1904
Anal. Chem. 1982, 54, 1904-1906
Table I. Accuracy and Precision of Plunger Movement in Millimeters
\n
nominal plunger travel
mean
std dev
2.540 5.080 12.700
2.539 5.078 12.690
0.003 0.003
obsd
0.007
Table 11. Accuracy and Precision of Aliquot Delivery 100 IOo
IO‘
4 02
1G3
P L
104
FREQUENCY (Hz)
Figure 4. Plot of standard deviation of 2 4 peak-to-peak sinusoidal signal vs. signal frequency attained with analog-to-dlgital converter/ phase-locked loop noise rejection circuit.
efficiency of noise rejection at frequencies harmonically related to 60 Hz. A 2-V peak-to-peak sinusoidal “noise” signal was electronically summed with the demodulator output. Twenty-five ADC readings each were taken at selected “noise” frequencies ranging from 1 to 10 kHz. The standard deviation of the ADC readings at each frequency was calculated and plotted as a function of frequency; the data are summarized in Figure 4. The plot rolls off a t about 10 Hz with a slope of -20 dB decade-l. These results suggest that noise rejection at harmonics of 60 Hz is approximately 45 d B without any signal averaging. The long-term stability of the LVDT-demodulator was measured under actual hot-cell operating conditions by recording voltage readings corresponding to the plunger position after delivery of a 1000-pLaliquot. For a period spanning 99 days (52 data points) a mean of +3.868 with a standard deviation of 0.004 V (i.e., 0.6 p L ) was obtained; the nominal value is 3.864 V. Because the reliability of solution pipetting is directly related tQ the accuracy and precision of the plunger movement, data related to these variables were collected. Fifteen measurements each were taken with a dial micrometer for linear distances corresponding to 100.0, 200.0, and 500.0 p L of plunger displacement. Each measurement was associated with a slightly different region of the drive screw, so that the results suggest pipetter performance over the entire operating range rather than only a small portion of it. Accuracy and precision for plunger motion obtained from these experiments are presented in Table I. Data for the actual delivery of variable sized aliquots of water were also obtained. Prior to obtaining these data, the system was calibrated. A slope of 0.9972 and intercept of -0.42 were used in the equation relating aliquot volume to steps of motor rotation. These variables are entered only during the power-up initialization dialogue. Each aliquot was delivered using a secondary displacement technique; i.e., the pipetter cavity was filled with colored mineral oil and only the tip
300 IIL
500
PE
700 ALL
av of delivered 100.5 300.9 501.2 701.9
aliquot, p L relstd dev, % re1 error, %
0.4
0.2
0.5
0.3
0.1 0.2
0.06 0.3
1000 M L
1002.1 0.02 0.2
contained solution to be aliquoted. This technique is routinely used to minimize cross contamination of samples. The volume of each aliquot was determined from its mass and the density of water. A summary of the accuracy and precision for these aliquot deliveries is given in Table 11. The remote pipetting system described herein enables sample aliquots to be accurately and precisely delivered in remote environments. The highly reliable, independent control afforded by a microcomputer facilitates operator interaction and provides accurate documentation of operations. The firmware-resident control programs allow quick instrument modifications to be made for changing applications at minimum cost. Although this system was developed for use in a hot-cell environment, it is equally suited for use in a glovebox environment; as such it is applicable in any situation where hazardous materials, chemical or biological, must be isolated from operating personnel. Details of the instrument may be obtained from the Technology Utilization Office, Oak Ridge National Laboratory, Oak Ridge, T N 37830, Remote Pipetter, Model Q-5780.
LITERATURE CITED (1) Maddox, W. L. In “Progress in Nuclear Energy, Series I X ” ; Stewart, D. C., Elion, H. A., Eds.; Pergamon Press: New York, 1970; Chapter
4. (2) Orseilo, S.; Pozzi, F.; Todini, M. Comltato Nazionole per I’Energia Nucleare Report RT CHI-(71)7, Rome, Italy, 1971. (3) Fortsch, E. M.; Wade, M. A. Anal. Chern. 1974, 4 6 , 2065-2067. (4) Ochsenfeld, W.; Welnlander, W.; Ertel, D. Proc. Remote Systems Techn., 25th, 1077. (5) Dykes, F. W.; Shurtliff, R. M.; Henscheid, J. P.; Baldwin, J. M. Proc. Remote Systems Techn., 27th, 1979. (6) Maddox, W. L.; Haga, F. E.; Fisher, D. J. Proc. Remote Systems Techn., 13th, 1965.
