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I LC R 237.4 550 ,100 of 79LN35R.D

i I I ( b )

5 10 15 20 25 Time ( m i n . )

Figure 4

HPLC chromatograms of degraded Nicardipine samples ( 2 mg/ml in mobile phase): (a) after three weeks under daylight and at room temperature; (b) after three months at 50 "C; (c) control sample.

4 Conclusions This work describes a chromatographic method which is appli- cable to the quantitation of Nicardipine and its related compounds

(present as minor impurities). Although the method is simple, and requires neither derivatization nor sophisticated apparatus, the chromatographic parameters must be set with care. The system suitability test should be run before each analysis to guard against deviation from the original conditions.

Both assay and purity methods have been shown to be good indicators of stability. The method is being applied to samples prepared from raw materials and excellent results have been obtained.

References

[l] P. 0. Greiner, D. Angignard, and J. Cahn, J. Pharm. Sci. 77 (1988) 387.

[2] A . T. Wu, I. J. Massey, and S. Kushinsky, J. Chromatogr. 415 (1987) 65.

[3] S. Higuchi and S. Kawamura, J. Chromatogr. 223 (1981) 341.

I41 A. T. Wu, I. J. Massey, and S. Kushinsky, J Pharm. Sci. 73 (1984) 1444.

[5] S. I. Kobayashi, J Chromatogr. 420 (1987) 439

Ms received: March 20, 1990

Flame Control Accessory for GC-FID Operation with Autosampler Injection Detlev Helmig: Norbert Schwarzer, and Jurgen Steinhanses Fraunhofer-lnstitut fur Umweltchemie und Okotoxikologie, W-5948 Schmallenberg, FRG

Key Words: Gas chromatography Automatic thermodesorption technique Flame ionization detector, FID Flame control

1 Introduction Automation in GC sample injection has increased markedly in recent years. Autosamplers, headspace devices, and automatic thermodesorption instruments are able to inject large numbers of samples for GC analysis, also overnight or at weekends. If GC detection is performed with FID, serious consequences may ensue if the FID flame is extinguished during the analysis series. Possible reasons for the extinction of the FID flame are, e.g., leakages of the fuel gases. In practice, however, the causes for the flame extinction do not always appear logical.

Most commercially available GC instruments do not have a sensing device to register flame extinction and thus the automatic inlection devices do not stop the analysis series The result is that large numbers of samples may be inlected without being

detected. If a thermodesorption technique is used, e.g., for the analysis of air samples, extinction of the FID flame during automatic analysis is very troublesome: all subsequent samples of the series will be lost as the analysis cannot be repeated because the complete sample is volatilized during the desorption step. Thus a new sample must be collected, involving many time- consuming working steps such as sample tube preparation (adsorbent cleaning), transport to the measuring site, and actual sampling (active or passive) itself. In most cases the composition of the new sample cannot be regarded as representative of the initial sample taken, since samplings were performed at different times and the air composition will usually have changed in the meantime. Thus sample losses, e.g. in work place monitoring, are often irreplaceable.

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Hence a simple, low cost supplementary device usable with most common GC instruments was developed in order to monitor the FID flame This instrument checks the burning of the flame and stops automatic sample inlection if the flame extinguishes, thus preventing the loss of subsequent samples

2 Experimental Possible methods of monitoring the burning of the flame are provided by, e .g , thermal or optical sensors for measuring the emitted heat or light of the flame, UV absorbance control near the flame position to measure unburnt Hz, or the sensitive monitoring of changes in the detector voltage. Only a few GC instruments with an integrated flame control accessory using one of these described techniques have been built so far, e.g. for initiating automatic re-ignition in the case of flame extinction [l-31.

The instrument described here is a supplementary device and does not require modification of the FID instrument itself. It monitors the voltage output of the detector. Extinction of the FID flame usually causes a distinct drop of the detector voltage output as illustrated in Table 1 for a selection of different GC instru- ments.

A schematic of the developed instrument is shown in Figure 1. It works by recording the difference in detector output voltage between burning and extinguished flame. After baseline stabi- lization, the detector output is set at a suitable working range of the GC integrator, e.g. in a range of 10-15 mV with the FID potentiometer or automatically on operation of the FID autozero function. A parallel output of the FID signal is taped on a BNC connector and smoothed by a low pass filter (R1 and Cl), The signal is transformed by a voltage follower and amplified by a factor of 50. The amplified signal is monitored by a voltmeter. Then the amplified voltage passes to a comparator. This compa- rator is adjusted to a defined voltage threshold by a potentio- meter. The voltage threshold can also be monitored on the voltmeter and thus adapted to the detector output. The threshold is set to about 5 mV below the stabilized baseline voltage. The comparator compares the actual detector output with the adjusted threshold value. If the detector flame is extinguished, the voltage falls below this threshold and the comparator opens. To avoid a reaction to short-term drops in voltage, e.g. from negative peaks, sparks, or line fluctuations, an adjustable cut-off

Figure 1

Block diagram of the FID control device (description given in the text).

Table 1

Measured voltage drop of detector output caused by flame extinction for several GC instruments.

