WO1998003264A2

WO1998003264A2 - Chemical sample treatment cassette and methods

Info

Publication number: WO1998003264A2
Application number: PCT/US1997/011828
Authority: WO
Original assignee: Amersham Pharmacia Biotech Ab
Priority date: 1996-07-09
Filing date: 1997-07-07
Publication date: 1998-01-29
Also published as: US5918273A; EP1015881A2; US6153437A; AU3653297A; EP1015881A4; US6082417A; US6039924A; US6173603B1; AU730411B2; US6168760B1; CA2259648A1; JP2000514563A; WO1998003264A3; US6427731B1; US5800784A

Abstract

This invention is directed to a cassette chemical immobilization and treatment system (10) and method for enabling the performance of various complex chemistries with minimal human intervention, near-zero dead volume, and flow-through protocols pursuant to a predetermined instruction set encoded on a multiple-address chemical treatment cassette assembly. The cassette assembly (12) comprises a plurality of analyte sample columns (13) ('mini-columns'), reagent wells (31) containing pre-packaged reagents including powdered, microencapsulated, liquid or lyophillized reagents, analyte loading funnels (19), an alignment assembly (15) for identifying a chemical treatment protocol. The mini-columns (13) are improved columns having high pressure interface capability to permit direct insertion of the mini-column into a high-pressure solvent line for use as a support column for HPLC analysis.

Description

TΓΠ E! CHEMICAL SAMPLE TREATMENT CASSETTE AND METHODS

BACKORouiro or TBS I VWJTIO

TE HNlCλXi πSUBl

The present invention relates to a cassette-type chemical sample treatment system apparatus and method for use in the treatment of less than one milligram quantities of amino acids, proteins, peptides, and the like pursuant to predetermined or preselected chemical, biochemical, and biomedical protocols. More particularly, the invention of this application relates to a cassette-type chemical sample treatment system and method having a cassette for holding a plurality of sample columns for immobilizing preselected samples and a plurality of reagent wells for retaining preselected reagents for enabling predetermined chemistries of the preselected samples with the preselected reagents, including column loading, treatment, and post-treatment analysis of the reacted sample with near-zero dead volume and minimal human intervention, the predetermined chemistries being specified by machine readable code integrated into the cassette.

BACXQλOUm AMi

With the exception of certain high-performance liquid chromatography (HPLC) , proteins and peptides are typically fractionated in aqueous buf ers containing amines, salts and, often, denaturants. Therefore, additional manipulations such as desalting by HPLC, precipitation, or dialysis are required to render the sample matrix compatible with protein and peptide chemistry or peptide sequencing protocol. Bach of these additional steps, however, involves potential losses, especially when only leas than milligram amounts of protein are present at low concentrations. It is generally understood that proteins include peptides, accordingly, for purposes of this invention, no distinction is made between peptides and proteins, and reference to one will also apply to the other.

Many of the steps involved with the aforementioned steps have been eliminated by immobilizing a protein or a peptide directly onto a solid support, washing away any interfering components, and leaving the protein bound to the support ready for either further on-eolumc chemistries or removed for analysis. Hewlett-Packard Analytical Instruments (Palo Alto, California, US) manufacturers a family of analytical devices (HP-Q1000A, HP-Q1004B, and HP- G1005Λ) which use sample columns containing a solid support upon which proteinβ or peptide^β might be immobilized. The HP-01004A is a Protein Chemistry Station (PCS) which permits on-column chemistries to proteins and peptidea immobilized on n yϋrophobj c nup ort. Tlio IIP-G1005 performs standard Edition degradation protocols on a peptide immobilized on a bi-phaβic support for the performance of peptide sequencing.

Poβt-tranβlational modifications will determine the stereochemistry and conformation of the peptide. Accordingly, there is a need to determine the nature of the side groups so that a coπformational analysis or a structure determination may be made. Such post-translational modification chemistries may be carried out manually or βemi-automatically whereby a sample 1B subjected to reaction with the appropriate reagents to give an appropriate indication in the event the post-translational modification is present. The HP-G004 Protein Chemistry station (PCS) represents the state of the art with reβpect to enabling semi-automatic performance of chemistries. The PCS has limitations, however, with respect to the types and complexity of chemistries that might be performed on this system. For example, the PCS is not able to perform bidirectional pumping; it can only pump down. Thiβ limitation precludes shuttling sample or reagents back and forth through the support. This feature would.be desirable, for example, when the reactivity of a side group is affected by the polarity of a solvent and the appropriate solvent is one that may separate the sample from the support. Second, the chemistries performed in the PCS require that the reagents be dispensed into a large funnel ln order to be introduced to the reaction. Without changlng-out the funnel between reagentβ, a significant risk of cross-contamination is created.

The IIP PCS βyβtem employs a hydrophobic column to immobilize the Bample during loading and during application of the chemistry. Figure 1 is a croβs- βectional diagram of single sample reaction chamber typical of the background art. Λ funnel a is press-fit attached to the inlet side of the hydrophobic column b, with the throat of the funnel c in communication with the top opening of the column d. The funnel/column assembly is loaded into a center cavity e of a clear ucite reaction chamber f , the assembly being secured by compressing and twisting the f nnel/column assembly in a single movement into a bayonet- type connection on the walls of the Lucite holder g, and the column being urged against the funnel by a spring k. Λ cap h is screwed onto the reaction chamber so as to seal the system. Inlet ports i,j in the cap permit the introduction of sample into the funnel and the introduction of a pressurized inert gas to pump the reagent through the column. During the chemistries performed on the PCS, the funnel is never changed-out. As a result, a serious concern with the eystem and method of the background art is that the side walls of the funnel may retain residues of previously introduced reagents, thus resulting ln cross- contamination or reagents.

After insertion and bayonet-locking of the unnel/column assembly into the reaction chamber, a protein or peptide solution sample is loaded in the 5 ml funnel attached to the column. The cap is screwed onto the holder and over the funnel so that preββurlzed nitrogen, or other inert gas, may be applied to the sample and pressure forced into and through the hydrophobic sample column. The sample loading process captures proteins and peptides on the hydrophobic portion of the sample column, while the sample solvent passes through.

It is possible to do multiple sample additions for larger volumes or to use a second solution to wash the sample. First, the holder cap removed, and a second sample or aqueous wash is added to the funnel, the cap reattached to the holder, and the holder pressurised with nitrogen, thus forcing the aqueouβ wash through the hydrophobic sample column.

Following the sample loading, sample preparation (rinses) , and possible sample pre-treatment, the funnel/column assembly is removed from the sample reaction chamber and is transferred to another reaction chamber in the Protein Chemistry Station, wherein the appropriate reagents are administered to perform the desired chemistries. A technician selects a computer program which directs the PCS, via a micro-controller interface, to dispense the appropriate reagent into the sample funnel pursuant to the selected program. The appropriate reagent Is directed through a tube and into a reagent port in the cap of the reaction chamber. The reagent flows from the reagent port into the sample funnel. Pressurized inert gas then forces the reagent out of the sample funnel and into the column.

All chemistries are carried out in the column or the funnel, and within the reaction chamber. Also, since only one reaction chamber may be loaded at a time in the PCS, preparation and treatment of multiple sample columns is a time consuming and tedious effort. Further, the PCS provides only four reservoirs for reagents, buffers or solvents, all of which must be a liquid. Additionally, there may be reactions, especially of blomedical interest, wherein a solid reagent, such as a lyophilised anzyrne, vaccine, hormone preparations, and the like which exhibit lower stability ln solution, tending to either degrade rapidly or require low storage temperatures in its hydrated state, may be needed in order to execute the desired chemistry. The devices of the background art are not able to accommodate these dry reagents. Accordingly, there is a need for an automated chemical treatment eystem capable of performing a multiplicity of both peptide and post-translational modification chemistries sequentially on a plurality of βampleβ, in an uninterrupted manner with minimal human intervention or direction. The automated chemical treatment eystem would also provide means to perform chemistries external to the sample columns and for reintroduction of the reacted sample or analyte to the sample column. Also, there is a need for an automated system that would provide means for " juβt-in-time" delivery of reagents requiring ju^βt-in-time aolubllization, such as adsorbed, lyophilised or other powdered reactantβ to the sample.

Since the same sample funnel is used for all reagents there is a risk of cross-contamination that may affect the outcome of sensitive chemistries.

Minute quantities of reagent may adhere to the walls of the funnel, only to be eluted into the sample column when the next reagent is introduced into the funnel . Where less than one milligram quantities of pep ides or proteins are being investigated, the presence of minute amounts of impurities or crosβ- contaminants may have a significant impact on the results. Accordingly, there

1B a need for a sample column/reaction chamber system that permits near-zero dead volume to minimize risk of cross contamination and the resulting inaccuracies such contamination may cause.

Once the protein chemistries are complete, the sample column from the PCS is removed from the βa ple reaction chamber and is transferred to the appropriate analytical measurement device in order to measure or characterize the results of the chemistries performed. Typically, the analysis iβ selected to characterize the products of a peptide cleavage.

Typically, HP-HPLC is used to analyze the reaction products. Ideally, it would be desirable to insert the sample column of background art directly into an HPLC sample column holder, thus integrating the sample column into the HPLC and transforming the sample column into a RP-HPLC column. In order to obtain adequate separation of proteins, however, column pressures greater than 1500 psi are required. The sample column of the background art rated to withstand pressures up to approximately 1000 psi. At pressures greater than 1000 pal, the non-tapered end of the sample column typically fails. In order to operate at the extremely high pressures required for protein separation, a special adapter is required, and is attached to the inlet end of the sample column to accommodate a high pressure line fitting. Fig. 2 shows a cross-section diagram of the sample column and adapter typical of the background art. The adapter 1 is inserted into the non-tapered and A of the sample column b. Since the βample column is a pre-column to the chromatography column of the HPLC, the adapter Is also a part of the HPLC pre-column. Accordingly, the column plus adapter of the background art is supplied in addition to the standerd chromatogragh column rather than as a substitute column. Consequently, the adapter 1 must also be packed with a hydrophobic support m, and once it is affixed to the outlet end d of the hydrophobic column, becomes an extension of the original hydrophobic column upon which the sample is immobilized. As a result, the adapter may be used only once and then must be discarded. Accordingly, there is a need for a sample column able to withstand tiie high pressures of nP-HPLC that might be directly incorporated, without an adapter or modification, into an HPLC receptacle BO as to integrate the sample column as the chromatography column of the RP-HPLC. The above single-eample procedures associated with the current state of the art device are incapable of performing chemistries or sequential, uninterrupted treatment of multiple samples and results, therefore, in extremely tedious protocolsand prone to crosβ-contamination. Further, a well recognized problem associated with the incorporation of the hydrophobic column into the RP-HPLC is that the coupling between the inlet port of the column and the RP-HPLC line is βuch that residual liquids are trapped ln the headspace between the end of the column support materia and the RP-HPLC coupling. It is well known in the art that a zero-head space is required in order to asβure the most accurate HPLC measurements. The existence of a non-zero head space introduces errors into the HPLC analysis.

