Temperature Control Of Enrichment And Separation Columns In Chromatography

Abstract

A method of analyzing samples includes loading a sufficient quantity of the sample onto a trap column to overload the trap column; heating an analytical column and the trap column to a greater temperature than the analytical column; and pumping a solvent, to the trap column, having a solvent composition profile that, in cooperation with a temperature differential, causes at least some of the components to elute sequentially from the trap column to the analytical column and focus on the analytical column prior to eluting from the analytical column; or optionally: loading a small-molecule sample onto a cooled portion of an analytical column; heating the analytical column; and pumping a solvent, to the heated analytical column, to elute the components from the analytical column. Chromatographic separation includes: a trap column; a separation column; a trap-column heater; a separation-column heater; a solvent pump unit; and a control unit can be used.

Description

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application Nos. 61/182,268 and 61/182,498, both filed May 29, 2009. The entire contents of these applications are incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates generally to chromatography. More specifically, preferred embodiments of the invention relate to apparatus and methods for liquid-chromatography of complex protein-related samples and of small-molecule samples.

BACKGROUND

High-performance liquid chromatography (HPLC) instruments are analytical tools for separating, identifying, and quantifying compounds. Traditional HPLC instruments use analytical columns constructed from stainless-steel tubing. Typically, the tubing has an inner bore diameter of 4.7 mm, and its length ranges from about 5 cm to about 25 cm.

In addition, the analytical column of an HPLC instrument typically has a fritted end fitting attached to a piece of tubing. Particles, typically silica-based, functionalized with a variety of functional moieties, pack the tube.

To achieve optimal separation efficiency, using the completed column, an appropriate flow rate of a mobile phase is important. For a 4.7 mm diameter column packed with 5 μm diameter particles, a desirable flow rate is typically between about 1 mL/min and about 2 mL/min. Minimizing the presence of unswept dead volume in the plumbing of the HPLC instrument is desirable for maintaining separation efficiency.

In an HPLC instrument, an injector is typically used to inject a sample into a flowing mobile phase as a discrete fluidic plug. Dispersion of a plug band as it travels to and/or from the column reduces the ultimate efficiency of the chromatographic system. For example, in a chromatographic system using 4.7 mm column tubing and a mobile phase flowing at 1-2 mL/min, tubing having an outer diameter of 1/16 inch and an inner diameter of about 0.010 inch is typically used to plumb connections between the various HPLC components (e.g. pump, injector, column, and detector). For these flow rates and tubing dimensions, it is relatively easy to machine port details to tolerances that will ensure minimal band broadening at tubing interfaces.

A desire to reduce mobile-phase solvent consumption, in part, has motivated a trend towards reducing column inner diameter. Thus, several scales of chromatography are now commonly practiced; these are typically defined as shown in Table 1 (where ID is inner diameter.)

	TABLE 1
	HPLC Scale	Column ID	Typical Flow range
	Analytical	4.7	mm	1s	mL/min
	Microbore	1-2	mm	100s	μL/min
	Capillary	300-500	μm	10s	μL/min
	Nano	50-150	μm	100s	nL/min

Microbore HPLC has often been practiced with equipment similar to that used for analytical scale HPLC, with minor modifications. Aside from requiring the exercise of a small degree of additional care in making fittings, microbore HPLC typically requires an operating skill level similar to that of analytical scale HPLC.

In contrast, capillary and nano-scale HPLC require relatively significant changes in HPLC components relative to analytical-scale HPLC. Generation of stable mobile-phase flows of less than about 50 μL/min is relatively difficult using standard open-loop reciprocating HPLC pumps, such as those commonly found in analytical and microbore HPLC systems.

For capillary-scale chromatography, stainless-steel tubing is usable for component interconnections; however, the inner diameter must typically be less than 0.005 inch (less than about 125 μm). Care is generally required in the manufacture of fitting terminations to avoid creation of even minute amounts of dead volume.

For nano-scale chromatography, tubing having inner diameters of about 25-50 μm is typically required to interconnect components of an instrument (e.g., to connect a pump to a separation column). Because stainless-steel tubing is typically unavailable in these dimensions, polyimide-coated fused-silica tubing is typically used. Although fused-silica tubing has excellent dimensional tolerances and very clean, non-reactive interior walls, it is fragile and can be difficult to work with. In addition, interconnection ports should be machined to exacting tolerances to prevent even nanoliters of unswept dead volume.

While the primary motivation to replace analytical-scale HPLC with microbore-scale HPLC may be the desire for reduced solvent consumption, moving to capillary-scale and nano-scale chromatography can support improved detection sensitivity for mass spectrometers, in addition to further reducing solvent consumption, when, for example, flows of less than about 10 μL/min are used. Moreover, capillary-scale or nano-scale systems are often the only options for the sensitive detection typically required for applications involving small amounts of available sample (e.g., neonatal blood screening).

Despite the advantages of capillary-scale and nano-scale chromatography, HPLC users tend to employ microbore-scale and analytical-scale chromatography systems. As described above, these systems typically provide good reliability and relative ease-of-use. In contrast, maintenance of good chromatographic efficiency while operating a capillary-scale or nano-scale chromatographic system requires significant care when plumbing the system (e.g., using tubing to connect pump, injector, column, and detector).

In practice, an operator switching from an analytical or microbore-scale system to a capillary or nano-scale system at times finds that better separation efficiency was achieved with the higher-flow rate (i.e., the analytical or microbore-scale) system. This typically occurs due to insufficiency in the operator's knowledge or experience required to achieve low band-spreading tubing interconnections. Moreover, use of smaller inner-diameter tubing at times can lead to frequent plugging of tubing.

Due the relative difficulty typically encountered with capillary-scale HPLC systems and, even more so, with nano-scale HPLC systems, such systems have primarily been used only when necessary, such as for small sample sizes, and when a relatively skilled operator is available. Thus, analytical laboratories tend to possess more analytical-scale and microbore-scale systems than capillary-scale and nano-scale systems, and do not realize the full benefits available from capillary-scale and nano-scale HPLC.

Proteomic analyses often utilize a trap column for sample enrichment and cleaning prior to separation of the sample in an analytical column. Often, different packing material chemistries are used for the trap and separation columns; sample components trapped on the trap column may be serially driven from the trap to the separation column during a gradient-based mobile phase elution process. The components can be initially focused at the head of the analytical column, due to the different chemistry, until the gradient attains a level that drives the component from the chemistry of the analytical column. It is also common to place the analytical column in an oven to provide a stable, elevated temperature, which promotes elution of sample components from the analytical column.

As noted, above, analyses involving small sample volumes, such as a volume of 50 μL, present challenges, even in an apparatus configured for nano-analysis. In particular, it remains difficult, if not impossible, to remove all significant sources of sample dispersion when handling such small sample volumes.

SUMMARY

Some embodiments of the invention arise, in part, from the realization that a trap-column-to-analytical-column temperature differential—in particular, where the trap is elevated relative to the analytical column—potentially provides improved sample separations, in particular, for complex protein-related samples. Some embodiments of the invention arise, in part, from the realization that small volumes of small-molecule-based samples can be focused at the head of a separation column, via cooling, to mitigate dispersion effects associated with upstream plumbing. Potential improvements include, for example, improved sensitivity and resolution. Moreover, temperature manipulation is optionally coordinated with trap-versus separation-column differences in chemistry and/or solvent-composition profiles to provide desirably sharp component peaks that elute from a separation column.

Moreover, some embodiments arise, in part, from a realization that an integrated high-pressure chemical-separation device, such as an HPLC apparatus or an ultra-high-pressure LC (UHPLC) apparatus, is advantageously fabricated, in part, from sintered inorganic particles. Some embodiments of the invention provide nano-scale microfluidic LC instruments that offer integration of a trap/enrichment column(s) and a separation column(s) on one (or more) ceramic-based substrates; some of these embodiments include features that support cooling of the enrichment column to enhance cycle time and/or improve enrichment-column performance.

