This invention relates to a funnel and a funnel apparatus, especially for ^' use in dispensing a. liquid in a controlled and substantially non-turbulent manner such as is useful as part of a method of density separation for separating a solid phase from a liquid phase. The invention also relates to such a method.

Background of the Invention The separation of solid and liquid components of. a mixture is of central importance to methods of heterogenous assay in which such a separation is necessary prior to the measurement of an assay parameter. In particular, heterogenous binding assays (such as im unoassays) rely upon the binding of a component of the assay to a solid phase and the subsequent removal of the solid phase with its bound component from the remaining liquid phase of the assay. Such assays may be conveniently conducted using the separation method described i British patent specification No.1566098. (The teachings of British patent specification 1566098 are incorporated herein by reference) .

In British patent specification No.1566098 there is described a method for separating solid and liquid components of a mixture comprising a solid and a liquid, in which a second liquid, having a density intermediate the density of the solid and the liquid components respectively, is delivered to the mixture to form a discrete layer above or below the mixture. Under the influence of gravity the solid component separates into the layer of the second liquid. Normally the second liquid is of higher density than the liquid component of the mixture and of lower density than the solid component of the mixture. The specification also describes an automated apparatus

for performing the method. The apparatus comprises a tubular probe for delivering the second liquid, anάr an automated control device for introducing the probe into the mixture contained in a receptacle such thaf an outlet end of the probe is positioned near the bottom of the mixture in the receptacle at least during delivery of the second liquid. The second liquid may in this way be delivered in a substantially non-turbulent manner to the bottom of the mixture by means of a pump, to form a discrete layer. The method described in British patent specification No.1566098 affords an alternative separation technique to the commonly used techniques of filtration and centrifugation. The operation of the method of British patent specification No.1566098 depends -upon the introduction of the second liquid into the mixture in a manner which allows the formation of a discrete layer. This requires a substantially non.- urbulent flow which is, in practice, not easy to achieve reproducibly. Non-turbulent flow is achieved in the apparatus described in British patent specification No.1566098 by the provision of a pumped flow of the second liquid through a tubular probe _* However, this is itself, to a certain extent, disadvantageous in that the pumping equipment complicates the apparatus for performing the separation method, requires periodic cleaning and maintenance, and may in the case, for example, of a peristaltic pump provide a non-uniform flow which may encourage mixing.

It is an object of this invention to provide apparatus which does not suffer from these disadvantages. Summary of the Invention According to a first aspect of the present invention we provide a funnel comprising, a reservoir

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for a liquid and an outle tube, charac erised, i that a constriction limits the flow rate of liquid through the outlet tube.

The constriction acts to reduce the flow of liquid passing from the reservoir under the influence of gravity through the outlet tube. The primary intended use of the funnel is to form a discrete layer of a liquid at the bottom of a mixture of a less dense liquid with a more dense solid material in a receptacle. The outlet tube is inserted into the mixture such that the outlet end of the outlet tube is close to, or at, the bottom of the mixture. The liquid which is to form the discrete layer is then placed in the reservoir and allowed to pass slowly through the outlet tube under gravity, the: rate of flow being limited by the constriction. In this way a non-turbulent flow is created reducing the risk of inadvertent mixing of the liquid with the mixture. The" 'funnel provides a simple, effective and cheap means for forming discrete liquid layers such as are required by the method of British patent specification No.1566098.

The constriction may be in the form of a uniform internal diameter of the outlet tube throughout its length or a tapering of the outlet tube.

Preferably however the constriction comprises a portion of the outlet tube having an internal cross-sectional area less than the remaining internal cross-sectional area of the outlet tube. Suitably the outlet tube may comprise a tube having an internal cross-sectional area which is uniform over most of its length, there being a short portion of constriction at some point along the tube. Preferably the constriction is located at the junction of the outlet tube with the reservoir.

In another aspect of the., invention we provide a., funnel for delivering, to a mixture comprising a solid and a liquid, a second liquid having a density intermediate those of the solid and the liquid, in which the funnel is arranged and dimensioned such that the second liquid introduced thereto is delivered therefrom by gravity towards the base of the mixture in a substantially non-turbulent manner to form a discrete layer beneath the mixture. The specific dimensions of the funnel depend upon the application for which the funnel, is used .and in particular upon the nature of the ^' liquid to be dispensed from the funnel.

