VOLATILE ELUENT PREPARATION

Abstract

There is provided a system for performing a chromatographic separation of an analyte, methods of using the system to separate at least one component of an analyte and an eluent generator of use in the system. An exemplary system comprises: (a) an eluent generator comprising: (i) a housing configured to be pressurizable by gas, comprising an annular void defined by the housing, and a gas inlet for the gas and a gas outlet for the gas in fluid communication with the annular void; (ii) a membrane permeable to the gas defining an eluent flow channel disposed within the annular void, the eluent flow channel having an eluent precursor fluid inlet and an eluent outlet; (iii) a source of gas in fluidic communication with the gas inlet; (iv) a source of the eluent precursor fluid; and (b) a chromatography column disposed downstream of and in fluidic communication with the eluent outlet.

Claims

1. A system for performing a chromatographic separation of an analyte, said system comprising: (a) an eluent generator comprising: (i) a housing configured to be pressurizable by gas, comprising an annular void defined by said housing, and a gas inlet for said gas and a gas outlet for said gas in fluid communication with said annular void; (ii) a membrane permeable to said gas defining an eluent flow channel disposed within said annular void, said eluent flow channel having an eluent precursor fluid inlet and an eluent outlet; (iii) a source of gas in fluidic communication with said gas inlet; (iv) a source of said eluent precursor fluid; and (b) a chromatography column disposed downstream of and in fluidic communication with said eluent outlet.

2. The system according to claim 1 further comprising a member selected from a gas input solenoid in fluidic communication with said source of gas and said gas inlet and interposed therebetween, a gas exhaust solenoid in fluidic communication with said gas outlet, and a combination thereof.

3. The system according to claim 2, wherein said member selected from said gas input solenoid, said gas exhaust solenoid and a combination thereof is controlled by a controller such that pressure of said gas within said eluent generator is controlled to a preselected pressure by said controller.

4. The system according to claim 1, further comprising a pump in fluidic communication with said eluent flow channel inlet and configured to supply said eluent precursor fluid thereto.

5. The system according to claim 1, wherein said membrane is configured to permit passage of said gas from said annular void into said eluent flow channel and prevent or retard passage of said eluent from said eluent flow channel into said annular void.

6. The system according to claim 1, wherein said gas permeable membrane is an amorphous fluoropolymer.

7. The system according to claim 1, wherein said source of gas provides a gas selected from CO.sub.2, a gas derived from a volatile acid, a gas derived from a volatile base, and ammonia.

8. The system according to claim 1, wherein said eluent is an aqueous eluent and said eluent precursor fluid is an aqueous eluent precursor fluid.

9. The system according to claim 1, further comprising a delay device in fluidic communication with said eluent outlet and said chromatography column and interposed therebetween, said delay device configured to retard flow of said eluent to said chromatography column and promote dissolution of said gas into said eluent prior to said eluent flowing to said chromatography column.

10. The system according to claim 1, further comprising a heating device configured to heat at least a portion of said eluent generator, thereby maintaining said gas in a gaseous state.

11. The system according to claim 10, wherein said heater heats at least said gas input or a component thereof.

12. The system according to claim 1, further comprising a suppressor configured to suppress ions of said eluent, said suppressor disposed downstream of said chromatography column and in fluidic communication therewith.

13. The system according to claim 1, further comprising a detector configured to detect at least one component of said analyte, said detector disposed downstream of said chromatography column and in fluidic communication therewith.

14. The system according to claim 1, further comprising a vacuum device in fluidic communication with said gas outlet.

15. The system according to claim 13, in which said detector comprises an evaporative type of detector selected from a corona charged aerosol detector, an evaborative light scattering detector, an electrospray ionization mass spectrometer, and a combination thereof.

16. The system according to claim 13 further comprising a gas removal device disposed in between said chromatography column and said detector.