Accordingly, one embodiment features a method of analyzing protein-related samples. The method includes: providing a complex protein-related sample; loading a sufficient quantity of the sample onto a trap column to overload the trap column; heating an analytical column and heating the trap column to a greater temperature than the analytical column; and pumping a solvent, to the trap column, having a solvent composition profile that, in cooperation with a temperature differential, causes at least some of the components to elute sequentially from the trap column to the analytical column and focus on the analytical column prior to eluting from the analytical column.

An alternative embodiment features an apparatus for chromatographic separation of a sample. The apparatus includes: a trap column; a separation column in fluidic communication with the trap column; a trap-column heater; a separation-column heater; a solvent pump unit; and a control unit. The control unit includes instructions that, when implemented, causes the apparatus to: load a sufficient quantity of a complex protein-related sample onto the trap column to overload the trap column; heat the analytical column and the trap column so that the trap column has a greater temperature than the heated analytical column; and pump a solvent, to the heated trap column, having a solvent composition profile that, in cooperation with the temperature differential, causes at least some of the components to elute sequentially from the heated trap column to the heated analytical column and focus on the heated analytical column prior to eluting from the heated analytical column.

Another embodiment features a method of chemical analysis, which includes: (a) providing a ceramic-particle-based and/or metal-based microfluidic substrate defining a trap column and an analytical column in fluidic communication with the trap column; (b) loading a sample on the trap column while the trap column is at a temperature in a load range; (c) heating at least a portion of the substrate containing the analytical column to provide a temperature above ambient during elution of the sample through the analytical column, such that the trap column is incidentally heated; (d) pumping a solvent to the trap column to elute the sample components from the trap column to the analytical column at the temperature above ambient, causing the components to elute from the analytical column; (e) cooling at least a portion of the substrate containing the trap column, after elution of the components from the analytical column, to return the trap column to a temperature in the load range; and (f) repeating (b) through (e) for each of one or more subsequent samples.

An alternative embodiment features an apparatus for chromatographic separation of a sample. The apparatus includes: a ceramic-particle-based and/or metal-based microfluidic substrate defining a trap column and an analytical column in fluidic communication with the trap column; a fluidic conduit having an outlet disposed to direct a fluid towards a location of the trap-column to cool at least a portion of the microfluidic substrate; a separation-column heating unit disposed to heat at least a separation column portion of the microfluidic substrate during separation of a sample; and a solvent pump unit for pumping a solvent composition to an inlet of the trap column.

Still another embodiment features a method of analyzing small molecules, The method includes: cooling at least a portion of an analytical column proximate to an inlet of the analytical column; loading, onto the cooled portion of the analytical column, a nano-scale sample comprising a plurality of different small-molecule components; heating the analytical column to promote elution of the loaded components; and pumping a solvent, to the heated analytical column, to elute the components from the analytical column.

Some preferred embodiments entail mass analysis.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and further advantages of this invention may be better understood by referring to the following description in conjunction with the accompanying drawings, in which like numerals indicate like structural elements and features in various figures. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention.

FIG. 1A is a block diagram of a portion of a chemical analysis device, in accordance with one embodiment.

FIG. 1B is a flowchart of a method of analysis of complex protein samples, in accordance with one embodiment.

FIG. 1C is a flowchart of a method of analyzing samples of small molecules, in accordance with one embodiment.

FIG. 1D is a flow chart of a method of chemical analysis using one or more microfluidic substrates.

FIG. 2 is a front view of an embodiment of the liquid chromatography module.

FIG. 3 is a view of the liquid chromatography module with an open cover to show a clamping assembly housed within.

FIG. 4 is a side view of the clamping assembly.

FIG. 5 is a view of an embodiment of an end housing of the clamping assembly.

FIG. 6 is a view of the left side of the microfluidic cartridge.

FIG. 7 is a side view of the microfluidic cartridge with the right side removed, showing a push block superimposed upon the microfluidic substrate in the microfluidic cartridge.

FIG. 8 is a side view of the microfluidic cartridge with the left side removed.

FIG. 9 is a side view of one embodiment of a microfluidic substrate within the microfluidic cartridge.

FIG. 10A is a view of an embodiment of a separation device having more than one microfluidic substrate.

FIG. 10B is a cross-sectional view of a portion of the device of FIG. 10A, illustrating, in more detail, fluidic-connection and alignment features.

DETAILED DESCRIPTION

As used herein, the term “complex protein sample” means a sample that includes at least hundreds of different proteins and/or peptides. A typical complex sample, for example, includes approximately 10,000 proteins, which may be digested, prior to chromatographic separation, yielding a complex protein sample that includes approximately 5 to 15 peptides per protein in the complex sample; such a complex protein sample will then, upon separation, yield thousands of chromatographic peaks. Some embodiments of the invention provide sharp chromatographic peaks for a complex protein sample, by stepping peptides, in sequence, off of a trap column, at least in part through manipulation of trap column and separation column temperatures. Various methods, according to some embodiments of the invention, are well suited to complex protein samples, as encountered, for example, in proteomics work.

As used herein, the term “small-molecule sample” means a sample that contains one or more different “small molecules”, which are organic non-polymeric molecules. The term “small molecule” is used herein in a conventional sense; a common definition of a “small molecule”, especially as found in the field of pharmacology, is generally restricted to a molecule that binds with high affinity to a biopolymer, such as a protein, nucleic acid, or polysaccharide, and, in addition, alters the activity or function of the biopolymer. The upper molecular weight limit for a small molecule is taken to be approximately 800 Daltons. The molecular-weight limit generally accommodates the possibility of rapid diffusion of small molecules across cell membranes, permitting intracellular access and activity. This molecular-weight cutoff is generally accepted as a necessary but insufficient condition for oral bioavailability.

Small molecules can have a variety of biological functions, serving as cell signaling molecules, as tools in molecular biology, as drugs in medicine, as pesticides in farming, and in many other roles. These compounds can be natural (such as secondary metabolites) or artificial (such as antiviral drugs); they may have a beneficial effect against a disease (such as drugs) or may be detrimental (such as teratogens and carcinogens).

Biopolymers such as nucleic acids, proteins, and polysaccharides (such as starch or cellulose) are generally consider as not falling within the definition of small molecules, although their constituent monomers—ribo- or deoxyribonucleotides, amino acids, and monosaccharides, respectively—are often considered to be small molecules. Very small oligomers are also usually considered small molecules, such as dinucleotides, peptides such as the antioxidant glutathione, and disaccharides such as sucrose.

The term “temperature differential” is used herein to mean that two, or more, entities, or portions of the same or different entities, have different temperatures. A “temperature differential” refers to the temperature difference and/or the particular absolute temperatures of the objects having different temperatures.

Various non-limiting embodiments of the invention relate, preferably, to chromatographic separations of complex protein samples or small volumes of small-molecule samples. These embodiments provide sharper chromatographic peaks through manipulation of column(s) temperature. Some embodiments include enrichment and separation columns, or only utilize one or more separation columns. Some embodiments, which in some cases are configured for separations of small amounts of sample, utilize ceramic-based or other suitable substrates, such as a bonded-titanium substrate. In view of this description, other embodiments and components will be apparent to one having ordinary skill in the compound-analysis arts.

Some embodiments include features that provide cooling and/or heating of one or more enrichment columns and/or cooling and/or heating of one or more separation (e.g., analytical) columns. In some cases, temperature control supports improved enrichment-column performance and shorter cycle time of sample analyses. In some preferred embodiments, a temperature differential, between a trap column and an analytical column, is provided during elution of a sample. In particular, a trap column, holding an enriched sample, is preferably held at a higher elevated temperature than the elevated temperature of an analytical column.

