chromatograph - Hindawi Publishing Corporation
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1. e Microcomputer Figure 1 A schematic of the computer the chromatograph and their interfaces Industry Group Eindhoven The Netherlands The central processor is an Intel 8080A The memory consists of 4K bytes of programmable read only memory EPROM for the system monitor and a number of commonly used sub routines 16 K bytes of EPROM for developed programs and 12K bytes of static random access memory RAM for storing data or programs under development Other cards are included for interrupt control for communication with peripherals and for a real time clock Seven segment displays on the front panel show elapsed time and chromatographic variables such as flow rate and pressure Fight light emitting diodes are also incorporated in the front panel to display the status of programmed variables such as the control byte for the automatic sampler which will be described later Software Our laboratories have extensive support for the 8080 including facilities for software development in either assembly language or a high level language Coral 66 We can use the multi access laboratory computer ICL 19045 or an Intel MDS microcomputer development system For the present work most software development was carried out on the main laboratory computer and output in machine code on paper tape which was then loaded into RAM using a high speed paper tape reader Programs could then be run debugged altered etc using the system m2. 3UJ UK This evaluation extended over a period of three months and follows the recommendations of Broughton vet al 1 2 The instrument The Kem O Mat Coulter Electronics Limited is a calculator controlled single channel discrete analyser which may be used for either kinetic or endpoint measurement analyses The throughput of the instrument varies with the methodology but can be a maximum of 110 samples hour at 37 C in the kinetic mode and 180 samples hour in the endpoint mode Results are calculated automatically and are printed in the units of choice The data are checked before calculation to ensure that linearity limits have not been exceeded and that the initial reagent absorbance was within predetermined limits The data for non linear analyses in the kinetic mode are printed in absorbance units The operator has the option to print any or all absorbances since all data are stored in the calculator s memory and can be recalled at the end of the analysis run All the key instrument functions are continuously Coulter Electronics Ltd Coldharbour Lane Harpenden Herts UK 140 monitored by the calculator and fault warnings are given by an audible alarm and printed message There is a comprehensive range of methodologies available for the instrument for which the manufacturer will supply the preprogrammed cassettes and the reagent packs or suggest a supplier for the latter The user has the facility to develop
3. ADI XX Se Sad ah KE Soe cxx xx as at A E A KEES EEE SAS SME XX X X X SHE E E E SE X X XX XX X X X Xxx X X XX X X Xx x Xx xXx x X X x ox x Xx xx x xx x xx E cx cxx xx xoxo xxx x xx x x x xx ese scoe oyee cooo c oeoa osse o o REMOTE FLOW STABILISATION TIME MINS AUTOSAMPLER TYFE SCSAMPLERD P CPURGE OR TYFE HOW MANY STEFS TYFE THE FOLLOWING ROUTINES ARE CONTROL KATEs 2 25 Y 3CSAMFLER PFOPURGE SCSAMPLER D PCOFURGED OR t OF FUMF Y ons br MCMOVE gt OR MCMOVE gt ACMOVED 1 5INGLE VIAL FROBE NOT WITHDRAWN 2 CONSECUTIUE VIAL SAMPLING S3 CONSECUTIVE VIAL WITH RINSE 4 CONSECUTIVE VIAL WITH FURGE ROUTI NE NUMBER 1 DATA HANDLING Y INTERNAL ADC SUGGESTED SLOFE DO YOU UISH TO SLOPE PEAK DOUBLING TIME SECS TOTAL TIMECSECS SENS WIDTH 3 3 Y SET 200 350 DELAY TIME XOSECS 100 MINIMUM AREA FLOW RATE 100 7 aom s FRELIMINARY RUN Wane 9995 oeeo cooo sese 0999 cone peos AREA eoo osos 9909 cece oseo cose ce Bene 2090 37659 ipia 49 cose 4699 soe seoa 100s osoo eves cone cos asse sore DO YOU WISH TO CHANGE FARAMETERS oeeo ceos 9949 vee osre esoe 0090 oses RET TIME 12 3 142 2 1793 3 203 1 seee sose sees acsee sete osso HOW MANY SAMPLES 1 SAMFLE NO oces onoo 0909 opoe eues P900 sote toes AREA 27031 1o30 37370 22822 END OF RUN erce Gees eee ses