Chemical procedures and treatments performed on a βample in preparation for analysis can become tedious, particularly where repetitive chemistries must be performed and time consuming where hundreds or thousands of samples are involved. Additionally, it also creates significant opportunities for errors in measurement, and for contamination of the sample or reagents. Further, if characterization of the sample requires several different chemistries to be performed there 1B an increased chance of error as the technician must now identify, track, and monitor the progress of each of the required protocols.

Although the present state of the art includes micro-controller interface with semi-automated apparatus, the technician must still determine which chemical procedure or protocol is to be performed on any particular sample, and key that protocol selection into the micro-controller. If the technician executes the incorrect protocol, the sample is ruined at best, or, at worst the erroneous analytical data recorded on that sample 1B included ln the data being accumulated.

There is a need to provide an automated means for sequential, uninterrupted performance of chemistries on a plurality of samples contained within or immobilized on a plurality of βample columns with minimal human intervention and reduced risk of performing incorrect protocols. SUMMARY OF THE! INVENTION Tills invention is directed to a caeaette-typβ chemical treatment system and method for enabling the performance of various chemistries with minimal human intervention, near-εαro dead volume, and flow-through protocols pursuant to a predetermined instruction set encoded on the cassette.

The cassette comprises a plurality of sample columnβ ("mini-columns"₎, reagent wells, sample loading funnels, alignment means for the sample columns, and a machine readable instruction code set for determining a chemical treatment protocol. All components are constructed from inert materials including but not limited to linear polyethylene, fluoro-copolymerβ, Teflon and the like.

The mini-columns contain a long inner chamber packed under pressure with a solid support, preferably silica, although other supports βuch as polymer beads, resins, and celluloβe may be used. Alternately, no solid support at all need be used when, for example, the βample is retained as a dry powder or beads, or if the sample Is mlcroencapsulated. In the preferred embodiment, a silica support is employed and derivatized to render it hydrophilli or hydrophobic. For protein chemistries, the silica βupport is derivatized with a lipophilic alkyl group thuε rendering the support non-polar. Consequently, lipophilic proteins diββolved or suspended in a more polar mobile phaβe will become immobilized on the hydrophobic support. Unlike the mini-columns of the prior art, both terminal endβ of the mini-columns of this invention are designed to accommodate the extremely high pressures (greater than 1200 psi) needed to perform reverse-phase HPLC of the immobilized proteins or other hydrophobic moieties. This is accomplished by designing a generally concave, preferably tapered opening at each end of the column so that an interface means may be attached thereto with near-zero dead volu n. The opening can be βpheroldally concave, parabolic, conioally tapered, and the like so long as a high pressure seal can be effected with a compression fit interface means. Suitable compression fit interface means include: a nozzle having a rounded tip for insertion and compression fit against the walls of the generally concave, preferably tapered opening; a nozzle having a conical tip for insertion inside the inner diameter of the longitudinal chamber for compression fit against the junction of the inner wall of the longitudinal chamber at apex of the generally concave, tapered opening with the conical nozzle; and the like to permit localized Interface wit the inlet and outlet ports to enable near-zero dead volume communication. The term "localised interface" ln the context of this invention refers to a near-zero dead volume interface wherein the surface area of the connection does not extend beyond the generally concave, preferably tapered walls of the mini-column opening, thus signi icantly reducing or minimizing the surface area in which reagents, solvents, and detached samples might contact. This is contrasted to the prior art where introduction of reagents and solvents is by dispensing the reagent or solvent into the earns Bample funnel rsultiπg in a significant risk of cross-contamination. In the preferred embodiment, the distal end of a rounded nozzle is inserted into the generally concave, preferably tapered opening, seated against the βideβ of the taper, and urged with sufficient force to provide a pressure tight seal so as to withstand column pressures up to 2000 pel. This feature permits the column of this invention to be directly integrated as the support column in a reverse- phase HPLC apparatus. The outlet end of the mini-column may also contain a generally concave, preferably tapered shoulder to mate with an alignment assembly, described below. Λs described above, each mini-column may be loaded with a hydrophobic support. The endβ of each column are fitted with a porous frit to permit liquid flow and to retain the solid support within the column chamber. The frit may be made from any inert material such as sintered glass, fluorinated polymers, and other polymers such as sintered polypropylene. A. novel feature of the cassette of this invention is that it includes a plurality of loading funnels to provide a funnel assembly. The funnels may be arranged in whatever configuration is deemed expedient in view of the ability of the treatment station to address each mini-column or reagent well opening with a nozzle. For example, If the funnel assembly is arranged as a rectangular or square array, the treatment station must provide for nozzle arrays corresponding to the X-ϊ location of each inlet and outlet port for each column and well. The inlet end or loading port of each mini-column 1B preβs- fit disposed in a connection sleeve extending from the throat of a corresponding sample loading funnel of the funnel assembly. The taper of the inlet port of the mini-column is coplanar with the taper of the conical bottom of the funnel βo that there is a virtually seamless transition from the funnel to the Inlet port of the mini column. This will ensure that no dead volume exits that might lead to possible cross-contamination. Alternately the mini- column may be Integrally caβt as an extension of the loading funnel. In the first embodiment, once the sample on the column has been treated, the column may be removed for insertion into the high pressure line of an external HPLC analyzer such that the mini-column is now the support column for the HPLC. This is contrasted to the column of the prior art wherein the mini-column iβ merely a pre-column, requires an adapter, and does not supplant the standard HPLC column. Alternately, an HPLC analysis capability may be built into the system of this invention. In this case, the mini-columnβ need not be removed from the ca^ββette and may be remain in place after treatment of the βample. The ^βame nozzle interface used to address each column now acts as part of the high pressure line for an integrated HPLC.

In order to ensure the mini-columns remain preβs-flt connected to the funnel assembly and to ensure that each mini-column is aligned with the axis of symmetry of it3 corresponding funnel, an alignment means is optionally provided. The alignment assembly of the cassette of this invention ensures that the longitudinal axis of all mounted mini-columnβ are coincident with the longitudinal axis of its corresponding loading funnel. Proper alignment 19 essential, as will be explained below, to ensure that both the inlet port and outlet port of each mini-column is properly aligned to receive the distal ends of dispensing and expending nozzles although self-centering is achieved by virtue of the generally concave, preferably tapered openings. Where the mini- column is not integral to the funnel assembly, an alignment assembly may be provided. Once each mini-column has been inserted into the funnel assembly, the alignment assmbly is positioned over the outlet ports of the mini-column. The alignment assembly is aligned with and removeably attached to the funnel assembly as a spatial reference. Precisely spaced through-holeβ having a tapered inner bore are positioned on the alignment assembly to mate with the tapered shoulder of outlet port of each mini-column. Each mini-column is now secured on each end , thus holding each mini-column in proper alignment. Alternately, an extended sleeve maybe cast as part of the funnel assembly so that insertion of the mini-column into the extended sleeve will result in a stable or robust alignment as well aβ providing βufficient preββ fit of the mini-column to prevent the mini-column from falling out.

Where the mini-column is an Integral part of the funnel assembly, bracing sufficient to ensure alignment of the mini-columns may cast as part of the integrated funnel assembly. For example, cross-bracing extending from the external wall of the integral mini column to the funnel assembly will provide a stable or robust alignment of the mini-columns.

A novel feature of this invention is the optional integration of a plurality of reagent wells into the funnel assembly. The reagent wells are open on each end and are supplied with frits, as in the mini-columns, to prevent material contained ln the wells from falling out. The reagent well may be packed with a suitable support for immobilizing the specific reagent contained therein. Alternately, the reagent may be in powdered form either as a soluble solid or as a lyophllized solid. The solid reagent may be provided as a microencapsulated reagent (this would permit use of liquid rsagentβ without having to provide a solid support), as beads of a predetermined size to permit solvent flow through and/or controlled βolubility rates, or, alternately, the solid reagent may be painted on the walls of the column chamber, thus permitting the free flow of solvents.

Alternately, the reagent wells may be used as an alternative way to introduce samples for chemical treatment. By packing the wells with a suitable solid support, sample may be immobilzed on the support. This would effectively double the number of ^βamples available on the cassette for treatment. Sample in other forms, such as adsorbed, lyophllized, powdered, microencapsulated, or free liquid, may be placed in the sample columns. Scanning the reagent wells with a scanning means will identify populated wellβ whose contents might participate in the predetermined chemical protocols.

