Abstract:
Apparatus for analysing a liquid sample ( 194 ) comprises beam generating means ( 1 ) for generating electromagnetic radiation, detector means ( 8 ) for detecting electromagnetic radiation from the beam generating means after the radiation has interacted with the sample, and sample retaining means ( 10 ) for releasably retaining sample in the path of the beam. The sample retaining means comprises a hydrophobic surface (for example, a coating on a plate  118 ) on which the sample is, in use, supported. There is also disclosed a method of performing photometric or spectrophotometric analysis of a liquid sample by sandwiching samples between two opposed hydrophobic support surfaces, passing a beam of electromagnetic radiation through one of the surfaces and then through the sample, and analysing the beam after it has passed through the sample.

Description:
FIELD OF THE INVENTION 
     This invention relates to apparatus for analysing a liquid sample, and to a method of performing photometric or spectrophotometric analysis of a liquid sample. 
     BACKGROUND TO THE INVENTION 
     The invention is of particular application to the analysis of low volume liquid samples, for example of volumes of five microlitres or less, such as would be used in the quantitative analysis of aqueous samples of DNA created in the laboratory. Since such DNA is a valuable resource, only a very small sample is usually available for assessment. 
     Typically, the analysis of a DNA sample involves passing a beam of light through the sample and measuring the amount of light absorbed at different wavelengths. Since the DNA has different absorbence characteristics from other proteins, the measurements can be used to provide a ratio of the concentration of DNA to that of other proteins in the sample. 
     U.S. Pat. No. 6,628,382 shows apparatus in which a sample in the form of a small volume liquid drop is sandwiched between two opposed anvil surfaces. The surfaces are moved together to compress the drop, at which point a first measurement is taken by passing light through the drop. The surfaces are then moved apart so as to draw the drop into a thin, concaved column in which condition a further measurement is taken by passing light axially along the column. After the measurements have been taken, the droplet cannot be fully retrieved since it will leave residues on the anvil surfaces which will need to be thoroughly cleaned in order to avoid problems of cross contamination with droplets subsequently placed in the apparatus. In addition, any dust in the sample droplet will affect the accuracy of the apparatus and the droplet, when drawn into the column, can lose significant amounts of liquid through evaporation and be affected by photobleaching caused by the ultraviolet light used in the measurement. These factors also affect the accuracy of the apparatus. 
     The invention seeks to avoid or at least mitigate one or more of the above-mentioned problems. 
     SUMMARY OF THE INVENTION 
     According to a first aspect of the invention, there is provided apparatus for analysing a liquid sample, the apparatus comprising generating means for generating electromagnetic radiation, detector means for detecting electromagnetic radiation from said generating means after the electromagnetic radiation has interacted with the sample and sample retaining means for releasably retaining the sample in the part of the beam, wherein the sample retaining means comprises a hydrophobic surface on which the sample is, in use, supported. 
     Since the sample is supported on a hydrophobic surface, it is a relatively straightforward matter to retrieve substantially all of the sample after the measurement has been made, for example a pipette could be used to remove the sample, the surface tension of the liquid of the sample assisting in the drawing of the whole sample into the pipette. Since the surface is hydrophobic, wetting of the surface by the sample will not occur so that the surface is clean after use. This reduces the risk of cross contamination between successive samples being analysed in the apparatus. 
     Preferably, the apparatus is operable to generate a beam of electromagnetic radiation, and to define a path for said beam which path passes through at least a part of a sample being analysed. 
     The electromagnetic radiation may, for example, be ultraviolet light. 
     Preferably, the surface is one of a pair of such surfaces between which, in use, a sample being analysed is sandwiched. 
     This enables a sample to be constrained in such a way that the length of the path of the electromagnetic radiation through the sample is accurately defined and reproduced from one sample to the next. 
     Preferably, at least one of the surfaces is moveable so that the surfaces have an open condition in which a sample may be applied to or removed from at least one of the surfaces, and a closed condition in which the sample is sandwiched between the two surfaces. 