RECEIVED for review May 22, 1981. Resubmitted March 3, 1982. Accepted May 26, 1982. Research sponsored by the Nuclear Fuel Cycle Division, U.S. Department of Energy, under Contract W-7405-eng-26 with Union Carbide Corp. Presented in part a t the 25th Conference on Analytical Chemistry in Energy Technology, Gatlinburg, TN, Oct 1981.
Isocratic Reversed-Phase Liquid Chromatography for Assay of 5’-Nucleotidase with Phosphoric Acid for Reaction Quenching LI-Wen Yu and Roger S. Fager” Department of Physiology, University of Virginia, School of Medicine, Charlottesville, Virginia 22908
The most commonly used methods for preparing tissue extracts of nucleotides or enzyme assays for nucleotidases for high-performance liquid chromatography involve stopping the 0003-2700/82/0354-1904$01.25/0
enzyme reactions with perchloric or trichloroacetic acid, neutralizing, removing the acid by precipitation or extraction, and filtering (I,2). For this reason, the sample handling often 0 1982 American Chemical Soclety
ANALYTICAL CHEMISTRY, VOL. 54, NO. 11, SEPTEMBER 1982 0024
1
0,000~
0048
1905
L
1 I25
25
5
I 5 ’ G M P l (mM1
b 0.04 -
d
5
mi,
Figure 1. UV absorbing artifacts caused by H3P04 and TCA. Shown are chromatograms of (a) 1.45 M H3P04, (b) 1 M TCA, (c) mixture of
standards (0.725 mM S’GMP, 1 mM guanosine, 1.25 mM cGMP) and 1.45 M H3P04 (7:lO by volume), and (d) mixture of standards and 1 M TCA (7:lO by volume).
W
0.03v)
i
takes more time than the HPLC analyses themselves. The increased sample handling also introduces questions of loss of the compounds of interest and unavoidable dilution errors. Since we have been carrying out detailed studies of photoreceptor nucleotidases which require large numbers of individual assays, we have investigated methods of simplifying sample preparation.
EXPERIMENTAL SECTION Materials. A Milli-Q ireagent grade water system (Millipore Co.) was used to purify the water. Trichloroacetic acid (TCA) solution was purchased from Sigma Chemical Co. Reagent grade phosphoric acid was purchased from J. T. Baker Chemical Co. Reagent grade methanol was purchased from Mallinckrodt Inc. Guanosine, guanosine 3’,8’-cyclic monophosphate (cGMP), and guanosine 5’-monophosphate (5’GMF’) were purchased from Sigma Chemical Co. Aqueous solutions were filtered through 0.45 pm pore size filters, and organic solvents were filtered through 0.2 pm pore size filters coated with Teflon. Separation Conditions. A Waters M-6000 pump was used to generate a smooth flow rate. A Waters Cl8 reversed-phase column was used with a short guard column and an in-line filter to protect the column. A 1J6K universal injector (Waters Associates Inc.) was used to lload the samples on the column. The outlet of the column was connected to a Model 153 monitor (Altex Scientific Inc.), set at 254 nm to monitor the nucleotides and nucleosides in the effluent. A 0.1 M sodium phosphate buffer at pH 2.5 with 5% methanol was used as the eluent. The flow rate was 2.0 mL/min. New Methods Developed t o Speed S a m p l e Workup-Replacement of Filtering. We have replaced hand filtering with centrifugation of the samples at top speed in an Eppendorf microcentrifuge 5412 (centrifugal force 12 800.g) with 1.5-mL polypropylene conical microcentrifuge tubes for 3 min and use of an in-line filter in the HPLC system (Mott Model No CRT 6150). This has proved adequate to protect the C-18 columns from particles. The in-line filters are good for approximately lo00 samples. Beyond this number, they start spreading chromatographic bands, probably by developing appreciable dead volumes, and must be replaced. RESULTS AND DISCUSSION As shown below, direct application of unneutralized TCA extracts are usable for some purposes. There are, however, two major problems. The first is there are ultraviolet absorbing components in commercial TCA which compromise
