Determination of the True Chloride Content of Biological Fluids and Tissues. II. Analysis by Simple, Nonisotopic Methods ERNEST COTLOVEI laboratory of Kidney and Electrolyte Metabolism, National Heart Institutes, National Institutes of Health, Bethesda, Md.
b Simple and reliable nonisotopic methods are described for determining the true chloride content of biological materials, which were validated b y direct comparison with an isotopic dilution method employed as a standard of reference. In all cases the final measurement was by an automatic, coulometric-amperometric titration. In analysis of biological fluids, direct dilution and titration i s accurate. In analysis of tissue, a solution for titration can be prepared from the tissue b y three types of methods: alkaline digestion of wet or dried tissue, followed by zinc hydroxide precipitation of protein, and perborate oxidation of sulfhydryl groups in the supernatant; dilute nitric acid extraction of fat-free, dried, pulverized tissue; or water extraction. The nonisotopic alkaline digestion method i s the most consistently accurate and widely applicable, and can serve as a secondary reference method for determination of true chloride in biological materials.
T
analysis of chloride in biological materials, particularly in tissues. has been a long-standing problem because of the substantial lack of agreement among different analytical methods, and the absence of a dependable means of deciding which methods, if any, yield a true measure of chloride content ( 3 ) . T o resolve this problem, a method employing the isotope dilution principle was developed which accurately mtasures the true chloride content of a biological sample. This irotope dilution method. described in the preceding paper ( 2 ) , can therefore serve as a standard of reference for evaluating other methods. Based on this reference, simple, nonisotopic methods have been developed, which are described in the present report together lr-ith the results of coinparisons with the isotopic dilution method. The nonisotopic methHE
Present address, Clinical Pathology Department, Tational Institutes of Health, Bethesda 14, Md.
ods. when performed as specified, provide a valid measure of the true chloride content of biological fluids and tissues. EXPERIMENTAL
All chemical analyses of chloride are performed with t h e coulometric-amperometric method of silver ion titration and t h e automatic titrator described b y Cotlove et al. (1, 6 ) . Titration is conducted in 20- X 40-mm. vials which fit the electrode assembly of the titrator. (+tomatic titrators which follow the original design are made by American Instrument Co., Silver Spring, &Id.. and by Buchler In+-umentr Inc., Fort Lee, Y. J., which also supply the titration vials and the gelatin-indicator reagent required by the method.) Glass or polyethylene stoppers and plastic tubing are used instead of rubber t o avoid introduction of sulfhydryl groups. A rocking platform is used for the extraction procedures. A wooden board is mounted on the arm of a vacuum windshield rriper (heavy d u t y model, for trucks). During extraction, the valve on the vacuum line is adjusted to give a slow up and down motion with a cycle of about 5 seconds. Reagents. Distilled and demineralized water was used throughout, a n d was confirmed t o be chloride-free by titration. Electrolytic S a O H (from Fisher Scientific Co.) war from a lot with a reported chloride content of O.OOO7,, which was confirmed by titration (bv measuring the intrinsic blank as desciibed belo+y. Methods. T h e analysis of chloride in biological fluids requires only dilution of a measured volume with t h e appropriate reagent solutions, followed by coulometric-amperometric titration. The analysis of tissues requires the preparation of a solution from the tissue containing all the chloride but no significant interferences, followed by titration of the solution. Analysis is usually performed on tissues n-hich have been initially prepared by drying. fat extraction, and homogenization. Apparatus.
