, the method can be divided into a numher of stages: sta,bility of reagents; oxidation of hydroxyproline; color formation and stability. STABILITY O F REAGESTS. Chloramine T. One batch of this material was received after having been in storage for several years, and was found to be inactive. If stored in a buffer solution mised with organic solvent, as reconimended by Stegemann ( 9 ) )it can, indeed, lose its activity in a fen- hours a t room temperature. But if stored in aqueous solution (pH 7.5), it, retains its activity for several weeks a t room temperature. Ehrlich's Reagent. Many authors have blamed impurities or decomposit,ion products in the solid p-dimethyl1962
ANALYTICAL CHEMISTRY
H y a r a x y p r o l ~ n et a k e r
~i
moles
Figure 1. Comparison of calibration curves for a number of methods
S a C l in the test solution had no ailamino benzaldehyde for variable results. This aldehyde has been received in preciable effect on the method. Departure from Seutrality of Test batches varying in color from JThite to Solution. Tenth molar HC1 had no pale yellow or green; the color was uneffect either, but the capacity for alkali related to the nominal purity of the of the buffer is ~ O T T - ,and 0 . l X NaOH material. When the aldehyde, even considerably lowered the color yield. though purified and colorless, was disconcentration of Chloramine T. solved and stored in organic solvents, Between Chloramine T concentrations the h i e and conditions of storage had of 7.1 and 17.9mlll at stage -4,there considerable effect on the method, was a barely detectable trend towards a especially in raising the absorbance of lolvering of the color yie!d obtained with the reagent blank. K h e n , as in the increasing Chloramine T concentration suggested procedure, the aldehyde was and an oxidation time of 20 minutes. dissolved and stored in aqueous acid in The concentration finally adopted, a dark bottle, the reagent blanks n-ere 12.5mX, was, however, higher than the low and reproducible. Removal of the minimum studied, to allow for reducible color from the solid aldehyde was unsuhstances that might he present, for necessary. I n addit,ion, dissolution of example, in hydrolyzates. the aldehyde was much easier in the Concentration of Organic Solvent. acid than in an acid organic-solvent K i t h concentrations of organic solwnt mixture. .such as used by Stegeniann ( 9 ) at stage OXIDATIOS OF HTDROXSPROLINE 1 (about 11% v , ' Y I . the color yield in(Stage 1 in Procedure). Chloramine T, rrcased with oxidstion time up to 20 the oxidizing agent introduced by minutes. Khen the organic solvent Stegemann (,Y), has bern adopted in the concentration wa. increaqed, the color present n-ork together with the citrate,' yield hecame relatiT-eiy independent of acetate buffer of p H d u e 6.0. Six oxidation time 1,etn.een 2 and 30 variables have, hon-ever, been investiminutes. \Then the concentration of gated in this oxidation stage, and the isopropanol in the prezent method v a s reagent has been modified accordingly: raised abol-e 62Yc ~ , , t ' vs .t itage 1, howconcentrations of salt in the test soluever, a cloudiness nppeared during tion. such as might be derived from the olidation, and the color yield decreased neiitralization of hydrolyzates of bioand became erratic. The concentration logical niatcrial; dppartiire from ncutralof isopropanol in the .suggested proity of the test wlution; concentration cedure (58.3yc v./v.) i- m a region of Chloramine T ; concentration of 1%-heresmall variations of concentration organic solvent; time of oxidation: do not affect the color J-ield. and presence of 2 mg. of alanine to Time of Oxidation. Pro\-ided the simulate nonspecific amino acids in organic solvent concentration a t stage hydrolyzates ( 2 ) . 1 \vas kept above shout 40% and hplow Salt in Test Solution. Vp to 0.1-11
?.
