Conpur. Gem. Vol. 12, No. 3, pp. 213-217, 1988 Printed in Great Britain

0097-8485/88 $3.00 + 0.00 Pcrgamon Prcaa plc

DEVELOPMENT OF A LOW-COST INTERFACE FOR COUPLING A MICROCOMPUTER WITH A FLUORESCENCE SPECTROPHOTOMETER.

COMPUTER-ASSISTED FLUORIMETRY

A. Mufioz DE LA PE~~A', J. A. MURILW~, J. M. VEGAS and F. BARINGO~ ‘Department of Analytical Chemistry and z Department of Medical Physics, University of Extremadura,

0607 1, Badajoz, Spain

(Received 21 Jufy 1987)

Ahstrct--A simple interface has been developed for combination of a Peckin-Elmer MPF-43 spectrofluorimeter with a low-cost microcomputer. Hardware and software designs of the interface for the Commodore 64 microcomputer are described in detail. The interface allows passive data transfer and the software provides for subsequent mathematical treatment.

Recent fluorescence techniques such as conventional or synchronous derivative fluorimetry are possible with the instrument. Excitation-emission matrices can also be generated as 3-D fluorescence spectra or contour plots. Sections through the contour plots at different paths allow the application of new techniques such as variable angle synchronous scanning fluorimetry and non-linear variable angle synchronous scanning flourimetry.

A variety of spectroscopic instruments have been in operation at many laboratories as valuable tools for fundamental research and chemical analysis. In order to cope with the recent increase of work load as well as complexity in operation of up-to-date soph- isticated instruments, computer technology has been adopted in laboratory surroundings.

Owing to the high cost of the new computerized- instruments, microcomputers are being interfaced to conventional instruments such as spectrofluorimeters or spectrophotometers to control the instrument, handle data collection and display the results (Smith, 1983; Ewen 8r Adams, 1984; Buschmann et al., 1984a,b; Bei et al., 1984; Susaki et al., 1983).

In this paper hardware and software designs are presented of a simple interface to couple a Com- modore 44 microcomputer with a Perkin-Elmer MPF-43 spectrotluorimeter. The interface utilizes passive data transfer and the software provides for subsequent mathematical treatment.

Conventional fluorescence techniques and syn- chronous derivative fluorimetry are both possible with the computer-assisted instrument. Excitation- emmission matrices can be generated as 3-D fluorescence spectra or as contour plots. Sections through the contour plots at different paths are provided to allow the application of new techniques such as variable-angle synchronous scanning and non-linear variable angle synchronous scanning fluorimetry.

HARDWARE DESCRIPTION

We used a Perkin-Elmer Model MPF43 spectrofluorimeter with a 150-W xenon arc lamp as

the excitation light source and an R-508 photo- multiplier (Hamamatsu Co.) as the detector.

For the microcomputer we used a Commodore 64 with a Commodore 1541 disk drive, a Commodore 1520 printer-plotter and a Riteman C + Commodore compatible printer. There is an expansion port at which the data bus, the address bus and other control signals are present. This allows different acquisition and control circuits to be connected easily.

Tbe electronic scheme, shown in Fig. 1, consists of an integrated circuit ADC 0808, an operational amplifier 1458, an invertor 7404 and an NOR port circuit 7402. The ADC 0808 is an &bit A/D converter based on the successive approximation procedure. In this implementation, the binary counters are replaced with a special successive approximation register (SAR).

Initially, the outputs of the SAR and the mutually connected D/A converter are at a zero level. After a start-conversion pulse is received, the SAR enables its bits one at a time, starting with the MSB. As each bit is enabled, the comparator gives an output signifying that the input signal amplitude is greater than or less than that of the output of the D/A converter. If the D/A output is greater, a 0 is set, otherwise a 1. An g-bit successive-approximation A/D converter takes only eight clock cycles to complete a conversion. The advantage is that the conversion time is fixed and usually faster than other types of converters.

The ADC 0808 sampling rate is 100 ps, and it has .an analog input between 0 and 5 V. Since the analog output signal of the photomultiplier of the spectrofluorimeter varies between 0 and 1 V, it is necessary to amplify this signal by a factor of 5 to obtain the maximal resolution of the converter. A

213

214 A. MuRoz DE LA PERA et al.

1458 I 8 4

14581 1 I el41 Connector

D5 QI Q7 D3 card I 7

Data acquisition system

Fig. 1. Outline of the B bit A/D converter interfaced to the Commodore 64 microcomputer.

1458 operational amplifier is used in a non-inverting configuration.

As we needed a slower frequency clock than that of the microprocessor, we constructed one using three IC 7404 invertors. The reference voltage of pins 11 and 12 of the ADC 0808 is supplied by the other 1458.

Connection to the Commodore 64 is through its expansion port. The digitized signal picked up by the data bus is stored in the 56832 position of address block I/O 1. The data are then accessible from BASIC by means of the PEEK instruction.

The conversion is begun on the falling edge of the start conversion pulse. This pulse is supplied by the R/W lines and by the I/O1 line when a writing operation is made to the corresponding memory address (POKE 56832, 0) through NOR ports and invertors. These also are responsible for enabling the data bus in a read operation (PEEK 56832).

The resolution of the converter is 1 part in 256. That corresponds approximately to 0.4% of the maximal signal. We synchronized the sampling rate with a start-up signal from the spectrofluorimeter (received through port I of the microcomputer) and with the scan rate of the monochromators. Syn- chronization is achieved through closure of two microswitches when the excitation or the emission manochromators are scanned. This allows us to record excitation, emission, and synchronous spectra. For synchronous measurements both excitation and emission monochromators are locked together and scanned simultaneously.

