GB2517997A

GB2517997A - Flow cell and method

Info

Publication number: GB2517997A
Application number: GB1316038.7A
Authority: GB
Inventors: John Creedy
Original assignee: PROGRESSIVE RES SYSTEMS Ltd
Current assignee: PROGRESSIVE RES SYSTEMS Ltd
Priority date: 2013-09-09
Filing date: 2013-09-09
Publication date: 2015-03-11
Anticipated expiration: 2033-09-09
Also published as: GB2517997A8; GB201316038D0; GB2517997B8; GB2517997B

Abstract

A method of providing a flow cell 101 having a diffuser portion 140 comprises providing a fluid flow cell 110 having a fluid entry portion 110In, a fluid exit portion 110Out and an inspection portion 115, and providing a porous diffuser portion 140 in the flow cell arranged whereby fluid flowing through the flow cell flows through the diffuser portion, whereby providing a diffuser portion comprises introducing a particulate diffuser medium into the flow cell and subsequently fixing the diffuser medium within the flow cell. The flow cell may be used for liquid chromatography and the inspection portion allows optical probing of the fluid flow. The diffuser (frit or sinter) reduces turbulent flow in the cell and provides a more laminar fluid flow.

Description

FLOW CELL AND METHOD

TECHNICAL FIELD

The present invention relates to flow cells employed in detector systems. In particular but not exclusively the invention relates to optical flow cells for use in liquid chromatography separation detection systems.

BACKGROUND

It is known to provide an optical flow cell coupled to a liquid chromatography (LC) separation system for performing optical inspection of a material or sequence of materials, desired product(s) or impurity(ies), eluted from an LC column in an elution stream. The material may for example be any dissolved or suspended material including, but not limited to, proteins, viruses or other bioparticles.

In LC separation systems, a flow of liquid from the LC column is typically transferred to the flow cell by means of circular cross-sectional tubing. Analytical systems utilise small diameter tubing and small volume flow cells, whilst preparative and process chromatography systems may use larger diameter tubing and may or may not utilise flow detectors with flow cells of larger volumes. Known optical flow cells typically have a larger circular or square cross-section than the delivery tubing and are formed from a transparent glass, quartz or other material allowing laser or other light to irradiate liquid flowing through the cell. Light that subsequently passes out from the flow cell is detected by means of a detector. A data logger logs data in respect of the intensity of the light that is detected by the detector. Optical detection systems used in LC separation systems are typically either one or two dimensional in light path design, i.e. having a light path that is linear or planar, with flow running in a direction orthogonal to the light path.

Such arrangements are commonly used in fluorescence detection systems and light scattering detection systems. Optical absorbance devices typically arrange the incident light source and detection in the same dimension, whilst light scattering or fluorescence detectors typically arrange detection at an angle or angles to the incident light path.

The incoming flow stream typically features sequential stepped or defined changes in corriposition as different materials are eluted from the LC column. The different materials are typically spatially separated from each other along the flow stream of carrier eluent, which may also be referred to as the mobile phase.

The present applicant has recognised that a problem exists with known flow detector systems, in both optical and non-optical detection modes. When a particular material is eluted from an [C column, the material can become distributed turbulently within the mobile phase as it enters the flow cell. This is due at least in part to a variation in flow velocity over a cross-sectional area of the delivery tubing and flow cell device. Linear velocity is found to decrease with increasing distance from a centre of the tubing due to the effects of wall friction. A core stream of eluent carrying material to be inspected at a centre of the tubular hose or tubing may therefore travel faster than eluent closer to the wall of the tubing. This increases the length of tubing occupied by the separated component and degrades the separation performance. This effect is particularly noticeable in analytical [C systems with small diameter tubing. Furthermore, as the liquid enters the flow cell and diverges to fill the flow cell, turbulent flow (vortexing') develops due to relatively abrupt deceleration of the liquid as the cross sectional area and/or geometry of the flowpath changes. The flow cell acts as a void space which mixes incoming material with the flow cell fluid volume in an irregular manner. The concentration of peak material described by the leading edge is diluted by the mobile phase in the flow cell as a peak enters the flow cell due to this turbulent mixing, and on exit the trailing edge of the peak is similarly diluted as mobile phase displaces it from the flow cell. Peak shape is thus significantly degraded when turbulence is present in the flow cell. This is commonly described as the wash out or flow through performance of a flow cell. This performance is severely compromised as a result of turbulence within a flow cell.