iGUAN0SlNEI I m M )
i 1 LL 3
10
-
0
008-
w
I 25
2.5
5
IcGMPI ( m M )
Figure 2. (a) Calibration of 5’GMP peak height vs. molar amount for Hap04 and TCA. Ten volumes of either 1.45 M H,PO, or 1 M TCA was mixed with 7 volumes of nucleoside or nucleotide solutions (this was the proportion used to stop the enzyme reaction) of concentrations
indicated in abscissa. The 1O-pLmixtures were injected on the column. The elution conditions were the same as shown in Figure 1 . (b) Calibration of guanosine peak height vs. molar amount for H,PO, and TCA. The experimental conditions were the same as in Figure 2a. (c) Calibration of cGMP peak height vs. molar amount for H,PO, and TCA. The experimental conditions were the same as in Figure 2a. sensitivity considerably. The second is that the TCA extract presents a much lower pH than that advised by the HPLC column manufacturers. While this is ameliorated to some degree by dilution with the eluent buffer, and, while in practice we have seen no effects on the column performance in the limited number of runs we have done, for large numbers of samples significant column breakdown would be expected, particularly a t the head of the column. Because of this ob-
1906
Anal. Chem. 1982, 5 4 , 1906-1908
served problem and this potential problem with TCA, we have investigated phosphoric acid quenching of nucleotidase reactions for direct application to the column. Chromatograms of blank injections of reagent grade TCA and reagent grade phosphoric acid are shown in Figure la,b. A 10-pL sample of each solution was injected on the column at the time indicated by the arrow. It is clear that either the TCA itself or UV absorbing contaminants produce spurious peaks in the elution profile, which would interfere with sample data, as shown in Figure Id. Figure 2 shows peak height vs. molar amount using either TCA or phosphoric acid as sample diluent for three test compounds. It is seen that for cGMP the calibration works well with both TCA and phosphoric acid, but that for 5’GMP and guanosine the calibration with phosphoric acid works well, while that with TCA has an artifactual offset which undercuts low-level determinations. This shows that direct injection of TCA extracts might be suitable for some compounds but that phosphoric acid is more universally applicable. We have been able to carry out enzyme assays at levels of 15 pmol at approximately &5% accuracy of product using this method. The simplified preparation has not led to degradation of the column, as we have run over 3000 analyses on the same C-18 column with only slight degradation of resolution and adsorption power.
Since phosphoric acid is not as strong an acid as TCA, the question comes up as to whether it can adequately quench enzyme reactions. We have used 5’-nucleotidase from bovine photoreceptors to test this. 5’-Nucleotidase is an appropriate enzyme to use for such a purpose, since it is quite hardy and resistant to denaturing conditions. A 200-pL portion of 1.45 M H3P04 was added to 125 pL of a 5’-nucleotidase incubation mixture. Three 100-pL aliquots of this mixture were withdrawn. After different periods of time, 200 pL of 1 M TCA was added to the individual aliquot. The amounts of product were not significantly different when the enzyme was double-killed by 1 M TCA after it had first been stopped by 1.45 M for three different periods of time, Le., 2 min, 1 h 15 min, and 5 h. Extraction or reaction quenching with phosphoric acid, microfuge centrifugation, and direct injection is, therefore, a convenient and rapid method of sample preparation which also avoids many of the problems of trichloroacetic acid.
LITERATURE CITED (1) Brown, P. R.: Miech, R. P. Anal. Chem. 1972, 44, 1072-1073. (2) Chen, S.-C.: Brown, P. 13.:Rosie, D. M. J. Chromatogr. Sci. 1977, 15, 218-221.
RECEIVED for review March 29,1982. Accepted May 17,1982.
