tion in grams of Zn per milliliter results in the usual linear plot. Because this procedure is somewhat cumbersome, especially when the Mylar pellet covers occasionally develop leaks, the bismuth reference previously used for lead analyses was tried as an internal standard for zinc. (The analyses of the standard blends with the zinc internal standard procedure also seem to be biased on the low side. This bias presumably was caused by almost unavoidable variations in effective sample thickness when two pellets were used.) The bismuth standard gives surprisingly good results and leads one to believe that relatively widely spaced wavelengths can be successfully used for analyses. The intensity of the Zn K , line at 1.436 A. (41.80’ 28) is ratioed to the Bi L, line a t 1.144 A. (33.01’ 28) which is
taken t o the 0.95 power. The Zn absorption edge is 1.283 A , ; the Bi edge is 0.923 A. Three milliliters of sample were added to give a sample depth of 1.5 mm. The accuracy of the zinc analyses in varying matrices is seen in Table 111.
(4) Gunn, E. L., “Advances in X-ray Analyses,” Vo1. 6, p. 403, Plenum Press, New York, 1963. ( 5 ) Hale, C. C., King, W. H., AKAL. CHEM.33, 74 (1961). (6) Jones, R. A4., Ihzd., 31, 1341 (1959). ( 7 ) Ihid.. 33. 71 (1961).
ACKNOWLEDGMENT
The authors are indebted to ;indrew Gemmell and Edwin Hodecker for machining the internal standard holders, to Allen Gerber for assisting in the analyses, and to Lawrence Gorry for aiding in the statistical procedures. LITERATURE CITED
(1) Davis, E. N., Van Nordstrand, R. A., ANAL.CHEM. 26, 973 (1954).
(2) Dniggins, C. W., Jr., Dunning, H. N,, Ibid., 31, 1040 (1959). (3) Ibid., 32, 1137 (1960).
Absorption and Emissihn in Analytical Chemistry,” p. 185, Wiley, New York, 1960. (11) LVoreZco Replr. 9, “Table of X-ray Mass Absorption Coefficients’’ (hlay 1962). (12) Yao, T. C., Porsche, F. W., ANAL. CHEM.31, 2010 (1959). RECEIVEDfor review April 21, 1964. Accepted September 16, 1964. Pittsburgh Conference on Bnalytical Chemistry and Applied Spectroscopy, Pittsburgh, Pa., 1964.
Determination of Zinc in Biological Materials by Atomic Absorption Spectrophotometry KEllCHlRO FUWA, PABLO PULIDOtl ROBERT McKAYt2 and BERT L. VALLEE Biophysics Research laboratory, Department of Biological Chemistry, Harvard Medical School, and Division o f Medical Biology, Department o f Medicine, Peter Bent Brigham Hospital, Boston, Mass.
b An atomic absorption spectrophotometric method for the direct determination of zinc in biological fluids has been developed, which does not require destruction or removal of organic material b y ashing or precipitation prior to analysis. The sample i s simply diluted wiih metal-free buffer or distilled water, and i s then aspirated directly into the hydrogen-air flame of the atomic absorption device. Correlation o f the data with those obtained b y colorimetric or Z d 5 measOther metals urements i s excellent. do not interfere. The limit of detection (1% deflection of the meter) i s 0.002 kg. of Zn per ml. The determination of zinc by atomic absorption i s 10 to 100 times more sensitive than the dithizone method now in common use, requires only one tenth as much time, and i s more precise. Accuracy was determined b y analysis of NBS samples, which also served to define optimal analytical conditions.
