counting. In this way one may discriminate against gamma rays scattered from the Ge(Li) detector into the NaI(Tl) detector (3). Confidence limits in the data depend upon the specific mineral and the number of samples run. Because there is no chemistry involved, confidence limits for each case are rapidly and accurately estimated from the counting data. For the Agllo photopeak in Figure 1, one standard deviation is *7y0 of the net intensity. Since the major analysis interference comes from the short-lived nuclides, 9.5-minute MgZ7 and 2.58-hour Mn56,samples may be r e analyzed for Ag following a several-hour cooling period. The measurement of Ag by neutron activation analysis is not new (1).
However, the ability to use neutron activation as an analytic tool requiring neither wet chemistry nor spectrum peeling is a major advance over previous approaches and is the result of using the Ge(Li) detector. The application of activation analysis and high resolution Ge(Li) gamma spectrometry to problems in the history of technology and other problems in geochemistry has been discussed elsewhere (4). Though the determination of the Agllo 660-k.e.v. gamma-ray intensity suffers from being located on a Compton edge, the sharp definition of the photopeak obtained with a Ge(Li) det,ector sets the limit of quantitative detectability a t about 15 p.p.m. Ag. The nondestructive aspects of the method and the short irradiation time allow the same
sample to be reanalyzed several times within a single day. Quantitative results are obtained in a matter of minutes following irradiation. LITERATURE CITED
(1) Anders, 0. U., Beamer, W. H., ANAL. CHEM.33, 226 (1961). ( 2 ) Evans, R. D., “The Atomic Nucleus,” p. 816, McGraw-Hill, New York, 1955. (3) Roulston, K. I., Nayvi, S. I. H., Rev. Sci. Znstr. 27, 830 (1956).
(4) Schroeder, G. L., Kraner, H. W., Evans, R. D., Trans. Aim. Nucl. SOC. 8, 327 (1965). (5) Tandberg, J., Proc. Phys. Soc. (London) 50, 87 (1938).
RECEIVED for review October 25, 1965. Sccepted November 26, 1965. This work was supported in part by the U.S. Atomic Energy Commission under contract AT(30-1)-952.
Ultramicro Spectrophotometric Determinatior of Calcium in Biologic Fluids D. S. HOWELL, J. C. PITA,
and J. F. MARQUEZ
Department of Medicine, University of Miami School of Medicine, Miami, Flu.
b
A sensitive spectrophotometric method for calcium determination (5) was modified to quantitate amounts of the range of 1 0-9 grams contained within 5 to 25 mpl. of a biologic fluid. Success of this method depended upon employment of a newly described ultramicro [Ultramicro in respect to the initial volume of fluid analyzed.] cuvette with satisfactory optical characteristics herein demonstrated and use of chlorophosphonazo 111 as a colorimetric reagent because of the high molar absorptivity of its calcium complex. The ultramicro cuvette consisted of a capillary glass tube with capacity of 2.8 pl. per cm. of light path oriented in the Zeiss PMQ spectrophotometer by a simple positioner and holder. Standard deviations of recovery values for calcium at a concentration range of 3 to 12 mg./IOO ml. were 0.07 to 0.15 mg./lOO ml.
T
HE PRESENT cuvette was designed for studies on fluid micropuncture aspirants from calcifying sites in rat epiphyseal cartilage but should have general direct applicability for nonbiologic problems wherein spectrophotometric determination of substances in the amount of 10-8 t o 10-10 gram is required. Several ultramicro cuvettes, previously designed by other investigators, have been reported suitable for the special problems for which they were designed as recently reviewed (1, 6).
