EP0036000A4

EP0036000A4 - Continuous flow automatic chemical analysis systems and components for use therein.

Info

Publication number: EP0036000A4
Application number: EP19800901707
Authority: EP
Inventors: Patrick Duffy
Original assignee: PRINCE CHARLES HOSPITAL DEVELOPMENT CENTRE TRUST; PRINCE CHARLES HOSPITAL DEV CT
Current assignee: PRINCE CHARLES HOSPITAL DEVELOPMENT CENTRE TRUST; PRINCE CHARLES HOSPITAL DEV CT
Priority date: 1979-09-19
Filing date: 1981-04-08
Publication date: 1983-08-03
Also published as: JPS56501383A; AU522965B2; AU6393680A; ATA903180A; WO1981000911A1; EP0036000A1; AT377493B

Abstract

A colorimeter including a phototransistor (16) a light source (18) of appropriate wavelength, a red blocking filter (20) for blocking light of wavelength greater than 660 nm, the filter being interposed in the optical path between the light source and the phototransistor, and a cell or cuvette (1) adapted to receive a sample liquid to be analysed also interposed in the optical path. An air segmented continuous flow automatic chemical analysis system including phase separation apparatus which comprises a membrane filter (52) clamped between two members (50, 51) each having surface grooves (56, 57) in opposed faces thereof which constitute together with the membrane filter ducts for the passage of liquid. One duct is for the passing through of an air segmented liquid sample stream and another duct is for the passing through of an air segmented liquid recipient stream. There is also provided pumping means (70) for pumping the sample stream through the phase separation apparatus and before discharge. The invention also relates to an automatic chemical analysis module for use with the above mentioned system and heating bath for use with the system.

Description

"CONTINUOUS FLOW AUTOMATIC CHEMICAL ANALYSIS SYSTEMS AND COMPONENTS FOR USE THEREIN"

THIS INVENTION is concerned with improvements in and relating to continuous-flow, automatic, chemical analysis systems.

The invention is especially concerned with modifications and improvements to components of continuous-flow analysis modules to enable them to effectively function in enzyme immunoassay systems and thereby to allow the construction of continuous flow analysis systems capable of: (a) The detection and quantification of antigens for which antibodies may be developed in animals by whatever means by continuous-flow, non-homogeneous enzyme immunoassay (NHEIA) methods.

(b) The detection and quantification of antibodies developed in host animals of whatever agent of continuous- flow, non-homogeneous enzyme immunoassay methods.

(c) The detection and quantification of micro¬ organisms or parasites by use of the intact micro-organism or products or components of the micro-organisms as appropriate components in continuous-flow non-homogeneous enzyme immunoassay systems.

Utilization of the basic system to provide for chemotherapeutic sensitivity determination of the micro¬ organisms is also envisaged. The contiguous-flow analysis module of the invention involves the following novel components or improvements, each of which individually constitutes a separate aspect of the invention: - (1) A phototransistor-based colorimeter. (2) A solid state oil heating bath. (3) A phase separating system.

These components may be integrated in a discrete analysis module, which constitutes a further aspect of the invention. Each of theseaspects will now be discussed and -2- ^' - - elucidated in detail.

(1) The phototransistor-based colorimeter.

The use of phototransistors in colorimeters with light-emitting diodes (LED's) providing red light at wavelenghts of 660 nM or greater has been reported.

The use of phototransistors with LED's at wavelengths less than 660 nM has not been reported to our knowledge, nor have phototransistors been used in colori¬ meters using light sources with a continuous spectrum and narrow- or broad-banded isolation filters. The reason fo this appears to be the loss of sensitivity which results from the use of phototransistors at these lower wavelengh e.g., from about 340 nM upwards. We have determined that the cause of this loss of sensitivity is leakage of light of wavelength greater than 660 nM from the lower waveleng LED's or through the filters used with continuous spectru light sources. This leakage of higher wavelength togethe with the increased sensitivity of phototransistors at the higher wavelength results in poorer performance. We have now found that the use of a red light blocking filter which blocks light of wavelength greater than about 660 nM in front of the phototransistor allows the development of a practical colorimenter utilizing low cost phototransistors as light transducers. Normally the red light blocking filter is coloured blue. Specifically we have found that a blue filter consisting of a copper sulphate solution of appropriate colour density provides a highly efficient filter for this purpose. Conventional blue glass blocking filters are not generally satisfactor as they allow leakage of light at wavelengths below about 900 nM. The copper sulphate filter does not leak in this way and has an additional advantage in that the optical density of the filter can be easily adjusted by varying the concentration of the solution. The use of the copper sulphate filter allows -3- . - construction of a very low cost colorimeter when the photo¬ transistor is used as part of an appropriate electronic circuit,