P
of methodology have been characteristic of investigations of the biological occurrence and function of zinc. The slow progress in the field can be related to difficulties in determining the small concentrations of the metal present in tissues and fluids. I s a consequence, the physical and cheniical methods employed for the determination of zinc and the awareness ROBLEMS
and avoidance of contaniination are the most important considerations required for the evaluation of biological data and the conclusions drawn from them. Thus, methods of high sensitivity, accuracy, and precision are essential (11). The microchemical determination of zinc in biological materials as the dithizonate (8) is generally performed as an extraction procedure, after ashing, washing with organic solvents, or precipi t’ation with trichloroacetic acid to remove protein or interfering substances (5, ‘7, 12). These methods, although accurate, are very time-consuming and tedious. K i t h the recent development of atomic absorption spectrophotometry (16,I?) it has become feasible to simplify and accelerate analytical work while improving its overall precision and accuracy. The general pertinence of the BeerLambert law to atomic absorption spectrophotometry and particularly to attainment of high sensitivity has been discussed (3). The niet,hod described here is based on the absorption of radiation a t 2138.6 A. by zinc present in biological materials, the emission source being a hollow brass cathode discharge tube (Westinghouse WL22607). The sample is aspirated directly into a horizontal aluminum oxide ceramic tube (Alunduni) through a modified Beckman hydrogen-air aspirator burner, and after
passage through a Hilger quartz prism spectrograph, the absorption of the radiant energy is measured by a 1 P 28 multiplier phototube coated with sodium salicylate to increase sensitivity of detection. The instrumental details have been described (3). The long path length of absorption provides a substantial increase in analytical sensitivity, such that the present method has a practical analytical sensitivity of 0.002 fig. of zinc per nil. Moreover, replicate analyses of saniples containing 0.3 p g . of Zn ger ml. have a coefficient of variation below 3.67,. Such concentrations are of the order of t,hose encountered in most biological material, and direct measurement of their zinc content iLs now feasible; dilution removed most substances which might potentially interfere. The present study was undertaken to define the optimal conditions for the determination of zinc in various biological samples. Previous efforts to employ atomic absorption spectrometry for the deterinination of zinc and other elements in biological materials have employed different procedures ( 2 , 6, 15, 18, 1 9 ) .
Recipient,, Lederle Internat,ional Fellowship. On leave of absence from Department of Physiology, Central University of J.enezuela, Caracas, Yenezuela. Research Fellow, American Cancer Society. VOL. 36, NO. 13, DECEMBER 1964
2407
Table I.
!
Serial Determination b y Atomic Absorption of a Standard Solution Containing 0.3 pg. of Zinc per MI.
No. of
Series A B C
readings
A + B + C
6 6 6 18
Log I,lI (mean) 0 234 0 237 0 231 0 234
Std. dev. f 0 007 f O 004 f 0 004 f 0 006
.6
Rel. std dev., 5 3 2 1 7 1 7 2 6 Y
m
0
Table II. Sensitivity Achieved with Absorption Tubes Used for Atomic Absorption Analysis of Zinc
Relative Tube sensitivitya Nortonh 1 .oo lIcL)anelc 0.85 1 53 llorganited a Relative absorption obtained on aspirating solution containing 0.220 p g . / ml. of zinc under standard conditions. h Sortcin Alundum (RA84R/P6487), 23 cm. long, 18-mm. i.d., 1-mm. wall. c 1Icl)anel-Hi-Temperature mullite, 25 cam. long, I7-mm. i.d., 3-mm. wall. d LIorganite recrystallized alumina, 2 5 cm. long, 13-mm. i.d., 3-mni. wall.