4. the end of an experiment after correction for baseline drift and overlap between peaks Correct coding of the algorithm was confirmed by loading a series of digital Gaussian curves into the microcomputer to simulate single and grossly overlapping peaks The smoothed version of the signal is available at a socket on the microcomputer after conversion to analogue form by the 12 bit DAC Performance User interaction One of our aims when writing the software to control this instrument was to offer the analyst a high degree of inter action and a wide choice of operational modes The computer asks a series of questions in plain English on the VDU to which the user can reply with the appropriate response at the keyboard This is illustrated by Figure 4 and Figure 5 From the keyboard the analyst can set the solvent flow rate and can choose the desired sampling mode with or without data handling or if manual injections are preferred the data handling procedure can be invoked alone Before sampling takes place the detector noise level is measured automatically and the user can then type in various parameters appropriate to the particular analysis in order to obtain the optimum separation and data handling accuracy After a preliminary analysis these parameters can be changed if necessary until the optimum conditions have been achieved The system can then be instructed to proceed with Volume 1 No 3 April 1979 6 4 Minutes its tas
5. Tilden amp Denton Advanced software concepts l d 0 O AAA ME LALL LRMEMLA A SS SS SSS SSS SS SSS YS SPE ed value STOP the two are compared to gt to see if the incremented value at LOCATION is larger then the STOP value if it is the program ends if not it repeats the process starting at BEGIN HERE It should be noted that whilst many variables have been pushed on the stack only the data will remain since each time a value is used it is popped removed from the stack If a different spectra region is to be scanned i e from 3000 to 6500 with 10 increments the variables need only be changed thus 3000 START 6500 STOP 10 INCREMENT and type SCAN system will now scan from 3000 to 6500 taking data every 10 steps While the code might look a little strange at first it quickly becomes very easy to work with The SCAN program of Figure 6 could be combined with other modules as shown in Figure 5 to perform some more complex experimental function Each module of the program can be easily tried out to ensure that it is operational before proceeding with the next Presently CONVERS is being used in the authors laboratories for a variety of spectrochemical investigations including laser excited optoacoustic spectroscopy Figure 7 and inductively coupled plasma optical emission spectro scopy Figure 8 Rather complex inte
6. cur when V2 is switched V4 is used only when multiple samples are to be Table 1 Pneumatic control functions Valve Controlled function Not energised 0 Energised 1 Vo Sampling valve Inject V Needle assembly Down V5 Direction of air flow Fill V3 Air supply On Pressure equalisation Table 2 Control sequence for repetitive sampling Comment Needle enters vial Air on line purged Loop filled Air off hold loop full Injection with pressure equalisation End of injection Outer tube vented Loop emptied Pressure equalised Rest then return to start 135 Mills et al Automation of a liquid chromatograph Manifold Sampling valve Pressure controller Column Loop 100 cm volume Septum Sample Figure 2 valve ce Pressure controller 1T0OcmP Figure 2b Figure 2 Pneumatic layout of autosampler a needle enters vial b the loop is filled and c transfer of sample to column A Sampling valve actuator Rheodyne Inc type 70 01 A Syringe actuator H Kuhnke Ltd type 37291 Vo Spool valve H Kuhnke Ltd type 87 030 01 V1 V2 Spool valves H Kuhnke Ltd type 44 250 1 V3 V4 Solenoid valves H Kuhnke Ltd type 65111 taken from a vial without the removal of the needle assembly between samples When open it rapidly equalises the pressure in the two lines The functions controlled by these valves and their corres pond
7. e coding is performed by two read only memories and the display is updated at the real time cock frequency Data processing algorithm For quantitative chromatography it is necessary to measure the area of each peak The algorithms that may be used to calculate the areas of discrete or overlapping peaks generally belong to two classes 1 relatively simple empirical methods 2 more complex methods such as curve fittings 3 4 that assume the recorded shape or some mathematical model of a peak e g Gaussian or Lorentzian The choice of algorithm is determined by the response time of the system the accuracy required the total available memory size and the computing power available In chroma tography where the minimum peak width at half height will be 1 second we are normally confined to the first group of algorithms because of the limited amount of real time processing that can be carried out in the 100 msec intervals between acquisition of data points Curve fitting techniques only become practical with the aid of hardware arithmetic devices One of our microcomputers has been subsequently fitted with an arithmetic unit Advanced Micro Devices AM 9511 and the hardware can now perform floating point calculations approximately two orders of magnitude faster Volume 1 No 3 April 1979 Pump Y Set flow Fon d and display J Auto sampler Y Select Analyse required number of samples Data handlin