17. A method of performing a chromatographic separation using a chromatographic system according comprising: (a) an eluent generator, comprising: (i) a housing configured to be pressurizable by gas, comprising an annular void defined by said housing, and a gas inlet for said gas and a gas outlet for said gas in fluid communication with said annular void; (ii) a membrane permeable to said gas defining an eluent flow channel disposed within said annular void, said eluent flow channel having an eluent precursor fluid inlet and an eluent outlet; (ii) a source of gas in fluidic communication with said gas inlet; (iii) a source of said eluent precursor fluid; and (b) a chromatography column disposed downstream of and in fluidic communication with said eluent outlet, said method comprising: (a1) flowing said eluent precursor fluid through said eluent flow channel while said annular void is maintained under positive pressure of said gas, said positive pressure selected to be of a magnitude sufficient to cause said gas to cross said gas permeable membrane, dissolving in said eluent precursor fluid, thereby forming said eluent; (b1) passing said eluent from said eluent outlet into said chromatography column; (c1) contacting said chromatography column with said analyte, thereby performing said chromatographic separation of said analyte.

18. The method according to claim 17, wherein said eluent precursor fluid is deionized water.

19. The method according to claim 17, wherein said eluent precursor fluid is aqueous NaOH or KOH.

20. The method according to claim 18 or 19, wherein said gas is CO.sub.2.

21. The method according to claim 17, wherein said gas is a member selected from ammonia, a gas derived from a volatile amine and a gas derived from a volatile acid.

22. The method according to claim 17, wherein said eluent format is an isocratic format or a gradient format.

23. The method according to claim 17, wherein said chromatographic separation is a modality which is a member selected from ion exclusion, ion exchange, chiral amine separation, amino acid separation, and protein separation, including monoclonal antibody separation.

24. The method according to claim 17, further comprising, prior to performing said separation, controlling said device to produce said eluent, wherein said eluent has a predetermined composition, said controlling comprising: (a) selecting said gas, and pre-determining; (i) pressure of said gas in said annular void; (ii) composition of said eluent precursor solution in said eluent flow channel; and (iii) flow rate of said eluent precursor solution through said eluent flow channel; and (b) controlling said eluent generator such that; (i) said pressure of said gas in said annular void is maintained at the predetermined pressure; (ii) said composition of said eluent precursor fluid is maintained at the predetermined composition; and (iii) said flow rate of said eluent precursor fluid in said eluent flow channel is maintained at the predetermined flow rate, thereby producing said eluent having said pre-determined composition.

25. The method of claim 24, further comprising pre-determining a rate of reaction of said gas with said eluent precursor, thereby determining concentration of a reaction product resulting therefrom in said eluent.

26. The method of claim 24, further comprising pre-determining a rate of dissolution of said gas in said eluent precursor, thereby determining concentration of said gas in said eluent.

27. The method of claim 24, wherein a parameter selected from: (i) said pressure of said gas in said annular void; (ii) said composition of said eluent precursor fluid; (iii) said flow rate of said eluent precursor fluid in said eluent flow channel; and (iv) a combination thereof is maintained for a pre-determined time after which it is varied, thereby producing said eluent in a gradient format.

28. The method according to claim 17, further comprising: outputting said eluent and said analyte from said chromatography column; evaporating said eluent; detecting said analyte with a detector, in which said detector comprises an evaporative type of detector selected from a corona aerosol detector, an evaporated light scattering detector, an electrospray ionization mass spectrometer, and a combination thereof.

29. The method according to claim 22, wherein said pressure of said gas is maintained constant in said isocratic format.

30. The method according to claim 22, wherein said pressure of said gas is varied in said gradient format.

31. The method according to claim 17, further comprising: outputting said eluent and said analyte from said chromatography column to a gas removal device; removing said gas with the gas removal device; detecting said analyte with a detector.