As in the iiii-colunuiB, the ends of the inside chamber of the reagent wells are generally concave, and preferably tapered to provide a flared opening to permit the pressure tight Beating of the distal ends of a dispensing and expensing interface means. These interface means Include the same means discussed supra; e.g., nozzles having rounded or conical shaped tips. Once the fully assembled and loaded cassette assembly is inserted into the treatment system of this invention, each column and well opening is addreβsed by a nozzle. The tip of the nozzle is of a geometry designed to fit within the generally concave, preferably tapered openings of each mini-column and reagent well, and provide a tight seal thus achieving near-zero dead volume resulting in minimizing the risk of cross-contamination. In contrast, the prior art eyBtems, which have no nozzle interface, or other direct flow-through communication Interface, haβ a significant amount of dead volume. A novel feature of thiβ invention is that the benefit of a funnel is attained for sample loading, however, near-zero dead volume is attained aβ well by the use of the nozzle interface. In accordance with the preselected chemical treatment protocol Identified for a particular cassette, the nozzles permit the free flow introduction of a solvent, treatment solution, or a sample-containing solution to the column or well with which it is in communication (dispensing) or for removal of a solvent, spent treatment solution, or βample containing solution from a column well with which it is communication (expensing). Another novel feature of the caββette of this invention iβ that each cassette has a machine readable code disposed on the cassette that iβ read by an appropriate device in the treatment station to automatically indicate to the treatment station in which the caββette iβ loaded the exact chemical protocols required for the samples in the cassette. The code will optionally indicate whether or not reagent iβ present in the reagent wells, whether the reagent wells contain additional samples rather than reagent, and which column and well addreβsea are to tretaed (in the event that not all column and well addressee are populated). Alternately, a βcanning means can scan each column and/or reagent well to identify tllOBβ column and reagent addresses that have material; i.e., solid support, reagent, solvent. Accordingly, empty columns and wells are not addressed and only those populated columns and wells identified by the Bcanner means participate in the identified chemistries. Scanning means suitable for identifying populated mini-columns and reagent-wells include either a light array having a single source or a plurality of sources and complementary light source detector array, or a mechanical probe inserted into the mini-column and reagent well openings to sense the presence of an obstruction such as a frit. In the preferred embodiment, the light source array is positioned over the mini-column and reagent well ports while the detector iβ positioned to detect whether the light iβ transmitted through the mini-column or reagent well. Those "empty" mini-columns and reagent wells where the light is transmitted through the column or well do not participate in the identified predetermined chemical protocols.

The machine readable code may be in the form of a bar code, a magnetic strip, an embedded diode, or a semiconductor memory chip. The device used to read the code will necessarily depend on the format and medium of the code and may Include a bar code reader, a magnetic strip reader, a radio transponder, or a data bus socket. The foregoing means for encoding aβ machine readable code the chemical protocol Information and the βcanning devices for reading the machine readable code are presented by way of example and not by way of limitation as any means whether optical, magnetic, electrical impulse, and the like may be employed to provide to the treatment station an indication of the desired chemical protocols. A further novel feature of the cassette of this invention is that once the caββette has been loaded into the treatment station of this invention the machine readable code is modified to Indicate that the desired chemical treatment protocol has been performed. Such modification may include modifying the code so that it becomes unreadable by the βcanning device, thus preventing execution of any treatment protocol. For example, a bar code may be disposed in a bar code holder that is βlideably inserted in a receiving groove in the caββette. Once the bar code is read, the bar code holder may be repositioned in the receiving groove so that part of the bar code is positioned in a pocket in the cassette, thus obscuring at least a portion of the bar code, rendering the bar code unreadable. This will serve to prevent the cassette from being inadvertantly processed a second time. As a further example, in the case of the semiconductor memory, a CMOS or static RAM may be u^βed to contain the required protocol instructions. Once the memory iβ read, the coded instruction set may be modified to indicate that the cassette has undergone the prescribed chemical protocols. An advantage of uβing a random access memory means is that the modification of the machine readable code may optionally include writing information to the memory to provide information as to date and time the protocols were executed, the name of the technician operating the eystem, any deviations to the protocol, addresβes of the columns and wells, and parametric information such as reagent volumes, operating temperatures and pressures, and the like. Optionally this information may be later downloaded to a permanent Information storage location.

The syβtem of this invention Includes the caββette assembly, a sample loading station, and a treatment station. By way of operation, the cassette assembly is loaded into the sample loading station and aligned in preparation of receiving the sample. An annular gasket iβ lowered onto the top rim of the βample funnel being loaded. The pressure resulting from mounting the loading et is such that the mini-column associated with the instant sample funnel is firmly pressed against the funnel opening to ensure a water tight fit between the narrow end of the minim-column and the sample funnel connection Bleeve up to about 40 psi. The outlet end of the mini-column is position over a drain tube. The sample solution 1B then pipetted into the first well. After the sample solution lias been introduced, a pressure cap having a centrally disposed plunger is lowered over the gasket so that the plunger extends into the sample funnel. An annular shoulder on the plunger seata against the gasket to create an air tight βeal up to about 40 psi. A port in the plunger permits an inert gas to pressurize the head space in the sample funnel thus forcing the sample solution into and through the mini-column. Alternatly, the sample solution may be forced into and through the mini-column by either providing a vacuum draw at the outlet end of the mini-column to suction the sample solution through, or the plunger on the pressure cap may directly push the sample solution through the mini-column; i.e., hydraulic preβsure. As the sample solution passes through the mini-column, any lipophilic moieties are Immobilized on the hydrophobic packing material. Similarly, if the packing material were hydrophilic, any lipophobic moietieβ would be immobilzed in that case. Superfluous sample solution lβ expired through the outlet port Into a drain line. The funnel iβ depreββurized, and the plunger and gasket removed. This process is repeated for all sample funnel/mini-column addresses in the array. Alternately a multi station sample loading device may be used whereby all sample funnel/mini-column addresses are loaded simultaneously. This embodiment, as well as the βingle station loading station embodiment, may optionally provide an Hample dispensing port in the plunger (s) to automatically diβpenβe the βample βolution into the βample funnel well(β) after the plunger lo seated. A further option includes a means for reading the machine readable code so that a microcontroller interface might load the cassette pursuant to a predetermined sampling protocol.

OBJECTS AND ADVANTAGES:

It ia an object of this invention to provide a cassette for a chemical treatment Bystem having a plurality of funnels for loading βample βolutionβ, a plurality of sample retaining means for holding a plurality of addressable, preselected samples and preselected reagents, to permit preβelected chemistries on the preselected samples in a sequential unterrupted fashion, the caβsette having a machine readable code integrated thereon to automatically identify to the chemical treatment system upon insertion of the caβsette into a chemical treatment station the chemistries to be performed on the preselected samples uβing the preβelected reagents, the cassette having very near-zero dead volume flow-through connection with a chemical treatment station.

It is another object of this invention to provide a βample loading station not requiring pre-isolation of the sample in a reaction chamber and that will enable rapid loading of the chemical treatment cassette of this invention.

It iβ another object of this invention to provide an improved sample column for sample immobilization or containment and for insertion into the cassette of this invention to permit flow-through chemistries with near-zero dead volume and to permit access to and use of the improved βample column as an HPLC column without the need for a high-pressure adapter.

It is another object of this invention to provide a sample treatment station for receiving the chemical treatment cassette of this Invention, for reading the machine readable code on the caββette, for uninterrupted, sequential accessing all pre-selected βantples and pre-βelected reagents, and for executing the chemistrleβ aa identified by the machine readable code.

It is another object of this invention to provide a chemical treatment system having a βample loading station, a chemical treatment cassette, and a chemical treatment station for performing automatic, near-simultaneous, flow- through chβmlstrieβ on a plurality of pre-selected βampleβ uβing a plurality of pre-βelected reagents, the chemistries being identified by machine readable code to a micro-controller, and the chemistries being executed by the microcontroller working in logical and electrical cooperation with the chemical treatment station.

-13- It is another object of this invention to provide a method for near simultaneous performance of chemistries on a plurality of preβelected samples, the sample^β being retained in a chemical treatment caββette, and the chemistries desired retained on machine readable code for automatic execution via micro-processor control pursuant to instructions contained in t_he machine readable code.

Still other objects will be evident from the specification claims and drawingβ of this application.

BRIEF DESCRIPTION OF DRAWINGSI

The invention is illustrated by reference to the drawingβ in which: Fig. 1 is a croβB-sectional diagram of a βingle-sample chemical treatment cartridge or reactor of the background art;

Pig. 2 is a croββ-βectional diagram of the βample column of the background art as connected to an HPLC high-preβsure adapter;

Fig . 3a-e are variouβ perspective views of the chemical treatment cassette of the chemical treatment system of this invention;

Fig. 3f is a cross section view of the multiple βample, chemical treate ent casstte cassette assembly of this invention; Fig. 4 iβ a cross-sectional diagram of the sample mini-column of this invention;

Fig. 5a,b are cross-secctional views of the chemical treatment cassette of the chemical treatment eystem of this invention showing the relationship of the sample columns and nozzles with respect to each other and to other elements of the chemical treatment casβette;

Fig. 6 is a crosβ-βection view of an equally preferred embodiment of the chemical treatment caββette of the chemical treatment eystem of this invention;

Fig. 7 iβ a croββ-βectioπ view showing how the nozzle iβ seated in the tapered opening embodiment of the mini-columns and reagent wells of the chemical treatment cassette of thlβ invention;

Fig. β iβ a croeβ-βection view of the chemical treatment caββette as loaded in the sample loading βtation;

Fig. 9 is a perspective view of the chemical treatment cassette and the upper and lower nozzle arrays of the chemical treatment station and their relative positionβ βpatial poβitionβ proir to loading the nozzles into the sample column and reagent well generally concave, preferably tapered openings;

Fig. 10 iβ a schematic overview diagram of the hydraulic layout of the chemical treatment βtation of this invention;

Fig. 11 is a cross-section diagram of the mini-column of this invention being in an HPLC in-line adaptor ; and

Fig. 12 is a flow diagram of the method of using the chemical treatemnt syBtem of this invention.

BEST MODE FOR CARRYING OUT THE INVENTION

The following detailed description illustrates the invention by way of example, not by way of limitation of the principles of the invention. This description will clearly enable one skilled in the art to make and use the invention, and describes several embodiments, adaptations, variations, alternatives and uses of the invention, including what we presently believe is the beet mode of carrying out the invention.

The three principle elements of the chemical treatment system of this invention include a sample loading βtation, a βample treatment βtation, and a chemical treatment cassette. Both the sample loading station and the chemical treatment station elements are configured in accordance with the geometry and configuration of the chemical treatment caβsette (hereafter "cassette"). The preferred embodiment of the cassette iβ easily seen in the exploded view as shown in Fig. 3a. The principle elements of the caβsette 10 include a sample funnel assembly 11, a plurality of mini-columns 13, a mini-column alignment assembly IS, and a receptacle for removeably retaining a medium containing a machine readable code 17.