Broadly, therefore, the colorimeter of the invention comprises a phototransistor, a light source of appropriate wavelength, a filter comprising a solution of copper sulphate interposed in the optical path between the source and the phototransistor, and a cell or curvette adapted to receive a sample liquid to be analysed also interposed or interposable in said optical path. The LED may be replaced by any other suitable light source, for example a tungsten lamp which emits a continuous spectrum. In this latter case, it may be desirable or necessary to provide further light filtration in the form of an isolation filter, which may be of the narrow- or broad-band type and which is selected according to the wavelength required for the particular analysis.

Such a colorimeter may be used, for example, as the colorimeter in a continuous-flow, automatic chemical analysis system in which a continuous stream of the sample liquid passes through a flow-through type cell . More especially, the flow system involves the continuous passage through the cell of individual slugs of sample liquids separated by gas bubbles, which also pass through the color¬ imeter. The amplified output of the phototransistor when combined with electro! ic or computerised methods allows removal of the interfering electrical signals resulting from passage of gas bubbles through the colorimeter. Thus, for example, the phototransistor may be connected to an appropriately designed electronic amplification device, the output of which is fed directly to an analogue computer system, or to a second electronic device designed to function as a peak sample and hold volt-meter. The output from this latter device may then be fed to a suitable computer device for further processing or may be fed directly to an appropriate chart or similar recorder.

fREA - - - -

The computer device just referred to may consi for example, of a microprocessor holding appropriate programmed instructions to provide for the conversion of the electronic signals to a voltage signal proportional 5 to the amount of colour present in the colorimeter. The programmed instruction set might be organised, for example, to allow direct comparison of this colour signal with the concentration of a particular analyte measured in a continuous flow automatic chemical analysis system.

10 (2) The solid state heating bath.

_~, This device comprised_ the combination of a meta preferably aluminium, containing vessel adapted to contain oil or any other suitable heat-transfer fluid, a heating circuit comprising one or more power transistors

15 and resistors which are in good thermal contact with the container, thereby to provide heating of the vessel and, hence, of its contained fluid.

Preferably, the transistors and/or resistors ar recessed into the external wall or walls of the vessel.

20 Control of the temperature of the block and the oil is achieved with a thermistor or other like thermal electric sensor appropriately placed in the fluid bath which controls the energy supply to the heating circuit by means of suitably designed electrical or electronic

25 control circuitry. Stirring to ensure even temperature in the fluid bath may be provided, for example, by means of a small electric motor driving a paddle wheel stirrer positioned to provide efficient stirring.

In use, an appropriately designed coiled glass

30 tube of the required length is positioned in the path so as to be immersed in the heat-transfer fluid and thereby to allow heating of a continuously flowing liquid stream which is passed through the tube, e.g. by action of an appropriate pumping device.

35. (3) The phase separating system. -5- The phase separating system according to this invention, is designed especially with a view to providing continuous flow separation of a non-homogeneous enzyme immunoassay solid phase from its liquid phase while still maintaining the integrity of the flowing streams necessary to allow the efficient performance of such immunoassays. The system is based on the continuous- flow dialyser modules known per se in the art. Briefly, such a module comprises a dialysis membrane clamped between two flat plates each having surface grooves in the opposed faces thereof which, with the membrane, constitute ducts for the passage of liquid. The ducts are thus superimposed and separated by the membrane to provide a dialysis cell. In the system of the invention the dialysis membrane is replaced by a membrane filter, e.g. of the "Millipore" type, having a pore size appropriate to the particular application. A standard sterilizing filter having a pore size of about 0.2 microns is often appropriate.

