Anal. Chem. 1987, 59, 2534-2535
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of mixtures of ionic or neutral and ionic compounds which are not well separated by simple reversed-phase HPLC.
4
3 1
I
Registry No. 1,62-44-2; 2,93-14-1;3, 59-42-7;4, 700-65-2;5, 299-42-3; ammonium trifluoroacetate, 3336-58-1. LITERATURE CITED
10
15 MINUTES
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(1) Garteiz, D.; Vestal, M. LC Mag. 1982, 334. (2) Voyksner, R.; Haney, C. Anal. Chem. 1986, 57, 991. (3) Covey, T.; Lee, E.; Bruins, A.; Henlon, J. Anal. Chem. 1966, 58, 1451A. (4) Snyder, L.; Kirkland, J. Introductlon to Madern UquM Chromatography; Wiiey: New York, 1979; Chapter 11. (5) Crowther. J.; Hartwick, R. Chromatographie 1982, 16, 349. (6) Floyd, T.; Clcero, S.;Fazb, S.;Ragilone, T.; Hus, S.; Winkle, S.;Hartwick, R. Anal. Blochem. 1986, 154, 570. (7) Bidlingrneyer, B. J. Chromatogr. Scl. 1980, 18, 525.
' Current address:
Finnigan-MAT, Rockville, MD.
Flgwe 1. Reconstructed ion chromatogram of a mixture of compounds 1-5.
However, the vacuum line between the thermospray source and the rough pump was found to clog with salt after a few hours of operation. Therefore, the HPLC column flow rate was limited to 0.5 mL/min. CONCLUSION Mixed-mode column thermospray LC/MS (discharge ionization) appears to offer an alternative approach to the analysis
J o h n R. Lloyd' Mary Lou Cotter David Ohori Alan R. Oyler* Research Laboratories Ortho Pharmaceutical Corporation Raritan, New Jersey 08869-0602
RECEIVED for review March 11,1987. Accepted June 29,1987.
AIDS FOR ANALYTICAL CHEMISTS Automated Sample Cell Cleaner D. E. Bautz and J. D. Ingle, Jr.* Department of Chemistry, Oregon State University, Corvallis, Oregon 97331 The majority of spectrophotometricand spectrofluorometric measurements are made on solutions in 1 cm path length sample cells. Many analytical procedures are based on monitoring the formation of an absorbing or fluorescing product by reaction of the analyte with suitable reagents. To simplify the procedure and to increase sample throughout, the sample and reagent solutions can be added to and mixed in a sample cell that is secured in a sample-cell holder. Addition of samples and reagents with automatic pipets (e.g., Eppendorf) or automatic syringe injectors has proved to be simple and rapid (I). Normally, one evacuates the contents of the cell with a vacuum aspirator and rinses several times with a blank solution from a wash bottle to prepare the cell for the next sample or standard. In continuous flow analysis systems, the sample flow cell is cleaned between sample plugs by the carrier stream without operator attention. With rapid and often automated addition of samples and reagents to the sample cell and microcomputer-based data acquisition, calculations, and reporting, we find that the emptying and cleaning of the sample cell between measurementa is the most time-consuming and tedious step during the analytical measurement. To address these limitations, a microcomputer-controlled cell cleaner was designed to perform the function of cell cleaning. This device allows spectrometric measurements with a conventional cell to be performed in a more automated fashion and frees the operator for more important tasks. The actual size of the cell cleaning system is small enough to be placed adjacent to or directly on top of the instrument
Table I. Instrumental Components
component three-way solution valve and pneumatic activator solid-state relays three-way air valve Pump
model
source
Dion ex, Sunnyvale, CA 7052-04-B04-F Grayhill, LaGrange, IL MBD-002 Skinner, New Britain, CT Lab pump junior Fluid Metering, Inc., Oyster Bay, NY 30520
containing the sample cell. The cell cleaner was used here to clean a fluorometric cell containing quinine sulfate (QS) and the resulting blank signal was measured as an indicator of the completeness of cleaning. INSTRUMENTATION A schematic of the cell cleaning device is shown in Figure 1 and the components are specified in Table I. A glass capillary tube (0.125 in. i.d.) is glued along an inside edge of the sample cell with the bottom end of the tube located -1 mm above the bottom of the cell. The other end of the capillary tube is connected with 0.125 in. i.d. Telfon tubing to the common input port of a three-way solution valve. The other two ports are connected to a pump and to vacuum. The pump is turned on and off by switching the ac power to the pump with a solid-state relay (SSR1) controlled by TTL logic
0003-2700/87/0359-2534$01.50/00 1987 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 59, NO. 20, OCTOBER 15, 1987 3-way
to house
vacuum
t o solvent rctervoi r
I
to C o m p u t e r
Flgure 1. Schematic of the automated cell Cleaning device.