The βample funel assembly 11 contains a plurality of funnels 19 integral to the aββembly and arranged in a even, regular funnel array. Fig. 3a shows the sample funnel assembly aβ a linear array 21. Fig. 3b βhowβ the mini- columns 13 inserted into the funnel assembly 11. The location of each mini- column may be stamped onto the alignment aββembly 15 to facilitate sample loading and for tracking purposes. Fig. 3c iβ a mirror-image perspective view of Figure 3b and clearly shows the plurality of through-holeβ 23 in the alignment asβembly by which the flared, generally concave, preferably tapered, free end of the mini-column may be aligned for later connection and interface to a chemical treatment βtation. The alignment holes 23 are tapered so as to receive the tapered flange of the free end of the mini-columns and to self align the columns as the alignment assembly 15 is snapped into place.

Fig. 3c βhowβ the inlet ports 29 of reagent wells 31, shown in phantom, that are integrated into the funnel assembly 11. As diβcuβsed above, the reagent wells may be used aβ a source of adsorbed, powdered, freeze dried, microencapβulated, and liquid reagents, solvents, salts, buffers, or other chemicals participating actively or passively in the chemistry performed on the sampleβ in the mini-columns. Also shown in Fig. 3c iβ the receiving slot 17 for the machine readable code means. In the preferred embodiment of the caβsette of this invention the machine readable code means iβ a bar code. Fig. 3d βhowβ how a bar code 25 is inβerted into the recelvelng βlot 17. Fig. 3e βhowβ the bar code 25 slideably received in the receiving slot. Λs shown in phantom, the bar code 25 has been displaced to the extreme bottom end 27 of the receiving βlot 17 rendering the bar code unreadable. A mechanism is provided in the treatment βtation of thiβ invention to displace the bar code to the extreme bottom end of the receiving βlot once the caβstte has been addressed by the interface nozzle arrays contained In the treatment station by which the flow-through chemistry is effected, thus preventing the caβsette from inadvertantly being used again.

A cross-section view of the chemical treatment cassette assembly iβ shown in Fig. 3f which clearly allows the inter-relationships between the various elements of the caβsette assembly. The sample mini-column 13 has a first narrow end 33 which is press-fit into a mini-column connection eleeve 35 formed by extending the throat of the βample funnel 19. A atop shoulder 37 disposed aimularly on the funnel end of the inside wall of the connection sleeve 35 provides a stop barrier to prevent the mini-column 13 from extending into the cavity of the funnel 19 and provides a sealing surface between the inlet port of the column and the throat of the funnel to prevent sample solution from leaking during sample loading. Further, the stop barrier extends over the edge of the mini-column narrow end 33 by an amount sufficient to provide a smooth transition from the sloped inside wall 39 of the funnel to the generally concave, preferably tapered wall 41 of the narrow end of the mini-column. This smooth transition from funnel to mini-column reduces the risk of sample or wash solvent reβidues forming on what would otherwise be a surface irregularity. Such residues may lead to errors and significantly affect the reaultβ of the chemistries involved. Fig. 3f also clearly shows the structure of the reagent wells 31. The wells have an inlet port 29, and an outlet port 43. A center chamber 44 runs longitudinally through the reagenet well and connects the inlet port 29 with the outlet port 43 within which chamber may be diβposed any reagents, solvents, buffers salts, enzymes, and the like, in either an adsorbed, powdered, lyophllized, microencapsulated, or liquid state, useful in the chemistries associated with the sample that is retained by the βample mini-column 13. Inert porous frits 45a and 45b are preββ fit into the ends of the central chamber to prevent loss of the materialβ contained in the reagent well central chamber. Fig. 3f alao clearly shows how the flange 49 of the flanged end 47 βample mini-column 13 i^β seated in the through hole 23 of the alignment assembly 15. The outside annular edge 51 of the flange 49 is tapered so that it mates with the taper of the inside wall of the through hole 23. Once the alignment asβembly 15 is snapped into place with the funnel assembly 11, the mini-co_lumn is held in an aligned position βtable enough to move and manipulate the ca^ββette aββembly 10 without loosening the mini-columns 13 from their connection sleeve^β 35. The mini-column 13 of this invention is shown more clearly in Fig. 4. It i^β comprised of a narrow end 33, a flanged end 47 having an annular flange 49 outwardly extending from the flanged end 47. The outside outside annular edge 51 of the flange 49 iβ tapered Inwardly βo aβ to be accomodated by and self-centering in an alignment hole 23 of the alignment assembly 15, the alignment hole also having a complementary tapered bore for receiving the outβide tapered mini-column flanged end 47. A logitudinal chamber 59 conneotβ a narrow end inlet port 53 with a flanged end outlet port 55. Both the inlet port 53 and the outlet port 55 have a boreβ 41 and 57 to facilitate leak-proof, high preββure seal with a nozzle Interface of the chemical treatment βtation of thiβ invention or external analyte analyzer. The chamber is typically packed with a solid support material 61 such as silica that has been derivatized with a lipophyllic polymer (e.g., a C18 compound) thus rendering the support hydrophobic. Other βupport materialβ may be used including polymer or reβin beads, cellulose, and the like. Further, depending on the sample to be immobilized, the support may be made hydrophillic. The support material is retained in the column by porouβ frits 63 and 65. These frits may be made of any inert porous material including sintered polyethylene, polypropylene, f luoropolymerβ, glass, and the like.

Fig. 5a is an exploded, cross-section view of the chemical treatment cassette assembly 10 showing how the nozzles of the chemical treatment station interface addresaeβ the cassette. Each address of the cassette includes a reagent well 31 with an inlet port 29 and outlet port 43, and a sample mini- column 13, also having an inlet port 53 and an outlet port 55. Aβ iβ clearly shown in Fig. 5a, a mini-column inlet nozzle 67 interfaces with the mini column inlet port 53 by seating the nozzle tip 68 againβt the generally concave, preferably tapered bore 41 of the mini-column inlet port 53. The nozzle 67 contains a through-bore 69 diβposed on the longitudinal axis of the nozzle, the through-bore having a rounded, polished distal end 68 in communication with the inlet port 53 of the mini-column through which solutions and solvents are either introduced to or removed from the mini-column.

Similarly, the mini-column oulet nozzle 73 interfaces with the mini-column

-ι«- outlet port 55 by seating the rounded, polished nozzle tip 74 against the generally concave, preferably tapered bore 57 of the mini-column outlet port. The nozzle 73 contains a through-bore 75 disposed on the longitudinal axle of the nozzle, the through-bore terminating at the rounded, polished distal end 74 to provide communica ion with the outlet port 55 of the mini-column through which solutions and solvents are either Introduced to or removed from the mini- column.

Similarly, the reagent well inlet port 29 interfaces with a reagent well inlet nozzle 7B having the same βtructure aβ the mini-column inlet port nozzle 67, and the reagent well outlet port 43 interfaceβ with a reagent well outlet nozzle 79 having the βame structure aβ the mini-column inlet port outlet nozzle 73, to enable hydraulic and pneumatic communication of the reagent well with the chemical treatment βtation.

Note that other interface means may be uβed including βlip fittlngβ, threaded fittings, gaβketted butt-joint fittings, and the like, however, the preferred embodiment uses the nαzzleβ aβ deβcribed above. Fig. 5b shows the nozzle tips inserted into the generally concave, preferably tapered bored openings of the iiilnl-column and reagent well inlet and outlet ports. Fig. 7 is a detailed cross-section view of the nozzle tip 74 of the mini-column outlet port nozzle 73 seated in the mini-column outlet port 55, and typifies the nozzle/port interface of this invention. The rounded, polished end 74 of the nozzle 73 is urged or pressed against the generally concave, preferably tapered wall 57 of the outlet opening 55 with a pressure sufficient to provide a leak- proof, seal between the tapered opening and the nozzle tip. The seal between the reagent well and the reagent well nozzleβ muβt withβtand up to 40-50 pel, whereas the mini-column seals muβt withβtand preβsureβ in excess of 1500 psi. The mini-column/nozzle interface muβt withβtand theβe higher pressures to provide in-line HPLC capability.

The adavantage of the column/nozzle Interface over the background art is easily discerned. A significant amount of dead volume is introduced by the system of the background art since the walls of their βample funnel provide an extensive surface area on which residues and other contaminants might collect. These residues pose a significant cross-contamination threat where multi-step chemistries are performed on less than milligram quantities of sample. By eliminating the βample funnel aβ a eanβ for dispensing reagentβ and βolventβ to the mini-column (as in the background art) the interface of thiβ invention achieves a near-zero dead volume βince there are no surfaces on which residues of prior solutions might collect, while still obtaining the benefit of the funnel for sample loading. Fig. 6 is a cro^βs-^βectlon, exploded view of an equally preferred chemical treatment ca^βeette a^ββembly wherein the mini-column 14 at each address i_β integral to the sample funnel aββembly. Thiβ embodiment is preferred when, for example, the chemical treatment βtation iβ capable of performing on-board, inline analysis of the reacted βample, thus vitiating the need to remove the ^βample mini-column from the chemical treatment cassette (in order to perform off-line analysis of the reacted sample immobilized on the column₎. Thi_β embodiment requires no alignment assembly as each integral column is prealigned and permanently affixed in place. Further, this embodiment permits the mini- column inlet and outlet nozzles to act as the high-pressure interface connection with the on-board analyzer, typically HPLC, thuβ elinating the need to have a ^βeparate analyzer dock with a separate interface assembly in the chemical treatment station.