A particular application of the funnel is in a heterogenous binding assay. In such an assay a sucrose solution, for example a 10% w/v sucrose solution, may be used as a second liquid. For use in such an application the volume of the reservoir is preferably from 0.5 to 10 ml, most preferably from 1 to 5 ml. The outlet tube is preferably of such a length that it may extend to, or almost to, the bottom of a sample tube such as is customarily used in binding assay procedures. In the case of a round-bottomed sample tube the outlet tube preferably extends into the sample tube at least to the point at which curvature of the bottom of the sample tube starts. Preferably the outlet tube is at least 5 cm in length, more preferably from 6 to 9 cm, for example about 7 cm in length. The outlet tube preferably has an internal diameter from about 0.25 to 5 mm and more preferably from 0.5 to 3 mm. The constriction may have an internal diameter of about 0.5 mm over a region between 1 and 2 mm in length. Preferably the volume of- the second . liquid is delivered from the outlet tube at a rate of about 2 ml in a period of 5 to 25 seconds, most preferably in

about 15 seconds.

' In applications where heterogeneous assay is to be performed there is often a need for assaying a number of samples simultaneously. In a further aspect of the invention therefore we provide a funnel apparatus comprising at least one reservoir for a liquid and two or more outlet tubes, wherein constrictions limit the flow rate of liquid passing through the tubes. The funnel apparatus of this aspect of the invention has the. advantage that the outlet tubes may be inserted into a number of suitable sample tubes by one movement of the assay operator (be that operator human or mechanical) . Preferably the outlet tubes and the or each reservoir are integral. Preferably the outlet tubes of the funnel apparatus form an array of one or more columns and two or more rows. An array of outlet tubes may in this way correspond to a row of sample tubes or to a two-dimensional array of sample tubes, for example a 4 x 5 array of sample tubes. In the apparatus each outlet tube is preferably provided with a separate reservoir, but there may be a single reservoir for all the outlet tubes. The outlet tubes may each have the features of the outlet tube described above with respect to the funnel of the first aspect of the invention. - -

The funnel or the funnel apparatus of the present invention may be constructed of any suitable material including glass or metals such as stainless steel to provide a re-usable apparatus. The funnel may be constructed of a plastics material and, for instance, in a moulded plastics form may provide a disposable apparatus. It will be appreciated that the nature of the material from which the funnel is constructed may affect the flow rate of the second liquid and with particular materials suitable adjust-

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- e - ments of the funnel dimensions, in particular the* inte'rnal diameter of the outlet, may be required to obtain satisfactory flow rates of the liquid.

In a further aspect of the invention we provide a method of separating solid and liquid components of a mixture comprising a solid and a liquid in which a second* liquid having a density intermediate those of the solid and liquid components of the mixture is delivered to the mixture using a funnel or funnel apparatus of the invention to form a discrete layer above or below the mixture and the solid separates into the layer of the second liquid.

The accompanying drawings are given for the purpose of illustrating embodiments of the invention. Description of the Drawings

Figure 1 is a vertical cross section through a funnel of the invention.

Figure 2 is a vertical cross section through a funnel apparatus of the invention. ^{' *} " Figure 3 is a vertical cross section through a further funnel apparatus of the invention.

Figure 4 is a plan view of a further funnel apparatus of the apparatus.

Figure 5 is a cross section of the funnel apparatus of Figure 4 on the line I-I.

Detailed Description of Embodiments

With reference to Figure 1 a funnel comprises an integral reservoir 1 and outlet tube 2 machined from stainless steel. The reservoir 1 comprises a tube of circular cross-section open at its top end 3. The outlet tube 2 is disposed symmetrically along the axis of reservoir tube 1 and comprises a tube of circular cross-section open at its lower end 4 and having a bevel 5 around its lower outer edge. The bottom of the reservoir 1 is provided by an inwardly and downwardly angled annular base 6 which joins with the top end of the outlet

tube 2 below the bottom of. the reservoir tube 1. The-, top "end of the outlet tube 2 has a section 7 of constricted internal diameter providing the outlet from the reservoir 1 to the rest of the outlet tube 2. The top end of the outlet tube 2 provides a horizontal flat annular surface 8 at the centre of the internal surface of the angled annular base 6 having a circular outlet 9 at its centre surrounded by a bevelled recess. The outlet 9 extends via the section 7 of constricted internal diameter into the rest of the outlet tube 2.