Turbulent flow conditions within the flow cell may be further generated by the change from inlet tubing size, which is typically circular in cross section, to the larger circular or square section required for optical measurements within the detection flow cell.

The present applicant has recognised that the turbulence and absence of laminar flow within the flow cell can result in failure to obtain reliable, accurate and sensitive optical or other inspection data in respect of materials that may be eluted from the [C column. This is particularly significant when they are present in low concentrations. Detector sensitivity depends on achieving a low baseline noise level. Baseline noise level is significantly increased by turbulent flow. Limits of detection for an LC system are typically described as three times baseline noise.

Turbulent flow within a flow cell causes excessive baseline noise and inefficient wash out characteristics which result in a substantial degradation of the separation detection sensitivity.

This can be sufficient to preclude use of flow detection for certain detection applications. The effects of turbulence on flow cell performance are not limited to optical detection systems, but have implications for other systems employing flow cells. The effects of turbulence may be significant in flow cells of a range of sizes.

In addition, turbulence at the leading edge and trailing edge of a peak in eluted material concentration increases the peak width of eluted materials at the detector flow cell which also degrades the upstream separation performance delivered by an LC separation system. As a peak is replaced with following eluent/mobile phase or a following eluted material, the same phenomenon of turbulence will occur with respect to the trailing edge of the peak.

It is an aim of embodiments of the present invention to address disadvantages associated with known LO separation flow detection systems in general. It is an aim of some embodiments of the present invention to improve sensitivity of existing LC detection systems and permit use of flow cells within systems which are currently unable to utilise an optical or other detection flow cell for one or more of the reasons described above.

SUMMARY OF THE INVENTION

Embodiments of the invention may be understood with reference to the appended claims.

Aspects of the present invention provide a flow cell for a liquid chromatography (LC) separation detection system and a method of flow cell construction. Some embodiments of the invention provide an LC detection system having a flow cell diffuser portion (or sinter or frit device element) capable of economical construction at low cost and providing reduced flow turbulence therethrough.

In one aspect of the invention for which protection is sought there is provided a method of providing a flow cell having a diffuser portion comprising: providing a fluid flow cell having a fluid entry portion, a fluid exit portion and an optical inspection portion; and providing a porous diffuser portion in the flow cell arranged whereby fluid flowing through the flow cell flows through the diffuser portion, providing a diffuser portion comprising: introducing a particulate diffuser medium material into the flow cell; and subsequently fixing the diffuser medium material within the flow cell.

The method may include ensuring the resulting diffuser portion or diffuser is homogeneously porous substantially throughout its structure.

Embodiments of the present invention have the advantage that a flow cell may be provided with a diffuser portion within a flow cell in a convenient, cost effective, reliable and reproducible manner. Embodiments of the invention enable the diffuser portion to be provided with a secure, closely fitting, homogeneous and open porous fit within the flow cell with substantially no leakage therepast. This is at least in part because the diffuser portion formed by introducing the particulate diffuser medium material into the flow cell and subsequently fixing the material in place. The resultant diffuser portion may be firmly fixed to pack the desired space with a homogeneously porous stable material which allows free passage of fluid including dissolved material and/or suspended particles to be analysed, optionally within substantially the entire diffuser portion. The particles may be particles having a size of up to around one micrometre in diameter. For the present purposes such particles are considered to be nanoparticles.

In some embodiments the diffuser portion may be referred to as a frit or sinter. Embodiments of the present invention relate to the economical manufacturing of a frit or sinter component in-situ within a preformed flow cell or during flow cell manufacture. Embodiments of the invention are useful in reducing turbulent flow and reducing detector noise caused by flow turbulence within a flow cell.

Advantageously, introducing the particulate medium material may allow provision of the particulate medium for a diffuser portion at the entry and/or exit portion of the flow cell of various geometries and sizes.

Providing the particulate medium material (and theretore the diffuser portion) in the entry and/or exit portion has the advantage that fluid niay be passed through the diffuser portion before reaching a region of the flow cell in which optical inspection of the fluid takes place.

Further advantageously, fixing the particulate medium niaterial may involve introducing a binder medium to the particulate medium material, the binder medium being arranged to bind to the particulate medium material. The binder niaterial may in addition bind to an inner surface of the flow cell.