Some embodiments of the invention are based on microfluidic substrates; in the following, more general embodiments are described first, followed by examples that are specific to microfluidic substrates. In the following, the term “analytical column” is used interchangeably with “separation column”, for convenience, and is not intended to limit all embodiments of the invention to columns of any particular dimensions, width or diameter, to any particular flow rates, or to any particular sample-volume sizes.

In some embodiments, a trap column is overloaded; during overloading, the band of enriched components grows broad as active sites are used up. Preferably, the broad bands of components are caused to focus or narrow as they move onto an analytical column. Various embodiments of the invention utilize temperature and/or chemistry variations to obtain desired focusing, sensitivity and/or resolution. Some preferred embodiments relate to protein or small-molecule analyses.

Regarding proteomics, some embodiments entail gradient elution, such as through the use of water and acetonitrile; as known in the art, different classes of peptides are generally released from a packing medium at different concentrations. After enrichment on a trap column, the release of a big band of a component from the trap can cause the big band to gradually enter the analytical column. Various embodiments provide focusing of such broad component bands into narrow bands in the vicinity of the head of the analytical column.

Some embodiments, in combination with temperature manipulation, utilize different chemistries in different columns. Some of these embodiments provide capillary- or nano-scale chromatography. For example, an analytical column optionally includes 1.7 μm diameter ethylene bridged hybrid particles (such as an ACQUITY UPLC® BEH TECHNOLOGY™ C18 column of 75 μm inner diameter and 100 mm length, and 1.7 μm particle size, available from Waters Corporation, Milford, Mass.) while a trap column optionally includes 5 μm diameter silica particles (such as a nanoACQUITY UPLC® 10K SYMMETRY® C18 column of 180 μm inner diameter and 20 mm length, available from Waters Corporation.) Using such columns, a sample component can be caused to come off the trap column at, for example, a lower acetonitrile strength than required for elution from the analytical column; the component will thus tend to form a narrow band on the analytical column prior to elution at a time when the solvent strength has further increased.

Some preferred embodiments of the invention thus use a chemistry mismatch with temperature manipulation and mismatching to obtain sharp component peaks, good sensitivity for substantially all components in a sample, and/or efficient analyses. As will be apparent to one of skill, in view of the description provided herein, solvent type and temporal composition profile are suitably chosen in cooperation with column-temperature manipulation and, optionally, chemistry manipulation, to support analysis of a particular type of sample. One of skill will also understand that the following examples merely illustrate various aspects of the invention, and are not intended to limit all embodiments to a specific collection of features.

FIG. 1A is a block diagram of a portion of a chemical-analysis device 500, which is used to illustrate some broad principles of some methods and apparatus, according to some embodiments of the invention. The device 500 includes a separation column 510, a thermal unit 511 in thermal communication with the column 510 for heating and/or cooling of all or portion(s) of the column 510, an optional cooler 512 in thermal communication with at least a portion of the separation column 510 proximate to an inlet of the column 510, an optional trap column 520, having an outlet in fluidic communication with an inlet of the separation column 512, an optional trap thermal unit 521 in thermal communication with the trap column 521, for heating and/or cooling of all or portion(s) of the trap column 520, and an optional control module 530 to control various operations of the device 500 such as heating and/or cooling by the thermal units 511, 521 and the cooler 512.

The device 500 is optionally used to implement various embodiments of methods of sample separation. For example, FIG. 1B is a flowchart of a method of analysis 600 of complex protein samples, in accordance with one embodiment of the invention. The method 600 includes loading 610 a sufficient quantity of a complex-protein sample onto a trap column to overload the trap column, heating 620 an analytical column, heating 630 the trap column to a greater temperature than the analytical column, thus providing a temperature differential between the trap and analytical columns; and delivering 640 a mobile phase having a solvent composition profile that, in cooperation with the temperature differential, causes at least some of the overloaded components to elute sequentially from the trap column to the analytical column and focus on the analytical column prior to eluting from the analytical column.

As one example, the delivered 640 mobile phase is based on a temporal gradient of acetonitrile/water, which serves to release different peptide components in sequence from the trap column and later, in sequence, from the analytical column. Preferably, the components are released from the trap column at a lower acetonitrile strength than the acetonitrile strength required to release the same components from the analytical column. Thus, a physically broad band of a component can be released from the trap column and become a physically narrow band on the analytical column, prior to release from the analytical column when a suitably high concentration of acetonitrile arrives at the analytical column.

As illustrated by the method 600, some preferred embodiments support analyses of complex protein samples through thermal manipulation of trap/enrichment columns and separation/analytical columns. The methods are particularly well suited to chromatographic analyses sample quantities that will tend to overload an enrichment column.

As understood by one of skill in the protein liquid chromatograph arts, such a sample will tend to load a broad band of sites of an enrichment column. Through some prior approaches of then passing the overloaded sample to a separation sample, broad bands of components eluting from the enrichment column tend to produce broad peaks eluting from the separation column. Thermal manipulation, however, according to some embodiments of the invention, promotes sequential elution from the enrichment column at solvent compositions that permit temporary binding at sites on the analytical column prior to subsequent elution from the analytical column as the solvent composition profile progresses.

Since sample components arrive at the analytical column in sequence, the components have a greater portion of binding sites available to them than in the case of the overloaded enrichment column. Thus, a broad component band, arriving at the analytical column can bind in a narrow band near the head of the analytical column, and elute as a relatively sharp band/peak from the analytical column. In this manner, some embodiments provide relatively good chromatographic resolution for large complex protein samples.

Moreover, as indicated above, different stationary-media chemistries are optionally selected to promote sequential elution of sample components and focusing on an analytical column of broad component bands eluting from an enrichment column. Thus, thermal manipulation and chemistry manipulation can cooperate with solvent profile selection to provide good chromatographic resolution.

Loading 610 of an enrichment column is optionally assisted by providing the column with an ambient or sub-ambient temperature. Thus, optionally, the trap column is allowed to equilibrate with a room temperature of, for example, 25° C., or is cooled to a sub-ambient temperature, as low as, for example, −5° C. Cooling is optionally used to increase the hydrophobicity of the trapping medium, to promote capture of the sample on the trap column. After loading 610, the trap column is heated 630 to a greater temperature than the analytical column. Preferably, during elution from the trap, the analytical column has greater hydrophobicity than the trap column, as provided, for example by temperature and chemistry differentials.

Preferably, as discussed, during elution, the trap is set at a higher temperature than the analytical column. For example, the trap is heated 630 to a temperature in a range of approximately 45° C. to 65° C. or greater, while the analytical column is heated 630 to approximately 35° C., during an elution phase.

For heating 630 of the analytical column, the analytical column is optionally maintained at a substantially constant temperature while a trap column alternates between relatively low and high temperatures, respectively, for alternating enrichment 610 and elution 640 steps of an analysis process. Thus, for example, during loading 610, the columns have temperatures of 20° C./35° C., and during elution 640, the columns have temperatures of 45° C./35° C.

A temperature differential is chosen, for example, to provide optimum narrowing of component bands, as relatively broad bands of components arrive at, and are temporarily retained on, the analytical column. More generally, enrichment trap chemistry and/or temperature are selected to provide trapping of substantially all components, or all desired components, of a protein-related sample. For efficient elution with good peak resolution, the chemistries and/or temperature and/or temperature differential are selected for the protein-related sample of interest.

During trapping 610, too high a temperature of the trap column can cause loss of compounds that exhibit little or no retention at the too high temperature; as one alternative, an enrichment temperature is chosen such that it is cool enough so that substantially all compounds, or all compounds of interest, are trapped. Preferably, the trap temperature is than increased for the elution 640 phase. Use of a same trap temperature during elution 640 potentially inhibits refocusing on the analytical column.

For example, a cool trap can require a high acetonitrile concentration to remove compounds from the trap. Such a concentration can be too great to provide desired refocusing on the analytical column; in particular, if the analytical column is at a higher temperature than a cool trap, compounds may run straight through the analytical column, retaining broad peak widths as developed on the trap column.