     Preferably, the surfaces are positioned one above the other and are substantially parallel to each other when in the closed condition. 
     Thus, insertion of the sample into the apparatus simply involves placing the sample on the lower surface when the surfaces are in the open condition, and the sample can subsequently be retrieved simply by being removed from the lower surface (substantially no residue remaining on the upper surface in view of its hydrophobic nature). 
     Preferably, the upper of said surfaces is mounted on a pivotal arm assembly so arranged that pivoting of the arm assembly moves the upper surface to achieve said movement between the open and closed conditions. 
     Such an assembly is convenient to use and can be of a robust and durable construction. 
     Preferably, the apparatus includes an interlock operable to permit operation of the beam generating means only when the surfaces are in their closed position. 
     This avoid the risk of the beam generating means operating when the surfaces are not in their closed condition and thus reduces the risk of inadvertent exposure of the user to radiation from the beam generating means. 
     Preferably, the interlock comprises a magnet and a magnetic switch, which are brought into and out of operative engagement with each other by the opening and closing of the surfaces. 
     It will be appreciated that “operative engagement” does not necessarily require contact of the magnet and the switch: such engagement will be achieved if the magnet is sufficiently close to the switch to change the state of the latter (from closed to open or vice versa). 
     The magnet may to advantage be fixed relative to one of the surfaces, the switch being fixed relative to the other. 
     The switch conveniently comprises a “Hall Effect Sensor”. 
     Preferably, the surfaces are constituted by hydrophobic coatings on upper and lower members. 
     Preferably, the lower member is transparent to allow the passage into the sample of the beam from the beam generating means, said beam being incident on the lower member from underneath. 
     The lower member preferably comprises a plate. 
     The apparatus may to advantage include an overhead reflector for reflecting the beam that has passed through the sample back to a region beneath the underside of the lower member. 
     Preferably, the reflector comprises a prism. 
     A face of the prism may constitute the upper surface, but the prism is preferably spaced from the member on which the upper surface is provided. 
     The upper surface is conveniently constituted by a hydrophobic coating on an upper transparent plate situated beneath said reflector. 
     Preferably, the plates are removable. To that end, the plates may, for example, be releasably snap-fitted into the apparatus. 
     This allows for relatively swift and easy removal and replacement of the plates if the hydrophobic coatings become worn. 
     The beam generating means and detector may each to advantage include a respective light guide means, respectively for emitting the beam towards the sample and receiving the beam transmitted through the sample. Both light guide means are preferably terminated below the lower surface. 
     Each light guide means may conveniently comprise a respective optical fibre. 
     The apparatus may to advantage include a moveable stop for defining a minimum distance between the surfaces, when in their closed condition, and drive means for extending and/or retracting. Automatically to vary the distances in accordance of a succession of measurements performed on the sample. 
     Preferably, the distance between the surfaces, when in their closed condition, is not more than 0.1 mm and is preferably not more than one millimeter. In this case, the diameter of the beam is preferably of the order of 1-5 millimeters, more preferably, 1.6 millimeters. Since the drop is sandwiched between two hydrophobic surfaces, it is formed into an oblate spheroid which can have a larger diameter than a concaved column. This allows a larger diameter of beam to be passed through the sample and thereby reduces the possible errors in the measurements caused by dust in the sample (since each particle of dust represents a smaller percentage of the total cross-sectional area being irradiated). 
     Preferably, the plate includes a well located in the path of electromagnetic radiation through the plate. 
     The well helps to centre the sample in the desired position, over the well, on the plate. 
     The well may to advantage be defined by a further coating being situated underneath the hydrophobic coating, and having an aperture which defines the well. 
     The coating may be opaque, and may for example be grey or black ink printed onto the plate. 
     According to a second aspect of the invention, there is provided a method of performing photometric or spectrophotometric analysis of a liquid sample of a volume not more than five microlitres, the steps of sandwiching the sample between two opposed hydrophobic support surfaces, passing a beam of electromagnetic radiation through one of the surfaces and through the sample and analysing the beam after it has passed through the sample. 