The loading station of this invention comprises an X-Y alignment means, a gasket loading meanβ and a plunger or presβurβ cap meanβ. Fig. 8 iβ a cross- section view of the chemical treatment caβsette of this invention ln the sample loading station. The cassette is positioned so that the gasket and presβure cap will preciβely engage the sample funnel. Fig. 8 shows the casβette positioned via an alignment means βhown diagramatically aβ an alignment pin 81 extending upwardβ from the loading platform 83. A plurality of alignment pins may be positioned on the platform to permit preciβe positioning of the caββette assembly. Alternately, the casβette may be locked onto a slldeable platform having predetermined stops to permit the precise positioning of each sample well 19 for single sample loading. Alternately, an alignment stop having a shape complementary conforming to the external βhape of the funnel aββembly may be used to position and Index the individual funnels during sample loading. Once positioned, an annular gasket 87 iβ positioned over the βample well and a βeal is made by exerting a downward presβure on the gasket to provide a pressure βeal. The preββure βeal also serves to urge the mini-column outlet port against a drain interface B5 thuβ providing a seal between the outlet port and the drain interface, and to urge the narrow end 33 of the mini-column 13 against the annular shoulder 37 in the connection sleeve 35, thu^β providing a preββure eealbetween the βample funnel 19 and the mini-column. Next, the βample βolution 16 is introduced into the βample funnel X9. The pressure cap B9 is then lowered onto the gasket and preββure applied to provide a seal between the pressure cap and the gasket. A centrally di^βpo^βed plunger 90 on the preββure cap extendβ into the funnel 19. Inert gas is then introduce into the head space IB between the sample βolution and the plunger 90 via a gas entry port 91 diβposed in the pressure cap, thuβ forcing the sample solution 1G through the mini-column 13 with the exceββ βolution passing through the mini-column and drained away through the waβte tube 86 connected to the drain interface B5. Once the ^βample βolution has passed through the column, the inert gas pressure is released, and the pressure cap and gaβket are removed, The caβsette is then repo^βitloned for sample loading of the funnel at the next address, or optionally, either a second sample βolution or a waβh solvent may be loaded. Thiβ proceeβ is repeated until all the mini-columns at the desired addresses have been loaded. It should be noted that the loader of this invention does not require that the βample funnel or the cassette be inserted in a special reaction chamber or holder in order to load the mini-column as is done in the background art. Further, the novel aspects of the loader of this invention may be extended to a multiple βample loading βtation whereby more than one addreβs may be loaded with an βa plα at a time. Alβo, an optional sample Introduction port 93 may be provided in the presβure cap βo that the gaβket 87, which serves to preββure βeal the mini-column with the funnel prior to Introduction of the βample, may be eliminated βince the pressure cap will now provide the force required to seal the mini-column against the funnel and the βample is not introduced into the funnel until after the pressure cap is in place. After the chemical treatment caββette has been loaded it iβ inserted into the chemical treatment βtation (CTS) of this invention. The CTS positions the caβsette so that the caβsette/CTS interface can be established. Positioning of the casβette is performed automatically by means well known in the art. Fig. 9 iβ a diagram showing the cassette 10 position between the nozzle interface arrays 95 and 96. After insertion into the CTS, the bar code 25 is read by a bar-code reader 99. The instructions contained in the bar code are sent to a micro-controller and the appropriate algorithm is acceββed and loaded for execution of the proceaβ steps and chemistries indicated by the bar code instructions. Once the caββette iβ transferred into position, interface nozzle arrays 95 and 96 are brought together and into contact with the caββette βo that the tips of the nozzleβ 67, 78, 73, and 79 are brought into pressure contact with their respective mini-column and reagent well ports. As the nozzle arrays are brought together, the bar code 25 is disabled by action of a tab or pin 96 on the slldeable bar code forcing it to a position in the bar code receiving slot 17 where the bar code iβ at laaβt partially obβcured. Once the nozzle/caββette interfaceβ have been eatabliβhed the materialβ in the reagent wells and the immobilized βample ln the minicolumnβ are now acceββible to the CTS βo that the predetermined chemistries can be performed.

The preferred embodiment of the CTS of this invention is shown schematically in Fig. 10. The laoded chemical treatment caββette iβ depicted as being addressed by the nozzle array interfaces. The mini-columns are addre^ββed by the mini-column inlet port nozzleβ 67 and outlet port nozzleβ 73, and the reagent porta are addressed by inlet nozzles 78 and outlet nozzles 79. Each reagent well may be selected individually for processing by adjustment of rotary valves 100, 101, 103, and 104, and each mini-column may be βelected individually by the appropriate adjustment of rotary valves 105, 106, 107, and 10B. Note that rotary valve inlet and out let pairs; i.e., 100 and 103, 101 and 104, 105 and 107, and 106 and 108, are synchronized βo that when the inlet port of a reagent well or a mini-column iβ βelected, the rotary valve for the outlet port may only be βet to that reagent well or mini-column.

Solvents, reagents, buffers, and other solutionβ are supplied from up to six reagent bottleβ llla-f, with the desired reagent being βelected by appropriate adjustment of the solvent valve blocks 109 and 110, and by appropriate βelection of preββure βwitch from the preβeurized valve blocks 122 and 123. These reagents are in addition to the reagents supplied in the reagent wells of the caaβette. All solventβ must pasβ through valve V4 102. Default position for all βolvent and reagent rotary valves iβ βet to direct the reagents to waβte 112. Reagentβ llla-f may be directed to the mini-columns by closing valve V 102. This will direct the selected reagent to the mini-column inlet selected by the rotary valves 105 and 106 with any waβte reagent paaβing through valve 118, valve 119 and into the waβte bottle 112.

Solvente may be directed to the reagent walls for rehydration or solvation of adsorbed, powdered or lyophilllzed reagantβ in the reagent wells by switching rotary valve V4 102. Solvent lβ now directed to reagent cell inlet rotary valves 100 and 101. Note that poβtion 6 of the reagent well and mini- column rotary valveβ iβ a paββ-through position. Thuβ if rotary valve 100 is βet at position 6, the βolvent will pass through to the reagent βelected by rotary valve 101. If desired. the βolvent may be pasBed- hrough and stored in the mixer-dlluter 114 by closing valve VB 115 and V4 102. This permits mixing various reagentβ and βolventβ with one another, including aolvated dry reagents from the reagent walls of the caβeette, prior to moving the mixture through line 124, valve 117 and valve 116, and to the mini-column rotary valveβ 105 and 106 for reaction with the immobilized βample.

The CTS of thiβ invention provide three areas where βolvent, analyte or βample βolutlonβ may be stored. Theβe areaβ include the mixer dlluter which, as deβcribed above, permitβ βolventβ and reagβntβ to be mixed prior to reaction with the βample or analyte. Reagβntβ, sample and analyte may also be accumulated (i.e., temporarily stored) in the heater coil 126 and in the shaking coil 125. It may be deeireable to remove the βample or analyte from the ^βupport packing in the mini-column for a number of reaβonβ. For example, where the hydrophobic ^βupport results in a conformational deformation of a protein or peptide side chain or otherwiβe affects the reactivity of the peptide or protein side chains, it might be deeireable to perform the desired reaction external to the column. In thiβ event, a βuitable lipophobic solvent is used to detach the protion from the βupport. Valves V10 119, and valve 117, and 116 are closed resulting in the sample or analyte, now in βolution, to be forced out of the inlet port of the affected mini-column, through valve 117, heater coil 126, valve 116 and into the mixer/diluter 114 wherein the desired reaction chemistry may be performed with reageπtβ already preβent, or introduced later. As another example, detaching the βample may be deeireable in the event the characteriβtica of the βupport material muat be changes; e.g., changing it from a hydrophobic βupport to a hydrophillic βupport in the middle of the chemistry being automatically executed by the CTS.

The CTS of this invention iβ able to mix βolventβ, reactantβ and buffers; to detach and shuttle the sample back and forth (i.e., pump-up and pump-down; to perform complex chemistries either on or off-column; and to βolvate adsorbed, powdered or lyophillized reageπtβ, all performed automatically, pursuant to instructions indicated to a micro-controller by a bar code, without having to remove the caββetβ from the CTS, without having to detach or reattach the CTS/casβette interface, or otherwiβe require human intervention. Further, the same chemistries may be performed on all sample mini-column addresses, or a βeparate completely independent set of protocols may be defined for each address, or for each block of addressee.

Further, an optional analyte analysis capability may be included in the CTS of thiβ invention. Any of the mini-columns may be converted to an in-line HPLC column, by βwithing rotary valve 117 to receive βolvent from an HPLC pump and by switching rotary valve 118 to direct the eluant to a detector. It i^β clear that the CTS iβ capable of performing a variety of analysis other than HPLC without having to remove the casaette from the CTS. However, if on-board, in-line analysis is not available, the mini-column may be removed from the caββtte and directly Inserted into an in-line, high-prβsβure adapter as shown in Fig. 11. The adapter 130 receives the mini-column 13 containing the analyte. Nozzles 131 and 133 are seated against the generally concave, preferably tapered inlet port 53 and outlet port 55 to form a high pre^ββure βeal. The mini-column of this invention doeβ not require that a βeparate column/adapter be provided in order to accomodate in-line HPLC.

-31 - The process of this invention as described supra beginning with the loading of the oasβette, followed by execution by the CTS of the βelected cheiiiiβtrieβ as predetermined by the inβtructions contained on a bar code affixed to the cassette, and concluding with either in-line onboard analysis, or an improved external analysis is summarized in Fig. 12.

It should be understood that various modi ications within the scope of this invention can be made by one of ordinary skill in the art without departing from tiie βpirit thereof. We therefore wiβh our invention to be defined by the βcope of the appended claims aβ broadly aβ the prior art will permit, and in view of the specification if need be.