As mentioned, some embodiments employ both selection of different chemistries and different temperatures to improve focusing and other characteristics; alternative embodiments use the same materials and differential heating only. Generally, a certain elevation of temperature lowers the percentage of acetonitrile at which a peptide will elute.

As one particular example, illustrative of some proteomics work, an analytical column having a relatively small inner diameter of 75 μm is used for analysis of a large sample volume. A trap column having an inner diameter of 180 μm is used, for example, to concentrate and purify the sample by, in part, washing salts from the loaded sample, and to provide quicker loading by use of the relatively wide diameter. A reference-protein sample, suitable for testing performance, is analyzed with a mobile phase composed of a varying mixture of mobile phase A (0.1% formic acid in water) and mobile phase B (0.1% formic acid in acetonitrile.) The trap column is a nanoACQUITY UPLC® 10K Symmetry® C18 180 μm×20 mm, 5 μm particles (available from Waters Corporation, Milford, Mass.)

The trapping flow rate is 10 μL/min, the trapping composition is 99.5% mobile phase A/0.5% mobile phase B, the load time is 1.5 min, the linear velocity is 14.89 mm/sec, the analytical column is a nanoACQUITY UPLC™® BEH TECHNOLOGY™ C18 column of 75 μm diameter×100 mm length and packed with 1.7 μm particles (available from Waters Corporation, Milford, Mass.), the flow rate is 0.300 μL/min, the gradient is 5% mobile phase B to 40% mobile phase B in 30 minutes, the sample is 100 fmol/μL of enolase in 0.1% formic acid in 97/3 water/acetonitrile, the injection volume is 2 μL in a “partial loop mode” for a 200 fmol load of enolase, a lock mass solution is 500 fmol GFP in 0.1% formic acid in 97/3 water/acetonitrile, and a lock mass flow rate is 0.5 μL/min.

The eluent, in this example, is delivered to a mass-spectrometry instrument portion of the apparatus. The example instrument is a Q-T of Micro™ (available from Waters Corporation, Milford, Mass.), the scan range is 400-1700 m/z, the scan time range is 0-40 minutes, the scan time is 0.88 sec, and the inter-scan delay is 0.1 sec.

A sample of 100 fmol/μL enolase in 0.1% formic acid in 97/3 water/acetonitrile may be prepared by diluting a stock of 1 pmol/μL enolase in 0.1% formic acid in 70/30 water/acetonitrile ten times. In one example, a 2 μL injection of the sample is made for enrichment, with the trap column approximately at or below ambient temperature. The trap and analytical columns are then both heated, the trap to a higher temperature than the analytical column, and the enriched sample is eluted through the trap and analytical columns. The trap is then cooled to prepare for enrichment of the next sample.

Some embodiments exploit temperature control features with other features that serve to reduce plumbing-related dispersion. For example a substrate having adjacent trap and separation columns (see FIG. 9) is optionally packed with trap and separation materials that are contiguous with one another.

As mentioned above, sample enrichment is utilized for any one or more of several reasons, for example: to limit injection volume, which can help to present a narrow band to the analytical column; to remove salts or other undesired components before passing a sample to an analytical column and then, potentially, to a mass spectrometer; and to more rapidly analyze large sample volumes (the trap column optionally has a larger inner diameter and lower back pressure than the analytical column.)

Generally, though a sample may not overload the analytical column, it may still overload a trap column, where, for example, the sample is a complex mixture, all components of which are simultaneously placed on the trap. Since components of the sample elute serially from the trap, the analytical column is potentially not overloaded. Thus, for example, the mass load of a fraction entering the analytical column does not overload the analytical column because the fraction is insufficient to saturate all of the active sites of the analytical column.

Some embodiments of the invention relate to small-molecule analyses, and, preferably, small samples sizes of such samples. Such samples generally do not require enrichment or cleaning, prior to separation, but can exhibit physical broadening of component bands due to dispersion as the sample travels, for example, from an injector to the head of an analytical column.

FIG. 1C is a flowchart of a method of analyzing samples of small molecules, in accordance with one embodiment of the invention. The method includes cooling 710 at least a portion of an analytical column proximate to an inlet of the analytical column, loading 720, onto the cooled portion of the analytical column, a nano-scale sample of different small-molecule components, heating 730 the analytical column to promote elution of the loaded components, and pumping 740 a solvent, to the heated analytical column, to elute the components from the analytical column. Preferably the analytical column has an inner cross-sectional dimension in a range of approximately 150 μm to approximately 500 μm. The cooled portion of the analytical column can be implemented as a distinct column connected to a primary analytical column, but suitably disposed relative to the primary analytical column to provide little or no dispersion of a sample as it travels from the distinct column to the primary column.

Some small-molecule samples, as will be understood by one of ordinary skill, will be relatively clean, and can be directly separated without first cleaning. The method 700 is optionally implemented with a single column, for both loading 720 and separation 740. Some embodiments entail capillary-scale chromatography; preferably, some embodiments entail capillary-scale chromatography, using, for example a 300 μm inner diameter analytical column. The associated small sample volumes (for example, sample injections of 100's of nL or less) can lead to significant dispersion in the plumbing that is common in typical liquid-chromatography apparatus. The method 700 provides a solution to difficulty in transferring a well controlled injection plug from an injector to the head of an analytical column.

Even a significantly dispersed injection plug can be sharpened on the head of an analytical column, through application of some embodiments of the invention. After focusing of a sample plug, the temperature is then raised to a level desired for separation and elution from an analytical column. The head only, or larger portions of a column, or the entire column is optionally cooled during loading 720.

Alternatively, the small-molecule sample is focused on a cooled column, which then releases the focused sample to an analytical column that is connected to the cooled column in a manner to preserve the focused plug.

Some embodiments of the invention entail microfluidic components, such as substrate(s) that contain column(s) and/or conduits and/or other microfluidic features. For example, FIG. 1D is a flow chart of a method 800 of chemical analysis using a ceramic-particle-based and/or metal-based microfluidic substrate(s) defining a trap column and an analytical column in fluidic communication with the trap column. The method includes: loading 810 a sample on the trap column while the trap column is at a temperature in a load range; heating 820 at least a portion of the substrate containing the analytical column to provide a temperature above ambient during elution of the sample through the analytical column, wherein the trap column is incidentally heated; pumping 830 a solvent to the trap column to elute the sample components from the trap column to the analytical column at the temperature above ambient, causing the components to subsequently elute from the analytical column; cooling 840 at least a portion of the substrate containing the trap column, after elution of the components from the analytical column, to return the trap column to a temperature in the load range; and repeating 850 steps 810, 820, 830, 840 for each of one or more subsequent samples.

The analytical column preferably has particles having a greater hydrophobicity than particles of the trap column. The temperature above ambient of the heated analytical column is preferably approximately 35° C. or greater. The analytical column optionally has an inner cross-sectional dimension in a range of about 50 μm to about 150 μm, for example, for protein analysis.

A microfluidic device, according to various embodiments of the invention, can take a variety of forms. The device can include more than one substrate; columns can be disposed on different substrates to facilitate control of temperature of the columns, for example, to restrict flow of heat from one column to the other, and/or, similarly, to provide different columns with different temperatures. A single substrate optionally includes features, such as thermal breaks, to restrict heat flow. For example, a substrate optionally includes a cut-out portion and/or portions of a relatively low thermal-conductivity material.

As noted above, some preferred embodiments of the invention entail High-Performance Liquid Chromatography (HPLC) instruments. As described next, some of these embodiments utilize microfluidic component(s). The herein described examples of HPLC embodiments having microfluidic features have an installation chamber for receiving a microfluidic cartridge having an electrospray emitter, and for bringing the tip of the emitter into operable communication with mass spectroscopy components of the HPLC instrument. The microfluidic cartridge houses one or more microfluidic substrates; a substrate is preferably a substantially rigid, ceramic-based, multilayer microfluidic substrate (also referred to herein as a ceramic tile), for example, as described in US Patent Publication No. 2009/032135, Gerhardt et al., which is incorporated herein by reference. For protein samples, the ceramic is preferably a High-Temperature Co-fired Ceramic (HTCC), which provides suitably low levels of loss of sample due to attachment of sample to walls of conduits in the substrate. Some embodiments dispose separation columns(s) and trap column(s) in different substrates.