8. ere removed but a small amount lt 0 1 of anthracene was still detected The analyses of the blank samples were made at maximum sensitivity eight times more sensitive than for the sample By a small change in the program the number of rinses from the same vial was increased to three This successfully removed all traces of carry over into the blank sample Figure 6 illustrates the results obtained using this combin ation of purging and multiple rinsing A blank injection to demonstrate the cleanliness of the system was followed by an injection of a mixture of mesitylene pentamethyl benzene and fluoranthene After purging rinsing and purging again a second sample this time the benzene naphthalene anthracene mixture was injected No trace of contamination by the constituents of the first mixture which have different elution times to the constituents of the second mixture can be seen in the second chromatogram Conclusions The automatic liquid chromatograph described in this paper is a reliable and flexible instrument for both routine and research applications Its simple straightforward design and construction incorporating a high proportion of readily available components has resulted in low hardware costs while the flexibility of software control allows a good performance to be achieved We have shown that it is capable of repetitive sampling with a precision of 1 relative standard deviation and effective rinse and pu
9. f the measured peak areas for all components of the mixture of less than 1 1 These results represent the precision of the complete chromatograph it is worth noting that the manufacturers of the sampling valve only claim less than 1 for the valve alone As a test of inter sample contamination an injection of the hydrocarbon mixture was immediately followed by an injection of n hexane The system was not rinsed or purged between these samples It was found that about 8 10 of the original sample 1420 ppm benzene 82 0 ppm naphthalene 3 38 ppm anthracene was carried over into the second blank injection This experiment was repeated with the addition of a one minute purge of each sample line before 139 Mills et al Automation of a liquid chromatograph eee anas Pr E ALERT n t Sn tt in Se DAE ENTRA ACE sie jg m tk rp m n TA ef A VTE c e t EA OR a PE SEA NI T T ad a TOUS AEE A P P e pe T AN QR T a P i ata SP RR a t rs RAR a S ER A A the n hexane was sampled This reduced the carry over almost completely for the more volatile benzene constituent to 0 276 of the original concentration There was not sufficient removal of the naphthalene and anthracene however to 1 6 and 4 7 respectively The addition of a rinse vial containing pure mobile phase was then tried The complete cycle was therefore sample purge rinse purge blank With only one rinse between samples all measurable traces of both benzene and naphthal ene w
10. g required gt Noise measured Enter parameters Was sampler selected 23 Y New parameters Manual Injection Auto injection Print results Print results More samples Parameters good N Y Analyse Samples and print results Figure 4 System operation showing user interaction than is possible by software alone Arithmetic definition of the parameters involved requires a combination of 8 bit 16 bit and 24 bit integer or floating point fp arithmetic The first two options provide adequate accuracy to define retention times peak boundaries etc but are not sufficient for integrated areas where large numbers must be handled In this instance floating point routines can be incorporated bearing in mind that a single fp multiplic ation on the 8080A requires about 5 msec out of the total 100 msec available for real time processing The data handling procedure used in this project is shown in Figure 3 Data points are intially summed in order to restrict the number of points across a peak and so optimise 137 Mills et al Automation of a liquid chromatograph a PS SSS aS SC ESA SSR SS SE SE ES SPR SS SES EPS SS ECE STEPS TELTE ELL Y 3 NDGAGX x wx ENN ELLE Paha ls pes Ce Yi PI CO YOR OS CS SES CO CR nS SOC SSO SO SO PR SS e AUTOMATIC LIQUID CHROMATOGRAFH
11. handling Analogue to digital converter The signal from the detector Pye Unicam Ltd model The Journal of Automatic Chemistry Mills et al Automation of a liquid chromatograph interrupt every 100ms Signal from chromatograp Signal processing Peak detection Storage of peak Real time parameters Operations Calculation of peak areas Figure 3 Block diagram of chromatographic data handling LC3 UV is digitised using a voltage to frequency converter and 16 bit counter before being processed in the micro computer The signal input is buffered using a low noise low drift amplier which can also provide switchable gains of 1 10 and 100 The voltage to frequency converter operates at a full scale frequency of 1 MHz for 10 Vinput and has a usable dynamic range of over 10 The analogue input circuitry is completely isolated from the interface to the microcomputer by a high speed optocoupler driven directly from the converter The real time clock signal from the microcomputer latches the 16 bit count into a register resets the counter and gates the data from the register onto the data highway as two 8 bit bytes on receipt of the device select signals The counter continues incrementing until receipt of the next real time clock pulse when it is again reset A row of light emitting diodes on the front panel allows the 16 bits of data to be shown as true binary information or as a baragraph display Th