-71-

Claims

1. A chemical treatment βyβtem for sequential, uninterrupted treatment of lesβ than milligram quantities of a plurality of chemical samples, comprising in operative combination! a) a chemical treatment cassette for retaining a plurality of chemical samples for chemical treatment, said chemical treatment caβsette further comprising: i) a funnel assembly comprising a plurality of integrated, individually addressable chemical βolution βa pling funnelβ for receiving chemical sample solutions, each of βaid sampling funnelβ having a firβt, open mouth end for Introduction of a chemical βample βolution, and a tapered, second conical end having a through-hole at the apex of βald conical end, βaid sampling funnels being arranged in an array to permit individual addressing for chemical treatment; ii) a plurality of βample mini-columns for retaining preselected chemical βampleβ, each of said mini-columns having a centrally dispoβed chamber extending longitudinally through said mini-column, at least one of βaid mini-columnβ having a material selected from at least one of a solid support, dry reagent, dry βample, liquid reagent, liquid sample, and porous frits disposed in βaid central chamber, said chamber terminating into a first open end, said firβt open end having a flared, tapered inlet port, βaid inlet port being in flow-through, phyβical connection with said through-hole in said conical second end of said funnel, βaid connection resulting in smooth, continuous transition of the taper from said conical βecond end of βaid funnel to aaid taper of βaid Inlet port, aaid chamber terminating into a βecond open end, said βecond open end having a flared, tapered outlet port, βaid outlet port being in flow-through communication with said inlet port, said firβt open end of each of βald funnels and aaid tapared conical βecond end of each of βaid funnelβ and permitting localized accβββ by a cαmpreββive fit interface means to said mini-column inlet port for near-zero dead volumn flow-through communication between βaid interface means and aaid mini-columns; iii) an optional plurality of reagent wells for retaining preselected reagentβ for reaction with said preβelected sample retained in βald min-columnβ, βaid reagent wells comprising a through-bore parallel to βald longitudinal axis of βaid mini-column, aaid through-bore terminating in a first end having a flared, tapered inlet port, and terminating in a βecond end having a flared, outlet port aaid reagent well inlet port being in flow-through communication with βaid reagent well outlet port; and iv) a machine readable code disposed on βaid chemical treatment cassette for identifying the chemistry protocols to be performed on the retained samples in said mini-columnβ; b) a sample loading station for loading sample solutions in said sample funnels, said sample loading station further comprising : i) a caββette alignment means for aligning said chemical treatment cassette to a predetermined X-Y position; ii) a sealing gaβket actuator for lowering an annular βealing gasket over said first end of said sampling funnel when said βample funnel iβ located at said predetermined X-Y position; ill) a preββure cap having an annular βhoulder and center plunger βection for prβββ-flt insertion into the mouth of said sample funnel to define a funnel headβpace, βaid shoulder engaging βaid annular gaβket to limit the extent of inβertion of said center plunger, βaid preββure cap having a through-bore gas inlet for introduction of pressurized gaβ into βaid funnel headspace to force said sample solution into said inlet port of βaid mlni- column; c) a chemical treatment βtation for performing predetermined chemiβtriββ on a plurality of preβelected retained chemical sampleβ located in βaid chemical treatment caβββtte, βaid chemical treatment station further comprising: 1) a code reading means for reading eaid machine readable code disposed on βaid chemical treatment casβette to identify the βpeclfic chemistry prσtocolβ to be performed on eaid retained chemical samples; ii) a scanning meanβ to identify mini-column addreββeβ wherein βaid central chamber of βaid mini-column iβ populated; iϋ) a micro-controller interface for communicating from βaid code reading meanβ to a micro-controller said identified specific chemistry protocols, said micro-controller accessing specific proceββ algorithms pursuant to the specific identified chemistry protocols, aaid proceββ algorithms defining and communicating to βaid micro-controller interface βtepwiβe micro- controller control inβtructionβ to enable execution of βaid identified specific chemiβtrieβ on βaid pre-selected chemical sampleβ at eaid populated mini-column addrββsaβ; iv) a cassette/treatment βtation interface .for enabling near-zero dead volume flow-through communication of βaid chemical treatment caββette with eaid chemical treatment βtation, βaid caββette/treatment station interface comprising: a caββette interface dock for precision alignment of βaid chemical treatment caββette, a first nozzle array comprising a plurality of nozzles spatially 80 arrayed to be congruous with said mini-column inlet portβ and reagent well inlet ports for flow-through localized interfacing with βaid mini-column inlet ports and eaid reagent well inlet ports, a βecond nozzle array having a plurality of nozzles spatially arrayed to be congruous with βaid mini-column outlet portβ and reagent well B5 outlet portβ for flow through communication with βaid mini-column outlet ports and said reagent well outlet portβ, βaid first nozzle array positioned above βaid caββette interface dock, βaid nozzles in said firβt array having nozzles tipβ extending downward towards said caββette interface dock, 90 βaid second nozzle array position below said caaβette interface dock, βaid nozzles in βaid βecond nozzle array having nozzle tipβ extending upwards to βaid caβsette interface dock, and said nozzles in eaid firβt array and βaid πozzlββ in βaid aecond array establishing said flow-through localized interface by first aligning eaid 95 chemical treatment cassette in said cassette Interface dock and by compression fitting said nozzle tips in said first nozzle array against aaid tapered opening of said mini-column inlet portβ and βaid reagent well inlet portβ, and by compression fitting βaid nozzle tips ln said βecond nozzle array againat βaid tapered opening of βaid mini-column outlet portβ and said reagent well 100 outlet ports; v) a chemical delivery βubβyβtem for providing automatic flow through chemistries on βaid preselected chemical samples further comprising: a solvent bank providing a plurality of reagents and solvents to 105 βald reagent wellβ for βolubilizing said reagβntβ retained therein and for reaction witli βaid chemical βa plββ purβuant to βaid identified specific chemistry protocolβ, a rotary valve array further comprlβing a mini-column inlet port rotary valve array for individual addreβaing of mini-column inlet ports, a 110 mini-column outlet port rotary valve array for individual addreeβing of mini- column outlet portβ, βaid mini-column inlet port rotary valve array and eaid mini-column outlet port rotary valve array being βynchronized to a βinglβ mini- column addreββ to permit active Interface with only one mini-column inlet and outlet port at a time, and a reagent well inlet port rotary valve array for 115 individual addressing of reagent well inlet ports, a reagent well outlet port rotary valve array for individual addressing of reagent well outlet portβ, βaid reagent well inlet port rotary valve array and βaid reagent well outlet port rotary valve array being βynchronized to a βingle reagent well address to permit active interface with only one reagent well inlet and outlet port at a 120 time, a plurality of accumulators for temporary storage of βolveπtβ and reagents, and temporary storage of detached chemical βample to enable chemistries to be performed on said detached sampleβ, and to enable chemistries not including βaid detached βampleβ to be performed in βaid mini-column, 125 a mixer/diluter reβervolr in communication with βaid βolvent bank, reagent wells, and mini-columns for temporary retention of at least one of a solvent, reagent, sample, and mixtures thereof, a pneumatic pressurizing system for bi-directional transfer of solvents, reagentβ, and chemical samples to and from eaid reagent wells, Bald 130 mini-columns, βaid accumulators, and said mixer/diluter, and for supplying inert gas to said chemical treatment delivery subβytem, and an optional valve meanβ for integration of an analyzer βubsystem into βaid chemical delivery subsystem to permit automatic analyais of the βample retained in βaid mini-columns without removal of βaid mini-columns from

135 βaid chemical treatment caββette and without diaengaging aaid cassette/treatment βtation interface.

2. A chemical treatment system as in claim 1 wherein said chemical treatment caββette further comprises an alignment means for aligning said mini- columns such that the longitudinal axle of βald mini columns iβ coincident with the longitudinal axia of βaid βample funnelβ.

3. A chemical treatment βyβtem aβ in claim 1 wherein a βolid βupport material is disposed in said central chamber of βaid mini-column, βald βolid support material being treated to Immobilize βaid chemical βample, saidβolid βupport being retained in βaid mini-columnβ by porous frits disposed in said

5 central chamber at βaid inlet port and and at βaid outlet port end.

4. A chemical treatment system aβ in claim 1 wherein aaid mini-columnβ are integral to βaid funnel aββembly to provide a unitary, one-piece funnel/mini-column assembly.

5. A unitary, one-piece sample mini-column for retaining a chemical βample for chemical treatment, βaid βample mini-column compriβing a centrally diβpoβed chamber extending longitudinally through a cylindrical column, βald chamber terminating into a firβt open end, βaid firβt open end having a flared, tapered inlet port, ^βaid firβt open end enabling βlip fit, flow-through, physical connection with a connection sleeve, βaid connection sleeve extending from a through-hole ln the tapered, conical end of a sampling funnel, βaid connection resulting in smooth, continuous transition of the taper from βaid conical end of ^βaid funnel to βaid taper of βaid inlet port, βaid chamber terminating Into a βecond open end, βaid βecond open end having a flared, tapered outlet port, βaid outlet port being in flow-through communication with βaid inlet port, both said tapered inlet port and outlet port providing means for localized, leak-proof communication with a compression fit interface meana .

6. A ^βample mini-column aβ in claim 5 further compriβing a βolid support material disposed in eaid central chamber, βaid βolid βupport material being tretaed to immobilize said chemical sample, said solid support retained in βaid mini-columns by porous frits dispoβed in eaid central chamber at eaid inlet port and at βaid outlet port.

. A chemical treatment cassette for retaining a plurality of chemical samples for chemical treatment, said chemical treatment caββette compriβin : a) an integrated, one-piece funnel aββembly compriβing a plurality of individually addreaβable chemical βolution sampling funnels for receiving chemical βample solutions, each of βaid sampling funnels having a firβt, open mouth and for introduction of a chemical βample solution, and a tapered, βecond conical end having a through-hole at the apex of βaid conical end, βaid sampling funnelβ being arranged ln an array to permit individual addressing for chemical treatment; b) a plurality of sample mini-columnβ for retaining preselected chemical samples, each of βald mini-columnβ having a centrally diβpoβed chamber extending longitudinally through said mini-column, at least one of said mini- columnβ having a material βelected from at leaβt one of a βolid βupport, dry reagent, dry βample, liquid reagent, liquid βample, and porouβ fritβ diβpoaed in βaid central chamber, said chamber terminating into a first open end, said firβt open end having a flared, tapered inlet port, βaid inlet port being in flow-through, physical connection with aaid through-hole in βald conical βecond end of βaid funnel, said connection resulting in smooth, continuouβ transition of the taper from said conical βecond end of βaid funnel to βaid taper of eaid inlet port, βaid chamber terminating into a βecond open and, said βecond open end having a flared, tapered outlet port, said outlet port being in flow- through communication with βaid inlet port, said firβt open end of each of said funnelβ and βaid tapered conical βecond end of each of βaid funnelβ and permitting localized access by a compreββive fit interface meanβ to said mini- column inlet port for near-zero dead volumn flow-through communication between said interface meanβ and βald mini-columnβ; c) an optional plurality of reagent wells for retaining preselected reagents for reaction with eaid preselected sample retained in βald mini- columns, said reagent wellβ compriβing a through-bore parallel to βaid longitudinal axle of eaid mini-column, βaid through-bore terminating in a first end having a flared, tapered inlet port, and terminating in a βecond end having a flared, outlet port βaid reagent well Inlet port being in flow-through communication with βaid reagent well outlet port; and d) a machine readable code diβpoβed on said chemical treatment cassette for identifying the chemistry protocols to be performed on the retained samples in an id miiii-columnβ .

