of samples was heated on a steam bath. Their composition and determinations, shown in Table I, indicate that the morpholine reaction is complete in 15 minutes a t an elevated temperature. I t was noted that condensers are required to prevent loss of the morpholine during heating. The reaction temperature on the steam bath is limited by the boiling point of the methanolic reagent (61 "C). Therefore, an attempt was made to further increase reaction rate by substituting butanol for methanol in each solution. This increased the solution boiling point to 78 "C. A 2:' factorial experimental design was made with the variabies stearic anhydride, IPS, and stearic acid concentration. Titrations were made colorimetrically. Details of the design are shown in Table 11. The results show a highly significant ( p 50.01) interference due to IPS, significant ( p 50.05) interference due to stearic acid, and significant interference attributable to the IPS-stearic acid interaction. As this approach was unpromising, use of methanol was re-introduced and the above design was repeated. Actual samples from the process are colored, and prevent colorimetric titrations. Therefore, this design was analyzed by potentiometric titration. In order to facilitate the titration, ca. 100 cm3 of methanol is added after heating. The end point was taken as the inflection point on the curve. The details and results of the design are listed in Table 111. The results indicate no significant interference. T o establish confidence limits for the determination, 23 data points were statistically determined. These data are
listed in Table IV. They include 3 samples containing only stearic anhydride, 4 samples with IPS and no stearic anhydride, 8 samples with both IPS and stearic anhydride, and 8 samples containing no IPS or stearic anhydride (blanks). The variances were pooled after it was determined that the variances of the 4 sets were not significantly different by pairs ( F test, p 50.05). The confidence limits were determined as f 0 . 0 4 4 gram. For a nominal 6-gram sample of stearic anhydride in IPS this f 0 . 0 4 4 gram stearic anhydride is equivalent to f0.73%. The mean of the pooled error data was not significantly different from 0.0 (by t test).
CONCLUSIONS The analytical method of Johnson and Funk, with the modifications described, can be used to measure stearic anhydride in isopropenyl stearate. The analysis, as modified, is not affected by IPS or stearic acid in the concentration range of 0-10% stearic anhydride. Analytical confidence limits of f0.73% were found, which is considered acceptable precision.
ACKNOWLEDGMENT The authors thank Leonard S. Silbert for his advice, and Howard I. Sinnamon and Nicholas C. Aceto for their guidance.
RECEIVED for review May 31, 1974. Accepted July 31, 1974.
AIDS FOR ANALYTICAL CHEMISTS Simplified Remote Pipetting System E. M. Fortsch' and M. A. Wade Allied Chemical Corporation, Idaho Chemical Programs-Operations
The analysis of highly radioactive samples, as in the processing of irradiated nuclear fuels, requires that accurate remote pipettings be made. Several types of remote pipets have been designed for use in shielded facilities ( I , 2). Usually, the design is influenced by limitations imposed by the facility in which the pipets are used and, as a result, a wide variety of pipetting devices are being used throughout the industry with a high cost of custom design and fabrication. Thus, there would be many advantages for a low cost, easily operated, relatively accurate, commercially available remote pipet. In recent years, hand operated semi-automatic pipetting devices such as the Eppendorf, Oxford, and Sherwood pipets have become commercially available. These cover the range of 5- to 1000-microliter volumes and have many features which make them attractive for remote use. These features are: low cost. no direct contact of sample with pipet mechanism, reasonable accuracy and precision, non-
'
Present address. Fluor Engineers & Constructors, 5559 Ferguson Drive. Lo' Angeles. Calif, 90022. (1) D. C. Stewart and H. A. Elion, "Progress in Nuclear Energy," Series IX, Vol 10, Pergamon Press. New York, N.Y., 1970, p 59. ( 2 ) F W. Dykes. >. P. Morgan, and W. G.Pieder, "The Remote Analytical Facility Model E Pipetter ' At. Energy Comm. Rep.. 100-14456, 1958.
Office, 550 Second Street, Idaho Falls, Idaho 8340 1
wettable disposable tips, simple construction, and ease of operation. In the normal operation of these pipets, two spring tensions are used to control the distance a positive displacement plunger travels. One spring is used to draw a measured volume of sample into the disposable pipet tip and for expelling the bulk of the sample. The second spring is used as a "blow-out" to completely expel any liquid which remains in the tip following the depression of the first spring. Orsello, Pozzi, and Tedini ( 3 ) have adapted the Eppendorf pipet to remote use by a mechanical device which uses a lever-screw system to operate the pipet. The lever which drives the pipet button is operated by manipulators from outside the shielded cell. This paper describes an air-controlled device for operating the Eppendorf pipet remotely. The Eppendorf pipet was chosen because its critical parts (plunger and cylinder) are made of plastic and glass and will resist the corrosive atmosphere of a hot cell. The system uses controlled air pressure acting on a small air cylinder to perform the pipetting functions. The system is small, inexpensive, and requires very few remote operations for either its use or maintenance. (3) S.Orsello, F. Pozzi, and M. Tedini, "Una Nuova Telepipetta Rernotizzata Per Microprelievi: Di Soluzioni: Altarnente Radioactive," Comitato Nazionale Energla Nucleare, preliminary unpublished report, 1972.