434
ANALYTICAL CHEMISTRY
Only those cuvettes readily wettable by aqueous solutions and positioned with the long axis parallel to the incident light beam were considered feasible for current needs. Craig, Bartle, and Kirk (4)diminished the volume of the microcell of Kirk, Rosenfels, and Hanahan (7) to about 20 pl. by use of a capillary glass tubing to which were fitted end windows made of removable cover slips. This model, adapted to the Beckman DU spectrophotometer, required a photomultiplier and an elaborate positioning device. The ultramicro cuvette and positioner, described herein, are a simplification of the unit described by Craig, Bartle, and Kirk (4)both in respect to construction and operation. With final volumes of 2.5, 5.0, and 8.4 pl., these simplified cuvettes permitted quantitation of solutes following dilution up to 500-fold from original samples, and by using the same cuvette and windows for blank, sample, and standard, errors from differences in respect to glass thickness (9) were avoided. With any given chromophore in this system, the molar absorptivity becomes critical because of the small starting samples. This factor largely determined the choice of the reagent in the present calcium method as discussed below. EXPERIMENTAL
Apparatus. The Optical System. A spectrophotometer was needed for which signal-to-noise ratio permitted
utilization of a cell with crosssectional area of about 0.2 sq. mm. For design of the absorption cell, advantage was taken of the Zeiss PMQII spectrophotometer optical and mechanical characteristics although other equivalent instruments would be satisfactory. Figures 1 and 2 show the dimensions and disposition of the ultramicro cuvette and the positioner in the Zeiss (No. 507425) special sample changer for two cells. This holder is provided with a screw-in bar for directing the sliding piece with a forked spring pressing the rear section of the positioner. The positioner is a black Bakelite cylinder turned to exactly the outside dimensions of the MS5 Zeiss cells except near one extremity a t which the diameter is reduced to provide shoulders for the resilient fork (Figures 1 and 2). The bore along the axis of the positioner snugly accommodates the capillary tube. Length of the ultramicro cuvette was not critical inasmuch as the same one was utilized with blank, standard, and sample. For most of the analytical work, a 3.1-cm. light path was employed. An ultramicro cuvette with total capacity: 8.4 ~ l . (2.8 pl. per cm. layer depth) was cut from capillary tubing of polyethylene or Pyrex glass (Corning Glass Corp.). The ends were polished with a fine grinding stone under a dissecting microscope and only those with smooth surfaces selected for use. The plane of each end was approximately parallel to that of the slit, and the end of the cuvette facing the detector was painted with acrylic black enamel. The ultramicro cuvette was replaced precisely to
-I
CUVETTE HOLMR CL CUVETTE
4" g. - - - - - - - - - - --.J
L..
7 1 1 1
]
'
LJ ~-----. ----- I
U e # t r gbrrl 2 ~ 2 x Q l 5 m m .
--
-
/$plm+ic
Figure 2. Relationship of ultramicro cuvette positioner to cell holder
ring
.
O.D. *
aam.
wall
0. I mm.
i
iositioner
Table I. Absorbance of T-1824 Dye at 4.5 X lO-'M (A) and 1.10 X 10-6M (B) Both Following 10 Refillings of the Ultramicro Cuvette
Solution A
the same rotational and longitudinal position by alignment of reference marks, engraved both on the positioner and on the external surface of the plastic ring of the cuvette (Figure 2). The plastic ring and adjacent parts, painted black, served to block the detector from stray light. To the PMQII spectrophotometer mas attached the Zeiss microcell equipment (No. 507425) for ube of microcell LIS5 (0.2 ml. per em. light path). .A diaphragm was supplemented to reduce the incident beam diameter to approximately 0.6 nmi. The collimation was found highly critical but readjustment was necessary only once for different positioners. The instrument sample changer is provided with two horizontal, parallel bars that form a V-shaped bed to support the l I S 5 cells of which the external diameter is identical to that of the positioner. Optical alignment was performed by fixing the cuvette, positioner, and the holder in place and adjusting the bars of the sample instrument changer to obtain maximum transmittance of light. At 550 mp. about 75% of incident power passed through the system. K i t h the positioner tightly pressed by the resilient fork, the optical alignment was permanent. Use of Ultramicro Cuvette. During manipulations, the ultramicro cuvette was inserted in the positioner forming a single unit with the holder. Guided under a dissecting microscope, fluid samples were delivered into the ultramicro cuvette from a micropipet with care to prevent bubbles. A small drop of solution for analysis was also placed on Corex or Quartz cover glasses fixed to both ends of the cuvette
by contact with the liquid column. Data were obtained (Table I) on repetitive transmissions duplicating these manipulationr of general usage. The mean absorbance a t 620 m l . and standard deviation (S.D.) of 10 successive fillings with a solution of T-1824 dye (Microchrome Xo. 231 Edward Gurr, Ltd., London) a t 4.5 X lO-7-V (Solution -4)and 1.10 x 10-6U (Solution B), respectively, was such as to exclude these manipulations as a source of important error. Optical Properties of Ultramicro Cuvette. The effect of cuvette length on absorbance of solutions of Table I registered linearity (Figure 3) although a slightly wider capillary was needed to maintain linearity a t the 5-cm. light path length. Further, optical properties were investigated in respect to conformity to Beer's law using two conventional standards. First, for a solution of K2Cr20,in 0.010N H2S04 the molar absorbance was observed in this ultramicro system as previously reported for macro systems (16) to be 3100 a t wavelength 380 mp. A11 further experiments were accomplished by duplicating the absorbance measurements for the same solution in the macro and ultramicro cells. When dilutions of the 0.0383Jf stock solution were analyzed in the macro cell, linear dependence of absorbance on concentration was complete, but in the micro cuvette, a negative deviation from Beer's law occurred for concentrations higher than 1.2 ml. in 1.5 liters (curve I, Figure 4). Nevertheless, when the same dilutions were prepared from a stock solution one fourth as concentrated (O.O096M), complete linearity
B
R
S.D.
