Anal. Chem. 1987, 59, 1064-1066
1064
(2) Ting, G. Ph.D. Dissertation, University of Tennessee, 1973. (3) Linga, H.; Stojek, 2.; Osteryoung, R. A. J. Am. Chem. SOC. 1081, 103. 3754. 1
Scheffler, J. B.; Hussey, C. L.; Seddon, K. R.; Kear, C. M.; Armitage, P. D. Inorg. Chem. 1083, 22,2099. Tremillon, B.; Bermond, A,; Mollna, R. J. Electroanal. Chem. Interfacial Nectrocbem . 1978. 74, 53. Berg, R. W.; Hjuler, H. A.; Bjerrum, N. J. Inorg. Cbem. 1984, 23,
557. Stojek, 2 . ; Linga, H.; Osteryoung, R. A. J. Nectroanal. Cbem. Interfacial Electrochem. 1081, 119, 365. Laher. T. M.; McCurry, L. E.; Mamantov, G. Anal. Chem. 1985, 57, 500. tivistendahi, J.; Klaeboe, P.; Rytter, E.; Oye, H. A. Inorg. Chem. 1084,
23. 706. Mamantov, C. B.; Laher, T. M.; Walton, R. P.; Mamantov. G., I n Light Metals 7985;Bohner, H. O., Ed.: The Metallurgical Society of AIME: 1985;pp 519-528. Rytter, E. Extended Abstract; Fall Meeting of the Electrochemical Society, Las Vegas, NV, 1985: No. 485. Marassi, R.; Chambers, J. Q.; Mamantov, G. J. Electroanal. Chem.
Interfacial Electrochem. 1076, 69, 345. (13) Levich, B. G. Theoreticalphvslcs ; North-Holland: Amsterdam, Holland, 1971;Vol. 2, pp 357-358. (14) Hvistendahl, J.; Rytter, E.; Oye, H. A. Appl. Spectrosc. 1083, 37, 182. (15)Harrison, T. R. Radiation Fyrometry and its Underlying Principles of Radiant Heat Transfer; Wiley: New York, 1960;pp 28-38. (16)Griffiths, P. R.; de Haseth, J. A. Fourier Transform Infrared Spectroscopy; Wiley: New York, 1986: p 202. (17) Berg, R. W.; Ostvoid, T. Acta Cbem. Scand., Ser. A , in press.
P a u l A. Flowers Gleb Mamantov* Department of Chemistry University of Tennessee Knoxville, Tennessee 37996-1600
RECEIVED for review July 7, 1986. Accepted December 15, 1986.
A I D S FOR ANALYTICAL CHEMISTS Precision and Accuracy of Mechanlcal-Action Micropipets Byron Kratochvil* and Norine Motkosky
Department
of
Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2
Mechanical-action micropipets for delivery of known volumes of solution in chemical analysis have become popular in recent years. They are rapid and convenient, and the disposable tips reduce contamination and eliminate timeconsuming cleaning. A variety of commercial designs are available with fixed or adjustable volumes, with air-displacement or positive-displacement action, and in sizes ranging from 1 pL to 10 mL. The commercial literature generally stresses the importance of using high-quality plastic tips for air-displacement models because tip quality is reported to significantly affect the precision and accuracy of the volume of liquid delivered. A precision and accuracy of the order of 1%is usually specified, depending on the design and on the volume to be dispensed. More important than tip quality is operator technique. The purpose of this communication is to draw attention to potential errors that may be introduced into analytical measurements by insufficient attention to the way that mechanical-action micropipets are used. The precision of earlier designs of micropipets was evaluated by Emanuel (1). EXPERIMENTAL SECTION Several types of pipets were studied. They included a 50-pL air-displacement,fied-volume unit (Socorex);several continuously adjustable air-displacement units including 100-10oO pL in I-pL increments (Eppendorf and Gilson Pipetman), 40-200 pL in 1-pL increments (Oxford),and 10-50 WLin l-wL increments (Oxford); two positive-displacement units consisting of a Teflon-tipped plunger in a disposable glass capillary, one adjustable in 5pL steps from 5-25 pL (Socorex) and another adjustable in 0.1-1L steps from 5-30 pL (SMI); and a continuously adjustable positivedisplacement unit delivering 1-10 mL in 0.1-mL increments (Eppendorf). For the air-displacement types plastic tips in three price ranges were evaluated. Operating instructions for the air-displacement pipets were provided as follows: Fit a clean tip snugly onto the end of the pipet. With the pipet held vertically, press the operating button to the f i t stop, and immerse the pipet tip 3-5 mm into the liquid to be pipetted. Allow the button to return slowly to ita original position, Remove the tip from the liquid by sliding it along the