DIRECT DILUTION AND TITRATION OF BIOLOGICAL FLUIDS.The procedure employed is t h a t described previously (!, 5 ) . The biological fluid is diluted directly in the titration vial. Ordinarily, 0.1 ml. of fluid, plus 4 ml. of 0.11%'
H K 0 3 in 10% acetic acid, and 4 drops of gelatin-indicator solution, are titrated at the high titration rate. Smaller aliquots, or fluids with Ion- concentrations of chloride, may be titrated a t the medium or low titration rate (1). The concentration of chloride in the fluid can also be displaj ed on a digital register in milliequivalents per liter ( 4 ) . PREPARATION OF FAT-FREE DRIED TI~SGES. Tissues were obtained from normal adult animals : female mongrel dogs, male Osborne-Mendel rats immediately after eusanguin:ation, and bullfrogs, Rana catesbeiana, and n-ere prepared as described previously (21. WEIGHINGOF SVALL -IMOUNTS OF DRIED TISSUE. The following procedure is necessary to obtain accurate dry n-eights of w r y small amounts of ue. Weigh approximately on weighing paper an amount of dried tissue estimated to contain a n amount of chloride within the range of the methods described below-, and transfer the sample into a previously weighed, round-bottomed glass-stoppered tube (15-nil. capacity, Korites Glass Co.). Heat the tubes unstoppered at 105" C. for several hours, cool briefly in a desiccator, stopper, and reneigh to obtain the fully dry w i g h t . PREPAR.4TION O F -4 SOLUTION FROM THE TJSSVE FOR TITRATION. For this purpose, three types of procedures n ere found t o be suitable under specified conditions: (1) alkaline digestion of either fresh, dried, or fat-free dried tissue, follon ed by removal of interferences b y protein precipitation and perborate oxidation of sulfhydryl groups in the supernatant solution; ( 2 ) acid extraction of fat-free, dried, pulverized tissue with dilute nitric acid; or (3) ryater extraction of (a? fat-free, dried, pulverized tissue, or (b) fresh, n e t red blood cells, followed b y protein precipitation, or (c) fresh, met tissue (frog gastric mucosa). (1) Alkaline Digestion of Fresh or Dried Tissue. Using the procedure described above, JTeigh the qumple into a 15-ml. glass-stoppered, round-bottoiiied tube. Take an amount estimated t o contain: (a) 15 to 30 peq. of chloride, or (b) 5 to 15 peq., or (c) less than 5 peq. The corresponding procedures are as follows. (a) 15 t o 30 peq. of chloride. Add 5 ml of 1.5N K'aOH to each tube containing tissue. With each set of unVOL. 35, NO. I , JANUARY 1963
e
101
knowns prepare duplicate tubes of a method blank containing 5 ml. of 1.5N NaOH, and a method standard containing 5 ml. of 4 peq. per ml. of NaCl i n 1.5N NaOH. Cap each tube with rinsed aluminum foil and heat, in a boiling water bath for 30 minutes, shaking the tube intermitt'ently witho u t inverting, to effect complete dissolution of the tissue. Cool the tube t o room temperature, transfer the contents quantitatively to a 25-ml. volumetric flask with several rinses of water, then dilute t o volume. For precipitation of protein, transfer a 10-ml. portion to aqotlier round-bott'omed tube, add 1 ml. of 20% ZnSO1.7H20 in 2 S "03, stopper with a glass or polyethylene stopper, shake vigorously, and set aside for 1 hour. Centrifuge at 2500 r.p.m. for 20 minutes, and carefully transfer the supernatant, which should be clear, to a clean tube. Pipet duplicate 2-ml. portions of supernatant into tit,ration vials, and treat with perboratr (as described below) prior to titration. (b) 5 to 15 peq. of chloride. This procedure is simpler than (la), since protein is precipitated in t,he same tube that is used for digestion; hoivever, the dilution volume is slightly more variable. To each tube containing tissue, add 5 ml. of O.&V S a O H . Prepare duplicates of a method blank containing 5 ml. of 0.6N NaOH, and of a method standard containing 5 ml. of 2 peq. per ml. of NaC1 in 0 . 6 4 S a O H . Heat to dissolve the tissue as in ( l a ] , cool, and add to each tube 5 ml. of 47, ZnS04.7H20in 0.4&VH N 0 3 . Stopper, shake, set aside for 1 hour, centrifuge, and t'ransfer the supernatant to a new tube. Pipet duplicate 2-ml. aliquots of supernatant into titration vials and treat with perborate (as described below) prior to titration. (c) Less than 5 peq. of chloride. This procedure is the same as ( l b ) , except t h a t 3-ml. volumes are used of NaOH, of the NaCl standard, and of the zinc sulfate. Thus, the 2-ml. aliquot of the supernatant which is titrated represents one third of the sample. The lower limit of the analysis is determined by the accuracy desired, and by the sensitivity of the coulometric-amperometric titration (described below). Perborate oxidation. Sulfhydryl (or