EXPERIMENTAL
Glassware and polyethylene were cleaned as Iireviously described (9). AI1 water wa+ ohtained by iiassing tap water through a mixed cation-anion exchange resin ( I O ) . Reagents. STANDARD ZIKC SOLUTIOSS. A stock ,+elution of approximately 8000 p g , of %n I)er nil. in 1-V HCI was preliared hy dissolving a known amount of spectrogral)hically pure nietallic zinc (,Johnson Matthey Co., Ltd., London, England) in metal-free 6 S HCI. The standard wa:, stored in a tightly clohecl Iiorosilicate gla+ hottle. Zinc standard solut range 0.05 to 0.5 f i g . prepared monthly h y dilution of the standard with metal-free water and stored in tightly closed Imlyethylene hot t les. METALS. Solutions of other metals tvere prepared in a similar way from s1)ectrometrically Inire metals (,Johnson Itatthey (lo,> Ltd., London, England). ‘rR I C H L O R 0 A C ET I C h C I D . ‘rc cryetals were redistilled and stored as a 20y0 d u t i o n in metal-free water in a pol!.ethylene bottle, a t 4’ C. I ~ C F F I : R \ , XACL. Reagent grade chemicals were dissolved in metal-free water. the I I H \vas adjusted hetween 4 and 8, and the solution was freed of metals by reiieated extraction with 0.0037c dithizone in carbon tetrachloride until no fiirther color change of the dithizonr layer occurred. After repented trratnient with sinal1 aliquot. of carhon, tetrachloride to remove the remaining dithizone, the top of the qeiiaratory funnel xaq connected to a water aspirator ~ ~ u n and i p the remaining (‘(‘la removed liy suction, while air was sllowed to bleed in through the stopcock A \
2408
ANALYTICAL CHEMISTRY
to agitate the solution. The huffer solutions were then stored in polyethylene hottles. S A T I O N A L I3UREAU OF STANDARDS SOLUTIOSS. Zinc alloys whose metal content was certified by the Sational Bureau of Standards (15i-.\, 171, and 109) were treated in the same manner as the zinc rods used as analytical standards; after proper dilution, metal solutions were stored in polyethylene containers. IJRIKESAMPLES.Twenty-four-hour urine samples from normal persons as well as from patient> with liver disease were collected. Sam;.lrs wrre voided directly into ilolyethylene funnels inserted in bottles of the same material. Fractions (100 ml.) were stored at 4’ C. after the I I H was adjusted to 2.0 and diluted five- to tenfold in metal-free distilled water immediately before analysis. BLOODSERUM.Human serum saniides were obtained by drawing blond from an antecubital vein, using stainless steel needle:: and acid-cleaned sterilized glass syringes. A tenfold dilution in metal-free distilled water was performed Iirior to aspiration of the sample into the flame. MF:TALL~ESZYMES. Highly purified saniiiles of cartioxypelhdase of bovine pancreas were used as a representative of zinc-containing metalloenzynies. ‘The salt-soluble meta1lol:rotein was dialyzed against metal-free tiuffers in order to remove contaminating a< w l l as loosely bound metals. In all cases the enzyme saniiiles analyzed were fully active in hydrolyzing s1)ecific substrates. Protein concentrations were measured as indicated previously ( 5 ) . Chemical Zinc Determination. The dithizone method was accepted as the standard analytical method for zinc. Determinations using this method were carried nut for comparisnn, as previously described (6, 7 , 12). PROCEDURE
After preliminary treatment, conditioned by the nature of the sample, the zinc content was adjusted to a concentration between 0.05 and 0.5 pg. Iier nil. by dilution with metal-free water. The instrumental background, including dark current and flame noise, was adjusted to zero, while the signal from the hollow cathode, I,, was set to ail arbitrary reading of 80 l a . on a galvanometer scale calibrated to 100 divisions while water was asllirated into the flame. The adjustment was performed either by a fine regulation of the voltage
0
.I
Zinc.
.2
3
,IJQ
per ml.
4
Figure 1. Effect of hydrogen pressure on determination of zinc b y atomic absorption A 2 p.s.i. W 3 p.s.i. p.s.i. 0 5 p.s.i. Air pressure kept constant a t 10 p.s.i.