R
S.D.
Ultramicro cuvette 0.119 f0.0042
0.292 zk0.0018
Macro cuvette 0.080
0.195
Table II. Per Cent Error in Absorbance of T-1824 for Solutions Used in Table I
0.060
0.290
0.830
0.510
*0.0040 zt0.0018
2.2 0.60
was seen (curve 11). Furthermore, the absorbance for any point in this curve, accurately coincided with the corresponding point of the curve for the macro cell if corrected for differences in solute concentration and optical path length. Also, the ratios of micro t o macro absorbancies of T-1824 dye (Table I) coincided satisfactorily with corresponding light path length ratios. The absorbance values for about lo3 dilution of 0.037, p-nitrophenol in 0.010-11 NaOH are given in Figure 5. The molar absorptivity, 18,400 a t pH 10 and a wavelength of 400 mp is in correct agreement with previously reported data (11). Negative deviations from Beer's law, a t low absorption center concentrations (curve A) was traced to the use of too wide a band width ( I d ) . The other most likely error, stray radiation, would be considered important a t high abaorbances only ( I S ) . By increasing momentarily the detector signal-to-noise ratio, it was possible to reduce the band used (4 mp) and the effect (curve B ) was an amplification in the concentration range showing linear dependence. The validity VOL. 38, NO, 3, MARCH 1966
435
I
I
I
1.0
2.0
I
3.0
I
I
5.0
4.0
Light Path in crns. Figure 3.
Relationship of absorbance to length of light path For T-1824 dye dilutions (A and B of Table I)
for the common part of the curves A and B was in this way confirmed. In classical spec t r o p h o t ome t r ic analysis the maximum error of concentration determination is expressed (16) : ___ (S.D.) A =
A
[:“*434k][ p.3
.600
S.D.
-log
-
Figures (Table 11) derived for maximal error based on observations of a solution absorbance measured a t wavelength 660 mp and band width of 4 mp indicated that the current unit was suitable for biochemical methods within the limited operation already described. Ultramicrochemical Manipulations. The ultramicro samples were manipulated in a microconcavity slide (Clay Adams A-1475), protected from evaporation with dust-free oil (paraffin white oil, U.S.P.-Saybolt 340355 a t 100’ F. filtered through a fine Buchner type fritted glass funnel). Conventional distilled demineralized water was used in reagents and cleaning. Glassware was dried with compressed air passed consecutively through concentrated sulfuric acid and oil. All the operations were performed under a dissecting microscope a t 60-1OOX. The micropipets were made, pulling Pyrex capillaries 0.4 mm. i.d., and used connected to a micromanipulator. Although the ultramicro aspiration techniques, using an oil pump (not described herein) were developed in this laboratory, other conventional aspiratory systems should suffice. The micropipet tips were sharpened to a diameter between 25 and 30 microns and a small 436
plastic mark was placed on the external surface for calibration. Then a 0.030% solution of p-nitrophenol was aspirated to the mark, diluted in 20 or 30 pl. of 0.010M NaOH, the absorbance of
the resulting solution read a t 400 mp, and the volume corresponding to the micropipet evaluated by using the standard curve of Figure 3, as described by Lowry (8). Pyrex capillary tubing (Corning Glass Corp.) 0.6-mm. i.d.; 0.8-mm 0.d. was used for ultramicro cuvettes Pipets were made, pulling by hand, on an oxygen-acetylene flame from Vitreosil (R) tubing of 4.0mm. i.d., 6.0-mm. 0.d. These were used exclusively for final steps of the calcium procedure. Ultramicro Determination of Calcium. Because of the estremely small starting samples, a highly sensitive colorimetric reaction was required. Therefore a method, published by J. W. Ferguson et al. ( 5 ) ,for determining calcium and magnesium in alkaline earths extracted with organophosphor o w compounds, was modified and adapted to quantitate calcium in biological sample volumes ranging from 5 to 25 mpl. The molar absorptivity of the calcium complex of the reagent, 2, 7 bis-(4 chloro-2-phosphonobenzeneazo) - 1,8 - dihydroxynaphthalene - 2 , 6 disulfonic acid (chlorophosphonazo 111) was found as reported previously (5) to be 64,000 a t pH 7.0 a t a wavelength of 669 mp. The calcium present in the diluted microscopic fluid sample was precipitated as oxalate, according to the procedures of Clark and Collip (3) and Munson et al. (IO), substituting for ammonium oxalate and tetrabutylammonium salt. As described by Sendroy (12), no treatment of the sample with trichloroacetic acid prior t o
ANALYTICAL CHEMISTRY
.500
.400
P !i a U
m
.300
,200
.IO0
I
I
I
I
I
2.5 I .o I .5 2.0 ml. of Stock Solution in 1.50L of 0.01N H2S04 Figure 4. Absorbance of aqueous solutions of KZCr20, 0.5
1 3 .O
Starting standard solutions for dilutions were 0.038M for curve I and 0.0096M for curve II
,500
.400
w ,300 V
2
a
m
If:
0
:,200
4
.I00
0.0
Figure 5.
