Table I. Recovery of Lead Added to Fish Samples
a
Pb in muscle tissue Pb addeda Pb predicted“ 1.81 i: 0.14 0.00 i 0.00 1.81 i: 0.14 0.43 zk 0.01 2 . 2 4 ’ 10.14 1.81 f 0.14 0.90 i 0.01 2.71 f 0.14 1.81 f 0.14 1.60 i 0.01 3.41 f 0.14 0.77 f 0.07 0.00 rt 0.00 0.77 f 0.07 3.33 i 0.01 4.10’2 0.07 0.77 f 0.07 1.36 i 0.01 2.13 i- 0.07 0.12 f 0.10 0.00 k 0.00 0.12 =t 0.10 2.27 i 0.01 2.39’10.10 0.12 f 0.10 4.88 rt 0.01 5.00 =t0.10 8.84 =k 0.01 8.96 + 0.01 0.12 f 0.10 Lead in pg/g dry weight, deviations at 95% confidence level,
at the 95% confidence limit is about f 6 Z . The relative standard deviation for the lead in muscle tissue is 5 %. Over this range of lead concentration, it is concluded that the accuracy of the method exceeds the obtainable precision. These results are consistent with recovery experiments using 212Pb with the perchloric-nitric wet ashing technique of cocoa (11). The advantages of this method include superior sensitivity, accuracy, and reproducibility. There also is no need for large samples, tedious extraction, or preconcentration steps. There seems to be little matrix interference. Many other (11) T. T. Gorsuch, Analyst (London),84, 135 (1959).
Pb founda 1.81 f 0.14 2.28 f 0.08 2.72 i: 0.13 3 . 3 3 =t 0.05 0.77 f 0.07 3.99 f 0.01 2.10 =k 0.16 0.12 f 0.10 2.38 f 0.22 5.00 f 0.16 8.52 f 0.18
Pb found/Pb predicted 1.017’20.072 1.004 i 0.071 0.977 f 0.043 0.973’: 0.017 0.986 f 0.082 0.995’20.101 1.OOO i: 0.038 0.952 f 0.023
procedures (10, 12) do exhibit matrix interference. This is probably due to the fact that drying, ashing, and atomization is attempted in one programmed step. In this procedure, we have separated the dissolution, drying, and atomization steps and it is believed that this results in a minimization of any matrix interference. Since acid digestion is used, this method is readily amenable to the analysis of any type of tissue or bone sample. RECEIVED for review May 5,1972. Accepted July 14,1972. (12) J. Y . Hwang, S. B. Smith, Jr., C. J. Mokeler, and P. A. Ullucci, Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Cleveland, Ohio, March 1972,paper number 37.
AIDS FOR ANALYTICAL CHEMISTS Injection Port and Pressure-Relief Device for High-speed Liquid Chromatography R. M. Cassidy and R. W. Frei Trace Analysis Research Centre, Chemistry Department, Dalhousie University, Halijax, N . S . , Canada
A simple, efficient, and trouble-free injection port is shown in Figure la. Samples are injected by interrupting the liquid flow into D by means of a ball valve and opening valve A . A long-needle (10 cm) Unimetric syringe (No. 1010-T, Unimetrics Universal Corp., Anaheim, Calif. 92801) is passed through A and the sample is introduced into a glass wool p!ug
on top of the column. The syringe is then removed, A is closed, and solvent is allowed to flow into D. This procedure can be accomplished in less than 4 seconds. The characteristics of this injection port were compared to those of a continuous flow injection system. Both injection ports were connected to a 25-mm X 2.38-mm column packed with uncoated Zipax (E. I. du Pont de Nemours & Co., Wilmington, Del. 19898). The variance u 2 (band width = 4 o) of the peaks for unretained samples was determined with both a UV detector (Pharmacia Fine Chemicals Inc., Piscataway, N.J. 08854) and a fluorescence detector (2). The solvents and samples chosen were 1 (v/v) acetonitrile in nhexane and benzene for the UV detector and 20% (v/v) acetone in n-hexane and a fluorogenically labelled pesticide N,Ndimethylnaphthylamino - (3,4,5- trimethylphenyl) - 5 -sulfonate (3) for the fluorescence detector. Two flow rates were used in this study: 1.07 ml min-l and 0.57 ml min-l. The variance
(1) “Modern Practice of Liquid Chromatography,” J. J. Kirkland, Ed., Wiley-Interscience, New York, N.Y., 1971.
(2) R. M. Cassidy and R. W. Frei, J . Chromatogr., in press. (3) R. W. Frei and J. F. Lawrence, ibid.,in press.