sulfide) groups are released by alkaline digestion of the tissue sample. Since these groups can combine with silver ion. they must be oxidized prior to the titration. Oxidation is conducted in the individual vials by exposure to 20 pmoles of sodium perborate in approximately 0.1N alkaline solution. On each day of use, prepare a fresh solution of 0.2M NaB03 in 2.8X NaOH by completely dissolving 0.3 gram of NaB03.4H20in 5 ml. of 0.5N HNOs, and then adding 5 ml. of 6 N NaOH. T o each titration vial containing 2 ml. of supernatant from procedure ( l a ) , (1b.l. or (IC), add 0.1 ml. of the fresh alkaline perborate solution, and mix by gentle swirling. (Residual zinc ions in the supernatant form a whit,e precipitate of zinc hydroxide in the 102
ANALYTICAL CHEMISTRY
tubes of method blank containing 8 alkaline solution, but this precipitate ml. of water, and of method standard dissolves at the subsequent step of acidification. The zinc ions do not containing 8 ml. of 1.0 peq. per ml. of S a C l in water. To each tube add 1 ml. affect the titration.) Cover the vials of acid zinc sulfate (10% ZnS01.7HnO with polyethylene stoppers or Parafilm, in 0.25iV HzS04) and mix. Add 1 ml. and leave for 16 to 24 hours at room of 0.75N NaOH, shake vigorously, temperature. Before titration, add set aside for 1 hour, centrifuge, and to each vial 3 drops of gelatin- indicator solution (a blue color should appear carefully remove the clear, colorless supernatant. [An alternative preindicating a p H above 9.5, confirming cipitation with neutral zinc sulfate that the alkalinity was adequate for (1 ml. of 10% ZnSO4.7H20 and 1 ml. of sulfhydryl oxidation). Then add 0.5 0.5N YaOH) is also satisfactory but ml. of 1.3N H X 0 3 in 50% acetic acid yields a smaller volume of supernatant.] (a pink color should rcsult, indicating a Transfer to titration vials duplicate p H below 1.2'). Titrat'e a t the low 2-ml. aliquots of supernatant, and add titrat.ion rate. (The initial ampero0.5 ml. of 0.5N HNOl in 50% acetic metric current folloiving the perborate acid and 3 drops of gelatin-indicator treatment is 2 t'o 4 pa. instead of the usual level of 1 to 3 pa., but the titrasolution before titration a t the low tion is not adversely affected.) rate. (cj Fresh, wet tissue. Water es(2) Acid Extract,ion of Fat-free, traction of wet tissue other than red Dried, Pulverized Tissue. This form blood cells was performed only in the of initial preparation of the tissue is case of frog gastric mucosa (separated required to allow adequate extraction from the muscular layers by blunt of chloride. Weigh accurately in a dissectioiil . Three experiments were 15-mi. glass-stoppered, round-bottomed conducted using the gastric mucosa of tube an amount of fat-free, dried, the entire stomach, weighing between pulverized tiwue estimated to contain 3.5 and 4 grams. Extraction was con5 to 15 peq. of chloride, add 10 ml. of ducted with 38 ml. of n a t e ~containing 0.75N " 0 3 , secure the stopper with NaCl36, by continuous mixing on a tape. and place on t.hr rocking platform rotating platform for 16 hours, and for 16 hours a t room temperature. The a t 2' C. to inhibit the growth of microperiodic inversion of the 10-ml. volume organisms and minimize the elution of in the 15-nil. tube ensures adequate protein. X portion of the supernatant contact of all portions of the tissue was removed for triplicate titration powder with the solution. Centrifuge without removal of the small amount and remove the supernatant. Pipet of protein present, and the remainder duplicate 2-ml. aliquots of supernatant of the supernatant and intact mucosa into titration vials. Prepare vials of was subjected to isotope dilution analymethod blank with 2 ml. of 0.75X sis ( 2 ) . "Os, and of method dandard n-ith CALCCLATIOS O F CHLORIDE COX2 ml. of 1.0 peq. per ml. of NaCl in TENT OF TISSUES. The net second 0.75N "03. Before titrat>ion add to titration time of each sample is obeach vial 3 drops of grlatin-indicator tained by subtracting the average solution and 0.5 ml. of 2.5-47 sodium seconds for titration of the eorreacetate in 6-1'acetic acid. (The lattrr sponding method blanks from t h r gro\s solution is prepared by mixing 42 ml. seconds of each titration. The chloride of 6 N NaOH and 50 ml. of glacial acetic content of the tissue is calculated aq acid and diluting to 100 ml.) Titrate at the low titration rate. follows: (3) Water Extraction. (ai Fat-free, (total peq of std. j dried, pulverized tissue. This pro- peq. Cl/gram tissue = __-____cedure is similar to the nitric acid (av. net see. of std.) extraction except that 10 ml. of water (av. net sec.