V 4
(generally 800 volts n-hen the current placed on the hollorv cathode was 20 ma.) aplilied to the multiplier phototube or hy adjusting the entrance slit of the monochromator. The ,mni)les were then aspirated through the hydrogenair burner into a 23- X 1.8-em. .llundum tuhe, and the deflection of the niicroammeter or recorder was noted. Biological fluids containing high protein concentrations must first he diluted, since direct aslliration will otherwihe obstruct the burner. Dilutions to concentrations of apliroximately 0.1% protein have generally heen found suitable for direct ah1)iration. EXPERIMENTAL RESULTS
Analysis of the various potential sources of fluctuations in meter readings demonstrated that the dark current of the phototuhe was the sole source of irreprodiicibility. Once the hollow cathode tuhe had h e m lit for 30 minutcs, the normal time required to rea(-h operating cwnditions, fluctuation diminishes substantially and neither the flame nor the c~onsequenccsof asliiration of the sample eontrilnited additional noise. Table I s h o w thc repeatatiility of serial deteimiinations of a standard mlution of zinc. Relative stnndard deviations of each s e i h of are less than 5%. and both the overall mean of all of the analyses and the coeflicicnts of variation indicate excellent precision. The alisorhance is linearly related to the zinc concentration in the range of 0.01 to 0.5 p g . per nil. In contrast to nsitive proc,edurc?, the coefficient of variation increaw with increasing zinc concentrations, indicating that the
niethod performs optimally even at these minute concmtrations. Table I1 denimstrates the relative sensitivities of detection observed when different types of ceramic tubes are employed as absorption cells ( 3 ) . The sensitivity obtained with a Norton Xlunduin tube is arbitrarily set as unitv. The tube fashioned of AIorganite .stallized alumina, which is highly hed and has a slightly smaller diameter than the other two tubes, gave the best results. However, for most routine purposes, the more inexpensive mullite or Alundum tubes are satisfactory. Figure 1 demonstrates the effect of hydrogen pressure on the sensitivity of zinc. detection by atomic absorption. The sensitivity increases as hydrogen preswre is increased, but eventually level3 off, I'se of high hydrogen pressures, of course, results in high flame temperature and, hence, very hot tubes; therefore, adequate exhau.?t and air circulation should be provided. The internal diameter rather than the comliosition of the tuhex proved to be the major factor accounting for the difference in the limits of detrrtion arhicved. Tuhe:: of smaller diaiiirter arc more responsive to changes in hydrogen pressure, and, hence, result in grtatpr sensitivity of zinc absorption than the larger tubes, provided they are identical in coniposition. Figure 2 shows the sensitivity achirved with a horizontal flame with and without an absorption tube, as extreme examples of the sensitivities achieved; the dramatic increase in smsitivity due t o the use of the absorption tulle is apparent, documenting its indispensability for ineasurenients intending to achieve extremes of sensitivity.
.5 .4 Y
\
& .3 m 0
-I
.2
.I
0 .2 .3 .4 ,5 Z i n c , pg. per ml. Figure 2. Effect of recrystallized Alundum absorption cell on measurement of zinc b y atomic absorption 0
.I
To a urine saml)le containing 0.i5 of Zn per nil. a known amount of Zn6j was added. The sample wah extracted five times a t p H 5.9 with dithizone in carbon tetrachloride and then washed with CC1, to remove e x c w dithixone.
0 Cell used W No cell
pg.
probably due to the interaction of the strong acid with the brass: (zinc) burner parts. Sodium chloride, u p to 1. O M , interferes little or not at all if narrow slit widths are employed to reduce background due to sodium emission in the flame. h solution containing 100 p.p.m. of Ba+*, Ca+2, Co+*, Cr+3, Cu'2, Fe+2,
Table 111.
-1s measured by isotolie dilution, only 0.8176 of the total zinc>remained after extraction, a final concentration of 0.01 pg. of Zn per nil. of urine. Subsequent zinc determinations by atomic absorption of the dithizone-extracted urine gave a value of 0.016 p g . of Zn per ml.
Effect of Chelating Agents and Buffers on Determination of Zinc b y Atomic Absorption Log
Zinc, p.p.m.