A channel, formed in the layers of the substrate, operates, for example, as a separation column. Apertures in the side of the substrate—formed, for example, via laser etching—provide openings into the channel through which fluid may be introduced into the column. Fluid passes through the apertures under high pressure and flows toward the electrospray emitter coupled at the egress end of the channel. Holes in the side of the microfluidic cartridge provide fluidic inlet ports for delivering fluid to the substrate. Each of one or more fluidic inlet ports align with and encircle one of the fluidic apertures.

A clamping mechanism applies a mechanical force to one side of the microfluidic cartridge, urging the substrate against fluidic nozzles coupled to the installation chamber. The nozzles deliver fluid to the substrate through the fluidic inlet ports of the cartridge.

In addition to the substrate, embodiments of microfluidic cartridges preferably house internal circuitry and one or more temperature control units for heating and/or cooling the substrate(s), such as one or more portions of the substrate(s). In some embodiments, an aperture in the microfluidic cartridge housing provides a window through which pogo pins supply low voltage and other electrical signals to internal circuitry. Another aperture in the microfluidic cartridge housing optionally provides access for a cooling gas, directed at one or more substrates.

Other embodiments of the invention entail apparatus and methods that manipulate the temperature of tube-based columns, for example conventional separation columns and/or trap columns for improved separations. Preferred embodiments are applied to complex protein samples or small-molecule samples, in particular, small volume samples. The following described examples will be understood by one of skill to merely illustrate some optional implementations of the invention, while not limiting all implementations to any particular collection of features.

FIG. 2 shows a front view of an embodiment of a liquid chromatography module 12 having a housing 20. The housing 20 has an upper section 22 with an on-off switch 23 and with status and pressure indicators 24, a middle section 26 having a slot 14 for receiving the microfluidic cartridge and an arm portion 28, and a lower section 30 having an adjustment knob 32 extending from an opening in the housing. The adjustment knob 32 is coupled to an interior x-translation stage (partly visible) for moving a microfluidic cartridge along the x-axis. Adjustment knobs for y-translation and z-translation stages (not shown) also enable y-axis and z-axis adjustments of the position of the microfluidic cartridge relative to an mass spectroscopy (MS) (not shown.) Such adjustments provide, for example, positioning of an emitter tip outlet in relation to an MS inlet orifice.

Coupled to the arm portion 28 is a lever 34 that is rotatable about a pivot point 36 between a clamped position and an unclamped position. In FIG. 2, the lever is in the unclamped position. Counterclockwise rotation of the lever about the pivot point, approximately 180 degrees, moves the lever into the clamped position. At one end of the housing, an electrical cable 38 and an electrical signal conductor 40 enter the housing through an opening 42 in the front of the housing. The electrical cable 38 supplies a high voltage, and the electrical signal conductor 40 supplies a low voltage, to the microfluidic cartridge, as described herein. Not shown are the microfluidic tubing and one or more gas lines, which also enter the housing through the opening 42, for bringing fluid and gas, respectively, to the microfluidic cartridge.

FIG. 3 shows the housing 20 with its front cover 50 opened to expose a clamping assembly 60 within. A hinge along a base edge of the housing attaches the front cover 50 to a translation stage support 52. The translation stages and adjustment knobs are absent from the drawing to simplify the illustration. Other components residing within the housing include a circuit board 62. The translation stage support 52, clamping assembly 60, and circuit board 62 are coupled to a rear panel 64 of the housing.

The electrical cable 38 and an electrical conduit 66 couple to one side of the clamping assembly 60. The electrical cable carries a high voltage (e.g., 3000 volts), and the electrical conduit 66 bundles a plurality of low-voltage electrical conductors. Not shown are the microfluidic tubing and gas line that are also coupled to the same side of the clamping assembly 60 as the electrical cable 38 and electrical conduit 66.

The clamping assembly 60 has a slot 68 for receiving a microfluidic cartridge and a post 70 to which the lever 34 (FIG. 2) is attached. When the front cover 50 is closed, the slot 14 in the front cover 50 aligns with the slot 68 of the clamping assembly 60, the adjustment knob 32 of FIG. 2 (not shown in FIG. 3) projects through the opening 72 in the front cover 50, and the post 70 projects through another opening, which is obscured by the sidewall of the arm portion 28.

FIG. 4 shows a side view of the clamping assembly 60, with a microfluidic cartridge 16 passing through both slots in sidewalls 84 of the clamping assembly 60. The clamping assembly 60 has a body 80 coupled to an end housing 82. The arm 110 catches a nook in an upper edge of the microfluidic cartridge 16, preventing the microfluidic cartridge from sliding further through the slots 68. A high-voltage electrical cable 38 couples to the opening 106 and an electrical conduit 66 couples to a two-piece bracket 92 of a pogo-pin block. In addition, a coupler 112 connects to the opening 104 in the L-shaped retainer 100.

FIG. 5 shows an interior view of one embodiment of the end housing 82, including opposing sidewalls 84-1, 84-2, separated by back wall 86. The sidewall 84-1 has the slot 68-1, and the sidewall 84-2 has the slot 68-2. Installed in the back wall 86 are pogo pin block 88 and a fluidic block 90.

The interior side of the pogo pin block 88 has a recessed region 140 with a pogo pin electrical connector 142 projecting inwardly from a surface thereof. In this example, the electrical connector 142 has ten electrically conductive pogo pins 144 for conducting electrical signals. Each pogo pin 144 is an individual cylindrical, spring-loaded electrical conductor for transmitting electrical signals.

The interior side of the fluidic block 90 has a plurality of microfluidic nozzles 130-1, 130-2, 130-3 (generally, 130) projecting therefrom. In this embodiment, the nozzles 130 are three in number and arranged in a triangular pattern. The locations of these nozzles 130 are fixed with respect to each other. Fluid delivered by microfluidic tubes attached to the exterior side of the fluidic block 90 exits through one or more of these nozzles 130. Situated below the triangular pattern of nozzles 130, aligned with the nozzle at the apex of the triangle, is a guide pin 128 for guiding a cartridge.

The fluidic block 90 also has a coolant nozzle 131, for delivery of a coolant. For example, a N₂ delivery line is connected to the nozzle 131, or integral to the nozzle 131. To implement the above-described methods, a flow of N₂ is optionally directed at a substrate, via the nozzle 131, to cool the substrate, as desired. A temperature of the coolant is optionally controlled, as is, optionally, the flow rate and/or velocity, to achieve a desired cooling effect of the substrate, as determined, for example, theoretically and/or empirically.

Referring next to FIGS. 6-8, the microfluidic cartridge 16 works in conjunction with the fluid- and electrical-related components described above with regard to FIG. 4 and FIG. 5. As described below, in more detail, the microfluidic cartridge 16 houses an emitter, a microfluidic substrate, a heater, and circuitry, and operates as an electromechanical interface for the delivery of voltages, electrical signals, and fluids (gas and liquid) to the various components housed within the microfluidic cartridge 16.

This non-limiting example implementation of a microfluidic cartridge 16 includes a housing made by joining two casing sections 200-1, 200-2, for example, by snapping the halves together, or using glue or mechanical fasteners, or any combination thereof. The two casing sections are also referred to herein as the left and right sides of the microfluidic cartridge 16, with the terms left and right being determined by the orientation of the microfluidic cartridge 16 when it is inserted into the clamping assembly 60. It is to be understood that such terms as left, right, top, bottom, front, and rear are for purposes of simplifying the description of the microfluidic cartridge 16, and not to impose any limitation on the structure of the microfluidic cartridge 16 itself.