m
7
I2 4
Figure 2. Absorption spectra of blank and solution corresponding to 40 pg. c,f hydroxyproline per 50 mi. in 2.5-cm. cuvettes
62%, the color yield was relatively independent of time between 2 and 30 minutes. There was, however, a barely noticeable trend for the color yield to decrease with osidatioi time; a period of 4 =t1 minutes n-as t'ierefore adopted. Presence of .\lanine. I n the met,hod of Prockop and L-denfriend ( 7 ) the presence of 2 nig. of alanine a t the oxidation stage almost completely inhibited color formatio:i with 10 pg. of hydroxyproline. These authors therefore suggested the us(: of higher concentrations of Chloramine T (xhich lowered the color yield considerably in their method) and of alanine (which partly reversed this effect) in o procedure designed for tizsue hydrolyzates poor in hydroxj-proline. I n the present work the depression in color yield for 2 mg. of alanine iva? onlj about 37,. COLORFORXATIO?; (Stage 2 in Procadiire). A typical a1)sorption spectrum is shown in Figure 2 For the red cdor produced in the pre:,ent method togethw with that of a reagent blank, I-iitler all conditiolis the rolor yield incrcwed initially u-ith heating time and the11 fell. Both the. development, of the c o h and its fading n-ere dependent on the temperature of heating, the acid concentration. and, a t 1ovi2r concentrations of orgmic solveiit than suggested, on the solvent cciiicentration also. When esposed to ultmviole t light-e.g., in vitreous silica cuvettes-the fading of the color w\-nsaccelerated. The variables studied a t stage 2 were: concentration of isopropanol ; conceiitration of aldehyde; heating time; temperature of heating bath; and concentration of perchloric acid. Concentration of Isopropanol. Small variations of #:oncentration of isopropanol from the 74y0 suggested had litt'le effect on eithar bhe color yield or on the optimum heating time. Below a concentration of about 5070, however, the optimum heating time, the
I
I
"
0 5
1 3
M o /e $ / t i t r e
Figure 3. Variation of color yield with concentration of aldehyde at color development stage for 40 pg. of hydroxyproline
color yield, and in addition the stability of the color tended to decrease. Concentration of Aldehyde. The variation of color yirld with concentration of aldehyde a t stage 2 is illustrated in Figure 3. The concentration of perchloric acid in each case was made 0.26X higher than that of the aldehyde to allow for neutralization. Heating Time and Temperature. The heating curves (color yield plotted against time) for 40 pg. of hydroxyproline in 0.815J1 perchloric acid a t three heating temperatures are shown in Figure 4. The variation of color yield with heating time was leas a t 55" C. than at higher temperatures, but 60" C. gave sufficiently flat curves and a shorter and more convenient optimum heating time. ilt still higher temperatures not only did the optimum heating time decrease, but the color yield also. The relationship between optimum heating time and heating temperature is exponential T = - 34 3 loglot
+ 107.6
where t = time in minutes and T is temperature in " C. Values of optimum heating times of about 2 minutes a t 100" C., and about 9 hours at room temperature, fit the relationship fairly well. The overnight procedure B was tested with 0.6X perchloric acid at 20" C. and at 25" C. Expressed in terms of the maximum color yield with the rapid procedure -1at 60" C., the color yield of procedure B was 97% after 17 hours a t 20" C., and 98% after 24 hours. A t 25" C. the yield was 98y0 after 17 hours, but had dropped to 92% after 24 hours. The value of color yield is, therefore, relatively independent of temperature after 17 hours. I n laboratories where the temperature is normally loner than 20" C., longer time. or higher acid con-
centrations may be more convenient. Procedures could also be easily devised for color development in incubatorb a t 37" C., where these are available. Heating Time and - h i d Concentration. The heating curves a t 60" C. for 40 pg. of hydroxyproline a t five perchloric acid concentration. are shonn in Figure 5 . The product of optiniuin heating time and acid concentration, L l , is 21 (where t = time in minutes and -1 = acid concentration in moles per liter). X concentration of 0.81531 a t stage 2 gave the maximum color yield and a convenient optimum heating time. With color development a t 20" C. or 25" C., an acid concentration of 0 815.11 at stage 2 gave lower color yields than O..5Jf or 0.6-11. An acid concentration of 0.6X a t stage 2 wab, therefore, adopted for the O T ernight procedure B as being best suited to a development time of about 17 hours a t about 20" C. Derivation of Method. The present method mas derived from t h a t of Stegeniann ( 9 ) in a number of qtages in which the reagents and solvent.. and their concentrations R ere varied $0 as to give the maximum yield and stability of color. He added acid to hi.. solution after oxidation, and allon ed time for the destruction of Chloramine T. I n the present work, this delay n a j found to be unnecessary, and the combination of the acid and aldehyde solution reduced the procedure by one reagent and one time interval. Hydrogen peroxide mas the oxidant used by Seuman and Logan (6) and in at least 14 derived methods, and was investigated in these laboratories ( 1 ) . With such methods, the color yield depends markedly on the concentration of peroxide and a130 on that of hydroxyproline. I n addition, all traces of peroxide must be removed before the color developnient stage. I n contrast to the present methods, additional inVOL. 35, NO. 12, NOVEMBER 1963
