WO1995029009A1

WO1995029009A1 - System for dosing and dispensing particles

Info

Publication number: WO1995029009A1
Application number: PCT/GB1995/000931
Authority: WO
Inventors: Eric Mackie Gallie; Michael John Burke
Original assignee: BIOMEDIA INTERNATIONAL Ltd
Current assignee: BIOMEDIA INTERNATIONAL Ltd
Priority date: 1994-04-25
Filing date: 1995-04-25
Publication date: 1995-11-02
Anticipated expiration: 1996-10-25
Also published as: GB9408135D0; EP0757590A1; AU2313095A

Abstract

There is described an apparatus and method which permits accurate weighing and/or measuring of solid particulate matter. The invention may be utilised to enable a hollow structure (1) such as a container (for example a tube) to be at least partially filled with a pre-determined volume or weight of solid particulate matter, such as powder, in an accurate and reproducible manner. In particular there is described a method of measuring out a specific volume and/or weight of particles. The method comprises the up-take of a pre-determined amount of particles into a transfer vehicle (4), movement of the particles into the tranfer vehicle (4) being controlled by pressure (5, 6); and the volume of the transfer vehicle (4) dictating the amount of particles taken up. In this manner a specific volume of particles may be accurately taken up into the transfer vehicle (4) and the volume may be calculated to give a certain weight of particles, depending on the density of the particles in question. The particles taken up in the transfer vehicle (4) may be expelled therefrom, when required, by alteration of the pressure (5, 6).

Description

SYSTEM FOR DOSING AND DISPENSING PARTICLES

There is described an apparatus and method which permits accurate weighing and/or measuring of solid particulate matter. The invention may be utilised to enable a hollow structure such as a container (for example a tube) to be at least partially filled with a pre-determined volume or weight of solid particulate matter, such as powder, in an accurate and reproducible manner.

The invention and apparatus is principally capable of accurately and reproducibly dispensing defined quantities of particulate matter utilising weight to volume mass ratios. The invention has wide areas of application across all areas of industry, commerce, institutions, etc, including biotechnology and pharmaceutical industries and research and development areas.

The accurate measurement of volumes of liquids is possible by the use of pipettes. The pipettes rely upon the production of a negative (suction) pressure to draw up the required amount of liquid. A positive pressure is produced to ensure complete expulsion of the liquid volume when required. However, where the liquid is particularly viscous accuracy of measurement is adversely affected. In addition, there is no method for the accurate measurement of volumes of solid particulate materials. At present the only accurate method of weighing out a specific weight of solid particulate matter is to do so on weighing scales.

In various applications in biochemistry and chemistry reactions are preferably conducted on or at a solid surface. To provide a maximum surface area on which the reaction may take place, solid particles or beads can be used and these are conveniently placed into a suitable container, such as a column. The particles or beads may themselves be hollow allowing the reaction to be conducted at an internal surface. This approach is of utility in many areas of chemistry and biochemistry, for example column chromatography, affinity chromatography, assays such as enzyme linked immunoassay or RNA/DNA assays and also continual production processes, for example where micro-organisms are grown on solid particles and a continual feed of nutrient media is fed through.

Hereinafter the term "particle" will be used to refer to any finely divided solid matter, including powders, filings, crystals, beads and the like and shall not be construed as being limiting to matter of completely solid cross-section but may include hollow or partially hollow particles.

Presently the columns filled with such particles have an internal bore of a relatively large size, for example a bore of 1 cm, and are filled manually by a laboratory technician, who inserts the particles via a filler such as a funnel.

Manual insertion is however laborious and time consuming. Moreover, due to the static attraction between the particles themselves or the particles and the sides of the column, the minimum internal diameter of the column is severely limited in practice. In addition, keeping the particles uncontaminated can be very difficult by manual filling. Accordingly, despite the advantages of providing a solid phase reaction surface, there is as yet no effective way of introducing particles, particularly a pre-determined and accurate measurement of particles, into hollow structures such as tubes.

The present invention seeks to overcome the disadvantages encountered in the prior art and provides a method by which a pre-determined amount of particles can be measured out in a repeatable manner. The invention also provides a means by which the particles may be introduced into a hollow member, such as a tube.