- _of_ unknown) ___ are used for extraction and a few (grams of tissues) crystals (approximately 2 mg.) of thymol are added to the tube to inhibit growth of microorganisms. The titraThe "total peq. of std." is 20 peq. tion vials receive 2-ml. aliquots of the in (la), 10 peq. in ( l b j , 6 peq. in ( I C ) ; supernatant. For titration, the method 10 peq. in (2); 10 peq. in (3a), and blank vials each contain 2 i d . of water; 8 peq. in (3b). A slight correction is the met.hod standard vials each contain required with water extracts of wet 2 ml. of 1.0 peq. per ml. NaCl in water tissue because of the small increment in (these are the same as the customary dilution which is contributed by the water content of the sample. I n the reagent blanks and standards). T o each vial add 3 drops of gelatin-incase of (3b) the result is multiplied by dicat'or reagent and 0.5 ml. of 0.5iV (0.001 X the correction factor, 1 "03 in 50% acetic acid before titragrams wet tissue x per cent water in tion a t the low rate. tissue). Since the correction is a small one, the per cent lvater need only be (bl Fresh, wet, red blood cells or whole blood. 4 sample of red blood 65% for red blood approximate-e.g., cells and 83% for whole blood. cells or of whole blood is prepared by dilution with water, forming a hemolyThe accuracy of the method standard sate, and protein is removed by prein the alkaline digestion method is cipitation prior to titrat,ion. The checked by titration of the usual reagent Somogyi zinc precipitation is employed blank and chloride standard ( I , 5 ) ; based on the latter, the Zml. aliquot of (6). iiccurately weigh 0.1 t o 0.2 gram method standard which is titrated of red cells or about 0.1 gram of whole should contain 1.455 peq. of chloride in blood into round-bottom tubes. Add ( l a ) , and 2.000 peq. in (111) and in (IC). 8 ml. of water and mix. Also prepare
+
COULOMETRIC.AMPEROMETRIC TITRACHLORIDEWITH SILVER ION. Table 1.
Analysis of Chloride in Biological Fluids by Isotope Dilution and by Direct All solutions are titrated by the proTitration cedure previously described ( I , 6). C1 by direct titration" The coulometric delivery of silver ion Isotope % of is at a constant rate which may be dilution C1 Isotooe Sample selected by a switch t o be approxiRemarks meq./liter Meq./liter dilutioh C1 mately 0.015 peq. per second (low Human serum normal subject 106.2 106.0 99.8 titration rate), or 0.06 peq. per second Human serum normal subject 104.5 103.4 98.9 (medium titration rate), or 0.26 peq. Human serum 106.0 106.1 100.1 pooled, 10 patients Human serum, lipemic per second (high titration rate). The 1 . 5 % total lipid 104.3 104.1 99.8 Human serum, lipemic 1.8% total lipid 101.7 100.9 99.2 solution being titrated should have Human serum, lipemic 2.4% total lipid 109.1 107.1 98.2 final concentrations of approximately Human serum, lipemic 3 . 5 % total liDid 103.9 105.6 101.6 0.1N HNO,, 10% (1.75M) acetic Dog pancreatic secretion milk;, viscid120.8 119.8 99.2 acid, and 0.025y0 gelatin. The total Dog pancreatic secretion milky, viscid 89.0 87.3 98.1 volume should be nearly the same in Human urine 1.294 total protein 58.7 59.0 100.5 all titration vials, within ~ t 0 . 0 5ml. Cat urine severe dehydration 440.0 451.4 102.6 at the low or medium titration rate, a Relative standard deviation of 4 t o 20 replicates of analyses by direct titration was and within h 0 . 2 ml. a t the high rate. 1 0 . 1 to 3~0.87,. Duplicates of blanks and standards are titrated before and after the unknowns. The lower limit of sensitivity is deThe results of direct titration of selen content of fat-free, dried samples vias termined bv the minimal variabilitv sera averaged 99 i% (=k0.4 standard encountered in analysis of amounts in the titration times of the blank a n a deviation) of the corresponding isotope between 0.02 and 0.3 gram. These sample, which is 1 0 . 1 second standard dilution values. Direct titration was small samples of powdered tissue abdeviation at the low titration rate, accurate e ~ r nin samples which might sorbed appreciable moisture. whilc excorresponding to 10.003 peq standard deviation of the results. Attainment of posed to air during the few minutes be expected to cause difficulty: serum minimal variability requires optimum n itli high lipide content; viscid. proteinof wighing : water uptake a,vcraged conditions for titration : freshly polished containing pancreatic secretion; and 3.5% of dry weight, 11-ith a rnngr of electrodes, adjustment of the generator protein-containing, or highly concen0.5 to 8%. Longer exposurr t'o room electrode to full length and diameter, trated, urine. air with 50% humidity allon-ed a n identical volumes in all vials, triplicate Analysis of Tissues. Three types increasing uptake of mater u p to a n