ml. of Standard Solution in 1.OL of 0.010 M NaOH Absorbance of p-nitrophenol 0.03% standard stock solution
Different volumes diluted to 1 liter with 0.01 O M NoOH.
the oxalate precipitation was necessary. All the analyqes mere performed on fresh rather than frozen samples as recommended elqewhere (2, 17‘). Reagents for Determination of Calcium. Aillthe reagents were prepared according to Ferguqon et al. (j),except for the following modifications or additions: standard stock calcium solution; approximately 2.5 grams of calcinated CaC03 (primary standard) were precisely weighed and dissolved in 10 ml. of concentrated hydrochloric acid. The solution mas evaporated to dryness and the remaining solid dissolved in C02-free distilled water to make 1.0 liter. Standard calcium chloride solution: the stock solution was diluted 1: 10 with reagent water to a concentration of about 10 mg./lOO ml. “Control” dehydrated normal serum (from Hyland Laboratory in California) was used for serum standards and calcium recoveries. Chlorophoephonazo I11 : this compound, the syntheiis of which has been described (5, 11) \vas dissolved in reagent water to make 1 x 10-4.U solution which, after slow passage through resin column, mas stored in plastic bottles. The absorbance of this solution, mixed with the pH 7.0 special buffer was carefully verified before being utilized in any determination. Only Vitreosil pipets were used to manipulate this solution, 10-~111solution of analytical grade hydrochloric acid; 1% aqueous solution of tetrabutylammonium iodide and 1.0 gram of Ag,O in 100 ml. of distilled H 2 0 . After agitation for one hour, the solution was passed through a Xtillipore filter and stored in plastic bottles a t 5’ C. Saturated solution of tetrabutylammonium oxalate was prepared, dissolving equivalent weights of oxalic
Negative deviation from a linearity i s shown with absorbances above 0.300
acid and tetrabutylammonium hydroxide. The resulting solution was concentrated by lyophilization until crystals appeared; the pH was adjusted to 7.0 and after filtration, stored in plastic containers. Procedure for Calcium Analysis. Approximately 20 mpl. of starting sample or standard, collected in a, calibrated ultramicro pipet, was delivered and an aqueous dilution made under oil to 0.5 pl. This was quantitatively aqpirated into a micropipet containing 0.5 pl. of saturated tetrabutylammonium oxalate a t pH 7.0 and an aqueous dilution made to 1.0 pl. Contents within the flame-sealed pipet were mixed by successive slow cen-
trifugation, inversion, and centrifugation and incubated a t 50’ C. for 20 minutes in a water bath. After centrifugation a t 15,000 r.p.m. for 10 minutes, the CaC204 precipitate was washed twice with 1% aqueous tetrabutylammonium hydroxide; 1.0 p l . of 10-3il.I hydrochloric acid was added, and the capillary resealed. The precipitate was mixed, dissolved during centrifugation, and incubated as above for 10 additional minutes. Isolated droplets adjacent to the airspace were pooled with the fluid column following a final centrifugation. The resulting solution was transferred to a plastic vessel (20-pl. capacity). With Vitreosil pipets 12.5 pl. of borate-tetrabutyl-
0.300 W 0
Z U
0.200
I
0
I
I
0.5 Figure 6.
I
I .o 1.5 2.0 mfig. of Calcium in 20 m b I Ultramicro determination of calcium
I
2.5
3.0
Standards range from 3 to 12.0 mg./100 ml.