     Preferably, the beam, after having passed through the sample, is reflected towards a detector for analysing the beam. 
     The reflection is preferably achieved using a prism situated on the opposite side of one of the surfaces from the sample. 
     Preferably, the method involves passing the beam vertically upwards through a lower surface, through the sample, through the upper surface and then reflecting the beam back down to a region below the lower surface, at which a detector is located. 
     Preferably the beam has a diameter of the order of 1-5 millimeters and the ample has a cross sectional area which is at least as large as that of the beam. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The invention will now be described, by way of example only, with reference to the accompanying drawings in which: 
         FIG. 1  is a schematic view of apparatus in accordance with the invention for performing the method also in accordance with the invention; 
         FIG. 2  is an isometric view of the assembled apparatus (with its top cover removed); 
         FIG. 3  is an isometric view of a light source forming part of the apparatus; 
         FIG. 4  is an isometric view of detector means for the apparatus; 
         FIG. 5  is an isometric view of a beam splitter forming part of the apparatus; 
         FIG. 6  is an isometric view of a sample handling assembly for the apparatus, the assembly being shown in a closed condition; 
         FIG. 7  is an exploded isometric view of the assembly; 
         FIG. 8  is a cut away side elevation of the assembly; 
         FIG. 9  is an isometric cut away view of the assembly; 
         FIG. 10  is a front elevation of the assembly; 
         FIG. 11  is a section view (along part of the line A-A of  FIG. 10 ) of part of the assembly; 
         FIGS. 12   a  and  12   b  are isometric views of two plate holders of the assembly; 
         FIG. 13  is an isometric view of an optical fibre which extends into the assembly; 
         FIG. 14  shows the assembled apparatus with the cover in place; 
         FIGS. 15   a - 15   c  are a sequence of frames showing various stages in the operation of the sample handling assembly (the views of the apparatus being shown at a reduced scale); 
         FIG. 16  is an enlarged view of the apparatus; 
         FIG. 17  is a sectional end view of part of a modified version of lower plate for the assembly; and 
         FIG. 18  is a plan view of the plate. 
     
    
    
     DETAILED DESCRIPTION 
     With reference to  FIGS. 1 and 2 , the apparatus hereinbelow described is a spectrophotometric analyser of liquid samples (containing DNA and other proteins) supplied to the analyser in droplets of a volume of five microlitres or less. The apparatus comprises a xenon lamp module  1  that acts as means for generating an electromagnetic beam supplied to a beam splitter  2  a detector means in the form of a dual monochromator  8  and a sample handling assembly  10 . The beam splitter  2  splits the beam into two further beams, each of which is fed to a respective one of two optical fibres  4  and  6 . The fibre  6  is connected directly to one half of the dual monochromator  8  whilst the beam fed along the optical fibre  4  is also supplied to the monochromator  8 , but via the sample handling assembly  10 , in which the sample to be analysed is contained, and a further optical fibre  12  connecting the assembly  10  to said other half of the monochromator  8 . 
     The output of the monochromator  8  is connected by cable  14  to a Printed Circuit Board (PCB)  16  that provides the electronics and associated software to control the light source in the xenon lamp module, the sample handling assembly  10  and the monochromator  8 . Commands can be fed to the PCB via a keypad  18  whilst the results of the analysis conducted on the sample can be displayed on a display  20  or fed via a USB output  22  to a separate computer (not shown). Control signals for the display are fed along a cable  24  whilst the PCB also provides power for operating the display, fed to the latter via an inverter  26 . As indicated at  28 , the PCB also has connections for a separate printer or other type of recorder, on which the results of the analysis may also be recorded. 