blanks and standards titrated before of methods were selected because their equilibrium value equal to approsand after each group of 12 or fewer imately 207, of the dry neigh-e.g., results compared favorably with those unknowns, and a normal end point in of the isotope dilution analysis ( 2 ) . rat muscle. The pi,ocrdure described the titration of unknowns. All reagents should be as free of chloride as possible These nonisotopic methods differ in under Methods was adopted to obtain t o minimize the blank. the preparation of a solution from the accurate dry weights of small s:tmples. MEASUREM ME KT OF THE INSTRINSE tissue for final measurement, which in Alkaline Digestion of W e t or Dried BLANKOF A TITRATION. The difference all cases is coulometric-amperometric Tissue. T h e nonisotopic alkaline ciibetween the titration times of the usual titration with silver ion. gestion method has been the niost reagent blank and the intrinsic blank is The nonisotopic methods are deconsistently accurate as rvell as t h e a sensitive measure of silver ion-comsigned t o use very small amounts of most widely applicable of the simplibining contaminants in the reagents and tissue containing 5 to 25 peq. of chloride fied methods in measuring the true water used. At the low titration rate, or less, in contrast to the complete this difference averages 0.5 second or chloride content of a variety of tlifless when contamination is minimal and iqotope dilution method which requires ferent, t,issues. I n each tissue t h e the electrodes are properly cleaned. amount. of tissue containing 150 to alkaline digestion mctlioti g:i\.e the Contaminants in the method blank of 250 peq. in order to have sufficient same result as the isotopic dilution the methods described, as well as in chloride for all the procedures and analysis of thc same sample, xithin the usual reagent blank, can be tested measurrment~ in1 olved. Comparisons the limits of variation of the mcasureby the follo~vingprocedure. hrtween isotopic and nonisotopic methnients (Tables I1 and 111). I n 16 sets Lrave the titration vial in place after ods on individual fat-free, dried tissues of conipnrisons on various tissiies, t h e the usual automatic shut-off of the weir made by taking separate portione. alkaline digestion results averaged titration. Record the titration time. of the iaine homogeneous, pan-dered Turn the titration sn-itch to the first 100.095 of the isotope dilution cliloride, position, and set the adjwtable pointer .ample of the tissue for each analvsis. n-it'h a rnasinium avernge difference of of the meter-relay a t 10 pa. above the Comparisons on wet tissucs mere made 2.09; in one tissue (frog skeletal niuscle.) terminal amperometric current shown hy analyzing a small aliquot of a digest The nonisotopir alkaline digestion by the indicator pointer. Reset the or extract by a nonisotopic method, is simple to pcrforni and 1ia.s method time regiiter to zero, and turn the sample and the remaindcr of the same the advantage of tieing equally aptitration siritch to the second position hy the isotopic method. Only a fern plicable to wet, or dried t,issuc, whether to resume titration. The time t o comparisons on met tisquc were made fat-extracted or not, and whether develop the increment of 10 pa. rebecause of the difficulty of performing finely diyided or not. AIethod blanks quired for a repeated automatic shut-off is designated the intrinqic blank of the repetitive analvses on samples while arr included in the procedure t o detect sample being titrated. still fresh and uncontaminated, and and corrrct for any silver ion-conil)ining of obtaining rcpresentati! e small porcontaminants in the rcagcnts. 11-hich RESULTS AND DISCUSSION tions of tis-ueq (other than red blood is especially important, in analyzing Direct Dilution a n d Titration of crlls). niinutc amounts of cliloritli~. K i t h Biological Fluids. Coulometric-amI n analjsiq of replicates of minute the reagents selected for tliv pixwnt perometric titration of serum, panportions of drier1 tissur, consistrnt and series of analyses, the titration time creatic secretion, or urine, diluted with accurate results could be obtained of the method hlanks dit1 not differ reagent solution, accurately measured only if care nas taken to ensure that from the titration timr of the usual t h e true chloride content, as dethe samplca were thoroughly homoreagent blanks. Method standards termined on t h e same samples b y geneous and adcquatP1y dried. At are employed to confirm thc re1ial)ility isotopic dilution analysis (Table I). firyt, error due to unrecognized water of the procedure and t o corrrct for small TION OF
VOL. 35, NO. 1, JANUARY 1963
103
Table II.
