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 Three 100-pL aliquots of this mixture were withdrawn.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
ANALYTICAL CHEMISTRY, VOL. 54, NO. 11, SEPTEMBER 1982
1907
Table I. Loading Capacity for Different Quartz Tubes Tested during Digestion of Dried Fish with Nitric Acid (p.a.)" tube data wall thickness, amt of moment, kPm remarks o.d., mm mm vol, cm3 sample, g HNO,,mL 1.5 2 burst 15 1.5 11.3 0.23 1.0 2 burst 0.220 0.18b 1.0 2 0,15b 1.0 2 19.5 1.75 21.0 0.20c 2.0 2 0.26c 2.5 2 0.25c 3.0 2 0.25c 3.0 2.7 0.2f1~ 3.5 3.0 burst 27.5 1.75 43.0 0.20c 2.0 2.0 0.27c 2.0 2.0 0.26c 3.0 2.0 burst 4.0 3.5 22.0 O.2gc 23.5 3.4 4.0 4.0 20.0 O.3gc 26.0 4.7 a
Temperature = 150 'C, time = 1h.
Muscle.
Liver. I
-2
70-1
Flgure 2. Titanium cylinder wlth inner tube of PTFE: (1) Ti-cylinder, (2) PTFE-tube, (3) PTFE-lid, (4) aluminum plate (the Ti-PTFE tube requires a flat bottom to rest on).
1 -~
4 07 crn
-,
Flgure 1. Digestion b o m b (1) outer casing, (2) quartz tube, (3) PTFE lid, (4) screw lid, (5) PTFE disk, (6) PTFE ring for centering the quartz tube.
the Ti tube was coated with a layer of TiC (the tube was filled with graphite powder and heated in an oven). But the high blanks for lead still remained. However, better results might be achieved with a more sophisticated method of carbide production. ( 4 ) After the failure with the metal tubes, a PTFE tube was put inside a thin Ti cylinder, which eliminated the contamination. The construction of the bomb is shown in Figure 1. The parts are an outer casing and screw lid of acid-proof stainless steel, quartz tube, inner lid of F'TFE, and a thin disk of PTFE between the quartz tube and the bottom of the steel cylinder. Figure 2 shows the inner part consiisting of a Ti cylinder with an inner tube and lid of PTFE. The inner part of the steel lid is coated with PTFE to reduce the rislk of contamination and to reduce the friction in the thread. The metal surface is first carefully cleaned by sand blasting. The PTFE is dissolved in a solvent with a binder and accelerators to form a slurry and then sprayed on the metal and heated in an oven at 250 "C. RESULT8 AND DISCUSSION Several series of testa were undertaken to determine both the maximum amount of sample and reagent for different
tubes and the appropriate torque for tightening the screw lid; see Table I. The loading tests enabled an optimal wall thickness of the quartz tubes to be estimated: walls that are too thin would increase the hazards for inner tube failure and would restrict the sample weights; walls that are too thick could create sealing problems between the quartz and the P T F E and would also increase costs. An optimal wall thickness is proposed to be in the range of 2.5-3.5 mm. When the screw lid is turned, it is preferable to use a dynamometric wrench; if turned too hard the tube will break, if too loose the tube will leak. Maximum torque for the thin-walled tubes was determined to approximately 3.0 kpm (kilopond meter). For the thick-walled tubes, 3.5 and 4.0 kpm, respectively, torque was used; no attempt was made to break them by turning the screw lid harder. No calculations were made to determine the force of the PTFE lid, because of the difficulty in estimating the coefficient of friction in the thread. The crucial factor for tubes with very thick walls (>4 mm) is not wall strength, but rather the fact that very high pressure combined the large amounts of dissolved gases may cause sudden loss of fluid when the lid is opened. Thus the upper limit (tube explosion) has not been determined for the thick-walled tubes. The maximum load for the Ti-PTFE
1908
Anal. Chem. 1982, 5 4 , 1908-1909
Table 11. Contamination Test for Hg, Cd,and Pbu Hg, found ng
x,
tube
n
Ti-PTFE quartz 1 mL of HNO,, blank runs
7
0.80
0.17
7
8
0.84
0.22
6
3
0.91
0.02
3
s
I1
.,
Pb, found ng
Cd, found 3c, ng
S
n
0.87
0.57
7
0.16
0.20
1.00
0.65
6
0.11
0.98
0.12
3
0.19 0.08
S
0.00
a 1 mL of HNO, (p.a.) was heated at 150 "C for 1 h. n = number of independent measurements, X=mean value, s = standard deviation.