12. he vials are contained in a circular rack mounted on a turntable which can be rotated to any position in either direction by a stepper motor Philips type 9904 112 04002 controlled by an integrated circuit SAA 1027 The sample is introduced at the head of the chroma tographic column by a six port loop valve Rheodyne Co model 70 10 rotated by a pneumatic actuator The sample is transferred from the vial to the loop valve through a concentric needle assembly which is lowered by a syringe actuator piercing the septum seal into the sample vial Compressed air or an inert gas is connected to the outer tube thus pushing some of the sample through the inner needle to the sample valve Figure 2 is a schematic diagram illustrating these pneumatic operations Valves Vo V1 V2 V3 and V4 are all operated by 24 V d c solenoids and are two position valves which allow the use of a binary control sequence Vo controls actuator A which operates the sampling valve Vj operates cylinder A2 which moves the needle assembly V3 allows air to pass into the sample transfer system V will switch this air supply either to the inner needle to purge the sample line or to the outer tube to take up the sample The flow rate of gas through these paths is controlled by a pressure regulator Watts Ltd type 362 1 and a restrictor 400 mm x 0 25 mm i d A volume of 100 ml is included between the pressure regulator and V7 to absorb any surge in pressure which may oc
13. his own analytical systems on the instrument and prepare his own programmed cassettes Description The Kem O Mat consists of three modules in the air bath version Figure 1 and four modules in the water bath version The air bath version was evaluated in this study Its three modules are a control module to which is fitted the analyser module while the calculator is connected to the control module by electric cable The control and analyser modules together occupy 590 x 600 mm of bench space The calculator requires 550 x 365 mm of space A single 99 132V or 198 264V 50 Hz mains socket supplying 550 VA is the only service required The control module contains most of the operational logic the pump mechanisms and a LED display of the The Journal of Automatic Chemistry International Journal of firent Chemistry International Journal of Carbohydrate Chemistry The Scientific World Journal Organic Chemistry International X International Journal of Analytical CIUS S A m Advances in Physica Chemistry Hindawi Submit your manuscripts at http www hindawi com Journal of Spectroscopy Journal of l Theoretical Chemistry International Journal of Inorganic Chemistry d a Fa i E Journal of Hindawi Publishing Corporation http www hindawi com Volume 2014 International Journal of Fectrochemistry Chromatogra
14. ing bit states when not energised 0 and when energised 1 are summarised in Table 1 The autosampler and turntable are operated by an eight bit control word transmitted from the microcomputer via an interface circuit Bits 0 to 4 inclusive switch the solenoid valves in the pneumatic control system Each bit is used to switch a relay 131A 4 which in turn switches the corres ponding solenoid valve Bit 0 controls valve Vo etc The 136 valve Restrict d nt Pressure controller D Colum Loop 1OO cm volume Figure 2a valve HA Pressure controller 10cm volume Figure 2c remaining bits of the control word 5 7 are connected to the three inputs of the SAA 1027 integrated circuit used to control the stepper motor Bit 5 which is connected to the trigger input is pulsed to step the motor Bit 6 is connected to the set input an option which is not used in this application Bit 7 is connected to the input which determines the direction of rotation 1 anticlockwise 0 clockwise By transmitting an appropriate timed sequence of control words any one of a variety of operational modes can be effected for example multiple samples can be taken from a single vial or a number of different consecutive samples can be analysed Optional purge and rinse routines can be called to overcome inter sample contamination Table 2 shows a typical control sequence to sample repetitively from a single vial Data