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1963
Heating time ( I ) : m i n i
Figure 4. Variation of color yield with heating tima for 40 pg. of hydroxyproline in 0.815M perchloric acid at three temperatures
,
IO
io
I 3c
40
io
convenient or uncertain procedures are Healing t i m e ( 1 ) mins necessary. Figute 5. Variation of color yield with heating time for 40 n-Propanol was used by Seuman and pg. of hydroxyproline a t 60" C. at a number of concentraLogan ( 6 ) ,and in modifications of their tions of perchloric acid method, as the organic solvent necessary to keep the aldehyde in solution at In general, overheated solutions-Le., acidities low enough for the final color papers describing spectrophotometric those which had been heated for a time to be stable. I n agreement with methods for estimation of hydroxygreater than the optimum for maximum Bowes ( 2 ) this solvent gaTe erratic reproline have failed t o test their absorbance-decreased in absorbance sults and lower color yields than isoproassumptions about the nature of the with time of standing a t room temperapanol despite attempts a t purification. oxidation product of hydroxyproline. ture, and underheated samples increased Methyl-cellosolve (2-methoxy ethaIn general, pyrrole and pyrrole-ain absorbance, often up to the maximum nol), the solvent used by Stegecarbo.;ylic acid have been suggested value. Dilution of the solution slowed mann (9) and in modifications of his (8). I n the present work both these down this trend. With underheated method (7, 10) gives even lower color compounds have, in turn, been subsamples, moreover, the wavelengths of yields and is liable to form interfering stituted for hydroxyproline in the the absorption peak (Amx) and, as far peroxides with storage. method, but gave no color. If the as could be seen, of the whole of the Dilution of the colored solution to a Chloraniine T reagent and the Ehrlich's double-humped absorption band started constant volume was suggested by reagent solutions were first mixed to at lower values. These values of Lollar (5) and by Bowes (2) to minimize destroy the Chloramine T and the increased to that of the optimally solution containing either pyrrole or errors owing to evaporation of the , , A heated samples with time of heating pyrrole-a-carboxylic acid added last, solution and fading of the color. I n the and, more slowly, with time at room the normal color developed, but at a present work, however, dilution with temperature. Dilution of the solution rate differing markedly for pyrrole, organic s o l ~ e n trather than with mater slowed down this trend also. pyrrole-a-carboxylic acid, and the reduced the fading of the color even The variation of A, with heating further, and also reduced the moveoxidation product of hydroxyproline time was even more dependent on the obtained in the present method. The ment of the wavelength of the absorptemperature of heating and on the conimplication is that unlike the latter comtion peak. I'nlike most of the current centration of acid at this stage. Depound, pyrrole and pyrrole-a-carboxylic methods, the present procedure allows crease of heating temperature and inacid are unstable in the presence of the measurement of the color to be made crease in acidity both tended to result in Chloramine T at p H 6. over a period of several hours (if prohigher values of. , , ,A At temperatures The optimum heating time of pyrroletected from ultraviolet light), without of 100" C. and in 0.2561 HClOd, , ,A a-carboxylic acid, when added last, was affecting the accuracy of the result. values as low as 550 mp were recorded. in the region of 80 minutes as against An increase in the proportion of At 60" C. ,A, values varied from 551 25 minutes for the hydroxyproline oxiorganic solvent a t the color developmfi in 0.4M HClO, to 558 mu and, ocdation product. The color yield was ment stage was suggested by Grunbaum casionally, to 562 mu in 1.0 to l.5M only about 75% of the optimum for and Glick (3) and by Hutterer and acid. For isopropanol at a heating hydroxyproline. With pyrrole, color Singer (4). Though similar in outline, development started immediately a t temperature in the region of 60" C., and the present results differ from theirs, acidities of above 0.7M HC104, A,, was room temperature, but appeared to perhaps because of the variability of the proceed in two stages. A value of appreciably constant at 558 mp. With n-propanol used in their work. The 80% of the final absorbance rvas development of color as in the normal increase in