     In  FIG. 2 , the various cables connecting the PCB  16  to other elements have been omitted for the sake of clarity. The various components are mounted on an aluminium plate  29  itself mounted on a base tray  30  of a plastics material. The PCB  16  is situated towards the rear of the plate  29  and tray  39  and the sample handling assembly  10  is positioned further forward and extending above the top of the tray  30 . The tray  30  includes known types of fixing, such as  32  in the form of screw threaded cylindrical sockets via which a cover (also of a plastics material) can be removably attached to the tray  30 . The cover, as well as improving the appearance of the apparatus, protects the apparatus from liquid spillage and provides a mounting surface for the screen and keypad. The cover is shown at  34  in  FIGS. 12-14 . 
     Turning to  FIG. 3 , the xenon lamp module  1  comprises a housing  36  which contains a pulsed xenon source running at approximately 20 Hz but generates a series of light flashes to illuminate the sample over a broad range of wavelengths, from 190 mm to 1100 mm. Light could be provided by a variety of other lamp types, for example deuterium for ultraviolet operation or a tungsten filament lamp for visible and IR (infra red) use. Power for operating the xenon lamp in the present example is supplied by the PCB  16 , but the module includes power modification circuitry for supplying the correct voltage to the lamp. 
     The module  1  has an output aperture  38  through which light from the xenon lamp passes into an input aperture  40  of the beam splitter  2  which is shown in more detail in  FIG. 5 . The aperture  40  is formed in a vertically extending rectangular block  42  which is formed integrally (from a suitable plastics material) with a base  44  provided with passages  46  and  48  for fixing the base  44  to the plate  29 . 
     The aperture  40  is the end of a passage in the block  42 , which passage also accommodates two lenses  50  and  52  optically coupled to optical fibres  4  and  6  respectively, and a condensing lens (not shown) in between the aperture and the lenses  50  and  52  and hence in the path of the beam to be split. The condensing lens collimates the incoming light from the beam, and thereby uniformly illuminates the lenses  50  and  52 , thus splitting the beam between those lenses. 
     It will be appreciated that other types of beam splitter could be used, for example a beam splitter employing a semi-silvered mirror or a beam splitter using a mirror pattern (where the pattern is substantially smaller than the beam size). 
     Turning to  FIG. 4 , the monochromator  8  is of a known kind, that converts incoming broadband light into individual or narrow band width components, the intensity of each of which is measured by means of an appropriate detector (e.g. a CCD, photo diode array, photomultiplier or single photo diode) forming part of the monochromator. These components are contained in a housing  58 , the electrical connections to the device being made via connecting board  60 , whilst the inputs for the optical fibres  6  and  12  are provided at  61  and  63 . The monochromator is of a type that can analyse light from the two inputs to provide two sets of spectral data that can be compared as discussed below in the description of the operation of the system. The splitting of the incoming light from each source may be achieved by any one of a number of possible means, such as interference filters, diffraction gratings or prisms. In this example, the monochromator uses diffraction gratings, and is of dual Czeny-Turner construction. 
     The lamp module, beam splitter, monochromator and PCB are of general types already known in the art, and have not been described in more detail. However, the sample handling assembly shown in  FIGS. 6-9  differs from known arrangements in a number of important respects, and will now be described in more detail with reference to those figures. 
     The assembly  10  comprises a base block  62  which has a pair of opposed lower apertured lugs  64  via which the block  62  is screwed into the tray  30 . A vertical support portion of the base block  62 , reference  66  arises from the lugs  64  and defines a substantially horizontal support platform  68  from which a sidewall  70  rises. An identical side wall rises from the other side of the platform  68 . Both sidewalls have apertures  72  through which a table  74  (having registering apertures  76 ) is screwed onto the base block  62 . The table  74  has a pair of through bores  78  and  80  extending vertically from the bottom to the top of the table in line with its longitudinal axis. Each bore contains a respective one of two clamping blocks  86  and  88 , the block  86  being shown in section plan view in  FIG. 11 . The block  86  includes a longitudinal V-shaped groove  85  which pushes a collar  90  against the bore  80  to clamp the collar  90  in position in the bore  80 . The side of clamping block  86  opposite the groove has a circular depression  91  for receiving a screw that passes through a screw threaded hole  89  in the table  74  and can be tightened against the block to urge the latter against the collar, thereby to provide the necessary force for the clamping action. 