According to one aspect, the present invention provides a method of measuring out a specific volume and/or weight of particles. The method comprises the up-take of a pre-determined amount of particles into a transfer vehicle, movement of the particles into the transfer vehicle being controlled by pressure; and the volume of the transfer vehicle dictating the amount of particles taken up. In this manner a specific volume of particles may be accurately taken up into the transfer vehicle and the volume may be calculated to give a certain weight of particles, depending on the density of the particles in question. The particles taken up in the transfer vehicle may be expelled therefrom, when required, by alteration of the pressure.

In one embodiment of the invention, the particles are transferred into the hollow member by locating the transfer vehicle close to a store of the particles to be inserted and causing the transfer of a pre- determined amount of particles into the transfer vehicle by applying a suction pressure to the transfer vehicle. The amount of particles taken up into the transfer vehicle is directly related to the volume of the vehicle itself. The vehicle volume is pre- calibrated as required. Optionally the volume of the transfer vehicle is adjustable so that the amount of particles transferred in accordance with the invention is not fixed and can be adjusted for different applications.

The transfer vehicle is generally of hollow structure having opening(s) through which the particles are taken up and later expelled and a second opening which is adapted to be connected to an apparatus capable of applying a suction pressure and/or a positive pressure. In its simplest form, the transfer vehicle will have a single opening through which particles are both taken up and later expelled. Alternatively separate openings may exist in the transfer vehicle for take-up of the particles and for their expulsion. Each opening will require its own pressure means to facilitate movement of the particles. In one embodiment the transfer vehicle is essentially tubular, with an opening at each end of the tube. In this embodiment the volume of the transfer vehicle to be filled with particles is generally bounded by a membrane or filter which does not interfere with the pressure applied but does not permit the particles to pass through.

Alternatively, the transfer vehicle may be a blind ended hollow chamber, the particles being taken up and expelled via the open end of the chamber and the pressure being applied via a second opening, optionally in a side wall of the chamber which is covered by a membrane to prevent particle escape.

A variety of membranes and compound materials can be used in the membrane or filter area. Examples include PTFE membrane material of various porosities and derisities, Vyon HP (both high and low derisities) , carbon and cellulose fibre materials, perlite and diatomaceous earth materials, cotton matrices (for example in polypropylene cartridges) . The membrane or filter material may be hydrophilically or hydrophobically treated, if required.

In this embodiment, suction is applied continually to the transfer vehicle once this is filled with particles until expulsion of the particles is required. Maintenance of the suction pressure ensures retention of the particles within the transfer vehicle.

The transfer vehicle is important not only for measuring out the required volume/weight of particles, but also for accurate positioning so that the particles are expelled fully into the receptacle provided.

According to another aspect, the present invention provides a method of filling a hollow member at least partially with particles. The method comprises the accurate location of a frit within the hollow member; insertion of a pre-determined amount of particles into the hollow member via a transfer vehicle, movement of the particles being controlled by pressure; and, optionally, location of a second frit in the bore of the hollow member above the particles. In this manner a layer of particles is located accurately in the hollow member sandwiched between an upper frit and a lower frit.

The hollow member may be of any suitable shape, but typically is generally tubular, for example a hollow cylinder. The cross-section of the main axis (internal or external) is not limited to being circular and the cross-section size or shape may vary. Whilst the hollow member requires two openings (to permit entry of the particles and to provide pressure differential) these openings need not be at the extreme ends of the hollow member. Indeed in one envisaged embodiment the hollow member comprises a tube having a blind end and with a side-opening or top opening to a suction pump, the particles entering via the open mouth of the tube.

The term "frit" is used herein to refer to any material which can be used to plug the hollow member at the required points. The frit material must be at least partially porous to enable any pressure differential applied to one side of the frit material to be transferred onto the opposite side of the frit material. Suitable examples of frit material include commercially available membranes or filters. Of course, the pore size of the frit should be selected to prevent escape of the particles bounded by it.

Uptake and retention of the particles in the transfer vehicle may be performed as described above.

The particles are transferred from the transfer vehicle to the hollow member by application of carefully balanced suction and positive pressures. Firstly, a partial vacuum is created in the hollow member, for example by applying suction to an end of the hollow member opposite or adjacent to the particle entrance. The suction pressure is present throughout the hollow member as the frit is permeable. The suction is balanced by a positive pressure now applied to the second end of the transfer vehicle. This positive pressure replaces the previous suction, and changeover between the negative and positive pressures should preferably be as smooth as possible. Once transfer of the particles has been completed, a second frit may then be inserted into the hollow member. Where the frit is a close or interference fit with the internal diameter of the hollow member, any particles located on the sides of the internal bore of the hollow member will be pushed downwards to form a compact and distinct body of particles.