organic solvent a t the reached within 3 minutes a t a , , ,A procedure but a t room temperature, oxidation stage was found, in the presvaried from 553 mp after 1 hour to of 545 mp. The inflection a t about ent work, to reduce the procedure by , , ,A 525 mp n-as less marked in the spectrum 562 mfi at about 9 hours. yet another critical variable, making I n one experiment two identical of this color than of any other. The the oxidation relatirely independent of absorbance then increased gradually to colored solutions were diluted, one with time. an organic solvent and the other with its final value (107% of that given by an Absorption Spectra of Final Color. water. The first solution was relatively equivalent molar concentration of I n the development of the method, hydrolyproline) over a period of 3 stable in absorbance and in A,, the absorption spectra of t h e colored The hours, while the value of, , ,A changed second, however, began with a lower solutions were measured i n a Cary absorbance; the absorbance decreased slowly to 562 mp, and the shape of the recording spectrophotometer, first spectrum returned to normal. If the more rapidly than that of the first immediately after the heating stage, colored solution was heated at 60" C. increased solution, and the , , ,A and then a t intervals. These spectra varied with the conditions and time for 25 minutes, a lower absorbance was gradually over a n hour until the, , ,A obtained (89% of the value reached at of heating, with dilution, and with the of the first solution had been passed. room temperature) but the, , ,A value Oxidation Product of Hydroxytime interval between the heating stage subsequently remained steady at 560 rnp. and the recording of the spectrum. proline. Many of the authors of
1964
ANALYTICAL CHEMISTRY
It is clear, therefor€, t h a t the oxidation product of hydroxyproline intermediate in the present method is neither pyrrole nor pyrrole-o -carboxylic acid. The dyestuff formed by condensation with p-dimethylamir o benzaldehyde does, however, appear to be the same for these three compounds. It is likely, though by no means certain, that the variations in A,, and perhaps some of the variations in absoabance are due to a n association of the dyestuff which is dependent on the dielxtric constant of the medium and whici responds slowly to changes in the environment. ~i*-Pyrroline-4-hydri,sy-2-carbo x y l i c acid has also been suggested by Radhakrishnan and Jleister as a product of the oxidation of hydroxyproline ( 8 ) , but has not been directly tested in the present work. Thiay found that this compound, when trezted with dilute mineral acid, was jlot%Tly transformed into pyrrole-a-carbor ylic acid. This fact was used in the present work to inTestigate the nature of the hydrosyproline oxidation product. Four aliquots of hydroxyproline, each Kith its corresponding blank, were taken through the method r-ith the following variations. (The blanks all gave equally
low absorbances.) Aliquot KO.1 was heated for 25 minutes (the optimum) in stage 2 of the method, and aliquot No. 2 for 35 minutes. The 10-minute overheating resulted in a 3% decrease in color yield. Aliquots No. 3 and 4 were taken through the oxidation stage, then the perchloric acid and propanol were added and, after an hour at room temperature, the solid aldehyde. Aliquot KO.3 was then heated at 60" C. for 25 minutes, and S o . 4 for 35 minutes. The 10-minute overheating again produced a 3% decrease in absorbance, though the color yields of aliquots No. 3 and 4 were 10% lower than those of KO. 1 and 2, respectively. Most compounds related to pyrrole are somewhat unstable in acid solution, and therefore, i t is not surprihing that the color yield was decreased by alloning the intermediate oxidation product to stand in acid for a n hour. If, however, this intermediate mere hydrosypyrroline carboxylic acid, then the treatment with acid should have transformed it, at least in part, into pyrrole-a-carboxylic acid. This latter has an optimum heating time in the present method of about 80 minutes at 60' C., and the color yield would, therefore, have increased rather than
decreased, for an increase in heating time from 25 to 35 minutes. The inference is that the oxidation product of hydroxyproline in the present work is not AI-pyrroline-4-hydroxy-2-carboxylic acid. LITERATURE CITED
( 1 ) Bergman, I., Tuck, G. C., Safety in
Mines Research Establishment, Sheffield, England, unpublished work. (2) Bowes, J. H., J . SOC.Leather Trades'
Chemists 4 3 , 2 0 3 (1959). (3) Grunbaum, B. W., Glick, D., Arch. Biochem. Biophys. 6 5 , 2 6 0 (1956). (4) Hutterer, F., Singer, E. J., ANAL. CHEM.3 2 , 5 5 6 (1960). ( 5 ) Lollar, J., J . Am. Leather Chemists' Assoc. 53, 2 (1958). (6) Neuman, R. E., Logan, bl. A4., J . Biol. C h e m 184, 299 (1950). ( 7 ) Prockop, D. J., Udenfriend, P., Anal. Biochem. 1 , 228 (1960). (8) Radhakrishnan, A. N., Illeister, A., J . Biol. Cheni. 226, 559 (1957). (9) Stegemann, H., Z. Physiol. Cheni. 3 1 1 , 4 1 (1958). (10) Woessner, J. F., Jr., Arch. Biochena. Biophys. 93,440 (1961).