Introducing the binder medium may comprise introducing the binder medium through the entry or exit portion. Typically this binder medium is a fluid (either liquid or gas) although other forms of medium such as solid, gel or other form are also useful in some embodiments.

Optionally, introducing the binder medium comprises introducing the binder medium into the flow cell together with the particulate medium.

It is to be understood that the binder medium may be in the form ol a particulate medium mixed with the diffuser medium. The binder medium may be a substantially dry particulate medium. It may be in the form of a solid, a gel or any other suitable medium.

Optionally the binder medium comprises a particulate medium.

The binder medium may comprise a liquid binder medium.

The method may involve introducing liquid binder medium to the flow cell through the entry or exit portion from below through the particulate diffuser medium niaterial retained in position during the binding/curing process.

This feature has the advantage of reducing the risk of unwanted contact between the binder medium and the optical inspection portion of the flow cell.

It is to be understood that with certain materials it may be particularly important to avoid direct contact between an internal wall of the flow cell defining part of the optical inspection portion and the binder medium. Accordingly, by introducing the binder medium to the particulate medium from below, the binder medium is required only to pass into the flow cell a sufficient distance to saturate the diffuser medium. It is to be understood that the particulate medium may be retained in the entry or exit portion of the flow cell at least in part due to gravity until the binder medium has fixed the particles. The binder medium may therefore be forced or drawn in an upwards direction through the entry or exit portion of the flow cell against gravity.

The method may comprise causing the binder medium to fix the diffuser medium within the flow cell.

The method may comprise subsequently passing a fluid through or into the flow cell and out from the flow cell via the entry or exit portion in which the diffuser portion is being fixed.

This feature has the advantage that a fabricator may ensure that the diffuser portion remains porous to passage of fluid both during and after the binding/curing process.

The method may comprise drawing fluid or gas/air through the flow cell whilst particulate medium is being fixed by the binder medium.

This feature has the advantage that a fabricator may ensure that the diffuser portion is porous to fluid. This is because the action of drawing a fluid through the diffuser portion may force the binder medium to be displaced whilst the diffuser medium remains in place, causing the diffuser portion to become porous and/or ensuring that the diffuser portion remains porous.

It is to be understood that certain binder media including media comprising volatile hydrocarbons may evolve vapours during a curing or setting process. The vapours may have an adverse effect on one or more properties of a flow cell. For example, in the case of an optical flow cell having a light transmitting portion allowing optical inspection of fluid, one or more optical properties of the light transmitting portion may be degraded by contact with the vapours.

For example, cyanoacrylic "superglue" adhesive may be used as the binder medium for certain particulate media.

This adhesive evolves vapours during curing and the vapours are known to cause degradation of silica glass and certain plastics materials upon contact. The vapours may cause deposits to form. Control of the vapours during curing can therefore be important in certain applications. It is to be understood that by passing (optionally, drawing or injecting) fluid through the flow cell and out from the flow cell via the entry or exit portion in which the diffuser particles are being fixed, any vapour associated with the binder medium may become entrained in the flow of fluid such as air or other gas and drawn out from the flow cell via the entry or exit portion. This may assist in reducing a risk of unwanted contact between volatile components and the optical inspection portion of the flow cell. In addition it may be useful to select low viscosity, low bloom/low odour cyanoacrylate adhesives or alternative binding chemistries.

By controlling the flow direction of the binder medium, the penetration distance of the particulate medium and the direction of flow, excess binder medium can be removed immediately following saturation of the particulate medium. This may assist in reducing a risk of unwanted contact between the vapours and the optical inspection portion of the flow cell.

The method may comprise forming the flow cell to have an optically transparent portion.

Alternatively, the flow cell may be formed to be substantially opaque to visible and/or infra-red radiation.

In some embodiments the flow cell may be formed to comprise a translucent portion.

In a further aspect of the invention for which protection is sought there is provided a flow cell formed by the method of any preceding aspect.

In one aspect of the invention for which protection is sought there is provided a kit of parts for making a flow cell according to the method of any preceding aspect comprising: a flow cell body; a particulate diffuser medium; means for fixing the diffuser medium within the flow cell; and instructions for making a flow cell according to the method of a preceding aspect.