Acid Digestion Bomb for Biological Samples Roland Uhrberg The National Swedlsh Environment Protection Board, Water Quality Laboratory, Box 8043, 750 08 Uppsala, Sweden
The most common way of digesting biological and mineralogical samples is to heat them with a mineral acid like HN03, HzS04,HC104, and H F or a mixture of these acids. This can be done in two different ways: open or closed digestion, the closed system generally being preferred. The advantages of a closed system are (1)less risk of contamination, (2) no losses of volatile elements, (3) less reagents needed, and (4) much faster digestion. Many different bombs have been described in the literature (1-5). These bombs are generally based on the same principle, an outer casing of stainless steel, an inner part of P T F E (polytetrafluorethylene),and a sealing of PTFE against PTFE. At our laboratory we found that this kind of bomb construction with PTFE against PTFE seals caused problems. The PTFE material softens even at moderate temperature increases, often resulting in deformation of the seal and leakage of the sample. The present bomb was developed during work with fish digestions for mercury analyses. The considerable strength of glass tubes used in Carius digestions led to the present construction. The aim was to construct a bomb which is safe to use and which allows acceptable digestion without losses, as well as being easy to manufacture and handle.
EXPERIMENTAL SECTION Instrumentation and Metal Analysis. Atomic absorption measurements for P b and Cd were made on a Hitachi Model 170-70 Zeeman effect atomic absorption spectrophotometer (6) (Naka Works Hitachi Ltd., Japan) flameless unit with a double channel recorder. For Hg a modified Perkin-Elmer Mercury Analyzer System Coleman 50 unit and a Perkin-Elmer 56 recorder were used. The Tecator digestion 20/40 control unit was used for heating the bombs (Tecator Instrument AB, Helsingborg, Sweden). Hg Analysis. The cold vapor technique was used for mercury analysis. For a lower detection limit, the following modifications 0003-2700/82/0354-1908$01.25/0
were made to the original instrument. The Hg vapor was flushed out of the solution with a Nz-gas stream and concentrated on a piece of Ag-coated quartz wool inside a quartz tube diameter 8 mm). The quartz wool was heated with a Perkin-Elmer (heated graphite atomizer) HGA 70, the graphite tube being used as a heating mantle around the quartz tube, to drive off the enriched Hg. The gas flow was turned off during the heating step after which it was turned on again, resulting in a fast Hg-peak on the recorder. Air volumes were minimized and the original plastic cuvette was replaced by a quartz cuvette, enabling detection of 0.1 ng of Hg. The equation of the calibration curve is y = 3 0 . 3 ~ + 1.8, x = Hg (ng), y = part of the scale on the recorder paper, (range 50 mV on the recorder). Correlation coefficient, r = 0.9993. Other Metal Analyses. The graphite cuvettes were soaked in a 6% Ta solution (7) before use; (NH4),HP04was utilized as a matrix modifer (8). This procedure lowered the detection limits and increased the reproducibility;higher ashing temperatures can also be applied. To a 200-wL sample was added 400 wL of 0.1 M (NH4)zHP04;if necessary the sample was first diluted with distilled water. Calibration curves were prepared with slightly acidified solutions with the same concentration of (NH4),HP04as added to the samples. Bomb Construction. The bomb is based on the following principle: an outer casing of “acid-proof“stainless steel (17% Cr, 13% Ni, and 2.7% Mo), which can withstand high pressures, and an inner part of inert (against acid) material. Four different types of inner tubes were tested: (1) An inner tube of quartz with a lid of PTFE. Tubes of different wall thickness were tested, three with thin walls (1.7-1.8 mm) and three with thick walls (3.4, 4.7, and 7 mm). (2) Some metals we very resistant to nitric acid (the acid used as oxidant in these tests), e.g., Ti, Ta, Zr, Rh, Pt, and Ir. Inner tubes of the metals Ti, Ta, and Zr were tested. All gave high blanks for lead; but for mercury and cadmium the blanks were acceptable. (3) Carbides for some of these metals are very resistant to chemical attack. In an attempt to reduce the lead contamination, 0 1982 Amerlcan Chemical Soclety