In the prior art dialysis module, the usual arrangement is to pump the sample stream from the preceding stage of the system through one of the ducts and thence to waste. The recipient stream is pumped through the other duct from which it passes to the next stage, the two flows being synchronised.

If such a system is used with the filter module of the present invention, however, all of the sample stream tends to pass across the filter membrane which disrupts the synchronousflow of the sample and recipient streams, and hence the integrity of the individual slugs of the sample and/or recipient liquids. Accordingly, in the present invention, the sample stream, after passage through the module and before discharge, is passed back through the pumping device with a pumping -6- volume selected by the user, thereby to maintain a closed system. Variation of the volume is achieved by, for example, varying the diameter of the pumping tube used in a peristaltic pump. By thus varying the volume of active pumped return stream from the module, a greater or lesser (as desired) amount of unbound enzyme-linked component from the non-homogeneous enzyme assay incubation mixture is passed across the filter to the recipient stream, in which the enzyme activity may then be subsequently measured. By way of example, if the sample stream rate is 1.0 ml/min and the return stream pumped at 0.5 ml/min. and the recipien stream entry flow rate is 0.5 ml/min, the above mentioned closed system will ensure that the recipient stream exit flow rate is 1.0 ml/min thus ensuring passage of 0.5 ml/min of filtered fluid from the sample to the recipient stream. Thus, on-line continuous-flow separation of the bound and free phases of the incubation mixture, which is essential for the performance of non-homogeneous enzyme immunoassay, is efficiently achieved. (4) Automatic chemical analysis module.

The above-described components- (i), (2) and (3) may be integrated into an automatic chemical analysis module in accordance with this invention.

In its simplest form such a module may comprise a suitable container, typically a box, which houses one or more of the solid state heating devices and are more of the phototransistor colorimeters just described, together with their associated electronic circuitry, which may include an integral micro-processor. Also associated with these components will be the necessary plumbing for the hydraulic system which typically consists of one or more coiled glass tubes which in use are immersed in the heating bath(s) and one or more dialysis or phase separation modules, together with appropriate interconnecting tubing. Conveniently, these components may be mounted on or suspende from (as appropriate) the lid of the box for ease of access, removal or replacement.

Such analysis module may then be utilized, in conjunction with the pumps and other anciliary equipment known per se in automatic analysis. By suitable design of the hydraulic system, the module may be used for a wide range of chemical analysis and the "bubble-through-the- colori eter" capabilities of the integral colorimeter provides for enhanced sample number throughput capability with this module.

By way of example, the following description shows the use of the module in a continuous-flow, non-homogeneous, enzyme immunoassay, specifically the analysis of dilantin in serum. An appropriate aliquot of serum is mixed with 1 ml of incubation mixture consisting of a drTantin-|3-gal actosidase derivative and Sepharose-bound, anti -dil antin antibodies. The continuously flowing stream at 1 ml per munute is segmented with air by an appropriate pumping device and is mixed on the surface of the module in a glass coil before entering a first heating bath at 37° for the time of passage of five minutes. On exit from this heating bath the stream flows to the top side of a phase separation module containing a 0.2 micron illipore type filter and is then returned to the pumping device where 1 ml per minute of liquid continues to be pumped. An air segmented 1 ml per minute recipient stream of buffer is pumped on to the lower side of the phase separating device and on exit from this device is joined by a 1 ml per minute continuous stream of para-nitrophenyl galactoside in buffer.. The stream is heated at 37° for five minutes in a second heating bath before the colour developed measured by passage through a colorimeter with a 405 nm filter. The electronic signal disturbances from passage of the air bubble through the colorimeter are removed by appropriate means and the resulting signal displayed on a chart recorder or alternatively handled by a computer system.

One of the aspects of the invention already mentioned is concerned with the detection and quantific- ation of antigens by continuous flow NHEIA methods. The essential requirements for the performance of this system are the following:

A(i) An antigen to which has been covalently coupled an appropriate enzyme suitable for use in an NHEIA. A(ii) An antibody directed against the antigen in

A(i) above which may be used directly, chemically modified for example to a Fab fragment, or covalently coupled to an appropriate solid phase e.g. a Sepharose particle. A(iii) An appropriately designed continuous flow NHEIA system module together with appropriate continuous flow analysis components.