B. A chemical treatment cassette as in claim 7 further comprlaing an alignment meanβ for aligning said mini-columnβ βuch that the longitudinal axle of βald mini columns iβ coincident with the longitudinal axis of βaid βample funnelβ.

9. A chemical treatment caββette aβ ln claim 7 wherein a βolid βupport material iβ diβpoβed in βaid central chamber of said mini-column, said βolid βupport material being treated to immobilize βaid chemical sample, βaidβolid support being retained in said mini-columnβ by porouβ fritβ diβpoβed in βaid central chamber at βaid inlet port end and at βaid outlet port end.

10. A chemical treatment cassette as ln claim 7 wherein said mini- columnβ are integral to βald funnel aβaembly to provide a unitary, one-piece funnel/mini-column assembly.

11. A chemical sample loading βtation for loading sample solutionβ in a sample funnel of a chemical treatment caββette, comprlaing in operative combinationi a) a caββette alignment meanβ for aligning the chemical treatment caββette to a predetermined X-Y position; b) a sealing gaβket actuator for lowering an annular sealing gaβket over a firβt open end of a sampling funnel when βaid sample funnel iβ located at βaid predetermined X-Y position, aaid sealing gaβket actuator exerting co preββive force on said sealing gasket and aaid βample funnel to

•2B- provide leak-proof βeal between βaid βealing gaβket and said open end of said funnel, βaid annular βealing gaβket permitting introduction of sample βolution or solvent into said funnel without having to diβengage aaid βealing gaβket actuator from said sampling funnel; and c) a pressure cap having an annular shoulder and center plunger section for press-fit insertion Into the mouth of said sample funnel to define a funnel headspace, said shoulder engaging said annular sealing gasket to limit the extent of inβertion of said center plunger, said preββure cap having a through-bore gas inlet for introduction of a pressurized gas into said funnel headspace to force said βample βolution into the through-hole of the conical second end of βaid βampling funnel.

12. A βample loading βtation aβ in claim 11 wherein βaid βample loading station further compriβeβ a plurality of βaid βealing gaβket actuators, and a plurality of βald preββure cape for βimultaneouβ βample βolution introduction to a plurality of sample funnelβ.

13. A chemical treatment station for performing predetermined chemistries on a plurality of preβelected retained chemical aa pleβ located in che ioal treatment casβette having a plurality of individually addresβable βample mini-columnβ for retaining preβelected chemical samples, the mini- columns having tapered inlet ports and outlet ports, and optionally having a plurality of reagent wells for retaining reagents, the reagent wells having tapered inlet ports and outlet portβ, βaid chemical treatment βtation compriβing: a) a code reading meanβ for reading a machine readable code disposed on the chemical treatment caββette to identify the predetermined specific chemistry protocols to be performed on the retained chemical βampleβ; b) a βcanning means to identify mini-column addresβeβ wherein βaid central chamber of βald mini-column iβ populated; c) a micro-controller interface for communicating from βaid code reading eanβ to a micro-controller aaid identified βpeclfic chemistry protocols, said micro-controller accessing βpeclfic proceββ algorithms purβuant to the βpecific identified chemistry protocols, βaid process algorithms defining and communicating to βaid micro-controller interface βtep-wiβe microcontroller control instructions to enable execution of aaid identified specific chemistries at βaid populated mini-column addresses; d) a cassette/treatment station interface for enabling near-zero dead volume low-through communication of the chemical treatment cassette with said chemical treatment βtation, eaid caββette/treatment atation interface comprising: 5 1) a cassette interface dock for precision alignment of the chemical treatment cassette,

11) a first nozzle array comprising a plurality of nozzles spatially arrayed to be congruous with mini-column inlet ports and reagent well inlet portβ for flow-through localized interfacing with the mini-column inlet 0 portβ and the reagent well inlet portβ, ill) a βecond nozzle array having a plurality of nozzles spatially arrayed to be congruouβ with the mini-column outlet portβ and reagent well outlet ports for flow through communication with the mini-column outlet ports and the reagent well outlet portβ, 5 iv) βaid first nozzle array positioned above said casβette interface dock, βaid nozzleβ in βaid first array having nozzles tipβ extending downward towards said cassette interface dock, v) βaid βecond nozzle array poβition below βaid caββette interface dock, said nozzles in said βecond nozzle array having nozzle tips 40 extending upwards to said caβsette interface dock, and vl) βaid nozzles in said firβt array and eaid nozzles in said βecond array eatablishing a flow-through localized interface by firβt aligning βaid chemical treatment caββette in said caββette interface dock and by compreββion fitting βaid nozzle tips in βaid firβt nozzle array againβt βaid 45 tapered opening of βaid mini-column inlet portβ and βaid optional reagent well inlet portβ, and by compreββion fitting βaid nozzle tipβ in βaid βecond nozzle array againβt βaid tapered opening of βaid mini-column outlet portβ and βaid optional reagent well outlet ports; and e) a chemical delivery subsystem for providing automatic flow 50 through chemiβtrieβ on the preβelected chemical βampleβ further compriβing 1

1) a βolvent bank for providing a plurality of reagentβ and solvents to said optional reagent wells for solubilizing βaid reagentβ retained therein and for reaction with the preaelected chemical βamplββ purβuant to βaid identified pre-determined βpecific chemistry protocols, 55 ii) a rotary valve array further comprising a mini-column inlet port rotary valve array for individual addreββing of mini-column inlet portβ, a mini-column outlet port rotary valve array for individual addreaelng of mini-column outlet portβ, βaid mini-column Inlet port rotary valve array and βaid mini-column outlet port rotary valve array being βynchronlzed to a aingle - 60 mini-column address to permit active, flow-through localized interface with only one mini-column at a time, and a reagent well inlet port rotary valve array for individual addressing of reagent well inlet portβ, a reagent well outlet port rotary valve array for individual addressing of reagent well outlet ports, βaid reagent well inlet port rotary valve array and βaid reagent well -65 outlet port rotary valve array being synchronized to a single reagent wall address to permit active, flow-through localised interface with only one reagent well at a time, ill) a plurality of accumulators for temporary storage of solventβ and reagents, and temporary storage of detached chemical sample to

70 ^' enable chemistries to be performed on βaid detached βampleβ, and to enable collateral chemiβtries not including said detached samples aβ a reagent to be performed in the mini-columns, iv) a mixer/diluter reservoir in communication with said solvent bank, reagent wells, and mini-columns for temporary retention of at 75 least one of a solvent, reagent, sample, and mixtures thereof, v) a pneumatic pressurizing syβte for bi-directional transfer of βolventβ, reagents, and chemical βampleβ to and from aaid reagent wells, βaid mini-columnβ, βald accu ulatora, and eaid mixer/diluter, and for supplying inert gas to said chemical treatment delivery βubβytem, and 80 vi) an optional valve means for integration of an analyzer subsystem into said chemical delivery subsystem to permit automatic analysis of the sample retained in said minl-columnβ without removal of eaid mini- columns from said chemical treatment caβaette and without disengaging said cassette/treatment βtation interface.

14. A chemical treatment station as in claim 13 wherein βald analyzer subsystem is a high performance liquid chroma ograph.

15. A chemical treatment syβtem for treatment of leββ than milligram quantitieβ of a chemical sample, compriβing in operative combination:

5 a) a chemical sample retaining meanβ for holding a plurality of chemical samples for chemical treatment, βald chemical βample retaining meanβ further compriβing: i) a βample funneling eana for receiving a plurality of chemical βample βolutionβ, said βample funneling means permitting individual 10 addresβing of each of βaid plurality of chemical sampling βolutionβ for chemical treatment; ii) meanβ for retaining an individual preaelected chemical βample, βaid Individual βample retaining meanβ being in flow-through, phyβical connection with βaid funneling meanβ, βaid meanβ permitting near-zero dead volume compression fit with an interface meanβ; iii) an optional plurality of reagent retaining means for retaining preβelected reagents, βaid reagent retaining meanβ permitting flow- through communication there through; iv) a machine readable code disposed on said chemical sample retaining means for identifying the chemistry protocols to be performed on the retained samples in eaid individual sample retaining means; b) a sample loading means for loading βa ple solutions in βaid sample funneling meanβ, βaid βample loading means further comprising:

1) an alignment means for aligning said chemical sample retaining means to a predetermined X-Y poβition; ii) a βealing gaβket actuator meanβ for loading a βealing gasket on said sample funneling meanβ and for providing compreββive force on βaid funneling meana when βaid βample funneling means is located at eaid predetermined X-Y position; iii) a preββure cap means for introduction of pressurized gaβ into βald βample funneling meanβ and for forcing a aample βolution through aaid Individual sample retaining means; c) a chemical treatment station for performing predetermined chemistries on a plurality of preselected retained chemical βampleβ located ln aaid chemical βample retaining meanβ, βaid chemical treatment atation further compriβing:

1) a code reading meanβ for reading βaid machine readable code diβpoβed on eaid chemical treatment caββette to identify the specific chemistry protocols to be performed on aaid retained chemical samples; ii) a scanning meanβ to identify populated mini-column addreββeβ; ill) a micro-controller Interface means for communicating from βaid code reading meanβ to a micro-controller aaid Identified βpecific che iβtry protocols, βaid micro-controller accaββlng βpecific proceββ alςorith β purβuant to the βpecific identified chemiβtry protocols, βald proceββ algorithms defining and communicating to aaid micro-controller interface step-wlβe micro-controller control instructions to enable execution of eaid identified specific chemiβtrieβ on βald pre-eelected chemical samples at said populated mini-column addreββeβ; iv) a chemical caaatte/treatment interface meanβ for enabling flow-through localized communication of aaid chemical βample retaining means with said chemical treatment βtation, βald interface means comprising: a docking means for precision alignment of said chemical βample 5 retaining meanβ, an interface meanβ for establishing flow-through localized Interface between eaid chemical treatment βtation and said chemical sample retaining means ; iv) a chemical delivery subsystem for providing automatic 0 flow through chemiβtrieβ on βaid preβelected chemical samples further comprising: a βolvent storage means for providing a plurality of reagents and solvents to eaid reagent retention means for solubilizing βaid reagents retained therein and to βaid chemical βample retaining meanβ for treatment of 5 said chemical sampleβ in said individual sample retaining means pursuant to said identified βpecific chemistry protocols, a valve means for individual addressing of said Individual βample retaining means and for individual addressing of eaid reagent retention means, a plurality of accumulator meanβ for temporary atorage of solvents 0 and reagentβ, and temporary βtorage of detached chemical βample to enable chemistries to be performed on βaid detached samples, and to enable chemiβtrieβ not including βaid detached βampleβ aβ a reactant to be performed in βaid individual βample retaining meanβ a mixer/diluter reβervoir in communication with aaid βolvent bank, 5 reagent wells, end mini-columns for temporary retention of at least one of a solvent, reagent, sample, and mixtures thereof, a pneumatic preββurizing system for bi-directional tranβfer of solvents, reagents, and chemical samples to and from βaid reagent wells, said mini-columns, eaid accumulatorβ, and βaid mixer/diluter, and for supplying O inert gas to βaid chemical treatment delivery βubβyte , and an optional valve meanβ for integration of an analyzer subsystem into βaid chemical delivery βubβyβtem to permit automatic analyβis of the sample retained in βaid mini-columnβ without removal of βaid mini-columnβ from βaid chemical treatment caββette and without disengaging βald

B5 cassette/treatment station Interface.