Buffer or chelating agent
0 .i22 l O - 3 M EI)TA
INTERFERENCES
Table 111 shows that the presence of either one of two chelating agents, EDTh or o-phenanthroline,does not decrease the absorbance. In fact, 10-3Jf o-l)hcrianthrc.line appears to increase the reading slightly. The effects of phosphate and tris (hydroxyinrthylaiiiinoniethane) buffers on the deteriiiination of zinc by atomic ahsorption are also shown in Table 111. P h o y h a L e drastically lowers the recovery of zinc,. whereas triq buffer, in much highei. concentration, had virtually no effect. Uuffer.; such as 0.05.11 glycine at pH 10 or 0.05AlIsuccinatr at pH 6 did not interfere, while carbonate buffers and trichloroacetic acid markedly decreased the recovery of zinc. Dilute HC1 ( < O . l L \ ) was found to be a suitable diluent but more concentrated solutions caused high readings, as previously reported (4). This is
Lit, hIg+*, or XiT2as the chloride did not affect the absorbance readings of a standard containing 0.2 pg. of zinc per ml. Accuracy of Method. The accuracy of the method was ascertained directly by cornparkon with the expected values of the certified SI3S alloys, dissolved a3 described. Table I T shows the results: 98.8: 101.3, and 105.27, of the known zinc content of the three known standards was found. These results provide a primary standard of accuracy for both the instrument and the method. Zinc in Urine. N o h t methods for the determination of trace metals in urine require prior r e ; n o v J of organic matter (?). Talde V e x p r e w s the results of an experiment designed to study the problem of the proper blank to be used in urinary zinc analyse3 by atomic absorption.
10-3Lllo-phenanthroline
1O-4Atlo-phenanthroline
0 261 0 0 0 0 0 0 0 0 313
a
05M S a sutcinate, pH 6 0 05,21 K gllcinate pH 9 0 05M tns, pH 7 5 05M &'a phosphate pH 7 0 10M Na phosphate, pH 7 0 15M ?;a phosphate, pH 7 0 O5M S a carbonate, pH 9 0
0 25c TCA 1 0 5 TCA
;:/I>
Log I,/I
Mean 0 268
0 267 0 294 0 279
0 0 0 0
007 008
o
n
004
f f i i 163 i
0 158 0 159
0158
0153 0 133
0056 0116 0197 0 178 0136
IC
Std. de"?-
=k
i i 3~
of
control 1
on
700 111 106 100
006 010
0 006 0 004 0004 0004 0 004
97
98 97 94
f f 0004 f 0007
82 34
f 0004 i 0 004 i 0002
100
71 90
79
Six deteiminations performed in all instances
Table IV.
Accuracy of Zinc Measurements b y Atomic Absorption Analysis of NBS Samples
Sample 157-A, Cu-Xi-Zn alloy 109, Zn spelter 171, R'Ig base alloy
% Zn 29 09 100 1 05
Knonn Zn content (after dilution), wg./ml. 0 290 0 380 0 304
Zn found, wg /nil 0 287 0 385 0 320
Accuracy,
VOL. 36, NO. 13, DECEMBER 1964
c
9b h 10 1 3.'
10.5 2
2409
The residual concentration of zinc found after exhaustive extraction is in close agreement with that previously reported on the occasion of establishing and validating a chemical method for the determination of zinc in urine ( 7 ) .
Table V. Preparation of "Zinc-Free'' Urine b y Dithizone Extraction To a 300-ml. sample of urine containing 2 2 5 pg. of zinc as measured b y dithizone, 1 1 8 pg. of zinc containing 63 pc. of ZnG5 w e r e a d d e d to allow measurements of the radioactivity content of the dithizone extrocts. Total Zn = 343 pg. = 1 OO%, equivalent to I .I 4 pg. of Zn/mI.