15. k without further attention until the required number of samples has been analysed Performance of the auto sampler The two most important performance characteristics of the auto sampler are its precision and the extent to which one sample is affected by residual contamination from the previous sample carry over In order to test these char acteristics mixtures of benzene naphthalene and anthracene in n hexane were analysed The separation column 250 mm x 4 8 mm i d was packed with Lichrosorb SI 60 B D H Ltd n hexane was used as the mobile phase and the wave length of the UV detector was set to 254 nm Two modes of automatic sampling were investigated a repetitive sampling from one vial b sampling the same mixture contained in consecutive vials The initial results in both modes were unsatisfactory the relative standard deviations of the peak areas obtained were high especially when sampling from consecutive vials The answer to these problems was found in simple modific ations to the software An overall improvement was made by the addition of a procedure to pre wet the transfer tubes with sample before the final sampling operation The use of a purge before the consecutive vial sampling routine to drive out the residue from the previous sample improved the results for this mode of operation After these changes were made the analysis of ten samples in each mode gave in both cases a relative standard deviation o
16. n report of the analytical results with accurate peak area measurements even for peaks which are poorly resolved Although liquid chromatographs incorporating micro processors for control and data handling purposes are commercially available these instruments are so far relatively inflexible This paper describes in detail the automation of a liquid chromatograph using an inexpensive general purpose microcomputer which has previously been applied in atomic absorption spectrophotometry 1 and for column switching in HPLC 2 Figure 1 illustrates the interconnection of the chromato graph and the microcomputer which controls the mobile phase flow rate operates an automatic sampler and analyses data from the detector The control and data handling functions are integrated in a program which enables the user to communicate with the instrument in plain English via a visual display unit VDU or teletypewriter keyboard A variety of operational modes is offered giving the analyst an opportunity to establish the best conditions for a partic ular separation before leaving the instrument to perform its given tasks without further interaction The microcomputer Hardware The computer is a general purpose instrument constructed using a set of ready made circuit cards Philips Science and The Journal of Automatic Chemistry Mills et al Automation of a liquid chromatograph CPU Real time clock Interrupt control Column
17. onitor resident in EPROM Some aspects of the control and data handling programs will be described in later sections in general the control programs were written in assembly language the data handling program in Coral 66 A 24 bit software floating point mathematics package was used for multiple precision arithmetic The complete program occupies approximately 8K bytes of memory Pump control The pump used in this instrument Pye Unicam Ltd model LC3 XP is a precision metering device with its own sophisticated electronics for constant flow control The flow rate is normally selected using front panel switches but can be set using a signal to a remote control socket at the rear of the pump It is this latter option which is used in this applic ation A purpose built interface converts a 14 bit control word into the appropriate pulse width modulated signal to Volume 1 No 3 April 1979 control the pump In this way a potential precision of better than 0 001 ml min can be achieved The required flow rate can be typed in from the keyboard as a decimal number or the flow can be programmed to change during a run A maximum pressure for the system can be selected before a run In the event of this pressure being exceeded perhaps because of a blocked column the pump will be switched off Automatic sampler The automatic sampling system can handle up to twenty four samples each in a glass vial sealed with a bonded silicone PTFE septum T
18. phy Research International Journal of Quantum S Hindawi Publishing Corporation http www hindawi com Journal of d Analytical Methods e in parens Bioinorganic Chemistry and Applications