GC instrument detector type voltage drop

Perkin Elmer F22 Perkin Elmer X300 Perkin Elmer 2300 Perkin Elmer 2000 Perkin Elmer 8500 Dani 3200 Dani 8521 Carlo Erba Mega 5160

FID

FID FID FID FID FPD* FID

F P D ~ ) -9 mV -7 mV

-13 mV -16 mV -13 mV -70 mV -80 mV -12 mV

a) Flame photometric detector

delay is inserted. The delay time of this module is typically set between one and two minutes. If the FID signal again exceeds the comparator's voltage threshold within the set time, the cut-off delay is reset. If the duration of the voltage drop exceeds the set time a number of options are performed by a tandem-arranged switch circuit: The alarm stage is indicated by an acoustic alarm (buzzer) and by a LED. The automatic sample injection is stopped by opening the autosampler's ready loop and the Nz(1) supply of the GC is interrupted by opening the power supply of the magnetic valve of the cry0 dewar. Furthermore, two magnetic valves inserted in the Hz and synthetic air supply of the FID are switched off and thus stop the gas flow. All options of the switch circuit can be reset manually by a reset option if the detector voltage is set above the comparator's voltage threshold.

3 Results The instrument has been routinely used for about one year in a measuring system consisting of a Perkin Elmer automatic ther- modesorber ATD 50, a Carlo Erba Mega 5160 GC equipped with a Carlo Erba FID 440 and a Carlo Erba Cry0 520 device. The response of the instrument depends essentially on the accurate adjustment of the voltage threshold at the beginning of a series of analyses. Since the detector zero current and the baseline stability depend

FID Signal -

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on various GC operating parameters such as column bleeding, oven temperature, fuel gas composition, and detector tempera- ture, the voltage threshold must be checked regularly to compen- sate drift effects. If temperature programming is used for GC operation the adjustment must be performed at the lowest oven temperature, where the zero current is usually the lowest.

With the help of the flame control accessory, sample losses by flame extinction were greatly reduced. Only those samples are lost which are actually in GC analysis when the flame is extinguished. In addition to reduction of sample losses, an unnecessary waste of the fuel gases and the oven cooling agent (liquid nitrogen) are prevented. Furthermore, switching off the Hz supply is of special interest for safety reasons since it prevents leaking of unburnt Hz. As shown by the data given in Table 1, the instrument is universally applicable for most of the common GC instruments. Since the control accessory is designed as supplementary device which only has to be linked to the detector output, the autosamp- ler ready loop, and with magnetic valves inserted in the fuel gas

supply, it can easily be moved between different GC instruments in a laboratory. Although the instrument has not yet been tested in conjunction with a flame photometric detector, the observed voltage drop for this detector type should also permit use of the device.

Note: A patent application for this circuitry monitoring the operation of a flame ionization detector was registered with the German Patent Office on April 6, 1990.

References

111 T. G. Andronikashvili, V. G. Berezkin, and Z. A. Gvelesiani, Soobshch. Akad Nauk Gruz. SSR 120 (3) (1985), 541-543 (CA 104 (18): 151341d).

[2] Hokushin Electric Works, Ltd., Jpn. Kokai Tokkyo Koho JP 56/114755A2 [81/114755], 9 Sep. 1981, 3 pp. (CA 96(6). 45559n).

131 Yokogawa-Hewlett Packard, Ltd., Jpn. Kokai Tokkyo Koho JP 55/104752 [80/1047521, 11 Aug. 1980, 7 pp. (CA 94(2): 10709t).

Ms received: October 1 , 1990

Comparison of Different Sample Preparation Methods for Bacteria Identification by Capillary Gas Chromatography L. Weber Department of Food Hygiene, University of Veterinary Sciences, PO. Box 2, H-1400 Budapest, Hungary

Key Words: Capillary gas chromatography Microbial identification Bacterial acid methyl ester formation

1 Introduction Gas chromatography is now routinely applied for the identifica- tion of bacteria through the whole cell fatty acid profile [ 11. Genera and species can be distinguished by qualitative and quantitative differences in acid abundances. Bacteria, or more generally microorganisms, are grown under controlled conditions and harvested. The lipid fraction is transformed into bacterial acid methyl esters (BAMEs) which are extracted from the medium and analyzed by gas chromatography.

Numerous methods and several reagents have been reported for the preparation of fatty acid methyl esters. Some of them have been applied to produce BAMEs and in this contribution the results are compared. In order to judge if a particular procedure is useful the following two aspects should be given special atten- tion: (a) none of the steps in the sequence of operations should affect the qualitative and quantitative results; (b) sample prepa- ration should be carried out with minimal sample transfer; if possible in a single vial.

BAMEs can be analyzed by using either a packed column or a capillary column [2]. Based on CGC on apolar methyl silicone phases automated Microbial Identification Systems have been commercialized [ l , 31. However, erratic elucidation of some fatty acid's raised doubts as to the usefulness of apolar columns. When

chemotaxonomy is the aim of the CGC analysis the use of highly polar columns with cyanopropyl silicone phases is recommended

The fatty acids present in bacteria range in length from 9 to 20 carbons. They are odd and even carbon numbered saturated acids, C16 : 1 and C18 : 1 monounsaturated acids, is0 and anteiso branched chain acids, C17 and C19 cyclopropane acids and C10, C12, C14 and C16 2-hydroxy and 3-hydroxy acids [2]. Salmonella enteritidis tested in our experiments contains most of the common bacterial fatty acids.

141

2 Experimental

2.1 Preparation of BAMEs

Cultures of Salmonella enteritidis were grown and harvested as described [ 11. For sample preparation the following methods were applied: Method A 11, 31: The cell mass is saponified with 3.75 M methanolic NaOH at 100 "C. The mixture is then acidified with 3.25 M methanolic HCl. Methylation is performed at 80 "C and the BAMEs are extracted with n-hexane : ethyl ether. Before injection the organic phase is washed with a 0.3 M NaOH solution.

0 1990 Dr. Alfred Huethig Publishers Journal of High Resolution Chromatography 851