wall of the vessel for 1cm or so. Check for droplets of liquid on the tip. If droplets are present on the outside, replace the tip with a new one. Deliver the contents immediately into the receiving vessel by touching the tip to the inside wall and slowly pressing the button to the first stop. Hold the button in this position for a few seconds to allow the inner wall of the tip to drain, and then press it slowly to the second stop to eject the last portion. Remove the pipet from the wall with the button still depressed to avoid drawing liquid back into the tip. If droplets of liquid remain on the inner surface of the tip, discard the sample and replace the tip. For the positive displacement tips the instructions were similar, except that the second stop for delivery is not present in the design. Precision and accuracy of the pipets were tested primarily by weighing water delivered, but some tests were conducted by titration of pipetted replicates of standard 2.5 or 6 M HCl with 0.05 M NaOH. Each operator used a separate tip for each set of measurements. The water was delivered into weighing bottles, 50-mL conical flasks, or 14.5 X 45 mm glass vials; the containers were stoppered immediately after delivery of each portion. Tests for weight loss through evaporation indicated that losses were less than 0.1 mg in the 5-10 s required to deliver the next sample and were negligible from well-stoppered containers. Thus errors from evaporation become significant only if small volumes, of the order of 10 pL or less, are being measured. Measurements were made by undergraduate students after careful instruction as well as by experienced graduate students and laboratory technicians. Masses were converted to volume by division by the density of water at the appropriate temperature. RESULTS AND DISCUSSION Calibration data on several 50-pL air-displacement pipets (same model) were collected over a period of 2 years from students using the pipets for standard additions in the determination of trace copper in nickel by atomic absorption. All the students had several months of previous experience with conventional volumetric pipets and other volumetric equipment and many had experience in other courses or jobs with mechanical-action micropipets. All were given written and oral instruction in use of the pipet before they performed the calibration operation. The results for one group of 74
0003-2700/87/0359-1064$01.50/0@ 1987 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 59, NO. 7, APRIL 1, 1987
Table I. Summary of Calibration Data for a 100-1000-pL Adjustable Air-Displacement Micropipet
volume, p L found from setting wt of water 100 150 250 500 7 50 900 1000
97.8 96.7" 148.0 2498' 489 504" 754" 892 894" 983
re1 std dev, ppt
no. of measmts
operators
16 5 8 6 6 1 2 3 2 5
86 12 6 12 85 12 12 16 11 72
7 2 1 2 8 2 2 2 2 6
no. of
"Values based on titration of pipetted aliquots of 2.5 M HCl
with 0.05 M NaOH. Table 11. Summary of Calibration Data for Two Positive-Displacement Adjustable Pipets volume, p L found from setting w t of water
re1 std dev, ppt
no. of
measmtsa
Pipet A, 5-25 25 15 5
24.3 14.4 4.5
a
pL
13 20 73
44 45 45
Pipet B, 5-30 30 15 18 5
29.4 14.6 17.3 4.7
no. of operators
12 13 11 55
3
3 3
pL
3
27 12 5 25
1 1 3
Measurements per capillary tip ranged from 5 to 25.
students were as follows: average, 49.2 pL; standard deviation, 0.8 pL. The results for a second group of 89 students were as follows: average, 48.8 pL; standard deviation, 1.5 pL. Additional data by selected students on other types of pipets are summarized in Tables 1-111. Table I shows delivered volumes for a 0.1-1 mL air-displacement pipet to be generally low, although the agreement among replicates for a given operator and tip is satisfactory in most instances. (Data for one operator, which were unusually low and could be rejected at the 95% confidence level, are not included in the table.) Where the precision was poor the delivered volume was often particularly low; this suggests that the tips were not draining reproducibly. The precision of calibration volumes based on titration of HCl delivered by the pipet was of the same order as that based on delivery of water. Table I1 compares results for positive displacement micropipets from different manufacturers, one adjustable in 5-pL increments from 5 to 25 pL and the other continuously adjustable from 5 to 30 pL. In each case the volume delivered tended to be low, and the operators often reported difficulty in removing the last droplet of solution from the tip during dispensing. This biased the results low by 0.3-0.7 pL over the entire range of the pipets.