Extraction
extractedTo of initial Total Zn, pg. zinc content
Initial sample 343.00 I 327 01 I1 10,97 I11 0.65 IV 0.38
v
100.00 95.34 3.20 0.19 0.11 0.03
0.10
Fina1,zinc remaining in aqueous phase
2.77
0.81
( = 0 . 0 1 pg.lm1.O)
Total zinc recovered 342,03
09.68
Atomic absorption analyses of extracted urine = 0.016 pg./ml. a
Table VI. Effect of Aqueous Dilution on Zinc Determinations in Normal Urine
Sample
Vrine$a ml.
Zn detd., pg./ml.
Zn, pg.!ml. urine
1 0.8 0.6 0 4 0 25 0 2
0.76 0.59 0.46 0 32 0 195 0 156
0.76 0.74 0.76 0 77 0 78 0 78
1 2 3
4 5 6
Indicated quantities of urine brought to 1 ml. with water and analyzed.
Table VII. Replicate Determinations of Zinc in Urine and Serum
No. of analysis
x =
0 78
8 9
0.76 0 78 0.78 0 76 0.76 0 78 0.75 0.80
1 27 1.26 1.25 1.25 1.22 1.22 1.18 1.22 1.18
0,774 0.015 2.04
1.227 0.031 2.6
24 1 0
ANALYTICAL CHEMISTRY
U
\
s 0
0
1
.2 .3 Zinc, pq. per ml.
0
.I
Figure
.4
3. Calibration curves
Zinc standards in distilled water compared with Dithizone-extracted urine, zinc a d d e d Higher intercept indicates residual amount of zinc present, measured both isotopically and b y atomic absorption
and 6 , have been described which vary in the S-terminal sequence but not in zinc content. 1 1 1 contain 1 gram atom of zinc per mole. These carboxypeptidases were chosen a' qtandards to examine the accuracy of the method when applied to biological material having an invariate, knoirn zinc content. Carboxypeptida-es are not soluble in water and inuit be diluted in 1-M sodium or lithium chloride to maintain the enzyme in solution. The enzymes were diluted to concentrations of 0.1% or less and the resultant salt-containing solutions were analyzed by direct aspiration into the flame. *Inalyses by atomic absorption were compared with the known value -i.e., gram atom per molecular weight 34,300 -demonstrating excellent accuracy of the atomic absorption procedure. Since atomic absorption utilizes much smaller quantities of enzyme than
Accuracy of Zinc Determinations in Human Serum and Recovery of Added Zinc b y Atomic Absorption
Table VIII.
pg. of pg. of Zn/ml. Zn/ml. of urine of serum
1
Std. dev. Rel. std. dev., %
A standard calibration curve in an aqueous medium is compared with that obtained employing the zinc-extracted urine as the solvent in Figure 3. The slopes of the two calibration curves are identical, indicating that recoveries approach 100yo under these conditions. The intercept with the y axis denotes the residual amount of zinc in the two solvents. In addition, Table VI shows that dilution of urine with metal-free distilled water followed by analysis of the mixture does not change the final result significantly. Repeatability. S i n e replicate determinations of zinc were performed on a normal urine sample (Table VII). h mean value of 0.774 =t0.015 p g . of Zn per ml. was obtained. The coefficient of variation was 2.04%. Zinc Determination in H u m a n Serum. Subsequent to dilution with metal-free water, human serum was aspirated directly into the flame; no other preliminary chemical treatment was performed. The mean value of nine replicate determinations was 1.227 + 0.0319 pg. of zinc per ml., with a coefficient of variation of 2.6%, validating the reproducibility of the method (Table VII). The accuracy of serum analysis was assessed by comparison of data obtained by the dithizone (Table VIII, second column) and the atomic absorption methods (Table VIII, third column). In Table VI11 the third column shows the atomic absorption data and the fourth shows the accuracy on comparing the third and fourth columns. The fifth column shows the atomic absorption results on adding 0.264 pg. of zinc per ml. of serum and the sixth shows the per cent of the added zinc recovered. Zinc Metalloproteins. Carboxypeptidase A, an enzyme from bovine pancreas, contains 1 gram atom of zinc per mole of protein of molecular weight 34,300 ( I S , 14). Dependent on the mode of isolation, four different types of this enzyme, designated cy, p, y ,
Results of chemical analyses of serum (second column) served as standards.