     The clamping block  88  is identical to block  86 , and clamps a collar  92  in position in the same way as the block  86  clamps the collar  90 . 
     Each collar houses a respective one of two identical sapphire ball lenses  94  and  96 . 
     The optical fibre  12  is held against the lens  96  by means of a glass ferrule  98  which is firmly held within the collar  90 . The ferrule  98  tightly holds the end of the fibre  12  which extends through the ferrule. The top end of a sheath  100  also extends into the base of the ferrule  98  to help secure the fibre in the ferrule. A similar arrangement of a ferrule  102  and sheath  104  connects with the lens  94  to the fibre  4 . 
     The front edge of the table  74  carries a mounting bracket  106  in which a magnet  108  is mounted. A pair of vertical slots  110  extend along part of one side of the Table  74  as far as a horizontal recess in which the holes  89  are accommodated. An identical arrangement of vertical slots and a horizontal slot are provided on the opposite side of the table  74 . The slots each accommodate a respective tang such as tang  112  which is vertical and forms part of a lower sample plate holder  114 . The lower end of each tang  112  carries on its inner surface a latching formation that releasably engages in the horizontal recess in which the holes  89  are provided (or the corresponding slot on the opposite side of the table  74 ) so that the holder  114  can be releasably snap-fitted onto the table  74 . 
     The holder of  114  has a rectangular peripheral frame  116  in which a glass plate  118  is mounted. The frame  116  includes an outer peripheral ridge  120 , the inner edge of which is, in use, spaced from the plate  118  so that there is defined between the plate and the ridge a trough into which excess sample liquid may fall if the user has applied too much sample to the plate  118 . The plate  118  carries on its upper surface a hydrophobic coating (for example, DuPont&#39;s Teflon AF and a cross marking  122  to indicate the desired position for the sample droplet. 
     The end of the table  74  opposite the bracket  106  is attached to two mounting blocks  126  and  127  via which an upper arm assembly  128  is pivotally mounted on the table  74 . The assembly  128  is also attached to a rotary dashpot  130  which provides damping for rotational movement of a cog  132  which meshes with a corresponding cog on the dashpot  130 . The cog  132  has a rectangular central aperture into which a correspondingly shaped end portion  134  of a hinge pin  136  extends. The other end portion of the hinge pin  136  is similarly shaped, and engages an identical cog and dashpot arrangement attached to the block  127 . Thus the hinge pin  136  is rotationally keyed to the dashpots. However, the hinge pin is also mounted on the blocks on ball race bearings  137  and  140 , each lining a respective aperture (such as aperture  142 ) in one of the blocks  126  and  127 . Accordingly, the hinge pin  136  can rotate on the ball race bearings  137  and  140  about its elongate access, and hence is rotatable relative to the blocks  126  and  127  and thus the table  74 . 
     The upper arm assembly includes an upper carrier arm  138  having apertured fixing lugs  144  and  146  through which the pin  136  passes, and at which the pin  136  is rotationally fixed to the arm  138 . Accordingly, the carrier arm  138  is pivotable relative to the table  74  about the axis defined by the pin  136 . 
     The carrier arm  138  includes a passage which runs substantially perpendicular to the pivot access and is situated adjacent to the side of the carrier arm  138  in which the lug  144  is provided. This passage accommodates the forward end of drive means comprising an electric motor/gear box assembly  148  having a mechanical output  150  connected to an actuator  152 . The actuator fits over the output  150 , and includes a guide disc  154  from which an eccentric rod  156  projects forwardly. The disc  154  provides axial location of the actuator  152  within the passage in the carrier arm  138 , whilst allowing the actuator to rotate in response to the operation of the motor  148 . The rod  156  engages in a diametric through bore  158  in a cylinder  160  from the bottom end of which a stop in the form of a pin  162  extends. The cylinder  160  is a sliding fit in a vertical bore in the arm  138 , so that the operation of the motor  148  can move the pin  162  vertically from a position in which it protrudes beneath the arm  138  to engage the plate  118  into a position in which the pin is substantially fully retracted into the arm  138 . 