Table I11 shows results for three differently priced tips provided by a single distributor. From the results it was concluded that no significant difference was discernible among the tips, even though small droplets could sometimes be seen adhering to the walls of the inexpensive tips. The more expensive tips provided somewhat improved precision for replicates with a single tip, but neither between-tip precision nor accuracy was significantly better. Table IV shows results for a 1-10-mL positive-displacement adjustable pipet. A t the 10-mL setting the pipet showed a precision of one part per thousand. A t the 1-mL setting, however, the precision was only 20 parts per thousand and the volume delivered was low. For reproducible results with this pipet at any setting, considerable care must be taken to remove the last drop from the tip when dispensing. Practice is particularly necessary before the precision shown in Table IV is obtained. Volumes delivered by all the pipets tested tended to be low. To test whether rinsing of the tip of a 50-pL air-displacement pipet after delivery might bring values into line, 50-pL portions of 6 M HC1 were pipeted into distilled water with and without rinsing and titrated with a standard solution of 0.05 M NaOH. Six titrations with an unrinsed tip gave a molarity for the HCl of 6.00 f 0.04, while six titrations with rinsing of the tip gave a value of 6.01 f 0.02. We conclude that rinsing of the tip affects the volume delivered by only a few parts per thousand or less and cannot account for the consistently low results observed. Some workers eject micropipet contents directly, without touching the receiving vessel. To evaluate this technique, three experienced operators who use this technique routinely were tested with three different adjustable air displacement pipets: a 100-1000-pL pipet used at 500 pL; a 40-200-pL pipet used at 150 pL; a 10-50-pL pipet used at 50 pL. The average volumes delivered, as determined by weighing water, were 504 f 0.5, 501 f 2,502 f 1; 146 f 1, 148 f 0.4, 148 f 0.5; and 48.5 f 0.9, 47.6 f 0.1, 47.5 f 0.3. (Each value is the average of six determinations with one tip by one operator.) In the hands of experienced users direct injection appears to be at least as precise as touching the tip to the receiving vessel. Volumes delivered still tend to be low, however. A procedure has been described for comparing the precision of different batches of pipet tips by pipetting 5 mL of 0.05 M NaOH and 0.1 mL of 0.1% aqueous Phenol Red into cuvettes and measuring the relative standard deviation of sets of 30 tips (2). The method measures the overall standard deviation of three operations, two pipettings and an absorbance reading, and so does not allow assessment of the standard deviation of the micropipetting operation alone. Thus comparison of relative standard deviations of two types of micropipet tips is only possible by assuming that the relative standard deviations of the other operations remain the same. Also, the absolute deviation of the average from the nominal value cannot be assessed. Therefore a procedure in which the weight of water delivered is measured seems more direct and useful. A major point arising from this study is that the precision of replicate measurements for a variety of micropipet designs is of the order of 10 parts per thousand or less, but accuracy varies from tip to tip and depends to a major degree on the
Table 111. Summary of Calibration Data for Three Tip Qualities for a 50-pL Fixed-Volume Air-Displacement Micropipet
a
type of tip
vol found, p L
re1 std dev, ppt
no. of measmts
no. of operators
no. of tipso
most expensive intermediate cost least expensive
48.8 49.2 49.5
16 11 8
90 28 41
4 3 4
16 5
Five to six measurements per tip.
1065
8
Anal. Chem. 1987, 5 9 , 1066-1069
1U66
found from wt of watern
re1 std dev, ppt
each tip used is calibrated by the operator. Manufacturer's claims for precision can generally be met by individual users, but volumes delivered tend to be below nominal values. We conclude that care should be taken in precise work to ensure that the operator employ consistent, careful technique when using mechanical action micropipets.
0.95 (tip 1) 0.97 (tip 2) 4.99 (tip 1) 5.01 (tip 2)
21 10 4 2 1
The assistance of students and staff at the University of Alberta in the collection of the data reported here is gratefully acknowledged.
Table IV. Summary of Calibration Data for a Positive-Displacement Adjustable 1-10-mL Pipet (Single Operator) volume, mL setting
1 5 10 a
10.02 (tip 1) 10.03 (tip 2)
1
ACKNOWLEDGMENT
LITERATURE CITED
Each value is the average of six measurements.
technique of the operator. We recommend that tips be calibrated by weighing the water delivered in all situations where an accuracy of a part per hundred or better is necessary. In summary, analytical data obtained by using mechanical-action micropipets may possess considerable bias unless
(1) Emanuel, C. F. Anal. Chem. 1973, 45, 1568. (2) Bo-Rad Cafalog 1886; Bio-Rad Laboratories: Richmond, CA, 1984; pp 26-27.