VOL. 38, NO. 3, MARCH 1966
437
Table 111. Recovery of Calcium from Standards and Reconstituted Human Serum with Increments of CaClz Added
Results are expressed as standard deviation from mean of theoretical recovery Ca S.D. Samrange, Sample ples mg./100 mg./100 standard (n) ml. ml. 0.10 CaC12 20 3.0- 5 . 0 0.07 CaClz 20 5.0-10.0 0.15 Serum 8 3.0- 6 . 0 Serum 10 6.0-12.0 0.11 ammonium borate buffer (pH 7.0) and 2.5 pl, of 1 x 10-4M aqueous solution of chlorophosphonazo I11 were added. The resulting solution was mixed by “buzzing” (6). The absorbance was read in a Pyrex capillary ultramicro cuvette a t wavelength 669 mp against a reagent blank with band width of 8 mp, Variation and Recoveries. hbsorbances of calcium chloride standard solution showed complete conformity to Beer’s law in the concentration range of 3 t o 12.0 mg./100 ml. and U ~ Jto 20 mg./100 ml. with a slightly larger bore capillary with starting samples of 20 mpl. (Figure 6). Recoveries of calcium (as chloride) added either to a 3 mg./100 ml. calcium chloride standard or to a basal solution containing a variety of biologic constituents including protein, Mg+z, Xa+, C1-, in amounts found in human plaL qma were analyzed (Table 111). For each theoretical level of calcium, the standard deviation of the recoveries was calculated. The poorest result was on the reconst,ituted serum in low concentra-
438
ANALYTICAL CHEMISTRY
tion ranges-95 to 105% (Table 111). The met,hod was also tested in macro scale on the same sera in comparison t o the procedure of Munson et al. (IO) with satisfactory standard deviations. CONCLUSIONS
The simplicity of design and operation of the current ultramicro cuvettes should render a variety of macrobiochemical methods, illustrated herein for calcium, suitable for accurate analysis on fluid samples of 5 to 20 mpl. congram of the subtaining lo4 to stances to be quantitated. Limitations of the method involved principally that chromophoric reagents must have a high molar absorptivity and that the absorbances observed with the ultramicro curvette be kept in the range of 0.1 to 0.3. The same ultramicro cuvette was employed in testing blank, standard, and sample with exact orientation in a positioner. Absorbances in the visible and near ultraviolet region of the spectrum were measured on highly diluted samples. The spectrophotometer used had a signal-to-noise iatio adequate for cells with 0.2 sq. mm crosssectional area. Standard deviation of recovery value5 for calcium at a concentration of 3 to 12 mg./100 ml. were 0.07 to 0.15 mg./lOO ml. ACKNOWLEDGMENT
The authors are indebted to C. V. Banks of Iowa State L-niversity for a supply of chlorophosphonazo 111.
LITERATURE CITED
(1) Boltz, D. F., Mellon, M. G., ANAL. CHEM.36,256R (1964). (2) Chen, P. S., Jr., Toribara, T. Y., Ibid., 26, 1957 (1954). (. 3.) Clark, E. P.. ColliD. J. B.. J. Bid. Chem. 63,461 (1925):’ (4) Craig, R., Bartle, -4.,Kirk, P. L., Rev. Sci. Instr. 24, 49 (1953). (5) Ferguson, J. W., Richard, J. J., O’Laughlin, J. W., Banks, C. V., ANAL. CHEY.36, 796 (1964). (6) Glick, D., “Quantitative Chemical Techniques of Histo and Cytochemistry,” Vol. 11, p. 17, Interscience, New York, 1963. (7) Kirk, P. L., Rosenfels, R. S., Hanahan, D. J., ISD. ENG. CHEM.,ANAL. ED. 19, 355 (1947). (8) Lowry, 0. H., Roberts, X., Leiner, K., Wu, >I. L., Farr, A,, J . Biol. Chem. 207, 1 (1954). (9) Meites, L., Anal. Chim. Acta 27, 131 (1962). (10) RIunson, P. L., Iseri, 0. A,, Kenney, A. D., Cohn, V., Sheps, 11.C., J . Dental Res. 34, 714 (1955). (11) Nemodruk, A. 4.,Novikov, Yu. P., Lukin, A. M., Kalinina, I. D., Zh. dnalit. Khim. 16, 180 (1961). (12) Sendroy, J. Jr., J . B i d . Chem. 152, 539 (1944). (13) Slavin, W., ANAL.CHEM.35, 561 (1963). (14) Strobel, H. .4.,“Chemical Instrumentation,” p. 162, Addison-Wesley Publishing Co., Inc., Boston, Mass., 1962. (15) Svehla, A., Erday, L., Talunta. 10. 719 i1963). (16) ’Vandenbeit, J. AI., J . Opt. SOC.Am. 50, 24 (1960). (17) Wilk, A. L., King, C. T. G., Xature 198, 187 [ I 96s).
RECEIVED for review November 5, 1965. Accepted December 16, 1965.
