A New Approach to Automating an Analytical Gas Chromatograph P. B. Stockwell and R. Sawyer Laboratory of the Government Chemist, Cornwall House, Stanford St., London, S W I , figland Some routine laboratories use gas chromatography extensively and in order to meet a specific requirement in our own laboratory a fully automatic liquid injector has been designed. This has resulted in the development of an automatic gas chromatograph which handles discrete samples and mixes them with internal standard and controls the gas chromatograph. The injection system is a modification of one designed for preparative use. This requires that a slug of sample/standard mixture is forced through a capillary onto the column using an overpressure of the carrier gas. A typical application and some results are given.
A FULLY AUTOMATIC gas chromatograph is a potentially valuable instrument in any laboratory carrying out many repetitive analyses. An outline specification for such an instrument requires the inclusion of the following features; it should handle discrete samples, mix them with internal standard where necessary, control the gas chromatographic conditions, calculate the results, and present them in a form suitable for reporting. Currently available automatic gas chromatographic units have been reviewed, with particular reference to the requirements of some of the analyses carried out in our laboratory. A paper has been published, which summarizes conclusions on the applicability of available commercial units ( I ) to these problems, together with proposals for the design of instruments for specific purposes. Three potentially useful systems have been described and this publication gives a detailed description of a system developed for liquid samples when not less than 2 to 3 ml of sample is available for analysis. In our laboratories, samples of tinctures and essences are analyzed for ethanol content by a manual gas chromatographic technique (2). The amount of work involved in this analysis is such that a fully automated analytical system was considered to be desirable. Thus the preliminary design studies for an automatic gas chromatograph have been aimed at this particular application. Complete automation with computer compatible output has been achieved and will form the basis of a further publication. A patent application for the equipment described has been filed in the U.K. (3). Development of the System. The system can be represented by the schematic block diagram shown in Figure 1. Techniques of mixing and dilution using continuous flow principles are well documented. Initially the main area of our experimentation and development was centered on the sample to gas chromatograph interface, since this seemed to present the greatest problem in producing a satisfactory unit. Designs for two types of interface had previously been published, first those using sliding valve systems, notably due to Kipping and Savage (4) and Evrard and Couvreur (5); second, one using a mechanically operated micro-syringe (1) P. B. Stockwell and R. Sawyer, Lab. Pract., 19, 279-284 (1970). (2) J. R. Harris, Analyst, 95, 158-164 (1970). (3) P. B. Stockwell and R. Sawyer, U. K. Patent Application 10550/69. (4) P. J. Kipping and G. A. Savage, Reprints to 7th Int. Symposium on Gas Chromatography and Its Exploitation, 1968, paper 17. (5) E. Evrard and J. J. Couvreur, Chromatogr., 27, 47-53, (1967).
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which formed the basis of the recently introduced HewlettPackard range of automatic gas chromatographs. In the sphere of preparative gas chromatography however, a novel injection interface has been designed by Heilbronner (6). This was later used by Boer (7) in his automatic preparative unit which is now marketed by the Pye Unicam (U.K.) group of companies. Basically the system requires that a slug of sample to be injected is forced by an overpressure of carrier gas onto the top of the column. In the preparative gas chromatograph, the sample is held in a sealed container and injected repetitively onto the column as each preceding injection is completely eluted. A diagram of the set up for preparative chromatography due to Boer is shown in Figure 2. By modification of the injection vessel and subsequent changes in the overpressure and the capillary system, a suitable unit for analysis of discrete samples has been devised. The size and shape of the vessel and the other important parameters have been determined by experiment. Detail of the overpressure system and the injection vessel will be discussed later. The sample quantity injected V , as a function of time, is obtained from the Hagen-Poiseuillelaw :
where d L
internal diameter of dosing capillary dosing capillary length y = dimension factor 17 = sample viscosity p s = overpressure p c = column pressure = =
The overpressure is determined from (ps - pc), a function of the vapor pressure of the sample since the pressure drop is offset by a back pressure developed by the vaporized sample. In these experiments the quantity of sample injected has been minimized so as not to overload the analytical columns. This quantity, for constant viscosity, varies almost linearly for injection times of 2.5 to 5 seconds. For a fixed injection time, the quantity of sample injected varies proportionally to the fourth power of the diameter of the capillary and inversely to its length and to the sample viscosity. For continuous running in an automatic gas chromatograph, d and L are constant and therefore for a fixed injection time with a constant overpressure the quantity injected is limited only by sample viscosity. In routine analysis of a range of, for example tinctures and essences for ethanol content, wide variations of sample viscosity are experienced. In order to obtain an acceptably constant viscosity, it is necessary to mix the samples before injection, with sufficient internal standard mixture to swamp these variations, and this requirement must be met in the design of a satisfactory unit. An advantage of the injection system is that the capillary is flushed constantly with hot carrier gas, thereby reducing any memory effect between samples to an insignificant amount. The injection vessel has been (6) ~, E. E. Heilbronner. E. Kovats, and W. Simon, Helu. Chim. Acta., 40,241 (1957). (7) H. Boer, J. Sci. Instrum., 41, 365-369 (1964).