The pressure differential required to cause movement of the particles first into the transfer vehicle and then into the hollow member is conveniently caused by evacuation of the surrounding gas to produce a suction (negative) pressure or by forcing gas into the appropriate component to cause a positive pressure. Generally the method of the present invention will be operated under atmospheric conditions and the surrounding gas will be air. However, in certain situations (for example where the particles being transferred are oxygen sensitive) the invention may be conducted under atmospheres of other gases, for example nitrogen.

The method of the present invention can be made sufficiently accurate to permit production of icro- columns. The filled columns may optionally be disposable. The size of the column may be typically about 2 mm internal diameter but can be lower, for example 0.5 mm diameter. Conversely, if larger columns are required then the method of the present invention can be easily adapted to cope with larger volumes.

The location of the frit material may be achieved by any conventional means but is conveniently achieved by the use of a two-stage punch.

In such a case, the first main part of the punch may be a hollow cylinder with a sharpened nozzle (of, for example, hardened steel) which, when pressed over a sheet of frit material, punches out and retains a piece of frit corresponding to the internal diameter and shape of the punch. To avoid blunting of the punch nozzle, the frit material can be conveniently placed over a surface made of defor able material, for example rubber. Thus the punch cuts through the frit material and contacts the deformable surface.

The second part of the punch may comprise a plunger located within the bore of the punch itself. When the punch nozzle is in position over the hollow member, the plunger is depressed to a pre-determined distance, transferring the frit a corresponding distance along the hollow member. The position of the frit may be controlled by the length of plunger within the punch or by the use of adjustable or replaceable stops within the punch apparatus. Alternatively, for example, pneumatic or hydraulic activators can be used to control the depth of depressing the punch and/or the plunger.

It is preferred that the shape of the frit corresponds to the internal bore of the hollow member. It is also preferred that the frit is of a slightly larger size than the internal bore of the hollow member so that a snug fit between the frit and the sides of the bore are produced. For example if the internal bore of the hollow member was 2 mm in diameter, then a frit of diameter 2.1 mm would be satisfactory.

In the pick-up of a specific amount of particles (optionally for transfer into the hollow member) , the particles may be picked up by the transfer vehicle from a store or hop of particles. Preferably, the level of the particles contained within the particle store is monitored, for example by the use of photoelectric cells, and the particle levels may be detected and refilled automatically as required. For accurate measurement, the transfer vehicle may be positioned over and not come into physical contact with the particle store. A vacuum supply connected to the transfer vehicle may be switched on as the transfer vehicle approaches the particle store or after the transfer vehicle is in position in the store.

For accurate measurement, the level of vacuum may be precisely controlled according to the density of the particles and the volume required. If the vacuum produced is too low then there is a risk that the pressure will be too weak to cause sufficient transfer of particles to completely fill the transfer vehicle.

Alternatively, if the vacuum applied is too strong then the particles may be compressed within the volume of the transfer vehicle and thus the amount of particles transferred could be too great. For example, to transfer 5 mg of powder, a vacuum of approximately -350 to -500 mbar may be sufficient. However, for larger or less accurate particle transfer, the level of vacuum need not be so precisely controlled.

Again, where the amount of the particles being picked- up is relatively large or where accuracy is not crucial it is possible for the open end of the transfer vehicle to be dipped into the particle store. However, this is not preferred for accurate measurement since some compacting of the particles may result.

If the amount of particles to be picked-up must be governed extremely accurately, it is possible to wipe off any excess particles adhering to the tip of the transfer vehicle in a separate wiping operation, for example using a spring-loaded shutter, prior to release of the particles.

Where the transfer vehicle is being used to measure out a specific volume of particles, reversal of the suction pressure in the transfer vehicle to produce a positive pressure may be sufficient to cause complete expulsion of all the particles from the transfer vehicle.