The right casing section 200-1 has a grip end 202 and an emitter end 204. A curved region 206 within the grip end 202 provides a finger hold by which a user can grasp the microfluidic cartridge 16 when inserting and removing it from the liquid chromatography module 12.

The right casing section 200-1 has a rectangular-shaped window 208, within which resides a push block 210. The surface of the push block 210 lies flush with the surface of the right casing section 200-1. As described further below, the push block 210 is not rigidly affixed to the right casing section 200-1, and can move slightly in, out, up, down, left, or right; that is, the push block 210 floats within the window 208. In one embodiment, the push block 210 is made of metal.

Disposed below the push block 210 is an opening 212, which extends completely through both casing sections 200-1, 200-2. Hereafter, the opening 212 is referred to as a through-hole 212. At the emitter end 204 is a nook 214 in the top edge of the microfluidic cartridge 16. Within the nook 214, a movable fin 216 projects through the top edge between the casing sections 200-1, 200-2.

FIG. 6 shows the left casing section 200-2 of the microfluidic cartridge 16. Like the right casing section 200-1, the left casing section 200-2 has a grip end 202 with a curved region 206 and an emitter end 204. Approximately central in the length of the left casing section 200-2 are three nozzle openings 220 in a triangular pattern that matches the triangular arrangement of the nozzles 130 of the fluidic block 90 (FIG. 5.) The triangular pattern is desirable, to define a circular load pattern. The center of the circle is preferably aligned to the axis of motion of a plunger (not shown) of the clamping assembly 60 (FIG. 6) to provide a load balance on the metal-plate bosses 260 of the push block 210; when the clamping assembly 60 is closed, the plunger presses against the push block 210, which, in turn, presses against the substrate, which, in turn, presses against the nozzles 130 of the fluidic block 90. A third fluidic port is used, for example, for a calibration port; an operator can run a calibration fluid. The other two ports provide access to, for example, the analytical column and the trap column.

Concentrically located behind each nozzle opening 220 is a microscopic fluidic aperture in the side of a microfluidic substrate housed within the microfluidic cartridge. The fluidic conduits of the microfluidic nozzles 130 of the fluidic block 90 have much larger inner diameters than the size of the microscopic apertures in the substrate, which facilitates alignment therebetween. In one embodiment, each microscopic fluidic aperture has a 0.003″ square cross section, and each microfluidic nozzle 130 has a 0.013″ orifice (lumen with a circular cross section) that aligns with and circumscribes the microscopic fluidic aperture on the substrate, such as a 0.003″ via (aperture with a square cross section.)

The microfluidic nozzles 130 utilize a polymer-to-ceramic interface, relying only on the compressive stress provided by the clamping assembly 60 (FIG. 6) to provide a fluidic seal; that is, the clamping assembly 60 provides a greater pressure at the polymer-to-ceramic interface than the operating fluidic pressure. For example, for an operating fluidic pressure of 5,000 psi—or alternative pressures, such as 15,000 psi—are implemented with a clamping load of 130 pounds across the total surface area of the nozzle-to-substrate interface, producing an effective fluidic seal for the selected operating pressure.

The left casing section 200-2 also has a coolant-nozzle opening 221, central to the three nozzle openings 220, and disposed to receive the coolant nozzle 131. In this example, when the cartridge 16 is clamped in an operating position, the coolant nozzle 131 is disposed with its outlet proximate to the surface of the substrate(s) in the cartridge 16.

A coolant is optionally used, for example, to enhance sample enrichment and/or sample focusing and/or decrease sample cycle time. For example, a nano-flow apparatus is optionally utilized with a solvent gradient that lasts for approximately 1.5 hours. The separation-column portion of a substrate is optionally maintained at a temperature of 45° C. during separation. Once a sample run is completed, a coolant gas is optionally directed at the substrate, as described above, to provider quicker cooling to support quicker sample turnaround. For example, a gas flow optionally sufficiently cools a substrate in approximately 2 minutes, which would often be satisfactory relative to a sample run time of 90 minutes.

Directly above the apex of the triangularly arranged nozzle openings 220 is a rectangular depression 222 within the left casing section 200-2. The depressed region 222 surrounds a rectangular-shaped window 224 through which an array of electrical contacts 226 is accessed. The electrical contacts 226 are electrically conductive pads for making electrical contact with the pogo pins 144 of the pogo pin block 88 (FIG. 5). The array of electrical contacts 226 is part of a flex circuit overlaid upon the microfluidic substrate, as described further below in connection with FIG. 13.

At the emitter end 204, the left casing section 200-2 has a gas inlet port 225 for receiving a gas nozzle and a high-voltage input port 228 for receiving the tip (pogo-pin) of the high-voltage electrical cable 38 (FIG. 4). A plurality of holes 234 hold alignment pins 236 that are used to align the casing sections 200-1, 200-2 when the halves are being joined.

The left casing section 200-2 further includes a rectangular-shaped groove 230 along its bottom edge. The groove 230 has an open end 232 at the emitter end 204, extends laterally therefrom, and terminates at the through-hole 212 situated below the nozzle openings 220. In addition, the groove 230 receives the guide pin 128 (FIG. 5) when the microfluidic cartridge 16 is inserted into the slot 68 (FIG. 4) of the clamping assembly 60. When the guide pin 128 reaches the through-hole 212, then the microfluidic cartridge 16 is fully installed in the chamber 120 and in position for clamping.

The substrate 250 is optionally formed in the following manner. Five green-sheet layers, for multiple substrates 250, are pressed together, after desired patterning. Vias for fluidic apertures are laser etched in one or both sides of the pressed sandwich. Edge portions are defined by laser etching. After firing, individual substrates 250 are snapped apart. Edges, or portions of edges, are optionally polished.

FIG. 7 shows a right side view of the microfluidic cartridge 16 with the right casing section 200-1 removed to reveal various components housed within and to show various features of the interior side of the left casing section 200-2. The various components include a microfluidic substrate 250 and a shutter 254, both of which are coupled to the left casing section 200-2 as shown.

A flex-circuit assembly 258 is folded over a top edge of the microfluidic substrate 250, and includes the array of electrical contacts 226. As described with respect to FIG. 6, these electrical contacts 226 are accessible through the window 224 in the left casing section 200-2.

The shutter 254 has a fin 216 (FIG. 8), and has an extension 217 that protects an emitter tube 264 by partially enveloping the tube 264, when the cartridge is not installed in the clamp assembly 60. The emitter tube 264 is part of an emitter assembly that is described, in more detail, below.

The flex-circuit assembly 258 includes a control circuitry portion 257 and a heater portion (hereafter, heater 270). As noted, the flex-circuit assembly 258 folds over a top edge of the microfluidic substrate 250 and covers a portion of the opposite side of the microfluidic substrate 250. An integrated circuit (IC) device 272 is mounted on the control circuitry portion of the flex-circuit assembly 258. In one embodiment, the IC device 272 is a memory device (e.g., EPROM) for storing program code and data. The heater 270 covers a separation column within the microfluidic substrate 250. Mounted to the heater 270 is a temperature sensor 274.

The flex-circuit assembly 258 is constructed of multiple stacked layers (e.g., three, four, or five). The polymer substrate of each layer holds different interconnectivity or circuitry. One of the layers contains resistive traces of the heater 270. Electrical contacts at the two ends of the resistive traces connect to two pads 259 on the control circuitry portion 257. Another layer of the flex-circuit assembly 258 has vias that electrically contact the ends of the resistive traces, another layer has contacts to connect electrically to electrical components 272, 274, and still another layer has pogo-pin contact pads 226 (FIG. 13). Through the flex-circuit assembly 258, each of the electrical components 272, 274 and resistive traces are electrically coupled to the contact pads 226. The gas inlet port 225 opens into a well 276 that channels injected gas into a gas tube that delivers the gas to the emitter end 204.