In alternative forms the constricted section 7 may be located at a different point within the length of the outlet tube 2. Also the internal diameter of the outlet tube 2 may be uniform throughout its length, and the outlet tube may taper along its length internally and/or externally.

In use, the outlet tube 2 is inserted into a bindi g assay sample tube (not shown) containing a mixture comprising a liquid and a solid and hangs suspended with the base 6 resting against the mouth of the assay tube with the lower end 4 held just above the bottom of the assay tube. 2 ml of a liquid for example 2 ml of 10% w/v sucrose solution, is then pipetted into the reservoir 1. Under the action of gravity the liquid passes fro the reservoir 1 through the constricted section 7 into the outlet tube 2 and is delivered from the bottom end 4 thereof to the base of the mixture in the assay tube to form a discrete layer of liquid beneath the mixture. The 2 ml volume of liquid is delivered under the action of gravity over a period of about 10 to 20 seconds, the flow rate being determined by the size of the constriction at 7, ensuring substantially non-turbulent delivery of the second liquid to the mixture.

After layering,, solid present in the mixture separates under the action of gravity into the layer of the liquid, passage of solid through the liquid effectively washing the solid and removing traces of liquid, from the mixture, which adhere to the solid. Further layering steps may be employed to provide improved separation of solids from the liquid component of the ^■ ■ mixture. Subsequent liquid layers may be of the same or greater density than the first layer added.

With reference to Figure 2 a funnel apparatus comprises a plurality of funnels moulded in one piece from a plastics material. Each funnel 20 shown generally has a reservoir 21 and an outlet tube 22. The outlet tube 22 has an upper section 23 of constricted internal diameter substantially as described for the single funnel of Figure 1. The. reservoirs 21 are contained within a large feeder reservoir bounded by walls 24. In use the separate outlet tubes 22 are located in a corresponding array (for example a 4 x 5 array) of assay tubes (not shown), with their lower ends resting against the internal curvature of the bottoms of the assay tubes. A large volume of liquid for example 10% w/v aqueous sucrose solution, is introduced into the feeder reservoir at ^" 25 and overflows into the separate funnel reservoirs 21. The liquid is then delivered from the funnels 20 under the action of gravity as described above to form discrete layers of liquid below the mixture contained in the assay tubes. The constricted section 23 of outlet tube 22 controls the rate of flow of the second liquid ensuring substantially non-turbulent delivery to the mixture. Alternatively 2 ml aliquots of second liquid may be pipetted into each reservoir 21 in turn.

With reference to Figure 3 a further, version of _s the funnel apparatus comprises a single feeder reservoir 30 having an array (for example a 4 x 5 array) of outlet tubes 31 moulded in on piece from

5. suitable plastics material. Each outlet tube 31 provides an outlet from a circular conical recess (of approximate volume 0.5 ml) in the base of the reservoir 30 through a section of tube 32 of constricted internal diameter. This apparatus is used in a similar fashion to the apparatus of Figure

2. The constricted sections of the outlet tubes.32 control the rate of flow of liquid through the tubes

31 and its non-turbulent delivery to the bottom of the mixture in the assay tubes. The required volume of liquid is introduced to the reservoir 30 and flows away therefrom simultaneously via the outlets 31 under the action of gravity. In an alternative form of the funnel apparatus of Figure 3, walls 33 (shown in broken lines) are provided to separate the conical recesses from one another and provide a plurality of reservoirs 36. The total volume of liquid required is added over a short time period to one reservoir 36 and overflows therefrom into the remainder of the reservoirs 36. With reference to Figures 4 and 5 a further version of a funnel apparatus comprises ^' twenty individual funnels (one of which is shown generally as 40) arranged as a 4 x 5 array. Each funnel 40 comprises a reservoir 41 having a collecting portion 42 for directing liquids towards the reservoir 41. At the base of the reservoir 41 is an outlet tube 43. The top end of the outlet tube 43 is provided with a constriction 44 for limiting the flow rate of liquid passing through the outlet tube 43 from the reservoir under the influence of gravity. The collecting portions 42 which are generally square

in plan and are formed with common connecting walls

(foE example 45) which link the funnels 40 together.

Ribs (for example 46) provide mechanical stability for the funnel apparatus. The funnel apparatus is of integral plastics construction. The method of use of the funnel apparatus is as described above, separate aliquots of the liquid being pipetted into the separate collecting portions 42.