Serum
Atomic* absorption, pg.jml.
Accuracy.
Sample
Chemical method," rg./ml.
1 2 3 4
1.20 1,43 1.27 0.52
1 . 2 2 i 0.03 1 . 3 6 f 0.06 1.23 f 0.04 0.54 0.09
101,6 95.1 96.8 103.8
+
76
+ 0 264
Recovery atomic absorp.,
1.509 1.719 1.402 0 744
101.7 105.8 93.8 92.5
fig. of Zn, pg./ml.
70
Bovine serum 0.282 98.9 0.03 alb. a Average of two determinations. * Serum diluted 1 :10 with metal-free water and six sequential determinations performed. Identical readings obtained after storage of diluted serum for 5 days at 4' C.
emission spectrographic or chemical procedures, this feature of the method is an essential advantage when dealing with materials available in limited quantities. The zinc content for all preparations as measured by atomic absorption was very close to the known value (Table IX). DISCUSSION
Sensitivity, accuracy, precision, and speed are the essential considerations determining the choice of a method for the measurement of zinc in biological material. The size and type of the sample needed, the effort and time required to prepare it for analysis, and the time required for the analytical procedure are critical factors indicating the suitability of a method for general use. The data presented here demonstrate the suitability of the atomic absorption method previously described ( 3 ) . For all biological materials analyzed, including urine, serum, and proteins, as well as chelating agents, which bind zinc very firmly, the method proved extremely simple and rapid; it should find wide application. The method described is simple, practical, efficient, and relatively free from interferences. Moreover, analysis is precise and rapid, being comparable to conventional flame photometry in terms of speed. Monochromators and detectors other than those employed here can be used, and even greater sensitivities can be achieved when alternative equipment, or conditions are employed. Selection of the optimal absorption cell size and type and optimal hydrogen pressures are important to achieve maximal sensitivity (Figure 1, Table 11). If it is reflectant, the type of cell material is less important than its dimensions. I n our hands 15 mm. has constituted a nearly optimal inside diameter and a length of 20 to 30 em. represents a good compromise to achieve adequate sensitivity while holding the dimensions of the apparatus to convenient and minimal size. While long, narrow Vycor tubes result in great sensitivity, such cells become contaminated easily and must be cleaned frequently. The increase in sensitivity achieved by means of very long tubes inay become so great as to present an analytical hazard. Contamination may become a serious problem, since at the low levels of total zinc determined contamination may become a significant percentage of the total amount of metal determined (3). The use of a 26-cm.