signals. The three-way solution valve position is controlled by a pneumatic activator. Another solid-state relay (SSR2) controls a three-way air valve and allows either high-pressure air (60psi) or the atmosphere to be connected to the three-way valve activator. The three-way solution valve and activations circuitry are mounted in one box and SSRl is mounted to the Pump. To activate the sample cell cleaner, a logic 1 (+5 V) signal is applied to the control input of SSR2. This switches the position of the three-way solution valve so that the capillary is connected to vacuum. When the control signal to SSR2 is logic 0 (0 V),the three-way solution valve is connected to the pump. The rinse cycle occurs when a logic 1signal is applied to the control input of SSR1. Normally the inlet of the pump is connected to the blank solution and the flow rate through the capillary tube is approximately 25 mL/min. Any microcomputer with 1/0 capability can be used to provide the appropriate logic signals to activate the SSRs. In this case, two 1/0 lines from port B of the 6522 1/0 chip on a Rockwell AIM-65 microcomputer are employed (these 1/0lines can source several milliamperes). A short machine language program was written to change the signals on the 1/0lines a t the appropriate times to evacuate the cell and then fill the cell with blank solution ( ~ 2 . mL). 5 The number and duration of the evacuation and filling (rinse) steps can be set to any desired values. The software was designed to make it easily adaptable for use as a subroutine in a larger data acquisition program.
EXPERIMENTAL SECTION Reagent grade chemicals were used throughout the study. Solutions were prepared from deionized water from a Millipore Milli-Q system fed by house deionized water. The blank solution was 0.05 M H2S04. The test solutions were 100 bg/mL QS and 50 ng/mL QS in 0.05 M H2S04. A standard 1-cm cell was placed in a spectrofluorometer that has previously been described (I). A magnetic stirring bar in the sample cell was operated continuously. The excitation monochromator wavelength and spectral band-pass were set to 365 and 17 nm, respectively. The fluorescence emission was isolated with an interference filter with maximum transmission at 450 nm and a 20-nm half-width. To evaluate the performance of the cleaning device, the following procedure was used. First, 2.5 mL of a 50 ng/mL solution of QS was added to the sample cell and the fluorescence signal
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was recorded. Next the cell-cleaning program was initiated with a 5-s evacuation step followed by a 12-sblank filling step. The blank signal was measured. The evacuation, filling, and measurement steps were repeated until the blank signal reached a constant level (i.e., the variation in sequential blank signals was random and limited by the blank noise). The same sequence of measurements was repeated with manual evacuation and rinsing of the cell. A wash bottle containing the blank solution and a disposable pipet attached to a vacuum aspirator were used for cleaning. Approximately the same volume of rinse solution was used to attempt to duplicate the conditions used for the automated procedure.
RESULTS AND DISCUSSION With a 50 ng/mL QS test solution, it takes three evacuation/fiiig steps to achieve a constant blank signal with either the automated or manual cell cleaning procedure. After one evacuation/filling cycle, approximately 95% of the QS is removed. The second cycle removes approximately 99% of the initial QS test solution. In another study using a 100 pg/mL QS test solution, the number of evacuation/filling cycles necessary to reach a stable blank signal was determined to be eight for the automated method and six for the manual method. Apparently, the efficiency of removal of the contents of the sample cell for one evacuation/filling cycle is not as good as with manual operation. This may be due to the use of the same tubing for both rinsing and evacuation. Small droplets or a film of the previous contents of the sample cell may adhere to the inside of the tube and be flushed back into the sample cell during the next rinse cycle. For routine measurements, the computer was programmed to carry out a cell-cleaning sequence consisting of a 5-s evacuation step followed by three rinse/evacuation cycles. The duration of each rinse step was 12 s. Each evacuation step was 5 s except for the final evacuation step. Here the duration was increased to 8 s to ensure complete evacuation of the sample cell. Repetitive measurements of the fluorescence signal from a 50 ng/mL QS test solution were made. Between each measurement, the above automated cell-cleaning sequence was implemented and a blank measurement was obtained. The relative standard deviation in the net fluorescence signal was 0.6% and was essentially the same as obtained with an equivalent manual procedure. With manual or automatic cell cleaning, an adequate number of rinse cycles must be used to obtain reliable measurements. The total time for the three-cycle automated cell cleaning sequence is 60 s. This is slightly longer than the time required for an equivalent manual rinsing sequence (approximately 30-40 8). However, the operator is free to prepare for the next analysis during the automated cleaning sequence and the cell-cleaning procedure is performed in an identical fashion for all samples. The time for filling the sample cell is the rate-limiting step. This time could be decreased by using a higher rinse solution flow rate.
LITERATURE CITED (1) Wilson, R. L.; Ingle, J . D., Jr. Anal. Chem. 1977, 49, 1060-1065.
RECEIVEDfor review January 16,1987. Accepted July 1,1987. Acknowledgment is made to the NSF (Grant No. CHE-8401784) for partial support of this research.