In a further aspect of the invention for which protection is sought there is provided a flow cell comprising: a fluid flow cell body having a fluid entry portion, a fluid exit portion and an optical inspection portion; and a porous diffuser portion comprising a particulate diffuser medium fixed within the flow cell body and arranged whereby fluid flowing through the flow cell flows through the diffuser portion.

Optionally, the diffuser portion comprises a binder medium and a particulate diffuser medium, the binder medium being arranged to fix the particulate diffuser medium within the flow cell.

The flow cell may have an optically transparent portion.

Alternatively, the flow cell may be substantially opaque to visible and/or infra-red radiation.

In some embodiments the flow cell comprise a translucent portion.

In a further aspect of the invention for which protection is sought there is provided a particle detection system or apparatus comprising a flow cell according to a preceding aspect.

In a further aspect of the invention for which protection is sought there is provided a liquid chromatography system or apparatus comprising a flow cell according to a preceding aspect.

In an aspect of the invention for which protection is sought there is provided a method of providing a flow cell having a diffuser portion. The method may comprise: providing a fluid flow cell having a fluid entry portion, a fluid exit portion and an optical inspection portion; and providing a porous diffuser portion or portions in the flow cell arranged whereby fluid flowing through the flow cell flows through the diffuser portion(s). Providing a diffuser portion may comprise: introducing a particulate diffuser medium into the flow cell; and subsequently fixing the diffuser medium within the flow cell.

Within the scope of this application it is envisaged that the various aspects, embodiments, examples and alternatives, and in particular the individual features thereof, set out in the preceding paragraphs, in the claims and/or in the following description and drawings, may be taken independently or in any combination. For example features described in connection with one embodiment are applicable to all embodiments, unless such features are incompatible.

For the avoidance of doubt, it is to be understood that features described with respect to one aspect of the invention may be included within any other aspect of the invention, alone or in appropriate combination with one or more other features.

BRIEF DESCRIPTION OF THE DRAWINGS

One or more embodiments of the invention will now be described by way of example only, with reference to the accompanying figures in which: FIGURE 1 is a schematic illustration of (a) a known flow cell installed in an [C system and (b) a flow cell according to an embodiment of the present invention; and FIGURE 2 is a schematic illustration of a process of fabricating a flow cell according to an embodiment of the invention.

DETAILED DESCRIPTION

FIG. 1(a) shows a known flow cell 1 connected in a liquid chromatography ([C) separation system (not shown). The flow cell 1 is formed from an optically transparent glass material. In the example shown the flow cell is formed from quartz glass although other materials are also useful such as silica glass, plastics materials or any other suitable material allowing optical transmission. The flow cell 1 has a cell body 10 having a fluid inlet conduit lOIN, an optical inspection portion 15 and a fluid outlet conduit bOUT. The inlet and outlet conduits lOIN, 100UT are connected to respective inlet and outlet hoses 51N, 5OUT of the [C separation system.

Fluid entering the flow cell body 10 through the inlet conduit lOIN passes through a diverging portion 12 of the cell body 10 to the inspection portion 15 which has a substantially uniform, square (rectangular) cross-section. A wall of the inspection portion 15 is substantially flat and of uniform thickness in order to reduce distortion and internal reflections of laser or other radiation passing therethrough. A converging portion 18 is provided between the inspection portion 15 and outlet conduit 100UT.

As shown in FIG. 1(a) fluid flowing through the inlet hose SIN has a velocity profile P which is at a maximum in a central region of the hose SIN and decreases with radial distance from the central region. Liquid entering the flow cell body 10 diverges within the diverging portion 12 and turbulent flow is established.

The present applicant has recognised that stepped changes in concentration of a given material in eluent flowing from an LC column can become increasingly diffusely distributed in a flowstream with distance from the column, and even more diffusely distributed within the flow cell body 10 as the fluid passes therethrough. This can result in failure to detect certain materials in the flowstream due to excessive signal noise caused by turbulence in the optical section of the flow cell.

The present applicant has conceived the idea of providing a diffuser portion which may be referred to as diffuser element, a sinter or a frit in some embodiments, within the flow cell in order to reduce turbulence of the flow of liquid therethrough. It is to be understood that the internal volume of the flow cell 1 is relatively irregular due to the requirement to connect cylindrical tubing of relatively small cylindrical cross-sectional area to a flow cell volume of relatively large cross-sectional size and with either cylindrical or substantially flat walls permitting optical inspection. The provision of a diffuser portion within the flow cell volume that will reduce turbulence and enhance laminar flow is consequently a non-trivial task.