     A further spacer pin  164  is partially accommodated in a vertical bore  166  in the arm  138  and is upwardly biased by means of a spring (not shown). An actuator comprising a cam  168  mounted on a screwhead  170  extends into a horizontal bore  172  from the front of the arm  138 , and through the bore  166 , so that the cam  168  bears against the top of the pin  164 . Accordingly, the degree of extension of the pin  164  can be manually adjusted by means of the screw head  170  accessible from the front of the arm  138 . The actuator passes through bearing races  174  and  176  which enable the actuator to rotate about its elongate access in the bore  172 . 
     At the front of the arm  138  there is provided a Hall Effect sensor  178  mounted in a bracket  180 , and arranged to co-operate with the magnet  108  so that the xenon lamp module  1  is prevented from operating unless the magnet and sensor are close together (i.e. unless the upper arm  138  is in the closed position). 
     The arm  138  also carries an upper holder  182  which is releasably snap-fitted onto the upper arm  138  by a mounting arrangement similar to that provided between the table  74  and the lower holder  114 . Above the central portion of the holder  182  there is provided a roof prism  184  which extends into a cavity in the upper arm  138  and is attached to the latter either by an adhesive or by means of a clamping mechanism. The roof prism  184  has a lower face which is in registry with an elongate window  186  in the upper holder  182 . The window  186  is, in turn, in registry with an upper glass plate  188  which is attached to the underside of the holder  182 , and which carries a hydrophobic coating of (for example) DuPont&#39;s Teflon AF on its underside. A handling assembly lid  189  is attached to the top of the arm  138 , and is shown in  FIGS. 12-14 . As can be seen from  FIG. 14 , the lid  189  includes a handle portion  190  to enable a user to raise or lower the upper arm, and a hole  192  in registry the slotted head  170 . 
     To place a sample on the apparatus for measurement, the user must first open the lid  189  of the liquid handling assembly  10 , as shown in  FIG. 15(   a ). This causes the upper arm  138  to pivot about the hinge pin  136 , and thus raises the upper arm  138  and the elements attached to it (including the plate  188 ) away from the table  74  to expose the upper surface of the plate  118  as shown in  FIGS. 13(   a ) and  13 ( b ). 
     The user then places a sample droplet  194  on the cross  122  on the upper surface of the plate  118  (as shown in  FIG. 15(   b )). During these steps, the safety interlock system provided by the Hall Effect sensor  178  and the magnet  108  prevents the operation of the xenon lamp module  1 . This is because the switch is connected to the PCB  16  which is so configured as to prevent the operation of the lamp module unless an enabling signal is received from the switch, and this does not happen when the lid is open. 
     The user then shuts the lid  189  ( FIG. 13(   c )) so that the droplet is squashed between the upper and lower glass plates. The gap between the plates corresponds to the sample path length is pre-set at one millimeter. This is the distance Z indicated in  FIG. 8 . This distance is maintained by the pin  162  which is initially in its extended position. Since the surfaces of the plates  118  and  188  in contact with the sample are hydrophobic the integrity of the sample is maintained, the sample taking the form of a bead which spreads out over the plates whilst compressed. The closing of the lid  189  also causes the switch  178  to close so that the lamp module  1  is enabled. 