alumina tube results in a 15-fold increase of sensitivity when compared to that of a vertical flame, even when the latter is operated under optimal conditions (Figure 2). Thus, the present method has been designed to result in an absorption of 1% at 0.002 wg. of zinc per ml., the limit of detection; this represents a compromise between the factors discussed. Compared t o the commonly used “vaporizing type” of burner, “total consumption” burners reduce considerably the volume of sample required for analysis. This represents a great advantage, since in working with biological material, availability of the sample is usually an important limiting factor, as in the case of enzymes of high purity such as carboxypeptidase. Therefore, the methodology here described makes possible the performance of individual experiments on minute amounts of biological material. The small size of the aspirator burner represents a further advantage, since it allows the injection of much higher concentrations of absorbing atoms into the absorption cell. Caution must be used in aspirating strong acid solutions, as the brass burner parts of the Beckman burner are easily attacked ( 4 ) . Construction of this type of burner from a noncorrosive metal would overcome this difficulty. The excellent reliability of this method is clearly shown from the data with XBS standard samples (Table IV). The results obtained on diverse biological materials such as urine, serum, and proteins indicate that the complex matrix of organobiological constituents does not affect analytical accuracy. Such results are due to the high sensitivity of the method, which allows sufficient dilution of the sample so that complete combustion can occur during the passage through the absorption cell. The analytical results obtained with the present method have been compared with other methods in common use for the determination of zinc, such as emission spectrography, dithizone extraction, and isotope dilution ( I ) . The agreement is remarkable. The level of sensitivity at which this atomic absorption method operates approaches that achieved by isotopic labeling. The simplicity of sample preparation is another factor which renders the method very practical, easy, and convenient. Only a dilution step is needed and no further treatment such as ashing or extraction is required. Hence, the dangers of losses by ashing or contamination by reagents or handling are
Table
IX.
Zinc Analyses of Bovine Carboxypeptidase A“
Atomic absorption, fig. of Zn/gram of Accuracy, Sample protein % CPD-A 1895 f 49b 99 3 CPD-A 1950 f 62 102.0 CPD-A 1850 i: 48 96.8 a Carboxypeptidase A contains 1 gram atom of zinc per molecular weight of 34,300 (16-1’73, or 1910 fig. of Zn per gram, which is considered 100%. b Six determinations performed in all instances.
avoided. This remarkable dividend, permitting samples to be analyzed simply by direct dilution avoiding complicated preanalysis preparative procedures, is the start of a revolutionary era for the determination of zinc in biological materials. LITERATURE CITED
(1) Coleman, J. E., Vallee, B. L., J . Bwl.
Chem. 235, 390 (1960). (2) David, D. J., Analyst 85, 779 (1960). (3) Fuwa, K., Vallee, B. L., ANAL. CHEM.35,942 (1963). (4) Gidley, J. A. F., Jones, J. T., Analyst 86, 371 (1961). (5) Hoch, F. L., Vallee, B. L., J . B i d . Chem. 181, 295 (1949). (6) Honegger, N., Aertztl. Lab. 9, 41 1 1963). ( 7 ) Kagi, J. H. R., Vallee, B. L., ANAL. CHEY.30,1951 (1958). (8) Sandell, E. B., “Colorimetric Determination of Traces of Metals,” p. 620, Interscience, New York, 1950. (9) Thiers, R. E., in “Methods in Biochemical hnalvsis.” D. Glirk. ed.. Vnl V, p. 301, Interscience, pr‘ew York,‘1957; (10) Thiers, R. E., in “Trace Analysis,’] J. H. Yoe and H. J. Koch, Jr., eds., Wiley, New York, 1957. (11) Vallee, B. L., Physiol. Rev. 39, 443 11959). (12) Vallee, B. L., Gibson, J. G., 11,J.B i d . Chem. 176, 435 (1948). (13) Vallee, B. L., Neurath, H., J. Am. Chem. SOC.76, 5006 (1954). (14) Vallee, B. L., Neurath, H., J. Biol. Chem. 217. 253 1 1955). (15) Warkei, U’.E. C., Iida, C., Fuwa, K., .Vature 202,659 (1964). (16) yalsh, A., Advan. Spectr. 2 , 1 (1961). ( 1 7 ) malsh, A., Spectrochim. Acta 7, 108 (1955). (18) Willis, J. B., ANAL. CHEM.34, 614 (1962). (19) Willis, J. B., Methods Biochem. Anal. 11, 1 (1963). ~
~
RECEIVEDfor revlew July 28, 1964. Accepted September 14, 1964. Work supported by Howard Hughes Medical Institute and by Grants-in-Aid HE-07297 from the National Institutes of Health, Department of Health, Education, and Welfare.
VOL. 36,
NO. 13,
DECEMBER 1964
241 1