It is to be understood that flow cells for optical inspection of liquids are relatively fragile compared with flow cells formed from metallic materials or robust plastics materials. As noted above, optical flow cells are typically formed from optical glass materials (typically fused silica or fused quartz), requiring relatively high temperature processing during their fabrication.

The present applicant has considered a number of options for providing a diffuser portion within an optical glass flow cell in a manner suitable for mass production at reasonable cost.

Preformed or machined diffuser portions are not easy to install in a flow cell of narrow inlet and outlet diameter. It is also difficult to obtain a satisfactory seal between edges of the diffuser portions and the flow cell body 110 without compromising the free flow characteristics of the diffuser portion or inlet/outlet itself. Fluid flowing through the flow cell 101 tends to channel through leakage pathways between diffuser portion and flow cell body 110 creating turbulence and extended elution stream peak widths.

To address these problems, the present applicant has conceived that the provision within the flow cell 101 of a diffuser portion formed from a bed of loose particles that are subsequently fixed in position within the flow cell may overcome this problem. Tests demonstrate a remarkable improvement in optical flow cell performance. Formation of a diffuser portion according to an embodiment of the present invention may be done at relatively low cost, in a reproducible and reliable manner, and without a requirement to modify existing flow cells 1 before the diffuser portion may be installed.

FIG. 1(b) illustrates an improved flow cell 101 according to an embodiment of the present invention, formed in the manner described above. Like features of the embodiment of FIG. 1(b) to the cell 1 of FIG. 1(a) are shown with like reference signs incremented by 100.

The flow cell 101 differs from the cell 1 of FIG. 1(a) in that it is provided with a diffuser portion in the diverging portion 112 thereof. The diffuser portion 140 substantially fills the diverging portion 112 in the embodiment shown, terminating substantially at a boundary between the diverging portion 112 and inspection portion 115 where the cross-sectional shape and size of the cell 10 becomes substantially uniform.

The diffuser portion 140 is formed from a particulate diffuser medium, which in the present embodiment is a size fractionated particulate borosilicate glass material. In the example shown the particle size is in the range from around 0.25 to around 0.75mm although other sizes larger or smaller are also useful. The size selected is such as to permit free flowing insertion of dry material by way of a small aperture funnel into the outlet conduit 11 OOUT (or in reverse case inlet conduit 11OIN). The diffuser medium is retained within the cell 101 by an adhesive in which the particulate diffuser medium is embedded. In the example shown the adhesive is a cyanoacrylate adhesive although other adhesives are also useful.

The diffuser portion 140 presents a tortuous path for the flow of incoming fluid therethrough.

This causes localised and efficient flow distribution across the varying (enlarging) section of the flow path. This has been found to have the significant advantage of causing a dramatic reduction in turbulence of liquid flowing through the cell 101 and enabling the establishment of substantially laminar flow. This reduces the amount of noise associated with optical measurements perfornied on fluid flowing through the cell.

The presence of the diffuser portion 140 also results in a dramatic increase in sharpness of changes in concentration profile of liquid flowing through the cell compared with known flow cells 1 not having the ditfuser portion 140. This facilitates the reliable detection of material that flows through the cell 101 in relatively low volumes and at relatively low concentration.

A method according to an embodiment of the invention by which a diffuser portion 140 may be formed in a flow cell 101 is now described, the method being illustrated in FIG. 2.

A flow cell body 110 is provided formed froni an optical glass material, in the present embodiment a quartz material. The body 110 is cleaned and dried thoroughly to prevent adhesion of particles of diffuser medium such as glass or other sinter material to the inner optical surfaces.

The flow cell is oriented with inlet conduit 1 lOIN pointing substantially vertically downwards. A flexible retaining screen 150 formed from a porous gauze material is positioned over the inlet aperture 11 OIN_A and held in place with flexible tubing 1 50T. Alternatively a small porous plug of compressed polymeric foam or other porous material may be inserted to retain dry particulate diffuser rnediuni during the binding/curing process. Other nieans for retaining the rnediuni are also useful. The screen 150 is of appropriate mesh porosity to retain particles of diffuser material (which may be referred to as particles of sinter') such as glass or quartz material. In the present embodiment borosilicate glass particles are employed as described above. Regular shaped particles having average diameters in the range from 5Oum (micrometres) to l000um are found to be particularly useful. Other sizes, smaller and/or larger, niay be useful in addition or as an alternative.

