A microprocessor controlled liquid chromatograph/atomic absorption sampling system

ACKNOWLEDGEMENTS The author is grateful to Mr. W. A. White for technical assistance, to V. A. Howe & Co. Ltd., for the loan of the instrument and to the Department of Health and Social Securify for their permission to publish this article. REFERENCES [1 Anderson, N.G., (1969) Analytical Biochemistry, 28, 545. [2] Anderson, N.G., (1969) Analytical Biochemistry, 32, 59. [3] Anderson, N.G., (1969) Science, 166, 317. [4] Anderson, N.G., (1970) American Journal of Clinical Pathology 52, 778. [5 Henry, P. and Saunders, R.A., (1976) Annals of Clinical Biochemistry, 13, 384. [6] Young, D.S. and Gochman, N., in "Standard Methods of Clinical Chemistry" volume 7 Ed. Cooper G.R. 1972 Academic Press, p. 303. [7] Broughton, P.M.G., Buttolph, M.A., Gowenlock, A.H., Neill, D.W. and Skentelbery, R.G., (1969) Journal of Clinical Pathology 22, 278.


Introduction
The need for molecular characterization of metal-containing species at the trace level has led to the interfacing of high resolution chromatographic techniques with element-specific detectors [1,2,3]. One such system is the interfacing, of liquid chromatography (LC) to atomic absorption spectroscopy (AA). The resultant instrumentation (LCAA) is capable of specific characterization (speciation) of the metalcontaining compounds and detection of the separate species at the nanogram level [3]. The application of LCAA with a graphite furnace atomic .absorption spectrometer has been reported [1,2]. The use of a graphite furnace atomic absorption spectrometer as a detector for a liquid chromatograph has technical problems associated with it. However, the ability to monitor biological transport of metalcontaining species in the environment, speciate clinically important metal compounds, or to use metal complexation as a tag for nonmetal species which are otherwise difficult to detect [4], make this technique attractive.
This work presents a versatile controller for the sampling interface, using a 6800 based microprocessor system. This interface controller is software programable to operate either in the 'pulsed' mode, or in the 'total consumption' mode. The hardware and software descriptions are presented, as well as the results of sampling precision studies.

Three sampling modes
There are three basic modes for LCAA operation: survey, pulsed and total consumption. In the survey mode the sample is chromatographed, fractions collected and the collected fractions analyzed for the metal of interest. This is the simplest form of the LCAA experiment and requires only the 'human' interface. The pulsed mode operates during the chromatographic run; the eluent stream is periodically sampled and the sample dispensed into a graphite furnace for the AA analysis cycle as shown in Figure 1 Figure 1. Description of the "pulsed" sampling mode. A, actual concentration profile of metallospecies leaving the column. B, the measured concentration profile from the intermittent removal of aliquots of the eluent stream. C, a diagram of the eluent stream showing the aliquots which are removed and analyzed by the graphite furnace atomic absorption spectrometer. is used for this mode because the element concentration data can be obtained only once every 30-120 seconds depending on the element of interest.
This causes the chromatogram generated by the AA detector to have the appearance of 'pulsed' concentration varying with retention time or volume. The data obtained have the form of Figure lB. An example of a pulsed mode chromatogram is shown in Figure 2. The most direct application of the pulsed mode of LCAA operation is to determine the presence and concentration of metal-containing species with unknown retention times, or reaction mixtures where the number of products and their identity is unknown (but all products are known to contain the metal of interest). The drawbacks of pulsed mode operation are that. the mobile phase flow rates that can be used are very low. This is expecially true in the case of elements which have long AA analysis times (including the cooling of the graphite furnace).
The flow rate must be low enough so that the product of one analysis time, tanalysis, ( Figure 1B) and the flow rate does not exceed the peak width. Another related drawback is the small number of data points obtained to describe each peak's concentration profile ( Figure 1B). In general, the number of data points per peak is between 4 and 8; also the chromatographic peak shape given by these peaks is not necessarily indicative of the shape of the concentration profile. Therefore, the third sampling method is designed to overcome these shortcomings.
The total consumption mode analyzes the total chromatographic peak by storage of the peak-containing eluent stream and subsequent dispensing of aliquots of the peak-containing eluent into the graphite furnace. In this mode the chromatographic peak is totally consumed. The temporary storage of Vickrey & Eue Microprocessor controlled LCAA sampling system the peak-containing eluent in a capillary tube allows the chromatographic information to be retained and the analysis to be performed off-line. A flow scheme for the method is shown in Figure 3. This total consumption of the chromatographic peak by the graphite furnace analyzer lowers the limit of detection, and greatly increases the number of AA data points per chomatographic peak. Since the analysis is performed off-line the restrictions on the mobile phase flow rate are removed. Data from an experiment using this sampling method are shown in Figure 4. The drawbacks to this sampling method are that the retention time of the peak muse be known (at least approximately). Also the extra

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The Journal of Automatic Chemistry Vicktey & Eue-Microprocessor controlled LCAA sampling system column broadening effects due to the large 'dead volume' of the storage tube are not negligible, but the peak width increase can be corrected in the data analysis. The last two sampling modes described above are complementary. The pulsed mode is most useful for exploratory studies and the total consumption mode is more suited to routine analysis of samples when the identity of the components is known. The interface system used should be capable of operation in any of the modes described above.

Instrumentation and procedures
To this end, the microprocessor control LCAA interface has been developed and is shown diagramatically in Figures 5   and 6. The controller consists of (1) the Motorola 6800 based Heathkit microprocessor trainer, (2) external clock (3) electronic interface to the dispenser and to the AA, and (4) the dispenser system [1]. The microprocessor and peripheral interface adapter generate an 8 bit control pattern.
The second injection is a critical part of the analysis cycle for non-metals such as selenium and arsenic. The addition of a co-analyte such as Ni2+ has been shown to increase the sensitivity for these elements and reduces loss due to the volatility of these elements 1,5]. The syringe pump used for the second injection delivers 11.0 + 0.1 /.tl/sec of coanalyte. The same syringe pump is used for the total consumption analysis mode. In the total consumption mode, however, the analyte is pumped out of the holding tube into the sampling valve and the dispensing of the sample then proceeds in the same fashion as in the pulsed mode of operation. The software modification for total consumption analysis involves only increasing the second injection time to 4-8 seconds and performing the second injection prior to the sample injection.
The graphite cuvette volume in the currently employed

Conclusion
The use of an inexpensive microprocessor system adds a great deal of versatility to a previously "hard wired" LCAA sampling system [1]. Both pulsed and total consumption analyses are possible with only minor plumbing and software changes. The use of other microprocessor systems would require only a change in machine language. The system has the advantage of being inexpensive. The interface sampling system can be assembled for a component cost of approximately $500 (depending on the availability of surplus equipment).
With the many advantages of performing LCAA analysis on trace level metal-containing compounds, hopefully this technique will find widespread use with investigators in the clinical, environmental, and inorganic biochemistry fields.
The ENI Gemeni was assessed in this institute for its suitability as a general laboratory instrument using a procedure which has been developed during the last two years for the Committee for Evaluation of Kits and Instrumentation of the Australian Association of Clinical Biochemists.

Materials and Methods
The Gemeni is a miniature centrifugal analyser consisting of an analyser module, microprocessor and work station. The methods recommended by the manufacturer for use on the Gemeni were run in parallel with routine methods used in this laboratory. These routine methods were:  [2], Barnett and Youden [3], Logan [4], Broughton et al [5] and modified in the light of our own experience.

Precision
(a) Intrabatch imprecision was checked by the analyses of replicates in the same batch and duplicates on the patient comparisons.
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