16. A method for rapid, βequential, uninterrupted chemical treatment of lesB than milligram quantities of a plurality of chemical aampleβ, comprising the steps of: a) providing a chemical treatment caββette for retaining a 5 plurality of chemical βampleβ for chemical treatment, βaid chemical treatment caββette further compriβing: 1) a funnel assembly comprising a plurality of integrated, individually addressable chemical βolution sampling funnels for receiving chemical βample βolutions, each of said sampling funnelβ having a firβt, open mouth end for introduction of a chemical sample solution, and a tapered, second conicnl end having a through-hole at the apex of said conical end, oald sampling funnels being arranged in an array to permit individual addressing for chemical treatment; ii) a plurality of βample mini-columns for retaining preβelected chemical βamples, each of βald mini-columnβ having a centrally disposed chamber extending longitudinally through βaid mini-column, at leaβt one of said mini-columns having a material selected from at leaβt one of a βolid support, dry reagent, dry sample, liquid reagent, liquid βample, and porouβ fritβ disposed in said central chamber, βaid chamber terminating into a first open end, eaid firat open end having a flared, tapered inlet port, aaid inlet port being in low-through, physical connection with said through-hole in said conical second end of said funnel, said connection resulting in smooth, continuous transition of the taper from aaid conical βecond end of βaid funnel to βaid taper of βaid inlet port, βaid chamber terminating into a βecond open end, said second open end having a flared, tapered outlet port, eaid outlet port being in flow-through communication with βaid inlet port, βaid first open end of each of aaid funnels and βaid tapered conical βecond end of each of βaid funnels and permitting localized access by a compreββive fit interface means to βaid mini-column inlet port for near-zero dead volu n flow-through communication between βaid interface means and βaid mini-columnβ;

ill) an optional plurality of reagent wellβ for retaining preβelected reagents for reaction with βaid preaelected βample retained in aaid min-columns, said reagent wellβ compriβing a through-bore parallel to βaid longitudinal axiβ of βaid mini-column, aaid through-bore terminating in a firβt end having a flared, tapered inlet port, and terminating ln a βecond end having a flared, outlet port eaid reagent well inlet port being ln flow-through communication with eaid reagent well outlet port; and iv) a machine readable code diβpoβed on βaid chemical treatment caββette for identifying the chemiβtry protocolβ to be performed on the retained sampleβ ln βald mini-columnβ; b) providing a βample loading βtation for loading aample βolutionβ in βald βample funnelβ, βaid βample loading station further comprising: i) a casβette alignment means for aligning βaid chemical treatment cassette to a predetermined X-Y position; ii) a sealing gasket actuator for lowering an annular sealing gasket over said firβt end of βaid βa pling funnel when βaid βample funnel is located at βald predetermined X-Y poβition; iii) a pressure cap having an annular shoulder and center plunger βection for presβ-fit inβertlon into the mouth of βaid βample funnel to define a funnel headspace, βaid βhoulder engaging aaid annular gaβket to limit the extent of in^βertion of said center plunger, eaid pressure cap having a through-bore gas inlet for introduction of preββurized gas into βald funnel headspace to force eaid βample βolution into βaid inlet port of βaid mini- coluinn; c) loading a preβelected chemical aample βolution into each funnel of βaid funnel assembly; d) retaining a preβelected chemical sample in said mini-columns; e) providing a chemical treatment βtation for performing predetermined chemistries on a plurality of preβelected retained chemical samples located in βaid chemical treatment caβsette, said chemical treatment station further compriβing: i) a code reading meanβ for reading βaid machine readable code diβpoβed on βaid chemical treatment caββette to identify the βpecific chemiβtry protocols to be performed on βaid retained chemcial samples;

11) a scanning means to identify mini-column addreββeβ wherein βaid central chamber of βaid mini-column iβ populated; iii) a micro-controller interface for communicating from βaid code reading meanβ to a micro-controller βald identified βpeclfic chemistry protocols, said micro-controller acceββing βpecific proceββ algorithms purβuant to the βpecific identified chemiβtry protocolβ, aaid proceββ algorithms defining and communicating to eaid micro-controller interface βtepwiβe microcontroller control inβtructionβ to enable execution of βaid identified specific chemistries on βaid pre-βelected chemical βa plea at said populated mini-column addresses; iv) a casβette/treatment station interface for enabling near-zero dead volume flow-through communication of aaid chemical treatment caβsette with said chemical treatment βtation, aaid caaβette/treatment βtation interface compriβing: a cassette interface dock for precision alignment of eaid chemical treatment caββette, a firβt nozzle array compriβing a plurality of nozzleβ spatially arrayed to be congruous with said mini-column inlet portβ and reagent well Θ5 inlet ports for flow-through localized interfacing with said mini-column inlet ports and eaid reagent well inlet ports, a Bβcond nozzle array having a plurality of nozzles βpatlally arrayed to be congruous with eaid mini-column outlet ports and reagent well outlet ports for flow-through lcoalized interfacing with βald mini-column 90 outlet ports and βaid reagent well outlet portβ, βaid first nozzle array positioned above said caββette interface dock, eaid nozzleβ in βaid first array having nozzles tips extending downward towards βaid cassette Interface dock, said βecond nozzle array position below βaid cassette interface 95 dock, said nozzles in βaid βecond nozzle array having nozzle tipβ extending upwardβ to βaid caββette interface dock, and aaid nozzles in βaid firβt array and βaid nozzles ln βaid βecond array establishing βaid flow-through localized interface by firβt aligning said chemical treatment cassette ln βaid cassette interface dock and by compression

100 fitting βaid nozzle tipβ in βaid firβt nozzle array againβt aaid tapered opening of βaid mini-column inlet portβ and βaid reagent well inlet ports, and by compression fitting eaid nozzle tips in βaid aecond nozzle array againβt βaid tapered opening of βaid mini-column outlet portβ and said reagent well outlet portβ;

105 iv) a chemical delivery subsystem for providing automatic flow through chemistrleβ on eaid preβelected chemical βampleβ further compriβing: a βolvent bank for providing a plurality of reagentβ and solvents to said reagent wellβ for βolubilizing βaid reagentβ retained therein and for 110 reaction with βaid chemical samples pursuant to said Identified specific chemistry protocols, a rotary valve array further compriβing a mlnicolumn inlet port rotary valve array for individual adβreββing of mini-column inlet portβ, a mini-column outlet port rotary valve array for individual addressing of mini- 115 column outlet portβ, βaid mini-column inlet port rotary valve array and βaid mini-column outlet port rotary valve array being βynchronized to a single mini- column addreas to permit active interface with only one mini-column inlet and outlet port at a time, and a reagent well inlet port rotary valve array for Individual adβreββing of reagent well inlet ports, a reagent well outlet port 120 rotary valve array for Individual addressing of reagent well outlet portβ, βald reagent well inlet port rotary valve array and aaid reagent well outlet port rotary valve array being synchronized to a single reagent well address to permit active interface with only one reagent well inlet and outlet port at a tline , 125 a plurality of accumulators for temporary storage of solvents and reagents, and temporary storage of detached chemical sample to enable chemistries to be performed on said detached samples, and to enable chemistries not including βaid detached βa pleβ to be performed in βaid mini-column, a mixer/diluter reservoir in communication with eaid solvent bank, 130 reagent wells, and mini-columns for temporary retention of at leaβt one of a solvent, reagent, sample, and mixtures thereof, a pneumatic preββurlzing eystem for bi-directional transfer of solventB, reagents, and chemical βampleβ to and from βaid reagent wells, said mini-columns, eaid accumulators, and said mixer/diluter, and for supplying 135 inert gas to said chemical treatment delivery βubsytem, and an optional valve means for integration of an analyzer subsystem into βaid chemical delivery βubβyβtem to permit automatic analysis of the

Bample reatined ln said mini-columns without removal of eaid mini-columns from said chemical treatement caββette and v/ithout disengaging βaid

140 caββette/treatment atation interface; f) inserting βaid chemical treatment casβette into βaid chemical treatment sation) g) βcanning said mini-columnβ to identify populated addressee; h) reading βaid machine readable barcode to identify the

145 cheiniβtry protocols to be performed on βaid βampleβ;

1) communicating βaid identified protocols to a micro-controller; j ) accessing and loading the correct proceββ algorithm purβuant to the βpecific identified chemiβtry protocols for stepwlse micro-controller control of βaid cassette treatment interface and said chemical delivery

150 subbsystem; k) establishing flow-through communication of βaid chemical treatemnt caββette with βaid chemical treatment station by engaing the caββette/treatmant βtation interface;

1) executing βequeπtial, uninterrupted chemistry protocols on 155 said plurality of sample^β; and ) analyzing each analyte.