Where the particles are being transferred from the transfer vehicles to a narrow restriction (such as the hollow member) then negative pressures behind the restriction (ie inside the hollow member) may be desirable. Careful balancing of such negative and positive pressures is required to avoid blowing back of the particles or movement of the frit within the hollow member. Generally the vacuum applied to the lower opening of the hollow member (ie beneath the first frit, the particles being placed above that frit) is exactly balanced by the positive pressure applied to the transfer vehicle to expel the particles. A pressure of 2 to 3 psi may be sufficient where 5 mg of particles are to be transferred to a tube of 2 mm diameter. Generally the difference between the positive and negative pressures applied to hollow member and transfer vehicle respectively should be controlled to a maximum variation of 0.1 psi, but the matching of the two pressures will of course be less relevant for non-accurate measurements and also for larger volumes.

The present invention also envisages that two or more powders could be inserted into the tube in separate transfer operations.

Each stage of the operation described above may be automated, with the completion of each step being detected by automatic sensors with feed-back to a central processing unit, such as a microprocessor.

In another aspect, the present invention provides apparatus to perform the method of the invention.

The apparatus according to the first aspect of the present invention comprises a transfer vehicle adapted for connection to pressure means capable of applying negative and positive pressures to the transfer vehicle sufficient to cause pick up of a pre-determined amount of particles and to aid expulsion of said particles.

The apparatus according to the second aspect of the present invention comprises a two-part punch for production and insertion of a first frit and a second frit to the hollow member; a transfer vehicle adapted for connection to pressure means capable of applying negative and positive pressures to the transfer vehicle sufficient to cause pick up of a pre-determined amount of particles and to aid expulsion of said particles; and means to apply a negative pressure to said hollow member to aid entry of said particles therein.

In a further aspect the present invention provides a hollow member at least partially filled between pre- determined upper and lower loci with a pre-determined amount of solid particles. Conveniently the hollow member may be a column or tube and when partially filled in the manner described may be of utility for chromatography (especially affinity chromatography) . In one embodiment the partially filled column or tube may be used to provide a solid support surface for conducting reactions thereon, for example PCR reactions.

Where the cleanliness and/or sterility of the particles is to be preserved, then the apparatus described above should be non-shedding and non-oil lubricated. For example, the apparatus could conveniently be comprised of a stainless steel and PTFE components with no metal- to-metal contact. Any moving parts could be controlled pneumatically by air pressure to avoid contamination. Again, the apparatus could be fully automated with each operation being detected by sensors and in the overall control of a central control unit.

The apparatus of the present invention will now be described in further detail with reference to the accompanying drawings.

Figure 1 shows a cross-section of the apparatus according to the invention including a punch apparatus and filling station at which the hollow member is filled with powder;

Figures 2 and 3 are cross-sections showing more detail of the two-stage punch;

Figure 4 shows in cross-section one embodiment of the transfer vehicle including the powder store; and

Figure 5 is a cross section of the embodiment of the dispensing vehicle - illustrated in Figure 4.

Figure la shows a hollow member (1) in the form of a tapered tube. The hollow member (1) is held within a holder (2) located in rack (3). Both holder (2) and rack (3) have apertures which connect to the lower end of hollow member (1) . By means of these apertures, lower end of hollow member (1) is connected to a suction pump (not shown) via connector (7) .

Shown in position over hollow member (1) is transfer vehicle (4) located in suitable holder (8) . A membrane (9) located at a pre-determined position within transfer vehicle (4) determines the volume of particles to be transferred into hollow member (1) . In this embodiment, transfer vehicle (4) is of tubular construction with membrane (9) being located approximately one third of the way up its length. Below membrane (9) , transfer vehicle (4) has a pre- calibrated volume (10) in which particles are transported from the store to the hollow member.

The upper end of transfer vehicle (4) is connected to a connector (11) which allows separate connections to apparatus for inducing a negative pressure (5) and apparatus for producing a positive pressure (6) (both not shown) .

Illustrated in Figure lb is the two-part punch apparatus indicated generally at (12) . The punch (12) comprises two main components namely a hollow cylinder (13) with a sharpened nozzle (18) capable of punching out the required frit material. Depression of the whole punch apparatus causes nozzle (18) to cut into the frit material (15) located immediately below it. The frit material (15) is placed on a hard rubber block (16) which provides a cutting service for the nozzle (18) whilst being sufficiently deformable to prevent blunting of the nozzle ends. During the cutting operation, the second part of the punch, plunger (14) is retained within the hollow cylinder (13) and is sufficiently retracted to allow a space behind nozzle (18) for the retention therein of the piece of frit cut out of material (15) . The punch apparatus, including the frit material (15) is then located over hollow member (1) (not drawn to scale) and once in position plunger (14) is lowered causing expulsion of the cut out piece of frit material from within the hollow cylinder (13) .