RECEIVED for review May 21, 1986. Accepted November 24, 1986.
Enhancement of Osmium Detection in Inductively Coupled Plasma Atomic Emission Spectrometry J. M. Bazan Lawrence Livermore National Laboratory, Livermore, California 94550 Use of 18'Re as a cosmochronometer has been investigated primarily by Luck et al. (1-5). As part of a project to develop 187Re-1870s chronometry (6,7), it became necessary to develop a method for the determination of nanogram quantities of osmium. The instability of osmium in various chemical environments limited the type of separation and purification chemistry that could be used. Previously reported osmium detection limits obtained in inductively coupled plasma atomic emission spectrometry (ICP-AES) vary from 38 ng/mL to 1 pg/mL (8-10). This variation was generally attributed to the unexplained chemical behavior of osmium. Summerhays et al. (11)observed, when using nebulization, that the signal intensity of osmium inThey attributed this rise creased with time in 9 M "OB. in intensity to the formation in solution of the volatile species Os04. The signal intensity did not reach a stable value over the period of investigation. We decided to take advantage of the volatility of Os04 and to introduce it directly into the plasma torch. A stable, enhanced signal for Os04was achieved after identifying (1)a stable matrix solution, (2) an efficient oxidant, (3) the ideal temperature for the reaction, and (4) the optimum instrumental operating parameters. Two distinct apparatus were designed to produce Os04 for on-line chemistry. Each setup was used for a specific purpose. A continuous-flow generator (12,13)(Figure 1)was used for samples contained in relatively unlimited volumes, i.e., 110 mL, for osmium concentrations 11 pg/mL, and for determination of the optimum instrumental operating parameters. A discrete batch sparging apparatus (14-16) (Figure 2) was used for samples of < l o mL volume and for osmium concentrations 51 pg/mL. Another desirable feature of the batch system was the total recovery of the sample solution, which could then be used for further elemental analysis. Since both apparatus generated a dry-torch environment, and required identical argon gas flows, the optimum operating conditions determined by continuous flow were directly applied to the discrete batch sparging. This on-line chemistry
technique had several advantages: (1)increased sensitivity for the detection of osmium, (2) removal of potential interferences caused by nonvolatile elements, and (3) simplified sample preparation. The increase in signal intensity obtained when using the continuous-flow apparatus was a factor of 10 over conventional nebulization. In the discrete batch sparging technique the increase in intensity ranges from 10 to lo3 over nebulization and all the osmium appeared to be removed from the sample.
EXPERIMENTAL SECTION Reagents. A standard solution of OsC1, in 10% HCl(2.7 N) at 1000 pg of Os/mL was obtained from Spex Industries, Inc., Metuchen, NJ. Water for solution preparation was double deionized, first, in large batch mode and, second, with a Millipore deionization system. Periodic acid (H5106)was purchased from Eastman Kodak Co., Rochester, NY. All other reagents were analytical grade and were used as received. Instrumentation. A Jarrell-Ash Model 975 Atomcomp direct reading spectrometer equipped with a 2400-grooves/mm concave grating was used. The rf generator has a frequency of 27.12 MHz and incident power set at 1.15 kW with the reflected power less than 25 W. The osmium wavelength used was 225.585 nm. The two OSO,generators (Figures 1and 2) were used as the sample sources for the ICP torch. The demountable torch had a thickwalled capillary-tip sample channel with a tip inside diameter of 1.2 mm. The argon flow rates for the dry plasma torch maintenance were coolant gas at 16.2 L/min, plasma or auxiliary gas at 0.5 L/min, and sample gas at 0.72 L/min. Procedure. The continuous-flow apparatus (figure 1)required that the reaction chamber (6) be maintained at an elevated temperature during the flow of the reactants. This was monitored by a thermocouple (7). The multichannel peristaltic pump (4) delivered 1 mL/min of the oxidant solution (3) and 1 mL/min of the sample solution (2). These were pumped continously through the mixing valve (5),through the heated reaction chamber at 135-140 "C, and into the separation chamber. The flexible connecting tubing from the peristaltic pump to the mixing valve and through the heated reaction chamber was made of Teflon, since it is nonreactive with the OsO, produced in this technique.
0003-2700/87/0359-1066$01.50/00 1987 American Chemical Society