ANALYTICAL CHEMISTRY, VOL. 42, NO. 11, SEPTEMBER 1970
sample-GC interface I 7
sampler held on turn t a b le
1
1
transport mixing Internal rtd.
i
integrator
1 I
recorder
Figure 1. Block diagram of basic system
constructed in glass and is a scaled down modification of that used in preparative chromatographs. Jentzsch (8) experienced variations of injection size because of changes in liquid level in his preparative unit. In practice the liquid in our unit is brought to an approximately constant level before injection and when the overpressure is applied the liquid level does not fall below the inlet capillary tubing. Experimental Assessment of the Interface Unit. In our laboratories samples of medicinal compounds are examined for their ethanol content by a manual gas chromatographic method using an internal standard. The peak areas of ethanol and propan-1-01 are measured using a Honeywell integrator (Brentford, Middlesex), and it was considered that this analytical method would be suitable for a feasibility study. A bulk sample and internal standard was premixed and placed in a container. A probe was lowered into the container and the solution pumped into the injection/collection vessel and allowed to drain to waste uia a pressure restrictor; this provided a continuous flow of solution through the apparatus. A solenoid valve controlling the waste outlet of the injector vessel was closed so that a column of liquid was collected in the vessel. The valve was opened and the vessel was allowed to drain, the valve was again closed and the collection procedure repeated. (8) D. Jentzsch, J . Gas Clzromatogr., 5, 226-231 (1967).
A gas overpressure was then applied to the injector vessel for a set period, the length of which was controlled by a process timer, the overpressure was sufficient to ensure that an injection took place through the capillary. A wash out cycle in which the injection vessel was flushed with internal standard solution was also included in the sequences to simulate the conditions for the operation of a full analytical system. The unit was allowed to operate unattended for 12 hr and 47 injections were made from the same liquid mixture and the results are summarized in Table I. These results show that the repeatability of the ratio of peak areas is within an accepted level of accuracy for the integrator used. A mixture of water, ethanol, isopropanol, and normal propanol was injected in these experiments. From the preliminary study, it was considered that the prototype interface shown in Figure 3 formed a viable system for further work. A complete system to monitor samples, admix with internal standards, transport to the mixing vessel, to inject them, to allow the chromatograph to elute, and repeat the cycle continuously has been constructed in a working prototype form; this is described below. Complete Automatic Gas Chromatograph, It is convenient to consider the automatic gas chromatograph as consisting of five integral parts: Sampler, Sample mixing and transport system, Injection vessel and gas chromatograph interface, Overpressure system, and Electrical control and timing unit. SAMPLER.Five milliliters of samples were held individually in standard (15-mi) test tubes and sealed with parafilm (Gallenkamp, Christopher Street, London, E.C.2, England). The sampler has a probe which is able to pierce the caps; in operation the cap was pierced in such a manner as to split the parafilm open and to prevent a seal between the cover and the probe since under these latter conditions a vapor lock was generated in the interconnecting pump tubing and imprecise pumping of the sample occurred. An auto sampler marketed in the U.K. by Evans Electroselenium Limited (Halstead, Essex, England) has been modified to form a suitable sampler for the gas chromatograph. The probe system has a horizontal movement fitted so that it may descend in two different locations, first into the sample tube and second into a reservoir containing internal standard solution. Sample tubes were held in detachable quadrant racks, each holding 12 tubes, and the full circular turntable holding 48 tubes. The movement of the turntable was controlled by a search and find mechanism operating a photosensitive diode switch. When a sample tube