FIG. 8 shows a view of the microfluidic cartridge 16 from the left, with the left casing section 200-2 removed to reveal various components housed within and to show features of the interior side of the right casing section 200-1. The flex-circuit assembly 258, is wrapped, as noted, on this side of the microfluidic substrate 250, and includes the array of electrical contacts 226 illustrated in FIG. 6. In addition, the microfluidic substrate 250 has a high-voltage input port 290 for receiving the electrically conductive terminal of the high-voltage electrical cable 38 (FIG. 4). The high-voltage input port 290 is disposed near an egress end of a separation column within the microfluidic substrate 250, described below in connection with FIG. 9.

The microfluidic cartridge 16 includes a spray unit 340, which supports electrospray output of sample that has passed through the substrate 250. The spray unit 340 includes a retainer 341 attached to the substrate 250, the emitter tube 264, a spring (not shown) that urges the tube 264 against the substrate 250, and a retainer cap 342. The cap 342 is fixedly or removably attached, for example, via a snap or threads, to the retainer 341. The spring is disposed within the cap 342, when the spray unit 340 is assembled for operation.

The interior side of the right casing section 200-1 includes a ridge 292 of casing material that runs from the emitter end 204 and terminates at the through-hole 212. When the casing sections 200-1, 200-2 are joined, the ridge 292 runs directly behind the groove 230 (FIG. 12) on the exterior side of the left casing section 200-2. The ridge 292 provides structural support to the microfluidic cartridge 16. In addition, by being directly opposite the groove 230, the ridge 292 resists bending of the cartridge 16 by the guide pin 128 (FIG. 5) should a user prematurely attempt to clamp the microfluidic cartridge 16 before the microfluidic cartridge 16 has fully reached the proper position. In addition, no portion of the microfluidic substrate 250 lies directly behind the groove 230, as a precautionary measure to avoid having the guide pin 128 bend the microfluidic substrate 250 in the event of a premature clamping attempt.

The interior side of the right casing section 200-1 provides the other half of the gas well 276, the walls of which align with and abut those defining the well 276 on the left casing section 200-2. To enhance a tight seal that constrains gas to within the gas well 276, a fastener or pin 296 (FIG. 7) tightens the connection between the casing sections at the opening 298 adjacent the well 276.

FIG. 9 shows a left-side view of an embodiment of the microfluidic substrate 250. In brief overview, the microfluidic substrate 250 is generally rectangular, flat, thin (approx. 0.050″), and of multilayer construction. Formed within the layers of the microfluidic substrate 250 is a serpentine channel 300 for transporting liquid. The microfluidic substrate 250 includes a trap region 302 and a column region 304. In the embodiment shown, the microfluidic substrate 250 has three microscopic fluidic apertures 306-1, 306-2, 306-3 (generally 306). One of the fluidic apertures 306-1 intersects the channel 300 at one end of the trap region 302; another of the fluidic apertures 306-2 intersects the channel 300 at the other end of the trap region 302. In this embodiment, the third fluidic aperture 306-3 is unused. Alternatively, the third fluidic aperture 306-3 can be used, for example, as a calibration port; an operator can run a calibration fluid. The channel 300 terminates at an egress end of the microfluidic substrate 250. The fluidic aperture 306-2 at the “downstream” end of the trap region 302 is optionally used as a fluidic outlet aperture, for example, during loading of the trap region 302, and is optionally closed to fluid flow, for example, during injection of a loaded sample from the trap region 302 into the channel 300.

The microfluidic substrate 250 also has a high-voltage input port 290 (FIG. 8) and a pair of alignment openings 310-1, 310-2 (generally, 310) that each receives a peg that projects from an interior side of the left casing section 200-2. The alignment openings 310 help position the microfluidic substrate 250 within the microfluidic cartridge 16. The size of the alignment openings 310, with respect to the size of the pegs, allows the microfluidic substrate 250 some play within the microfluidic cartridge 16.

As described above, some embodiments of the invention including cooling features. In addition to, or alternative to, the above-described coolant-fluid-related features, other embodiments of the invention utilize alternative mechanisms to cool substrate(s) and/or portions of substrate(s). Any suitable cooling mechanisms, including known mechanisms, are optionally included. Some suitable mechanisms include Peltier devices, use two or more substrates, as described next, use heat sinks, and/or use heat pipes, such as a closed system that volatilizes a liquid disposed over the heat source and condenses the volatilized liquid in a cooled area, provide heat transport potentially at the speed of sound. Moreover, a substrate itself is optionally configured with channels to carry a coolant. Related to the multiple-substrate option, some substrates include a thermal barrier or gap, disposed in, or defined by, the substrate, to help thermally isolate trap and enrichment columns from one another.

Some embodiments include features to permit cooling and/or heating of a trap column and/or a separation column, for example, to implement some of the specific temperature ranges and temperatures of above-described examples.

Some embodiments include multiple microfluidic substrates. For example, rather than a single microfluidic substrate 250, the microfluidic cartridge 16 can house a plurality of interconnected microfluidic substrates. Some example embodiments, having multiple substrates, are described next, with reference to FIGS. 10A and 10B.

FIG. 10A shows a portion of a device, according to one embodiment of the invention, which has two (or more) fluidically connected microfluidic substrates 250-1, 250-2. The substrates 250-1, 250-2 include a separations substrate (also referred to herein as a column tile or substrate) 250-2 and a trap tile (or load substrate) 250-1. The microfluidic substrates 250-1, 250-2 are coupled to each other via couplers 322-1, 322-2, which are optionally aligned to ports in the substrates 250-1, 250-2 via fittings 325-1, 325-2, 326-1, 326-2 (generally, fittings 325, 326.) The trap tile 250-1 has a fluid-conducting channel 300-1 and the column tile 250-2 has a fluid conducting channel 300-2, which are connected via a coupler 322-2. The locations of the channels 300-1, 300-2 are illustrated, although one of skill will understand that the channels 300-1, 300-2 are internal to the substrates 250-1, 250-2.

This example of a trap tile 250-1 has two or three fluidic apertures. Coupled about each fluidic aperture is a fitting 320-1, 320-2, 320-3 (generally 320). The fitting 320-3 is optionally located at a dummy aperture; the dummy location may merely be used, for example, application of a clamping site. The fittings 320 serve to self-align the tips of the nozzles (e.g., nozzles 130 of FIG. 5) on the fluidics block 90 when the microfluidic cartridge 16 is installed in the chamber, as described in more detail below. These fittings 310 can be made of metal, plastic, ceramic, or any combination of these materials or other suitable materials. To couple the fittings 320 to the substrate 250-1, they can be glued, fastened, fused, brazed, or a combination thereof, for example.

The tile 250-1 has a notch, or open spot 318, which, for example, provides access for a nozzle to directly contact an aperture of the separations substrate 250-2. The aperture can be a dummy aperture, for example, for substrates that do not require use of a fourth nozzle contacting the substrate at the site of the dummy aperture. The notch 318 optionally assists alignment, orientation and/or substrate identifications, for example.

FIG. 10B shows a cross-section of the device of FIG. 10A, sliced through the trap tile 250-1 and column tile 250-2, two of the fittings 320-1 and 320-2 and two couplers 322-1, 322-2 respectively associated with the fittings 320-1, 320-2. The channel 300-1 of the trap tile 250-1 extends from the fitting 320-1 to the fitting 320-2. The couplers 322-1, 322-2 (generally, 322) connect the trap tile 250-1 to the column tile 250-2. Each coupler 322 has an associated a pair of alignment fittings 325, 326: one fitting 325-1, 325-2 on the underside of the trap tile 250-1, and the other fitting 326-1, 326-2 on the top side of the column tile 250-2. The coupler 322-2 (as noted above) provides a channel (or lumen) 324 for fluid passing through the channel 300-1 of the trap tile 250-1 to reach the channel 300-2 of the column tile 250-2. The couplers 322 can be made of metal, plastic, ceramic, or any combination of these materials and can be glued, fastened, fused, and brazed, or any combination thereof, to the tiles 250-1, 250-2.