In such a method, a suitable aliquot of the biological or other material containing the antigen required to be measured (or alternatively an appropriate standard containing a graduated known level of this same antigen) is mixed in a continuously flowing buffer stream containing suitable aliquots of A(i) and A(ii) above. After a suitable incubation period under appropriate conditions, during which the 1iqand-enzyme complex compete for binding sites on the antibody against possible ligand molecules derived from the sample, a portion of the unboun enzyme-1igand complex is separated on-line from the antibody. This separation is effected preferably by passa of unbound enzyme-1 igand complex across a porous membrane into a recipient stream. To provide for the separation, a solid phase of the coupled antibody enzyme-ligand derivatives is required. This may be obtained directly by previous coupling of the antibody to a solid phase, e.g. a Sepharose particle, or by subsequent addition to th flowing stream of a second antibody or binding material wh

- REAC

OMPI W1PO is directed against the first antibody and which is bound to a solid phase. The enzy e-ligand complex separated from the antibody-bound enzyme-ligand is then subjected to enzyme measurment techniques and this may include the use of the enzy ic cyclicising methods described in our copending Australian Patent Application no.

By comparison of the results from known levels of ligands with that resulting from the unknown test ligand, quantification of the test level of ligand is achieved. As an example the analysis of dilantin in serum may be carried out by mixing an appropriate aliquot of serum with 1 ml of an incubation mixture consisting of a dilantin-β-galactosidase derivative and Sepharose- bound anti-dilantin antibodies. The continuously flowing sample stream segmented with air by an appropriate pumping device, is mixed, e.g. in a glass coil, before entering a heating coil at 37 for the time of passage of five minutes. On exit from this heating coil the stream flows to a phase separation module containing a 0.2 micron millipore type filter and is then returned to the pumping device where 1 ml per minute, of liquid is discharged to waste. An air segmented 1 ml per minute recipient stream of buffer is pumped to the lower side of the phase separating device and on exit from this device is joined by a 1 ml per minute continuous stream of para-nitrophenyl galactoside in buffer. The stream is heated at 37° for five minutes before the colour developed measured by passage through a colorimeter with a 405 nm filter. In the detection and quantification of antibodies by continuous flow non-homogeneous enzyme immunoassay methods, in accordance with this invention, the essential requirements are the following: B(i) A purified antibody directed against a particular ligand from whatever source to which is covalently

-^TREΛ -10- ... ^{: '} .- ^• joined an appropriate enzyme suitable for use in a non-homogeneous enzyme immunoassay system and having an immunological specificity similar to that of the antibody which is required to be determined. B(ii) Appropriate components capable of binding to the antibody enzyme in B(i) above. This product must be capable of removal in a separation phase of a continuous flow non-homogeneous enzyme immunoassay syste and to this end the product may be linked chemically to a solid phase, for example, a Sepharose particle.

B(iii) An appropriately designed continuous flow non-homogeneous immunoassay system module together with appropriate continuous flow analysis components.

In use, a suitable aliquot of the biological o other material containing the suspected antibody or alternatively appropriate standard containing graduated known levels of this same antibody are mixed in a ^•continuously flowing buffer stream containing suitable aliquots of B(i) and B(ii) above, with for example, covalently bound Sepharose particles. After a suitable incubation period in an appropriately designed module during which enzyme antibody competes to bind the solid phase ligand with possible antibodies in an aliquot of the biological or other material, a portion of the unbou enzyme antibody is separated on-line from the enzyme antibody bound to the solid phase ligand by whatever means, for example by passage across a millipore-1ike membrane, into a recipient stream. The enzyme antibody so separated is measured by usual means, and this may include the use of the aforementioned enzymatic cyclic- i.zing methods. By comparison of the unknown enzyme rate with that resulting from known levels of antibodies quantification of the test levels of antibodies is achieved. Specifically, for example, to detect Brucella -11- antibodies of the Gamma G type, the following components would be used in the above system.

(a) Purified Brucella antibodies of the Gamma G class covalently joined to B-galactosidase. (b) Purified antigen from Brucella capable of combining with (a) and covalently linked to Sepharose particles.