In the embodiment as shown, a negative air pressure may be used to control the punch action the connection to the suction pump being via connector (17) .

Figure 2 shows in more detail the two-part punch (12) including the hollow cylinder (13) with punching nozzle (18) located over the frit material (15) positioned on a hard rubber block (16) .

In the embodiment of the two-part punch illustrated in Figure 3 the hollow cylinder (13) is retained by rim (20) within a housing (21) positioned within the interior bore of hollow cylinder (13) is the plunger (14). The plunger (14) is normally retained within the cylinder (13) with its lower end retracted a pre- determined distance within nozzle (18) . The positioning of the plunger (14) in the retracted position may be achieved by the use of a biasing means such as helical spring (22) which urges the plunger (14) into its retracted position. The plunger (14) is prevented from falling out of the two-part punch under gravity by lip (24) which contacts the other end of helical spring (22) and may further contact rim (20) of hollow cylinder (13) . Rim (24) also provides a contact with upper plunger (23) which when depressed causes plunger (14) to move out down the hollow cylinder (13) pushing any frit material retained therein into position within the hollow member (1) (not shown) .

In the embodiment illustrated, upper plunger (23) is fixed relative to the housing shown at (25) . The housing (25) together with upper plunger (23) is urged into a downwards motion by a lever action which compresses helical spring (26) . Upper plunger (23) contacts rim (24) of the punch plunger (14) urging this downwards and compressing helical spring (22) . Upper plunger (23) moves downwardly until housing (25) contacts stopper (26) which prevents any further downward movement. The height of stopper (26) thus determines the distance that upper plunger (23) and plunger (14) may move through and thus the distance that the frit material (not shown) is moved through by the action of punch plunger (14) into the hollow member (l) (not shown) .

Stopper (26) may be replaced by a pneumatic cylinder attached to upper housing (26) and which can provide the force needed to cause the downward action of upper plunger (23) and the consequent movement of plunger (14) and the frit material. This pneumatic cylinder can be pre-calibrated to determine the extent of the downward motion and thus the positioning of the frit material into the hollow member (1) (not shown) .

Figure 4 illustrates a particle storage chamber (30) containing a pre-determined level of particles (31) . The level of the particles (31) is constantly monitored by an optoelectric sensor (33) which feeds the data obtained into a microprocessor unit (not shown) . The output from the sensor (33) is used to control delivery of particles (31) via a delivery tube (32) . The transfer vehicle (4) is illustrated with a pre- determined volume bounded by membrane (9) . The transfer vehicle (4) approaches the particle store (30) by movement in the direction of Arrow A to within a distance of approximately 2 mm to the upper surface of particles (31) . As the transfer vehicle (4) approaches, a suction pressure is applied through the bore of transfer vehicle (4) from connector (11) which is used to connect to a suction pump (not shown) . The suction pressure applied throughout transfer vehicle (4) is transferred through membrane (9) towards the lower opening (34) . The pressure differential experienced by the particles (31) causes their uptake into the interior of vehicle (4) so that volume (10) becomes completely filled with the particles (31) . The amount of suction is carefully controlled at the suction pump and/or at connector (11) so that -the particles (31) do not become compacted within volume (10) although the pressure applied is sufficient to cause volume (10) is completely filled with particles ( 31 ) .

Once volume (10) is completely filled with particles (31) it is essential that the suction pressure is continual until expulsion of the particles (31) from the transfer vehicle (4) is required.

In the embodiment illustrated, movement of the transfer vehicle (4) is achieved by movement on the racks (35 and 36) by any convenient means, for example by a ratchet motion. Movement is permitted in the vertical (up and down) and also in the horizontal (side to side) directions. Once the transfer vehicle (4) has been filled with the required volume of powder (31) the particles will be transferred to a hollow chamber (1) (not shown) which is retained within a rack (2) and holder (3) .