Chloride Analysis of Fat-free, Dried Tissues by Isotope Dilution and by Three Nonisotopic Methods
Isotope dilution Pl
V I
meq./kg. Species FFDT Dog 430.0 106.3a Dog Rat 92.@ Human 155.5" 152.3 Rat 62.0 Beef Frog 40.2
Per cent of isotoae dilution chloride Alkaline Acid Water digestion extraction extraction
Tissue 100.5 f 1 . 4 98.7 f 1 . 0 Kidney 100.0 i 1.1 96.2 f 0 . 9 Liver 99.2 f 1.2 99.2 f 1.1 93.3 f 1 . 8 97.2 f 1 . 8 99.1 f 1.4 Liver 100.6 i 0 . 8 99.5 f 0.9 100.3 f 0.6 Red blood cells f 1.2 99.5 99.8 i 1.3 101.6 f 1.5 Red blood cells 98.0 f 0 . 3 9 9 . 1 f 0.4 97.4 f 0.9 Skeletal muscle 100.8 f 0 . 8 101.0 i 0.4 Skeletal muscle 102.0 f 1 . 0 100.9 f 2 . 0 69.1 100.1 f 1.5 99.1 f 1.5 Skeletal muscle 99.6 f 0 . 9 Skeletal muscle Dog Rat 48.8. 99.8 f 1 . 4 99.9 f 1 . 4 103.5 i 1.7 99.4 f 1.5 103.2 f 1 . 4 289.ha Stomach Frog 98.8 f 1 . 4 99.7 f 0 . 8 Tendon 99.6 f 1 . 3 Dog 219.P 100.8 f 1.3 Cardiac muscle Dog 99.6 i 0 . 8 99.4 i 1 . 2 109.0 100.0 f 2.9 100.7 f 2.2 101.7 f 2 . 1 Whole body 167.1 Rat a Based on axwage of tiyo analyses; remaining values in this column represent single analysis ( 2 ) . Averages and standard deviations of results by three nonisotopic methods are each based on 4 t o 7 analyses, and are expressed as percentage of corresponding isotope dilution values on same sample. Table 111.
Chloride Analysis of Wet Tissues by Isotope Dilution and b y Two Nonisotopic Methods
Isotope dilution C1 ax-. mey./kg.
Per cent of isotope dilution C1 Tissue Alkaline digestion Water extraction Human red blood cellss 52 8 99.8 f 0 . 8 9 9 . i i 0.6 Frog gastric mucosaR 51.4 99.5 f 1 . 2 99.0 f 1.6 Rabbit liver mitochondria 1.96 98.0 a Average of three samples each. Nonisotopic methods were performed on triplicate portions of each of corresponding samples, and results are expressed as percentage of isotopic dilution values (av. and std. dev.). Chloride content shown for wet mitochondrial preparation from rabbit liver corresponds to 0.063 peq. per mg. of nitrogen.
sources of variation. I n 13 comparison?, the net titration time of the method standards averaged 99.8% ( 5 0 . 6 standard dev.) of the values expected from the usual chloride titration st,andards. The nonisotopic alkaline digestion method, because of its reliability, simplicity, and wide applicability, can be employed as a secondary reference method in any laboratory to evaluate other procedures for measuring tissue chloride which might be more suitable for particular requirements. Acid Extraction of Fat-free, Dried, Pulverized Tissue. T h e chloride values
obtained b y coulometric-amperometric titration of dilute nitric acid extracts of dried tissue compared favorably with the isotope dilution values, averaging 100.O~o of the latter in thirteen tissues (Table 11). T h e variation in results b y the acid extraction method was slightly greater t h a n b y t h e alkaline digestion method. The chloride contents of five samples of calf rib cartilage mere measured by dilute nitric acid extraction in comparison with the nonisotopic alkaline digestion method and averaged 100.0% ( 1 3 . 5 standard deviation) of the results of the latter employed as the method of reference.