RECEIVED for review January 31, 1963. Accepted July 15, 1963. The illustrations to this paper are British Crown Copyright and are reproduced by permission of the Controller of Her Britannic Majesty's Stationery Office.
Analysis of Mixtures of Antimony and Bismuth Tellurides Containing Selenium and Iodine K. L. CHENG a n d B. 1. GOYDISH RCA laboratories, Princeton, N . J .
b There i s a need fot, determining the composition of the tellurides o f antimony a n d bismuth. Simpler a n d more r a p i d methods for defermining bismuth, antimony, tellurium, selenium, a n d iodine in such alloys have been developed. Tellurium i s determined gravimetrically as tellurium dioxide. Bismuth a n d antimony a r e determined volumetrically b y step (ethylenedinitri1o)tetraacetic acid titrations in the presence a n d in the absence of tarirate, which can mask antimony. Selenium i s determined photometrically with 3,3'-diaminobenzidine, a n d iodine i s determined photometrically by measuring the turbidity o f the silver iodide precipitate in a n ammoniacal medium after a simple distillation. If desired, each individual element m a y b e determined independently. All five elements m a y b e determined sequentially in a single sample when the sample supply i s limited.
M
such as those of bismuth and antimony, possess interesting thermoelectric properties. One of the important thermoANY TELLUHIUES,
electric materials is a bismuth telluride compound in which bismuth is partly replaced by antimony, tellurium is partly replaced by selenium, and the material is doped with small amounts of iodine which can affect the conductivity type ( n or p ) of the thermoelectric material. It is necessary to analyze various materials to ascertain their compositions and to obtain desired properties. Reed ( 7 ) reported methods of analyzing this type of material in which selenium and tellurium are determined gravimetrically by precipitation with sulfurous acid at different acidities. The antimony is titrated potentiometrically after its separation as antimony sulfide from the filtratp, from which selenium and tellurium have been previously removed, and bismuth is titrated with (ethylenedinitri1o)tetraacetic acid (EDTA) in the filtrate after selenium and tellurium have been removed. H e did not report the method of analyzing for iodine. Cluley and Proffitt (6) reported essentially similar methods of analyzing this type of material, and also reported a method of
iodometric determination of iodine after separation by steam distillation. This paper reports rapid methods of analyzing the material. Tellurium is determined gravimetrically as TeOz while bismuth and antimony are masked by EDTA in the presence of acetone. Selenium is directly determined photometrically with 3,3'-diaminobenzidine in the presence of tartrate (3). Both bismuth and antimony are titrated Kith E D T A in the presence of acetone. By masking antimony with tartrate, only bismuth is titrated by EDTA. Then the values for antimony and bismuth can be obtained by stepwise E D T A titrations with and without addition of tartrate ( 4 ) . The proposed methods are more rapid, simpler, and at least as accurate as those previously reported. Another significant feature of the proposed methods is that when the determination of only one element is desired, it can be done rapidly and independently without separation of other elements. If desired, iodine, tellurium, bismuth, antimony, and selenium may be determined in a single sample. VOL. 35, NO. 12, NOVEMBER 1963
1965