(c) A suitable aliquot of serum from, for example, a cow, diluted in a buffer containing Sepharose particles to which are joined rabbit antibodies directed against bovine Gamma M for the purpose of removal of Gamma M specific antibodies which might otherwise interfere with the assay.

(d) A continuous flow analyser module as per previous patent and containing a mil1 ipore-1ike 0.2 micron membrane.

(e) An appropriate subststrate for B-glactosidase together with cyclicizing enzymes, for example, β-glactoside, p-catechol , laccase, and NADH.

In the detection and quantification of micro organisms or parasites by continuous flow NHEIA methods, in accordance with the invention, the essential require¬ ments of the performance of this system, are the following: (i) A highly purified specific antibody directed against an antigen on the organism of interest and this may be covalently bound to a solid phase are directly orbound indirectly by a solid phase second antibody. (ii) An enzyme antigen complex which is specifically bound at high affinity to the antibody in (i) above. (iii) An appropriately designed continuous flow NHEIA system module together with appropriate continuous flow analyses components.

In use, a suitable aliquot of the biological or other material to be tested for the presence of the organism is sampled directly or following an initial pre-incubation^' step which may include incubation of -12- - :. _ - — aliquots of the test material in the presence of anti¬ biotics or other anti-growth factors directed against the organism in question. The aliquot or alternatively appropriate standards containing graduated known levels of the antigen are mixed in a continuously flowing buffer stream containing suitable aliquots of (i) and (ii) above. Following this, there is a suitable incubation period in an appropriately designed module during which ligand enzyme competes for binding sites on the antibody against possible ligand molecules derived from the sample portion. These possible ligand molecules may be intact micro¬ organism particles or could be particles resulting from the on-line disruption of intact organism by whatever means. After this incubation period, portion of the unbound enzyme ligand is separated across a porous membrane in an appropriate separation module and into a continuous-flow recipient stream. The unbound enzyme activity is then subsequently measured by established methods which may include the aforementioned enzyme cyclizing techniques.

The invention, in its various. aspects, will be further described and elucidated by reference to contained preferred embodiments.

Reference is made to accompanying drawings, in which:

FIGURE 1 is an exploded view of the colorimeter of the invention;

FIGURE 2 shows the heating bath;

FIGURE 3 is the electronic heating circuit for the bath of FIGURE 3;

FIGURE 4 shows the phase separation module; and

FIGURE 5 shows the hydraulic circuit for the module of FIGURE 4 and the complete analysis module.

It should be noted that the dimensions cited in the following description are by way of example onl^'y and -13- are not to be construed as limiting on^' the invention.

The colorimeter shown diagram atically in FIGURE 1 comprises a block 1 of "Perspex" (or like material) 12 mm wide, 18 mm high, 15 mm long and is drilled horizontally through the centre with a hole 2 of 0.75 mm diameter. At angles from the top of the block 1 through to within 2 ram of this transverse hole 2, connecting holes 3, 4 are drilled at each end of the transverse hole. Connecting grooves 6, 7 are drilled on each surface to a depth of 0.45 mm to all connections between the horizontal and vertical hole. Grooves 8, 9 are milled outside the resulting holes at each end of the transverse hole to accept appropriate "0" rings 11, 12 and suitably shaped 1 mm thick glass end plates 13, 14 are compressed against the "0" rings by restraining blocks (not shown) to provide a leak-proof assembly. Suitable nipples (not shown) are fixed to the openings of the transverse holes (3, 4) on the top of the block. The assembly thus comprises a modified u-tube assembly. At one end of the horizontal hole 2 a suitable phototransistor 16 is positioned in an approp¬ riately aligned holder 17. To the other end of hole 2 a light emitting diode 18 or a tungsten bulb in a holder 19 together with a copper sulphate blocking filter 20 and, if required, an appropriately sized narrow band isolation filter 21 are positioned.

The copper sulphate filter 20 consists of a hollow glass or plastic cell containing a copper sulphate solution.