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Figure I. tppenoon piper insrailed
Flgure 2. Control unit for remotely operated Eppendorfpipet
A, Eppendorl pipet: B. Locking pin for pipet air cylinder: C, Pipet air cylinder: D,Pipet holder: and E, Rotating sample holder
A, Pipet control unn; 8. Five-way selector vaive: C. Tip removal control valve
EXPERIMENTAL Apparatus. The apparatus is illustrated in Figures 1 and 2. Both of these figures are photographs of the actual installation in the Remote Anal$titical Facility of the Idaho Chemical Processing Plant. Figure 1 shows the Eppendorf pipet mounted inside the shielded cell and Figure 2 shows the control unit mounted on the outside of the cell. Two air lines lead to the pipet. The air line from the control unit leads to a Mead single acting air cylinder (C, Figure 1) that operates the pipet. The other air line leads from a 3way disk valve (C, Figure 2) to a Bimha single acting cylinder that is used to remove the pipet tips, The second cylinder is mounted behind the pipet and is thus hidden from view in Figure 1. The “heart” of this pipetting system is the control unit and it is illustrated schematically in Figure 3. It contains the valves and air restrictors necessary for the operation of the pipet. The differential air pressure required to operate the two spring tensions on the Eppendorf pipet is about 7 psi. Two pressure regulating valves (V-1 and V-2, Figure 3) are used to obtain these two air pressures. The low pressure valve (V-I) depresses the spring that controls the volume of liquid drawn into the pipet and the high pressure valve (V-2) depresses the spring that controls the tip “blow-out.” It is mandatory that the rate at which the pipet is operated (the rate the plunger is depressed and released) be carefully controlled. This control is ohtained by using adjustable Foxhoro air restrictors (FE, Figure 3). It is necessary to balance the pressure of the line and the air cylinders used. In this installation, the low and high pressure valves are set at 20 and 30 psi, respectively; the line pressure is 60 psi. A five-way valve (V-3, Figure 3) controls all the pipetting functions. The pipet holder (D, Figure 1)will accommodate different sized pipets. The equipment is designed so that the air cylinder can be swung out of the way, thus permitting easy changing of pipets. The locking pin (B, Figure 1) is removed permitting the cylinder to swing away. The pipet holder will accommodate pipets greater than 100 pl. An adapter is required to use 100-pl and smaller pipets. Operation. All operating functions of the pipet (with the exception of the tip removal) are controlled by the five-way selector valve on the control unit (B, Figure 2). The pipet tip is removed by operating valve C, Figure 2. Position 1 of the five-way valve provides a slow return of the pipet button. Position 2 applies air at low pressure and slow speed to drive the pipet button to its first discharge position. Position 3 applies air at an increased pressure which depresses the pipet button to the “blow-aut” position, Pasition 4 releases the pipet button providing a rapid return to the starting position. Stepwise pipetting operations are as follows: 1) Turn the selector valve to position 2. Immerse the pipet tip into the solution to be pipetted. 2066
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Figure 3. Valve and tubing schematic of pipet control unit V-I. Air regulator valve. low pressure (20 psi): V-2, Air regulator valve. high pressure (30 psi); V-3. Whtney 5-way Selector valva: V-4, Bimba 3-wey disk air valve: PI, Air pressure gauge: FE. Faxboro adjustable resfrictor: AC-1, Mead model H-I single acting air cylinder; AC-2, Bimba model 011-Psingle acting air cylinder
2) Return the selector valve to position 1. The plunger will slowlyrise drawing thesolution into the pipet tip. 3) Place a receiving container under the pipet tip and then turn the selector valve to position 2. The pipet button will slowly extend to its first position, discharging sample into the receiving container. 4) Turn the selector valve to position 3. Any remaining solutioh will be expelled as the pipet button is extended to its “blow-out” position. 5) Open the tip removal valve to eject the disposable tip. 6) Install a new tip with the manipulators. I t is necessary to have the selector valve in position 3 (the air cylinder fully extended) while the tip is installed, otherwise the pipet will be forced out of its holder. I ) Turn the selector valve to position 4 then to position 1. The pipet will return to the startingposition.