     The lamp then fires a single pulse of light which is conveyed to the monochromater  8  (and along optical fibre  4  to the sample handling assembly  10 ). The pulse passes from the end of the optical fibre  4  and through the ball lens  94  which collimates the light from the pulse to a desired diameter (1-5 millimeters) beam. The light then travels through the lower plate  118 , the sample compressed between the plates, the upper plate  188  and into the prism  184  which reflects the light back down through the plates (at a region where the sample has not reached) and into the lens  96  to be conveyed by the optical fibre  12  to the monochromater  8 . The light fed directly to the monochromater along the fibre  6  constitutes a reference channel, the light from the fibre  12  a measurement channel. The monochromater processes the light from both channels simultaneously: within the monochromater, the light is directed onto a diode array sensor via a series of mirrors and a diffraction grating. 
     If a signal is received through the measurement channel this confirms that the prism  184  is in place and that measurement can proceed. 
     The apparatus then fires an appropriate number of pulses from the lamp module  1  to ensure that a strong return signal (with a reasonable signal to noise ratio), is obtained by the diode array sensor. A measurement is then taken at the 1 millimeter pathlength position. 
     The signal that has been received by the sensor is then analysed by the PCB  16 . 
     The PCB then sends an actuating signal to the motor  148  causing the latter to retract the pin  162  into the arm  138 . This allows the lid to move downwards under gravity allowing the further pin  164  to rest against the lower glass plate  118 , thus reducing the path length to between 0.1 and 0.3 millimeters, depending on the position of the pin  164  as governed by the cam  168 . The initial check and measurement processes are then repeated with a reduced gap, and the motor  148  then operates for a second time allowing the pin  162  to return to its original, extended position, and hence to increase the path length to 1 millimeter. 
     The type of analysis employed on the signals obtained by the monochromater is already known, and is not therefore described in detail. 
     After the user has taken the measurements mentioned above, the user raises the lid  189  of the sample handling assembly  10 , thus disengaging the interlock (Hall Effect switch  178  and magnet  108 ), to ensure that the system is now non-operational and no bright UV flashes can be emitted by the xenon lamp. 
     If the user wishes to keep the sample for further use, it may be removed using a pipette. Alternatively, the sample may be removed using a laboratory wipe or cloth. 
     Any small residual traces can be removed by wiping the top and bottom plates with an absorbent wipe (ideally alcohol based). 
     The upper and lower plates of the sample handling assembly  10  may be renewed periodically by the user, in the event of the deterioration of either hydrophobic coating. The glass plates are permanently fixed within the holders  114  and  182 , which can be unclipped from the table  74  or (as the case may be) arm  138  and replaced with identical holders which carry replacement, coated plates. 
     If the holders are replaced, it is desirable to be able to calibrate the system. To that end, a calibration fluid could be applied to the lower glass plate in the same manner as an ordinary sample. The lid  189  can then be closed and the appropriate programme run by the user to calibrate the instrument. To do this effectively the user may be required to adjust the pin that controls the 0.1 millimeter-0.3 millimeter path length position (i.e. pin  164 ). The fine tuning of the pin position is done by the cam  168  of the actuator, by inserting a flat screwdriver through the hole  192  in the front of the lid  189 . If this is required, the user will be prompted to do this by the instrument&#39;s display. 
       FIGS. 17 and 18  show an alternative type of plate for use in place of plate  118 . 
     The alternative type of plate has a glass substrate  200  onto which a black or grey ink has been screen printed and dried to leave an opaque layer  202  of a thickness of about 20-40 microns. The ink layer  202  includes elongated apertures  203 - 206  which define a cross marking having a similar purpose to the marking  122 , and which extend radially relative to a central, circular aperture  208  about which the elongated apertures  203 - 206  are equiangularly arranged. The aperture  208  corresponds to the position at which the upwardly direction portion of the beam passes through the plate. A further circular aperture  210 , in the coating  202  is provided at the position of the return leg of the beam. The diameter of the aperture  208  is approximately 1.3 mm. 
     The Teflon AF coating  212  is applied to the upper surface of the layer  202 , the aperture  208  in which defines a very shallow well  214 , into which the droplet will preferentially extrude as the upper plate closes upon it. The well thus provides a means for centering a sample droplet should the latter not be accurately placed on the plate.