A stabilized electrical supply (not shown) is provided for the phototransistor or lamp 18 and the phototransistor 16 is connected to a suitable amplifier (not shown) the signal from which may be fed to a- recording or indicating device or to a computer for signal processing. The heating bath shown diagrammatically in -14- - V-^{" "^} FIGURE 2 comprises an aluminium (or other suitable) metal cylinder (30) 75 mm in diameter and 100 mm in height having a milled central cavity 31 of 63 mm in diam¬ eter to a depth of 94 mm. In the outside wall of the cylinder at regular intervals are milled grooves 32, 33 to allow placement respectively of two^' power transistors 34 and three heating resistors 36. (For clarity, only one of each component is shown in the drawing). The bath is provided with a lid 37 which supports a small electric motor 38 driving a central paddle wheel stirrer 39. The lid is provided with a small hole to allow a thermistor (not shown) to dip into the oil as well as two holes to allow placement of tubed glass coils (e.g., 41) of 50 mm diameter and of varying total length, in the bath.

Temperature regulation is provided by a solid state temperature controlling device which is capable of controlling the temperature in the oil contained in the bath over the range of ambient to 100° to an accuracy of 0.1°. The circuit of this device is shown in FIGURE 3.

The phase separation module is shown in FIGURE 4. It consists of two 50 mm square blocks 50,51 of "Perspex" or other suitable material, between which is sandwiched a sheet 52 of membrane filter material, e.g., a "Millipore" filter of 0.2 micron pore size. Other membrane filters of dialysis membranes may of course be used.

The inner faces 53, 54 of the blocks 51, 52, i.e. those faces which contact the membranes are provided with matching surface grooves 56, 57 which are basically U-shaped in plan. These grooves, which act as fluid passages in use, terminate at ports of passageways 58, 59, 61, 62 drilled between the opposed faces 53, 54 of the blocks and their respective end faces 63, 64. Fluid connections to the passageways 58 to 62 and hence -15- the grooves 56, 57 may be made by suitable nipples (not shown)

The hydraulic circuit of the phase separation system and the configuration of the whole automatic analysis module is shown schematically in FIGURE 5. The operation of the module will be explained by reference to the dilantin assay already described.

In FIGURE 5, the module consists of all of the integers enclosed by the dashed line. The other integer is a peristatic pump 70 of standard type, which as shown here has 7 pumping lines A to G. Operation of the system is as follows:

The incoming serum sample is pumped in through line B and is mixed with 1 ml of incubation mixture con- sisting of a dilantin-B-galactosidase derivative and

Sepharose-bound, anti-dilantin antibodies pumped in through line C. The continuously flowing stream at 1 ml per minute is segmented with air pumped in at line D and is allowed to mix in a glass coil 71 before entering a first heating bath 72 at 37° for the time of passage of five minutes. On exit from heating bath 72 the stream flows through to line H to the top of the phase separation module 73 and is then returned through line A to the pumping device where 1 ml per minute of liquid is discharged to waste. A 1 ml per minute recipient stream of buffer from line F, segmented with air from line E is pumped to the lower side of the phase separation module 73 and on exit (line I) from this device is joined by a 1 ml per minute continuous stream of para-nitrophenyl galactoside in buffer (line G). The stream is passed through a second mixing coil 74 and then to a second heating bath 76 where it is heated at 37° for five minutes. The stream then passes through the colorimeter 77 where the colour developed is measured using a 405 n filter. The electronic signal disturbances from passage of the air ^■ - bubble through the colorimeter are removed (as already described) by appropriate means and the resulting signa displayed on a chart recorder or alternatively handled by a computer system.

It will also be appreciated that the automati chemical analysis module may include any conventional colorimeter or water bath in place of the colorimeter a solid state heating bath specifically described above.

Claims

-17-

Claims

1. A cololorimeter including: a phototransistor; a light source of appropriate wavelength; a red light blocking filter which effectively blocks light of wavelength greater than about 660 n to a high degree, said filter being interposed in the optical path between the source and the phototransistor; and a cell or curvette adapted to receive a sample liquid to be analysed also interposed or interposable in said optical path.

2. A colorimeter as claimed in claim 1 wherein the light source is a light emitting diode.

3. A colorimeter as claimed in claim 1 wherein the light source is a lamp which emits a continuous spectrum and an isolation filter is also included.

4. A colorimeter as claimed in claim 3 wherein the lamp is a tungsten lamp.

5. A colorimeter as claimed in any preceding claim wherein the phototransistor is connected to an electronic amplification device.