104
ANALYTICAL CHEMISTRY
Acid extraction of wet tissue v a s tried but wa? unsatisfactory, since extraction of chloride was often incomplete. The protein precipitation produced by 0 . 7 5 s nitric acid caused clumping of tissue even if finely minced, and this greatly delayed extraction of chloride. The extraction method, using dilute nitric acid or mater, is dependent on adequate mixing, duration of extraction. and proper preparation of the tissue by fat extraction and fine subdiviqion. Pulverization of the tissue can readily be carried out on tissue driPd initially under vacuum, which expands it and makes it friable ( 2 ) . Water Extraction of Tissue. Titration of water evtracts of fat-free, dried, pulverized tissue gave satisfactory results for chloride content except for t h e distinctly reduced values obtained with liver tissue (Table 11). I n the two wet tissues examined, red blood cells and gastric mucosa, t h e water extraction process gave accurate results (Table ITI) The sample must be eytracted with water shortly after collection to a~ oid growth of microorganisms. Water extraction of tissue heated a t
105' C. usually elutes very little or no protein, which is denatured by this heating. The water hemolysate of met, red blood cells requires deproteinization prior to titration. The presence of excessive protein in the titration solution causes a delayed rise of amperometric current a t the end point. The resulting error is negligible below 1 mg. of protein per peq. of chloride (as in serum), but rises to about +5yo with amounts of protein up to 5 mg. per peq. (which may be present in water extracts of wet tissues or in some biological fluids). Even this small error may be largely corrected by measuring the intrinsic blank time of the sample, as described above, and subtracting the time of the intrinsic blank instead of the usual reagent blank. It is usually preferable, however. to remove excess protein by zinc hydroyide precipitation prior to titration, and thereby obtain a normal and accuratP end point. Sulfhydryl and Sulfide Groups. I n biological fluids uncontaminated with microorganisms, free sulfhydryl or sulfide groups are not ordinarily present in amounts detectable b y the coulometric - amperometric tibration. Titratable sulfhydryl groups appear, however, with the growth of microorganisms or after alkaline digestion, and are only partially removed by deproteinization. The presence of free sulfhydryl or sulfide groups is usually manifested by an initial amperometric current which is negative or decreased (1 pa. or more below the initial current of a chloride standard). Sulfhydryl groups in the protein-free supernatant can be oxidized b y treatment with alkaline perborate and are then no longer titrated by silver ion. Titratable sulfhydryl groups were not detected in uncontaminated water extracts of dried tissues or of two wet tissues examined, or in dilute nitric acid extracts of dried tissues. Direct titration of serum gave results cqual to those on protein-free supernatants of serum precipitated with zinc hydroxide, indicating that sulfhydryl groups of serum proteins undergo negligible combination nTith silver ion undcr the conditions of direct titration (1).
Coulometric-Amperometric TitraIt is important to
tion of Chloride.
emphasize t h a t in all t h e procedures described here t h e final measurement of chloride has been by reaction with silver ion, using an automatic coulometric-amperometric titration (1, 5 ) . This method is highly reproducible in titration of amounts of chloride as small as 1 peq. or even less, and is remarkably free of interferences which affect other titration methods, being unaffected by color or by other substances in biological sample. which have been appropriately treated. If alternate methods were used to measure chloride subsequent to the
preparative procedures descrilied here, i t would be necessary to validate the entire set of procedures used b y direct comparison with the isotopic dilution analysis.
and W.11. Wallace for providing some of the biological samples. LITERATURE CITED
(1) Cotlove, E., “Standard Methods of ACKNOWLEDGMENT
The author thanks Sordica D. Green for skillful technical assistance, and J. L. Gamble, Jr., D. S. Horell, J. G. Parker,
Clinical Chemistry,” Seligson, D., ed., Vol. 3, pp. 81-92, -4cademic Press, Yew York and London, 1961. (2) Cotlove, E., - 4 s ~ CHEM. ~. 35, 95 (1963). (3) Cotlove, E., Hogben, C. A. M.,
“Mineral Metabolism,” Comar, C. L. and Bronner, F., eds., Vol. 2, pp. 109-73, Academic Press, Xew York and London, 1962. (4) Cotlove, E., &hi, H. N., C’lin. Chein. 7,285 (1961). ( 5 ) Cotlove, E., Trantham, H. V.,Bowman, R. L., J . Lab. Clin. M e d . 5 1 , 461 (1958). (6) Somogyi, XI., J . Bid. Chem. 86, 655 (1930). RECEIVEDfor review June 27, 1962. Accepted Xovember 11, 1962.