19. ractive control and data acquisition programs have been easily implemented Memory requirements and operating speed have been found to be far superior to conventional approaches Additionally new system users have encountered a difficulty in utilising previously developed custom software for a particular experiment even when documentation was vague Discusston The authors hope that this short introduction to only a few of the concepts employed in CONVERS will generate interest in its capabilities A much more complete discussion is available in the form of a user s manual 3 available from the authors ACKNOWLEDGEMENTS The development of the CONVERS system was partially supported by the Office of Naval Research and a Alfred P Cloan Foundation Research Fellowship to M Bonner Denton REFERENCES 1 C Moore Astronomical Astrophysics Supplemental 15 1974 497 2 M B Denton J D Mack M W Routh and D B Swartz American Laboratory 8 69 1976 3 CONVERS An Interpretive Compiler developed by Scott B Tilden and M Bonner Denton Department of Chemistry University of Arizona Tucson Arizona 85721 USA pd The use of a microcomputer for flexible automation of a liquid chromatograph A D Mills I Mackenzie and R J Dolphin Philips Research Laboratories Redhill Surrey RH1 5HA U K Introduction Microprocessors are being used to add inexpensive automatic control and data handling facilities to a varie
20. rge routines can be readily constructed to ensure that all traces of prior contaminants are removed from the system The accuracy of the data handling algor ithm ensures the integrity of the quantitative results even for those samples which are poorly resolved We have demonstrated its use with procedures which would probably satisfy most users one of the outstanding advantages of software control is of course the ease with which procedures can be adapted or supplemented in order to cope with new tasks This is exemplified by the column switching application 2 which uses a peak detection algorithm developed from the data handling program used in this general purpose liquid chromatograph REFERENCES 1 Willmott F W and Mackenzie I Analytica Chimica Acta 1978 103 401 2 Willmott F W Mackenzie I and Dolphin R J in Schomburg G and Rohrschneider L Editors Chromatography 1978 Elsevier p 151 3 Brouwer G and Jansen J A J Analytical Chemistry 1973 45 2239 4 Anderson A H Gibb T C and Littlewood A B Analytical Chemistry 1970 42 434 5 Savitzky A and Golay M J E Analytical Chemistry 1964 36 1627 6 Steiner J Termonia Y and Deltour J Analytical Chemistry 1972 44 1906 An evaluation of the Kem O Mat programmable discrete analyser Geoffrey C Seymour Division of Clinical Chemistry Northwick Park Hospital and Clinical Research Centre Harrow Middlesex HA1
21. s ogee cons es gece 1 ees caes osso osos ase oos oec osi t eooo 1235 3 142 4 179 5 263 7 Figure 5 A listing of the dialogue between computer and user 138 eese esos osse 0000 tars 0005 eese beos 9400 osos core eses esee ocos ose oros oss aeos osoo ssa sooo t so RET TINE SENSITIVITY IS ANY PARAMETERS L B 118 1356 171 274 ee seee eese c9 90 toos C008 sose oeeo sone sees cot eese ooo wees oce t N p n 9 AVAILABLE 7 y 148 187 p 292 aee c s tn J B 128 XAREA 20 AREA 3 a 04 2 600 33 99 20 27 ees 9s 9 99 poe oo oss t aroa Duro Cars boni obeo esie base oses ote asii oore Gose poss DUI soi o 04 ooe ooie da c bte ves 30 00 ea Gl 30 62 wt x The Journal of Automatic Chemistry Mills et al Automation of a liquid chromatograph Rinse purge and rinse 6 4 Minutes Figure 6 Consecutive analyses showing the effectiveness of the auto sampler cleansing routine Identification 1 Mesitylene 2 Pentamethylbenzene 3 Fluoranthene 4 Benzene 5 Naphthalene 6 Anthracene the peak width with respect to the subsequent digital differ entiation The signal is then smoothed by a digital filter of the form described by Savitzky and Golay 5 6 Peaks are detected by comparison of the first differential of a signal with a preset threshold value Retention times boundaries and integrated signals are stored for each peak and output at
22. ty of chemical instruments With a microcomputer it is now possible to realise the flexibility formerly available only with a relatively large and expensive minicomputer in an instrument little different in size and cost from one controlled by inflexible hardware In many ways chromatography is an ideal process for such automation Most instruments are given a high workload and although many applications may be routine and repetitive the versatility of the technique requires an instrument which can easily be used in a variety of modes In addition to improving the convenience to the user automation of a liquid chromatrograph should enhance the performance of the instrument Some aspects of high performance liquid chromatography HPLC which can benefit in this way are as follows 1 Accurate control of solvent flow rate will compensate for changes in pressure drop and lead to more reliable retention times 2 The composition of the mobile phase can be accurately controlled in either isocratic or gradient elution chrom atography using for example a proportioning valve on the low pressure side of the pump 3 Automatic sampling can be operated in a variety of modes to process a number of samples without super vision It is also more precise than manual injection Present address Pye Unicam Ltd York Street Cambridge CB1 2PX U K 134 4 A built in data handling facility can present the analyst with an easily read post ru
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