6. A colorimeter as claimed in claim 4 wherein the output of the electronic amplification device is fed to an analogue computer system.

7. A colorimeter as claimed in claim 4 wherein the output of the electronic amplification device is fed to an electronic device designed to function as a peak sample and hold voltmeter, the output from this latter device being fed to a computer device for further processing or is fed to recording means.

8. A colorimeter as claimed in claim 6 wherein the computer device is a microprocessor which is adapted to provide for the conversion of the electronic signals to a voltage signal proportional to the amount of colour present in the colorimeter so as to allow direct

OMPI -18- comparison of this colour signal with the concentration of an analyte measured in a continuous flow automatic chemical analysis system.

9. A colorimeter as claimed in any preceding cla wherein the filter is a solution of copper sulphate.

10. A solid state heating bath including a vessel adapted to contain oil or other heat transfer fluid, a heating circuit comprising one or more power transistor and resistors which are in good thermal contact with th vessel thereby to provide heating of the vessel and of the fluid contained therein.

11. A solid state heating bath as claimed in clai wherein the transistors and resistors are recessed into the external wall or walls of the vessel.

12. A solid state heating bath as claimed in clai 10 or 11 wherein temperature of the vessel and of the h transfer fluid is achieved by a sensor contained in the fluid bath which sensor controls the energy supply to the heating circuit by means of control circuitry.

13. A solid state heating bath as claimed in clai 12 wherein the sensor is a thermistor. -

14. A solid state heating bath as claimed in any one of claims 10 to 13 wherein there is provided a padd wheel stirrer driven by an electric motor to stir the contents of the bath.

15. A solid state heating bath as claimed in any one of claims 10 to 14 whereby a coiled tube made of glass or other suitable material is positioned in the bath to allow heating of a continuously flowing liquid stream which is passed through the tube by a pumping device.

16. An air segmented continuous flow automatic chemical analysis system including:

(1) a phase separation apparatus adapted to provide continuous flow separation of a non-homogeneous - - enzy e immunoassay solid phase from its liquid phase wherein both phases are included in a non-homogeneous enzyme immunoassay mixture, said phase separation appar¬ atus comprising a membrane filter clamped between two members each having surface grooves in opposed faces thereof which together with the membrane filter constitute ducts for the passage of liquid, there being provided a duct for the passing through the apparatus of an air segmented liquid sample stream and another duct for the passing through the apparatus of an air segmented liquid recipient stream, and

(ii) means for pumping the sample stream through the phase separation apparatus and before discharge, the ducts and the pumping means forming a closed system to thus ensure the integrity of the recipient stream, the return stream flow from the phase separation apparatus being varied so as to provide a greater or lesser (as desired) amount of unbound enzyme component from the non-homogeneous enzyme assay mixture to the recipient stream thus allowing for the enzyme activity to be subsequently measured.

17. A system as claimed in claim 15 wherein the pump means is a peristaltic pump.

18. A system as claimed in claim 16 wherein the return stream flow is varied by varying the bore of a conduit carrying the return stream within the peristaltic pump.

19. An automatic chemical analysis module including: (i) a colorimeter including: a phototransistor; a light source of appropriate wavelength; a red light blocking filter which effectively blocks light of wavelength greater than about

660 n to a high degree, said filter being interposed in the optical path between the source and the phototransistor; and _θ:cell or curvette adapted to receive a sample liquid to be analysed also interposed or interposable in said optical path; (ii) a solid state heating bath including a vessel adapted to contain oil or other heat transfer fluid, a heating circuit comprising one or more power transistors and/or resistors which are in good thermal contact with the vessel thereby to provide heating of the vessel and of the fluid contained therein; (iii) a phase separation apparatus comprising a membrane filter clamped between two members each having surface grooves in opposed faces thereof which together with the membran filter constitute ducts for the passage of liquid, there being provided a duct for the passing through the apparatus of an air segmented liquid sample s.tream and another duct for the passing through the apparatus of an air segmented liquid recipient stream, said phase separation apparatus being connectable to pumping means whereby in use the sample stream may be pumped through the phase separation apparatus and before discharge, the ducts and the pumping means forming a closed system to thus ensure the integrity of the recipient stream.