RESULTS AND DISCUSSION The remote pipetting system was evaluated by calibrating three different sized pipets by hand and by the remote
NOVEMBER 1974
system. The pipets were calibrated by pipetting distilled water into stoppered test tubes and weighing. The results of these calibrations are given in Table I. These data show excellent agreement between the two methods. The precision of both the hand and remote systems is about 0.1% relative standard deviation for the 200- and 500-11 pipets. In the case of the 100-p1 pipets, the precision is 0.2% and 0.6% relative standard deviation for hand and remote pipetting, respectively. The data in Table I also show t h a t the stated value of the pipet may be slightly different than the actual value. These three pipets were within 1% of the stated values; an accuracy good enough for most work. For best accuracy, however, each pipet shoud be calibrated before use. The Model B remote pipets (2) which have been used a t the Idaho Chemical Processing Plant for many years have proved their value for routine measurements. They have been reasonably maintenance-free and have proved t o be quite reliable. However, solutions such as aqua regia, hydrolluoric acid, and any other material which corrodes stainless steel could not be pipetted. In addition, organic solutions could not be pipetted. In some applications, cross-contamination could not be avoided. The remote Eppendorf system does not suffer from these disadvantages and has expanded pipetting capabilities. In addition, it is cheaper to fabricate, install, and maintain. A comparison of the data obtained by the two methods of remote pipetting is presented in Table 11. These data were obtained over a long period of time by a large number of different operators and they show the remote Eppendorf system to be both more accurate and more precise than the Model B pipettor. At the 500-p1 level, the Model B system has an average relative standard deviation of about 0.8% compared to 0.4% for the remote Eppendorf system. The Eppendorf pipets are not trouble-free and do require periodic maintenance. Removing the pipets a t about one-month intervals and cleaning and lubricating according to manufacturers' directions will maintain a constant delivery. Although this paper describes the installation of a single pipet, multiple units can be installed. A two-unit sys-
Table I. Calibration of Eppendorf Pipets by Both Hand and Remote Procedure Pipet size, Pl
100
200 500
Pipet delivery, p l a Hand
Remote
100.2 f 0.08 200.5 f 0 . 1
0.2 2 0 0 . 7 5 0.08 503.8 0.2 99.8
*
*
503.9 f 0 . 3
Each value is the average of 6 determinations and the i value is the standard deviation of the mean ( S D i d n ) . a
~-
Table 11. Comparison of the Model B and Eppendorf Remote Pipetting Systems Found, plQ Stated value, pl
Model B"
Eppendorf
500
495.2 f 4 . 1 502.0 i 2 . 5
5 0 0 . 6 =k 2 . 0
503.6 k 3 . 1
*
a The i value is the standard deviation of a single analysis. The values for three different Model B Pipettors are given.
tem operating from a single selector valve is in use; more units can be installed if desired.
CONCLUSIONS The pipetting system described in this paper provides a simple, convenient, and inexpensive method for operating the Eppendorf pipet remotely. The results obtained in the use of this system confirm that while high precision pipetting may not be accomplished, precision satisfactory for most applications is attained. The system is versatile and can be used as a permanent or temporary adjunct t o existing equipment.
RECEIVEDfor review March 18, 1974. Accepted June 4, 1974.
Digital interlace for a Cary 14 Spectrophotometer Henry Longerich and Louis Ramaley' Department of Chemistry, Dalhousie University, Halifax, N. S., Canada
Digitization of spectral data followed by numerical data analy5is can make possible operations which would be extremely difficult or impossible by manual methods. Examples of such operations are base-line correction; conversion from wavelength t o wavenumber presentation; smoothing, differentiation, and peak finding ( I ) ; and resolution of complex spectra into component peaks (2). In addition, for instrument. in which the readout device, usually a meter or recorder, is the limiting part of the system, digitization can resuit in improvements in accuracy and precision.
' ?iUti?(Jr
IO
Many laboratories today possess minicomputers or computing calculators and high qualit> scanning spectrophotometers such as the Cary 11 (Varian Instruments, Palo Alto, Calif.). The advantages mentioned above can readily be obtained by providing an interface between the instrument and the computer. This situation has existed for some time; however, the authors have been unable to find much in the literature describing the intt.rfacicg of commercial spectrophotometers with digital computers. The one exception to this is a paper by Anderson ( 3 )which contains a section briefly describing. in general terms, an interface for a
whom correspondence should be directed.
(1) A . Savirzky and M. J. E. Golay, Anal. Chern.. 36, 1627 (1964) ( 2 ) L . M. Schwartz, Anal. Chern., 43, 1336 (1971).
(3) R. E. Anderson, Preprin? UCRL-72039, Lawrence Radiation Lab., Livermore, Calif.. Dec. 1969.
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