With the cell 101 in the orientation shown in FIG. 2, a required amount of diffuser material is introduced from the opposite end of the cell body 110 through the flow cell outlet conduit 11 OOUT. This is done by way of a material cone with appropriately small exit held inside the upper flow cell conduit 1100UT. Sinter grade is selected to permit free flow of dry particulate material into the conduit which is typically at least 4 to 5 tirries the diameter of the largest particles. Conduit diameter in the current example is between 1 and 2 mm diameter.

The diffuser material is caused to settle in the diverging portion 112 of the cell body 110, being retained in position by means of retaining screen 150 or alternative plug as described. Gentle agitation of the body 110 with the body 110 substantially upright causes a relatively homogeneously packed bed of dry particulate material to fully occupy the internal shape and volume of the diverging portion 112. The upper surface of the bed forms a substantially flat surface during agitation. As shown in FIG. 2 the bed extends within the inlet conduit 11OIN from level C to level B and upwards across the diverging portion 112 to Level A. With the cell body 110 retained in the orientation shown, a fluid adhesive such as cyanoacrylate "superglue" is introduced from below through the retaining screen 150 to flood the dry particulate diffuser material, i.e. saturate the material with adhesive. The adhesive is injected at a sufficiently low rate to avoid disturbance of the diffuser material.

When the particulate material has been fully wetted, and before the adhesive cures, the flow of adhesive is reversed whereby the adhesive is withdrawn through the saturated porous bed. Air is thereby drawn through the moist bed until the adhesive has cured sufficiently to fix the particles in a single homogeneous block. This process reduces and in some embodiments substantially prevents exposure of the optical surfaces to potentially damaging volatile components.

Tests have shown that polyacrylate adhesive may cure within 60s if moist air is drawn through the particulate material, since the curing reaction requires the presence of water molecules.

Flow is maintained until the curing process is complete.

Following curing, the tubing 150T and retaining screen 150 (or porous plug) may be removed. A diffuser portion in the form of a substantially homogeneous sinter is thereby formed, firmly fixed within the flow cell body 110.

It is to be understood that the process described above may be repeated in respect of the outlet conduit 1100UT if required. In this case the particulate diffuser material may be introduced through the exit conduit 1 bOUT such that it rests on the diffuser portion 140 that has already been forniecl, and a retaining screen or porous plug installed over or within exit aperture 1100UT_A. The flow cell body 110 may then be inverted so that the exit aperture 1100UT_A points vertically downwards and the particulate diffuser niedium settles in the lower portion of the cell body 110. Adhesive may then be introduced to fix the particles in the manner described above.

In some embodiments, curing of the adhesive may be by means of heat, light or UV radiation and/or by means of an alternative gas to air. However it is to be understood that drawing gas or other fluid through the diffuser portion during curing may be useful in ensuring sufficient porosity of the diffuser portion.

It has been found by the present applicant that a relative rapid change of direction of adhesive fluid flow may be required to be effected lollowing saturation of the diffuser particles with adhesive, in order to avoid excessive contact between volatile components evolved by the adhesive and inner optical surfaces of the cell body 110. Etching and other surface interactions may occur through contact with volatile components, which can degrade the optical performance of the detector cell. This issue may be less relevant in the case of non-optically critical detection systems.

It is to be understood that other methods of introducing adhesive are also useful, for example by injection through an aperture other than the one at which the diffuser portion is being provided.

It is to be understood that other flow cell designs which may or may not incorporate alternative tubing connectors may be fitted with a porous diffuser portion in a similar manner but may require modifications to the embodiment described herein.

In some embodiments, instead of injecting an adhesive into the flow cell body 110 to flood the diffuser particles, dry or solid particles of adhesive are mixed with particulate diffuser material before being introduced into the flow cell body 110. The diffuser particles are subsequently fixed by activating the particles of adhesive to bond the particles of diffuser material to one another and to the cell body 110, optionally by application of heat or other treatment such as exposure to UV light, other radiation, or other suitable treatment. The adhesive particles may be selected such that any heat required to activate the particles is insufficient to cause damage to the flow cell body 110 or any other feature of the flow cell 101. Gas may be drawn through the flow cell whilst the diffuser particles are being fixed, typically through the inlet or outlet aperture at which the diffuser portion 140 is being provided, in order to ensure sufficient porosity of the diffuser portion 140. Other arrangements are also useful.