THEVOLUME of scientific literature concerned with high-speed liquid chromatography (HSLC) has increased rapidly during the past 2-3 years (1) and the importance of HSLC for analytical purposes is now widely recognized. Due to the high pressures (up to 5000 psi) commonly used for HSLC, the cost of instrumentation is often high and a definite need exists for simpler and less expensive modifications of present instrumentation. In this paper, simple and inexpensive devices for sample introduction and pressure relief are described. SAMPLE INJECTION PORT
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was found to depend on sample volume (quantities of 1-10 p1 were used), but no differences could be found between the two injection modes with either detector. Interruption of the solvent flow for as long as 15 seconds caused no noticeable band broadening. The ball valve of the injection system (Figure la) has a pressure rating of 3000 psi but can be used to 5000 psi with additional packing adjustment. A number of advantages have been observed for this injection system in comparison to those previously reported (4, 5). The procedure for stopped-flow injection without a septum (5) is cumbersome and time-consuming, especially at high pressures. There is also a danger of introducing air into the chromatographic system. Since the liquid level in the ball valve A (Figure la) is maintained above the ball, no air can enter the system. Injection ports designed for high-pressure sampling through a septum ( 4 ) require high-pressure syringes and are plagued by problems of septum break-down except for the more expensive perfluoroelastomer septa. A sample loop can be designed for efficient injection of either large or small sample volumes but not both. These loops (Hamilton Co., Whittier, Calif. 90608) can be used up to 5000 psi, but are rather expensive. If desired, it is a simple matter to convert this injection port to continuous flow injection. Figure l b shows the modifications required for the top of the ball valve. A pressure-tight fit is obtained with a standard Swagelok nut. PRESSURE-RELIEF DEVICE
Pressure-relief valves are an important component of HSLC apparatus, especially for pumps which operate at constant flow rather than constant pressure. A simple and low-cost pressure-relief device, which has been found to operate satisfactorily for long periods, is similar to that in Figure 16, where J is part of a Swagelok tee in the solvent line and G is absent. A pressure-tight fit can be obtained with a standard Swagelok nut. As the pressure of the system is increased, H is forced through the hole in Z, until, at a critical pressure, H ruptures and the system is returned to atmospheric pressure. If plastic tubing is fitted over the Swagelok nut, the expelled liquid can be conveniently collected in an appropriate flask. The hole size and corresponding rupture pressure for a No. 0405 Supelco Septum (Supelco, Inc., Supelco Park, Bellefonte, Pa. 16823) are as follows: 1/4-in.,900 psi; 3/16-in.,1100 psi; l/$n., 1800 psi; 1/i6-in,,3000 psi. The reproducibility of the rupture pressure is =!=lo%for aqueous solutions. With the 1/16-in.hole, there is a lag in rupture of the septum if the pressure is suddenly increased. This problem can be solved by using larger holes for I and a thinner, stronger material for H. No attempts have been made to study other materials for H , so the upper pressure range of this device is not known. With nonaqueous solvents, the septum swells and becomes brittle but rupture occurs at lower pressures and the safety factor is not lost. During solvent change over, the unswept volume of this fitting can be purged easily by loosening the cap. (4) R. A. Henry, in “Modern Practice of Liquid Chromatography,” J. J. Kirkland, Ed., Wiley-Interscience, New York, N.Y., 1971, Chap. 2. ( 5 ) C. G. Scott and P. Bommer, J. Chromatogr. Sci., 8,446 (1970).
A
H
G J
Figure 1. (a) Sample injection port, (6) Adaptor for continuous flow injection ( A ) Whitey ball valve No. 4354-316 (Whitey Research Tool Co., Emeryville, Calif. 94608) ( B ) Stainless steel tubing (l/An. 0.d. X 3/32-in. i.d.) bevelled at top to allow smooth entry of syringe. The lower portion is machined to allow it to fit through the body of the Swagelok tee (C) Small hole in side of B to allow access of eluting solvent (D) Entrance for eluting solvent ( E ) Top of column with small glass wool plug (F) Swagelok 400-3-4-316 union tee (G) Short section of tubing as B ( H ) Septum (I) Stainless steel metal disk (1/8-in. thick) with small hole in center ( J ) Top of Whitey ball valve CUNCLUSIUNS
The two devices discussed above are reliable and efficient and can be adopted to advantage over similar systems. In addition, they are rugged and inexpensive. The materials for the injection port costs ~ $ 4 7which , is comparable to or less than commercially available models. For the pressure-relief device, the cost of the septum is negligible and a Swagelok tee costs ~ $ 1 0which , is well below the cost of any commercially available pressure-relief valve. ACKNOWLEDGMENT
The authors wish to thank R. A. Henry for helpful suggestions during the preparation of the manuscript.
RECEIVED for review March 23, 1972. Accepted June 5, 1972. This work is supported by grants of the National Cancer Institute of Canada and the Department of National Health and Welfare (Grant No. 602-7-141).
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