Figure 5 shows the transfer vehicle (4) in greater detail. In this embodiment, vehicle (4) consists potentially of a hollow cylinder with an upper lip (14) to engage with connecting apparatus (11) (not shown) . At a pre-determined distance along the internal bore of the cylinder making up the main part of transfer vehicle (4) a membrane (9) is located. It can be seen from the diagram that membrane (9) is a snug fit within the internal bore of transfer vehicle (4) and effectively bounds volume (10) into which the particles (31) will be taken up. The position of membrane (9) within the internal bore of transfer vehicle (4) is directly related to the size of volume (10) and the position of membrane (9) may be altered as required depending on the volume of particles to be transferred.

The embodiment illustrated in Figure 5 is made in two parts, the lower portion (41) and upper portion (42) fitting together so that the internal bore is continuous. The two components (41 and 42) are held together by a housing (43) which contains two sets of indentations (44) adapted to receive a spring clip (45) which may hold the transfer vehicle (4) movably attached to its holder. It is clear however that, if required, transfer vehicle (4) may be made in one piece although for easy construction the construction illustrated may be preferred.

Claims

l. A method of measuring a specific volume and/or weight of particles, said method comprising filling a pre-determined internal volume of a transfer vehicle with said particles; expelling the particles from said transfer vehicle; wherein movement of said particles into and out of said transfer vehicle is controlled by pressure.

2. A method as claimed in Claim 1 wherein said particles are urged into said transfer vehicle by suction pressure.

3. A method as claimed in Claim 2 wherein said suction pressure is applied during take-up and retention of said particles in said transfer vehicle.

4. A method as claimed in any one of Claims 1 to 3 wherein said transfer vehicle is adapted to be connected to apparatus capable of creating suction and/or positive pressure within the transfer vehicle.

5. A method as claimed in any one of Claims 1 to 4 wherein the internal volume of said transfer vehicle can be adjusted.

6. A method as claimed in any one of Claims 1 to 5 wherein said transfer vehicle has two openings and a pre-determined internal volume for containing said particles, the first opening being connectable to a pressure means and being covered by a membrane or filter to prevent particle escape, and the second opening being adapted for take-up and/or expulsion of said particles.

7. A method as claimed in Claim 6 wherein said transfer vehicle is an open-ended cylinder, wherein the two open ends form said first and second openings respectively.

8. A method as claimed in Claim 6 wherein said transfer vehicle is a blind ended hollow chamber, the first opening being located in a side wall of said chamber and said second opening being formed by the open end of the chamber.

9. A method as claimed is any one of Claims 1 to 8 wherein the particle-filled transfer vehicle is positioned so that expulsion of said particles occurs into a receptacle.

10. A method of at least partially filling a hollow member with particles, said method comprising location of a first frit within the hollow member; measuring a specific volume and/or weight of particles and inserting this measured amount of particles into said hollow member according to the method as claimed in any one of Claims 1 to 9; and optionally locating a second frit in the bore of the hollow member above the particles.

11. A method as claimed in Claim 10 wherein a partial vacuum is created in the hollow member to aid particle transfer.

12. A method as claimed in either one of Claims 10 and 11 wherein the internal diameter of the hollow member is about 2mm or less.

13. A method as claimed in any one of Claims 10 to 12 wherein the first frit is permeable to gas and wherein said partial vacuum is created by suction.

14. A method as claimed in any one of Claims 10 to 13 wherein said second frit is a close or interference fit with the internal diameter of the hollow member.

15. Apparatus adapted to perform the method as claimed in any one of Claims 1 to 9, said apparatus comprising a transfer vehicle adapted for connection to pressure means capable of applying negative and positive pressures to the transfer vehicle sufficient to cause pick up of a pre-determined amount of particles and to aid expulsion of said particles.

16. Apparatus adapted to perform the method as claimed in any one of Claims 10 to 14, said apparatus comprising a two-part punch for production and insertion of a first frit and a second frit to the hollow member; a transfer vehicle adapted for connection to pressure means capable of applying negative and positive pressures to the transfer vehicle sufficient to cause pick up of a pre-determined amount of particles and to aid expulsion of said particles; and means to apply a negative pressure to said hollow member to aid entry of said particles therein.

17. A hollow member at least partially filled between pre-determined upper and lower loci with a pre- determined amount of solid particles, said hollow member being filled according to the method as claimed in any one of Claims 10 to 14.