Table 111. Recovery Test for Hg" tube Ti-PTFE quartz
amt of Hg found, ng 103.0 100.0
98.6 102.8 100.0
99.0 mean
100.6, s = 1.9
To 1mL of HNO, containing 0.9 ng of Hg (cf. Table 11) was added 100 ng of Hg as HgCI, and the mixture heated at 150 "C for 1h. a
tubes has not been determined for the same reason by probably they are even stronger than the thick-walled quartz tubes. By use of these fragile inner parts and a much stronger shield of stainless steel the safety of the bomb increases-if the inner part bursts the pressure will slip out through the thread. The limiting pressure, causing the inner tube to burst, has not been determined. Nevertheless, an attempt was made with a tube (outer diameter 19.5 mm, wall 1.75 mm) which was half filled with water. The temperature was gradually increased to a maximum temperature of 280 "C (implying a pressure of 66 kp/cm2), but this was not enough to cause it to burst. The important thing is to know the amount of sample and acid that can be used a t a given temperature. Recommended cooling procedure is as follows: Place the bomb in water to half its height. This will cool the sample solution without causing large temperature changes in the PTFE lid which might lead to gas leakage. The bomb can then be opened after about 10 min. The contamination was measured in a series of analyses (blank runs) for Hg, Cd, and Pb, see Table 11. The losses was determined by recovery tests for Hg; see Table 111. The PTFE material will have a rather high Hg contamination (5-10 ng per run and tube) unless it is pretreated before the tubes are manufactured. The material was therefore prepared by heating at 250-270 "C for several days. As a further test of this bomb technique the NBS standard 1577 bovine liver was analyzed for P b and Cd, giving 335 pg/kg
of P b (range 60), 280 pg/kg of Cd (range 40), and 16.4 pg/kg of Hg (range 3.8),as mean of three analyses. The certified values are 340 f 80,270 f 40, and 16 f 2, respectively. The precision of the technique a t low concentrations is shown by analysis of sample of freeze-dried cod which gave 65 pg/kg of P b (n = 8, s = 15) and 6.2 pg/kg of Cd ( n = 10, s = 1.2). CONCLUSIONS There are three advantages of this bomb construction compared to others described in the literature. First, and most important, the seal is not achieved by pressing PTFE against PTFE, but the rim of a quartz tube against a PTFE lid, which makes it possible to apply a higher pressure on the seal without causing deformation. Further, the contamination is low because of the space between the seal and the outer steel tube, and, finally, it is easy to evaporate surplus acid by placing the quartz tube on a hot plate. The Ti-PTFE inner tube should only be regarded as a substitute for the quartz tube (e.g., if H F is utilized); in the long run the sealing of the quartz tube is superior to that of the Ti-PTFE tube. The results of the bomb tests show that this new bomb technique is a good method of achieving fast and safe digestion, low blanks, good precision and accuracy (on Bovine Liver 1577 standard reference material). ACKNOWLEDGMENT The author thanks Folke Nydahl at the Institute of Analytical Chemistry, Uppsala University, for reviewing the manuscript. LITERATURE CITED Kotz. L.: Kaiser, 0.:TschoDel, P.: Tola. G. 2.Anal. Chem. 1972, 260. 207-209. Bernas, 8. Anal. Chem. 1968, 4 0 , 1682-1986. Pausch, P. E. At. Absorpt. Newsl. 1971, 10 (l), 44. Lida, C.; Uchida, T.; Kojima, I. Anal. Chlm. Acta 1980, 173, 365-368. Parr Acld Digestion Bombs, Parr Bulletin 4745. Kozumi, H.; Yasuda, K.; Katayama, M. Anal. Chem. 1977, 4 9 , 1106-1 112. Zatka, V. I. Anal. Chem. 1978, 50, 538-541. Hodges, D. J. Analyst (London) 1977, 702, 66-69.
RECEIVED for review March 1, 1982. Accepted April 29, 1982.
Temperature Monitor with Dual-Channel Platinum Resistance Elements Michael A. Balm, Fred J. Schuetze, John M. Frame, and Herbert
H. Hill, Jr."
Department of Chemistry, Washington State University, Pullman, Washington 99 164
Often seemingly outdated laboratory equipment may be easily modified to serve a new purpose at little expense. Such is the case with a laboratory oven we have refurbished by the addition of a digital temperature monitor to house our ion
mobility spectrometer (I). Unlike the majority of gas chromatography detectors which may be conveniently heated by cartridge heaters, the ion mobility spectrometer requires a heated and insulated enclosure due to high voltages present
0003-2700/82/0354-1908$01.25/00 1982 American Chemical Society