High Temperature Diffuse Reflectance Spectroscopy WESLEY W. WENDLANDT, PRESTON H. FRANKE, Jr., and JAMES Deparfment of Chemistry, Texas Technological College, Lubbock, Tex.
b A high temperature diffuse reflectance spectra sample holder, capable of operation from ambient temperature to 500” C., in the 350- to 7 5 0 - m p wavelength region is described. The sample holder i s attached to the reflectance attachment of a Bausch & Lomb Spectronic 505 spectrophotometer. Operation of the sample holder is illustrated by the thermal deaquation of [Co(NH3)6H20] Cla and [Co(NH&H20] Brz. This new high temperature sample holder appears to offer a promising technique for the study of the effect of elevated temperatures on coordination and other inorganic compounds, paints and paint pigments, dyes, plastics, fabrics, and other materials.
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D
REFLECTAXCE SpeCtroScopy, in the visible wavelength region of the spectrum, is a useful technique for the study of coordination compounds ( 2 , 4, 6, IO, 11) and other inorganic substances (1, 3, 5, 7-9, 14). Kormally, the reflectance spectra are obtained on the powdered crystalline samples, either in the pure state or diluted with a matrix substance, a t ambient or low temperatures. The spectra obtained by this technique show good correlation with solution absorption spectra and single crystal spectra (6, 6). This technique can give useful information about unstable compounds for which single crystal spectra would be difficult, if not impossible, to obtain. Also, the technique is potentially useful for studying surface phenomena ( 5 ) because i t is the surface layer which is measured by reflectance. I n connection with our studies in coordination chemistry, it was necessary t o obtain the diffuse reflectance spectra of certain cobalt(II1) complexes a t temperatures from ambient to about 500’ C. This technique is IFFIJSE
P. SMITH
described and some further applications in analytical and inorganic chemistry are pointed out. EXPERIMENTAL
Spectrophotometer. A Bausch & Lomb Spectronic 505 ultraviolet a n d visible range spectrophotometer, equipped with a reflectance attachment, S o . 33-28-12, was employed. The high temperature sample holder is schematically illustrated in Figure 1. The main body of the holder was about 6.0 cm. in diameter b y 1.1 cm. thick and was machined from aluminum. The sample was contained in a circular indentation, 2.5 cm. in diameter by 0.1 cm. deep, machined on the external face of the cell. Two circular ridges were cut a t regular intervals on the indentation t o increase the surface area of the holder and to prevent the powdered sample from falling out of the holder when it is in a vertical position. The sample holder was heated by coils of Sichrome resistance wire wound spirally on an asbestos board and then ALUMINUM SbMHPLE BLOCK-
qJ 4-h THERM
....
THERMAL SPACER
REFERENCE CELL
Figure 1 . Schematic diagram of the high temperature reflectance sample holder, thermal spacer, and reference sample holder
covered with a thin layer of asbestos paper. Enough wire to provide about 15 ohms of resistance was used. T h e temperature of the sample was detected b y a Chromel-Alumel thermocouple contained in a two-holed ceramic insulator tube 0.35 cm. in diameter. T h e thermocouple junction made contact with the aluminum block directly behind the sample indentation. T o prevent heat transfer from the sample holder t o the integrating sphere, a thermal spacer was constructed from a loop of 0.25-inch aluminum tubing and \vet shredded asbestos. After drying, the thermal spacer was glued to the sphere and the sample holder attached by a spring-loaded clip. The power supply employed has previously been described (12). Output from the thermocouple mas recorded on a Varian RIodel G-22 strip-chart recorder as shown in Figure 2 . Fullscale span on t h e recorder corresponded to a sample cell temperature of 500” C. The reference sample holder, Figure 1, 17-as 6niilar in dimensions to the sample holder except that i t mas unheated. Sample Preparation. Difficulties n e r e experienced when some of t h e pure samples mere heated in t h e sample holder. J l a n y of t h e samples qintered or fused when heated, t h u s altering t h e reflecting surface. T o prevent this, t h e samples were finely powdered. b y grinding in a mortar and pestle or a Kig-L-Bug. and t h e n diluted with a suitable matriv material quch a s previously ignited aluminum oxide, powdered potassium chloride, or potassium sulfate. .\ reference sample of the matrix, contained in a similar sample holder, was placed in the reference port of the integrating sphere by a spring-loaded clip. Procedure. About 1 gram of a 20% mixture of t h e cobalt coordination compound in finely powdered potassium sulfate was intimately ground together and then firmly packed i n t o t h e sample holder indentation. T h e exposed sample surface was made as smooth a s possible b y use of a steel spatula blade. T h e holder was then VOL. 35, NO. 1, JANUARY 1963
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