Embodiments of the present invention have the advantage that a diffuser portion may be formed to have substantially any required shape through conformity of a particulate medium to a geometric form and configuration of an internal volume of a flow cell. Diffuser portions having shapes that would otherwise be impossible to prefabricate and install may thus be formed in a convenient and cost effective manner.

It is to be understood that the method described herein may be scaled to enable fabrication of diffuser portions of substantially any desired size. In the embodiment of FIG. 1(a) the flow cell body 110 has an internal volume of approximately 200 microlitres (ul). However the same principle of manufacture may be used for smaller devices and larger devices, and also with flow cells of alternative designs and geometries including commercial fluorimetry flow cells.

Embodiments of the present invention enable the in-situ fabrication of a diffuser portion within a prefabricated device of variable and imprecise geometry. Embodiments of the present invention are ideally suited to the custom fabrication of diffuser portions in flow cells formed by methods which may suffer variations in exact shape and size, such as cells formed from glass and quartz materials. Such devices would otherwise require custom fabrication of a diffuser portion to match the precise internal shape of the flow cell. The diffuser portions are essentially moulded' to the size and shape of individual cells by virtue of the manufacturing process, inherently providing a leak tight' seal between the diffuser portion and cell wall. By leak tight is meant that any preferential flow path between cell wall and diffuser portion is unlikely to be of a larger cross-sectional area than that of pores formed in the diffuser portion by gaps between particles.

This also includes internal leak paths resulting from any inhomogeneity within the packed diffuser material.

In some embodiments, the flow cell may be configured to allow introduction of a material into a flow stream of eluted material through the cell. In some embodiments an additional inlet may be provided, optionally upstream of the diffuser portion, to allow mixing of the material with fluid of which the flow stream is comprised. In some embodiments, in addition or instead the material to be introduced may be at least partially provided by the diffuser portion. Thus the material may be embedded in the diffuser portion. The material may be arranged to leach out from the diffuser portion, optionally to enter the flow stream by being dissolved by fluid of the flow stream.

In some embodiments the material may be introduced at a location between opposite ends of the diffuser portion. The material may be introduced in a carrier medium such as a carrier fluid.

Other arrangements are also useful.

Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", means "including but not limited to", and is not intended to (and does not) exclude other moieties, additives, components, integers or steps.

Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

Claims

CLAIMS: 1. A method of providing a flow cell having a diffuser portion comprising: providing a fluid flow cell having a fluid entry portion, a fluid exit portion and an inspection portion; and providing a porous diffuser portion in the flow cell arranged whereby fluid flowing through the flow cell flows through the diffuser portion, providing a diffuser portion comprising: introducing a particulate diffuser medium into the flow cell; and subsequently fixing the diffuser medium within the flow cell.
2. A method according to claim 1 whereby introducing the particulate medium comprises providing the particulate medium in or at the entry portion of the flow cell.
3. A method according to claim 1 or claim 2 whereby fixing the particulate medium comprises introducing a binder medium to the particulate medium, the binder medium being arranged to bind to the particulate medium and to an inner surface of the flow cell.
4. A method according to claim 3 whereby introducing the binder medium comprises introducing the binder medium through the entry and/or exit portion.
5. A method according to claim 3 or claim 4 whereby introducing the binder medium comprises introducing the binder medium into the flow cell together with the particulate medium.
6. A method according to any one of claims 3 to 5 whereby the binder medium comprises a particulate medium.
7. A method according to any one of claims 3 to 6 whereby the binder medium comprises a liquid binder medium.
8. A method according to claim 7 comprising introducing liquid binder medium to the flow cell through the entry or exit portion from below the particulate diffuser medium.
9. A method according to any one of claims 3 to 8 comprising causing the binder medium to fix the diffuser medium within the flow cell.
10. A method according to any preceding claim comprising subsequently drawing a fluid through the flow cell and out from the flow cell via the entry or exit portion in which the diffuser portion is being fixed.
11. A method according to claim 10 as depending through claim 3 comprising drawing the fluid through the flow cell whilst particulate medium is being fixed by the binder medium, the fluid comprising a gaseous fluid.
12. A method according to any preceding claim comprising forming the flow cell to have an inspection portion comprising an optically transparent portion.
13. A flow cell formed by the method of any preceding claim.
14. A kit of parts for making a flow cell according to the method of any preceding claim comprising: a flow cell body; a particulate diffuser medium; means for fixing the diffuser medium within the flow cell; and instructions for making a flow cell according to the method of any of claims ito 12.
15. A flow cell comprising: a fluid flow cell body having a fluid entry portion, a fluid exit portion and an inspection portion; and a porous diffuser portion comprising a particulate diffuser medium fixed within the flow cell body and arranged whereby fluid flowing through the flow cell flows through the diffuser portion.
16. A flow cell according to claim 15 wherein the diffuser portion comprises a binder medium and a particulate diffuser medium, the binder medium being arranged to fix the particulate diffuser medium within the flow cell.
17. A flow cell according to claim 15 or claim 16 wherein the inspection portion comprises an optically transparent portion.
18. A particle detection system or apparatus comprising a flow cell according to any one of claims l5to 17.
19. A liquid chromatography system or apparatus comprising a flow cell according to any one of claims lSto 17.
20. A method, a flow cell, a kit of parts, a particle detection system or apparatus, or a liquid chromatography system or apparatus substantially as hereinbefore described with reference to FIG. 1(b) or FIG. 2.AMENDMENTS TO CLAIMS HAVE BEEN FILED AS FOLLOWSCLAIMS: 1. A method of providing a flow cell having a diffuser portion comprising: providing a fluid flow cell having a fluid entry portion, a fluid exit portion and an inspection portion; and providing a porous diffuser portion in the flow cell arranged whereby fluid flowing through the flow cell flows through the diffuser portion, providing a diffuser portion comprising: introducing a particulate diffuser medium into the flow cell; and subsequently fixing the diffuser medium within the flow cell by introducing a binder medium to the particulate medium so as to bind the particulate medium together and to an inner surface of the flow cell; and drawing a fluid through the diffuser portion whilst the particulate medium is being fixed by the binder medium.2. A method according to claim 1 whereby introducing the particulate medium comprises providing the particulate medium in or at the entry or exit portion of the flow cell.3. A method according to claim 2 whereby introducing the binder medium comprises introducing the binder medium through the entry and/or exit portion.4. A method according to any preceding claim whereby introducing the binder medium comprises introducing the binder medium into the flow cell together with the particulate medium.5. A method according to any preceding claim whereby the binder medium comprises a particulate medium.6. A method according to any preceding claim whereby the binder medium comprises a liquid binder medium.7. A method according to claim 6 comprising introducing liquid binder medium to the flow cell through the entry or exit portion from below the particulate diffuser medium.8. A method according to any preceding claim comprising causing the binder medium to fix the diffuser medium within the flow cell.9. A method according to claim 2 or any one of claims 3 to 8 depending through claim 2 whereby drawing a fluid through the diffuser portion whilst the particulate medium is being fixed by the binder medium comprises drawing a fluid through the flow cell and out from the flow cell via the entry or exit portion in which the diffuser portion is being fixed.10. A method according to any preceding claim wherein the fluid comprises a gaseous fluid.11. A method according to any preceding claim comprising forming the flow cell to have an optical inspection portion comprising an optically transparent portion.12. A flow cell formed by the method of any preceding claim.13. A kit of parts for making a flow cell according to the method of any preceding claim comprising: a flow cell body; a particulate diffuser medium; means for fixing the diffuser medium within the flow cell; and instructions for making a flow cell according to the method of any of claims 1 to 11.14. A flow cell comprising: a fluid flow cell body having a fluid entry portion, a fluid exit portion and an inspection portion; and a porous diffuser portion comprising a particulate diffuser medium and a binder medium, the binder medium being arranged to bind the particulate medium together and to an inner surface of the flow cell to fix the particulate diffuser medium within the flow cell body and arranged whereby fluid flowing through the flow cell flows through the diffuser portion.15. A flow cell according to claim 14 wherein the inspection portion comprises an optically transparent portion.16. A particle detection system or apparatus comprising a flow cell according to any one of claims 12, 14or15.17. A liquid chromatography system or apparatus comprising a flow cell according to any one of claims 12, 14 or 15.18. A method, a flow cell, a kit of parts, a particle detection system or apparatus, or a liquid chromatography system or apparatus substantially as hereinbefore described with reference to FIG. 1(b) or FIG. 2.