Preferably, however, in some embodiments, the couplers 322 are formed of a deformable matter, and need not be permanently attached to either substrate 250-1, 250-2; in some embodiments that have a swappable trap tile 250-1, the couplers 326-1, 326-2 are fixedly attached to the column tile 250-2, to facilitate swapping of the trap tile 250-1. For example, a device optionally includes a cartridge having a housing with a slot, a door, or other means to permit access to the cartridge for removable and/or insertion of substrate(s).

A deformable material is optionally any suitable material or materials, for example, a material similar to or the same as the material of the nozzles 130. Mechanical pressure alone is optionally used to provide a fluid-tight seal between the trap tile 250-1, the couplers 322 and the column tile 250-2, using, for example, a clamping device, such as the clamping device described below. Alignment-assisting features, such as the fittings 320-1 and 320-2, are optionally included in the embodiment illustrated in FIG. 9 and in other embodiments, to align nozzles or fluidic connectors.

In a multi-substrate device, according to some embodiments of the invention, one or more of the substrates, such as the substrates 250-2, 450-2 remain in a cartridge housing, while secondary substrates such as the tiles 250-1, 450-1 are swapped for analyses of different samples. For example, samples may be loaded on several tiles, which are then swapped in a cartridge for sequential analysis.

Multi-substrate devices have additional advantages. For example, the packing of a column in a column tile and a column in a trap tile may progress more easily if the columns reside in different substrates rather than the same substrate.

Multi-substrate devices have other uses, in addition to those mentioned above. For example, different substrates are optionally maintained at different temperatures. For example, a temperature differential between a trap column and a separation column may be more readily controlled if the columns reside in different substrates. In particular, this may be the case where the substrates are formed of ceramic materials having a relatively high thermal conductivity. In some embodiments, active temperature control is applied to one or more of the substrates. For example, one or more of the substrates can have heating and/or cooling features, as described above.

Some preferred embodiments of the invention entail apparatus of reduced cost and size relative to existing apparatus, such as existing analytical equipment based on LC-MS. Miniaturization provides many potential benefits in addition to size reduction, for example: improving reliability; reducing the quantity and cost of reagents, and the cost of used-reagent disposal; and improve performance reducing dispersion in LC-related components. While preferred embodiments, described herein, relate to liquid chromatography, one of skill will recognize that the invention may be applied to other separation techniques.

While the invention has been shown and described with reference to specific preferred embodiments, it should be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the following claims. For example, multiple substrates are optionally attached to one another in a more or less permanent manner, for example, using glue or some other bonding mechanism.

Claims

1. A method of analyzing proteins, comprising:

providing a complex protein-related sample comprising a plurality of components;

loading a sufficient quantity of the sample onto a trap column to overload the trap column, wherein an outlet of the trap column is in fluid communication with an inlet of an analytical column;

heating the analytical column;

heating the trap column to a greater temperature than the analytical column, thereby providing a temperature differential between the trap and analytical columns; and

pumping a solvent, to the trap column, having a solvent composition profile that, in cooperation with the temperature differential, causes at least some of the components to elute sequentially from the trap column to the analytical column and focus on the analytical column prior to eluting from the analytical column.

2. The method of claim 1, wherein the trap column is packed with particles having a diameter of greater than about 2 μm, and the analytical column is packed with particles having a diameter of less than about 2 μm.

3. The method of claim 1, wherein the particles of the analytical column have a greater hydrophobicity than the particles of the trap column.

4. The method of claim 1, wherein the trap column is maintained at a temperature of greater than about 45° C. and the analytical column is maintained at a temperature of less than about 45° C.

5. The method of claim 1, wherein loading comprises maintaining the trap column at a temperature of about 25° C. or less.

6. The method of claim 1, wherein the analytical column has an inner cross-sectional dimension in a range of about 50 μm to about 150 μm.

7. An apparatus for chromatographic separation of a sample, comprising:

a trap column;

a separation column in fluidic communication with the trap column;

a trap-column heater;

a separation-column heater;

a solvent pump unit; and

a control unit, including instructions, which, when implemented, causes the apparatus to perform the steps of, loading a sufficient quantity of a complex protein-related sample onto the trap column to overload the trap column; heating the analytical column; heating the trap column, wherein the heated trap column has a greater temperature than the heated analytical column, thereby providing a temperature differential between the trap and analytical columns; and pumping a solvent, to the heated trap column, having a solvent composition profile that, in cooperation with the temperature differential, causes at least some of the components to elute sequentially from the heated trap column to the heated analytical column and focus on the heated analytical column prior to eluting from the heated analytical column.

8. The apparatus of claim 7, wherein the trap column is packed with particles having a diameter of greater than about 2 μm, and the analytical column is packed with particles having a diameter of less than about 2 μm.

9. The apparatus of claim 8, wherein the particles of the analytical column have a greater hydrophobicity than the particles of the trap column.

10. The apparatus of claim 7, wherein the temperature of the heated trap column is greater than about 45° C. and the temperature of the heated analytical column less than about 45° C.

11. The apparatus of claim 7, wherein the analytical column has an inner cross-sectional dimension in a range of about 50 μm to about 150 μm

12. A method of chemical analysis, comprising:

(a) providing a ceramic-particle-based and/or metal-based microfluidic substrate defining a trap column and an analytical column in fluidic communication with the trap column;

(b) loading a sample on the trap column while the trap column is at a temperature in a load range;

(c) heating at least a portion of the substrate containing the analytical column to provide a temperature above ambient during elution of the sample through the analytical column, wherein the trap column is incidentally heated;

(d) pumping a solvent to the trap column to elute the sample components from the trap column to the analytical column at the temperature above ambient, causing the components to elute from the analytical column;

(e) cooling at least a portion of the substrate containing the trap column, after elution of the components from the analytical column, to return the trap column to a temperature in the load range; and

(f) repeating (b) through (e) for each of one or more subsequent samples.

13. The method of claim 12, wherein the analytical column has particles having a greater hydrophobicity than particles of the trap column.

14. The method of claim 12, wherein the temperature above ambient of the heated analytical column about 35° C. or greater.

15. The method of claim 12, wherein the analytical column has an inner cross-sectional dimension in a range of about 50 μm to about 150 μm.

16. An apparatus for chromatographic separation of a sample, comprising:

a ceramic-particle-based and/or metal-based microfluidic substrate defining a trap column and an analytical column in fluidic communication with the trap column;

a fluidic conduit having an outlet disposed to direct a fluid towards a location of the trap-column to cool at least a portion of the microfluidic substrate;

a separation-column heating unit disposed to heat at least a separation column portion of the microfluidic substrate during separation of a sample; and

a solvent pump unit for pumping a solvent composition to an inlet of the trap column.

17. The apparatus of claim 16, wherein an outlet of the trap column and an inlet of the analytical column are substantially co-located, in the substrate.

18. The apparatus of claim 17, wherein a packing material of the trap column and a packing material of the analytical column are contiguous.

19. The apparatus of claim 16, wherein the analytical column has an inner cross-sectional dimension in a range of about 50 μm to about 150 μm.

20. A method of analyzing small molecules, comprising:

cooling at least a portion of an analytical column proximate to an inlet of the analytical column;

loading, onto the cooled portion of the analytical column, a nano-scale sample comprising a plurality of different small-molecule components;

heating the analytical column to promote elution of the loaded components; and

pumping a solvent, to the heated analytical column, to elute the components from the analytical column.

21. The method of claim 20, wherein the analytical column has an inner cross-sectional dimension in a range of about 150 μm to about 500 μm.

22. The method of claim 21, wherein the analytical column is disposed in a microfluidic substrate.