-
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injection
-bml
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Figure 2. Injection device (Boer)
refill
C1, C2 capillaries; P1, 2, 3, pneumatically controlled
on-off valves; V,needle valve
injection port column buffer
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Table I. Summary Normalized count for ethanol (C = 6000) 2862 2862 2862 2873 2834 2876 2876 2887 2892 2884 2870 2872 2862 2855 2859 2893 2876 2889 2864 2880 2877 2886 2866 2865 2881 2879 2888 2878 2895 2862 2876 2867 2872 2997 2977 2873 2890 2875 2862 2883 2880 2874 2886 2862
Ethanol (A) 2826 2709 2743 2944 3075 3042 3029 3053 3000 3006 3129 3093 3118 3108 3038 2941 3015 2881 2944 2883 2895 2867 291 1 2972 2844 2839 2854 2803 2772 2795 2839 2867 2916 2828 2810 2692 2964 2789 2760 2811 2816 2733 2745 2845
Integrator counts Propan-2-01(B) 4292 4047 4103 4418 4623 4532 4517 4581 4457 4484 4738 4657 4730 4698 4555 4394 4531 4314 4439 4316 4307 4280 4300 4467 4250 4234 4197 4188 4116 4152 4255 4300 4360 4209 4193 401 1 4129 4143 4123 4197 4202 4055 4095 4315
Propane-1-01 ( C )
Ratio, BjC
Ratio AJB
5859 5678 5714 6148 6425 6346 6318 6341 6224 6253 6541 6462 6537 6531 6375 6098 6288 5984 6167 6005 6037 5965 6094 6624 5922 5917 5887 5878 5745 5859 5922 6000 6091 5877 5861 5621 5738 5820 5787 5849 5866 5705 5697 5963
73.255 71.275 71.806 71.861 71.953 71.415 71.494 72.244 71.575 71.710 72.435 72.067 72.357 71.934 71.451 72.056 72.058 72,076 71.979 71.873 71.343 71.752 71.218 71.736 71.766 71.557 71.293 71.249 71.645 70.865 71.850 71.667 71.581 71.618 71.541 71.357 71.959 71.186 71.246 71.756 71.633 71.078 71.879 71.362 0.419
48.23 47.71 48.0049 47.885 47.859 47.935 47.942 48.147 48.200 48.073 47.837 41.864 41.697 47.558 47.655 48.228 47.948 48.145 47.737 48.035 47.954 48.064 47.768 47.751 48.0243 47.980 48.140 47.686 48.251 47.704 47.939 47.778 47.834 48.120 47.944 47.892 48,170 47.921 47.793 48.059 48.005 47.905 48.183 47.711 0.177
Standard deviation was advanced into the samplingposition, the mechanism caused the turntable motor to stop. Positions on the tray which do not contain a sample tube were ignored since the mechanism was not operated and the turntable motors to the next tube. After finding a tube the motor was not activated again until the full cycle has been carried out. A drip feed reservoir of internal standard was mounted at the centre of the turntable as shown in Figure 4 ; this solution was pumped through the mixing system during the “Wash” phase. At the start of the cycle, the probe was lowered into the sample tube piercing the parafilm cap. Sample was withdrawn and mixed with the internal standard solution, supplied by a second tube, and finally monitored into the injection/collection vessel. After injection the probe was raised from the sample by a microswitch which overides the drive motor control. A solenoid mechanism was used to move the probe horizontally over the internal standard reservoir and the probe was lowered into this to provide a washout sequence for the collection/injection vessel and associated pump tubes. At the end of the washout period, the probe was raised and returned to the test position. The operation of the probe, 1138