APPLICATION TO COMPUTER ASSISTED-FLUORIMETRY

In current commercial microcomputer spec- trometers the sequence of data measurement oper-

Computer-assisted fluorimetry 215

ation is controlled by software. However, it is difficult for users to change the sequence unless they are familiar with the assembly language of the micro- computer involved. We have used BASIC for our software so that a user can easily establish the desired sequence of measurement. The software can easily he adapted to any microcomputer based on the 6502 microprocessor. Only the memory addresses have to be changed. The software allows for monochromator scan rates of 60, 120, 240, and 480 nm/min, corre- sponding respectively to A/D sampling rates of 1, 2, 4, and 8 measurements per second. In other words, the spectra are sampled at each nanometer indepen- dently of the scanning speed of the monochromators.

In order to have access to high resolution graphics commands, the BASIC of the Commodore 64 has been extended by use of Simon’s BASIC expansion. This program provides a graphic display of the data on the screen, and automatically calculates the wave- length of maximum fluorescence and the relative fluorescence intensity at the maximum. Also, a cursor allows us to request the wavelength and the relative fluorescence intensity at any point of the spectrum. The program is interactive, easily modifiable, and easy to use.

Our system has provision for subtracting spectra from each other. Spectra may lx stored on the Commodore 1541 disk drive, may be output to the Commodore 1520 digital printer-plotter, and may be recalled from disk for further manipulation.

CONVENTIONAL AND SYNCHRONOUS DERIVATIVE FLUORIMETRY

We have written routines that calculate first and second derivatives and that perform noise reduction by smoothing based on the simplified least-squares procedure of data smoothing and differentiation of Savitzky & Golay (Savitzky & Golay, 1964; Steinier et al., 1972). Differentiation of spectra1 data is a powerful tool for analysing mixtures (Butler, 1979; O’Haver, 1979; Talsky et a/., 1978; Fell, 1980; Trav- eset et al., 1980). It is increasingly applied in pharma- ceutical and biomedical analysis for resolution en- hancement (Butler, 1979; Talsky et al., 1978; Gill et al., 1982).

Figure 2 shows the zero, first and second deriva- tives of the fluorescence spectrum of Rhodamine B.

The coupling of synchronous fluorimetry with de- rivative techniques was first suggested by John & Soutar (1976). This involves taking the derivative of the synchronous spectrum. In synchronous fluorimetry the excitation and emission mono- chromators are scanned simultaneously maintaining a constant wavelength different between the two monochromators. The result of synchronous fluorescence scanning is a narrowing of the spectral bands, owing to the synchronous multiplication of the simultaneously increasing and then decreasing

500 560 660

A EMISSION Mn)

Fig. 2. (a) Zero, (b) first and (c) second derivative spectra of a sample of Rhodamine B 5 x lWSM.

fluorescence spectra as a function of excitation and emission wavelengths (Vo-Dinh ei al., 1978; Lloyd & Evett, 1975; Lloyd, 1975; Vo-Dinh, 1978; Vo-Dinh & Gammage, 1978).

It is obviously interesting to combine synchronous and derivative fluorimetry to enhance minor spectral features and, because of the band narrowing effect, to increase the sensitivity as in recently reported deter- minations (Salinas et al., 1985; Cruces et al., 1986).

EXCITATION-EMISSION MATRICES

Specificity in luminescence spectroscopy is achieved insofar as each compound is characterized by distinctive excitation and emission wavelengths. The identification of individual compounds in com- plex mixtures is often difficult owing to the lack of structure in the excitation or emission spectra. A fingerprint of the mixture is obtained by collecting both emission and excitation spectra. We visualize the results in the form of a three-dimensional iso- metric plot (Fig. 3) or a two-dimensional contour plot (Fig. 4).

The spectrum shown in Fig. 3 is a projection containing 30 individual spectra superimposed with the aid of a “hidden line removal” routine. The 3-D spectra give excellent general pictures of the fluorescent properties of samples but it is difficult to extract detailed information from them.

In the contour plots of Fig. 4, the two normal axes represent the emission and excitation wavelengths, while the intensities are expressed as a series of contours. In Fig. 4 contours are drawn at 90, 80,d , . IO%, of the peak intensity.

216 A. MufJoz DE LA PER.4 er al.

Intensity

ITATION (run)

400 EMISSION (nm) 640

Fig. 3. Three-dimensional isometric ulot of the total fluorescence spectrum of Ovalene 1 x 10e5 M. The number of scans is 30 and the excitation inckment is 8 nm.

400 EMISSION (run)

Fig. 4. Contour plot of the excitation-emission matrix of Ovalens 1 x IO-’ M. Contours are drawn at 90, 80, . lo%, of the peak intensity.

Contour plots are more informative than are 3-D plots especially if quantitative data are needed. Sec- tions through them give useful information. Thus a vertical section, i.e. at constant emission wavelength, is equivalent to an excitation spectrum, and a hori- zontal section is equivalent to a conventional fluorescence spectrum. A 45” section, with a constant wavelength difference between the two mono- chromators, produces a synchronous spectrum.

A new analytical procedure known as variable angle synchronous scanning is also possible (Miller,

1984) The technique consists of selecting linear path at any preselected angle other than 45” through the excitation-emission matrix. It may give a higher degree of selectivity than does the synchronous ap- proach. Recently some pharmaceutical applications of the technique have been developed (Clarke et a!., 1985). Also the angle of the scan trajectory can be varied continuously through the excitation-emission matrix, describing any desired path under computer control. This new technique is known as non-linear variable angle synchronous scanning {Clarke et al.,

Computer-assisted fluorimetry 217

1985) and sometimes allows the resolution of over- lapping systems that cannot be resolved by linear scanning (Rubio et ai., i986).

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