turntable unit, and valve was controlled by a simple cam timer and microswitches. The electrical control unit was sited in the instrument housing of the turntable mechanism. SAMPLEMIXINGAND TRANSPORT SYSTEM. For reasons elaborated in the discussion section, a pumping and mixing unit using the peristaltic principle was adopted and this is shown in Figure 5 . A Technicon 15-channel peristaltic pump with Solvaflex pump tubes was used. When the sample probe was in a sample tube, sample, air, and internal standard were pumped through their respective tubes and into a mixing coil. The design of this mixing coil was similar to that described by Shaw and Duncombe (9). The mixed stream was then collected in a vessel with two outlets controlled by a three port two-way Trist Lucifer control valve (No. 33A05510). In the normal mode the vessel was drained by two pump tubes connected in parallel but when the valve was operated, [position (b), Figure 51 liquid was drawn into the collection/injection vessel. The rate of pumping into this vessel was greater than (9) W. Shaw and R. E. Duncombe, Ann. N . Y . Acad. Sci., 130, 647-656 (1965).
ANALYTICAL CHEMISTRY, VOL. 42, NO. 11, SEPTEMBER 1970
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Figure 3. Preliminary set-up
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solvent
iu
waste
the rate of withdrawal so that an amount of mixture accumulated in the vessel. When a sufficient volume had been built up, the valve was switched [position (a) Figure 51 and the liquid was drained from the injection unit. The cycle was repeated to build up a representative sample in the vessel for injection. An overpressure was then applied and sample mixture was injected onto the column. After injection the control valve reverted to the open position Figure 5 (a)and the probe was removed from the sample. A solvent washout procedure closely followed the sampling cycle. In this case the probe was positioned in the internal standard reservoir. Pump tube sizes for a typical manifold, with addition of internal standard, washout facilities, and injection onto a gas chromatograph column are shown in the diagram. INJECTION VESSELAND GASCHROMATOGRAPH INTERFACE. The sample injection vessel (10 ml capacity) was constructed in borosilicate glass as shown in Figure 6 . Simplifix couplings modified to fit the size of glass and tube used were fixed into position with “Araldite” to form gas tight seals. The injection port of a conventional gas chromatograph has been replaced by a modified Simplifix compression coupling. The coupling has a 1.5-mm stainless steel tubing, brazed into the side, and it has been drilled so that 3-mm tubing can be positioned opposite this inlet. The 1.5-mm tube forms the carrier gas inlet. A capillary tube from the injection vessel and the top of the column are positioned in the coupling to give the minimum dead volume without inhibiting the flow of gas through capillary and column. A stainless steel capillary, 0.5-mm i.d. x 3-mm o.d., was used and a 0.375-mm nichrome wire restrictor was inserted into the capillary to obtain injections compatible with 3-mm 0.d. columns. OVERPRESSURE SYSTEM.A diagram of the overpressure system is shown in Figure 7. The column inlet pressure and overpressure are all controlled using Negretti Zambra pressure regulators. Nitrogen carrier gas was fed into the “T” at the column inlet from the “buffer tank” with a volume of 500 ml. Normally an overpressure was allowed to vent to atmosphere via the solenoid valve V Iand a restrictor R1. A back pressure was maintained in the injection vessel through valves V, and V B the , “buffer” Bz and the restrictor Rz. The size of Rzwas adjusted to limit the bleed of the carrier gas into the sample injector system. On injection Vr,Vz,and V , operated for “t’’ seconds (2 to 2.5 sec), this had two effects; first, an overpres-
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Figure 4. Sampling probe and internal assembly Probe mechanism Turntable assembly Samples D. Internal standard
A. B. C.
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289
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