32. An eluent generator configured for generating an eluent of a pre-determined composition and for incorporation into a system for chromatographic separation of an analyte, said eluent generator comprising: (a) a housing configured to be pressurizable by gas, comprising an annular void defined by said housing, and a gas inlet for said gas and a gas outlet for said gas in fluid communication with said annular void; (b) a membrane permeable to said gas defining an eluent flow channel disposed within said annular void, said eluent flow channel having an eluent precursor fluid inlet and an eluent outlet, said outlet configured for coupling to a chromatography column and fluidically communicating therewith; (c) a first solenoid communicating fluidically with said gas inlet and controlled by a first controller; and (d) a second solenoid communicating fluidically with said gas outlet and controlled by a second controller, wherein said first controller and said second controller are programmed to operate cooperatively to control pressure of said gas in said annular void at a predetermined value, thereby generating said eluent of said pre-determined composition.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIG. 1 is the general eluent generator (“Engasser”) design for CO.sub.2 introduction into an eluent precursor.

[0016] FIG. 2 is an exemplary eluent generator of the invention.

[0017] FIG. 3 is a graph showing the pressure and resultant solution conductivity over time for CO.sub.2 permeating through a 160 cm long membrane.

[0018] FIG. 4 is a graph showing solution conductivity, pH, and CO.sub.2 permeation rate as a function of pressure through 2 different membrane lengths.

[0019] FIG. 5 is a graph showing solution conductivity vs. pressure of CO.sub.2 introduced into a solution containing NaOH through a 160 cm long Engasser. Conductivity was normalized to a solution of only NaOH.

[0020] FIG. 6 shows the experimental setup for dynamic titration of CO.sub.2 using NH.sub.3 and for generating NH.sub.4.sup.+/HCO.sub.3.sup.−/CO.sub.3.sup.2− buffers.

[0021] FIG. 7 shows titration of fixed NH.sub.3 with CO.sub.2. Note virtually identical CO.sub.2 pressures at the end point from ascending and descending pCO.sub.2 titration protocols. The minor difference arises due to hysteresis.

[0022] FIG. 8 shows isocratic elution of various organic acids on a Thermo Scientific™ Dionex™ IonPac™ ICE-AS6 column. The eluent contained up to 250 mM CO.sub.2 0.5 mL/min; Thermo Scientific™ Dionex™ IonPac™ ICE-AS6 column (9×250 mm); 160 cm Engasser; 16-140 psi (˜25 mM-250 mM CO.sub.2); 20 μL injection volume.

[0023] FIG. 9 is a graph showing peak retention time relationship with Engasser CO.sub.2 pressure for the ion exclusion separations shown in FIG. 8.

[0024] FIG. 10 is a graph showing gradient separation program and resultant background conductivity signal. The dip associated with the gradient can be seen nearly 40 minutes after being initiated.

[0025] FIG. 11 shows ion exclusion chromatograms of 12 organic acids with a CO.sub.2 eluent. The CO.sub.2 gradient chromatogram in the middle may be compared with isocratic separations using a relatively high CO.sub.2 pressure (140 psia, top) or relatively low CO.sub.2 pressure (16 psia, bottom) Thermo Scientific™ Dionex™ IonPac™ ICE-AS6 column (9×250 mm); 0.5 mL/min; 160 cm Engasser; 16-140 psi (˜25 mM-250 mM CO.sub.2); 20 μL injection.

[0026] FIG. 12 is a graph showing peak resolutions for the first 6 eluting ions using isocratic CO.sub.2 elution at 5 different CO.sub.2 pressures ranging from 16 to 140 psia (solid symbols) compared to a gradient in the CO.sub.2 pressure (the ordinate value corresponding to the dashed lines indicate the indicated peak resolution observed in the gradient chromatogram of FIG. 11).

[0027] FIG. 13A shows conductivity chromatograms for a standard mixture of 13 organic acids, using a Dionex™ IonPac™ ICE-AS6 column and a flow rate of 1.0 ml/min. The inset shows the current practice, a 0.4 mM heptafluorobutyric acideluent is converted to tetrabutylammonium heptafluorobutyrate by the Themo Scientific™ Dionex™ AMMS-ICE suppressor with a detector background of ˜24 μS/cm. An injection volume of 50 μL is used. The main figure shows a chromatogram according to the present invention. CO.sub.2 is introduced into water with a 160 cm engasser using a pressure of 20 psi to function as an eluent. A CRD is placed between the column and the conductivity detector and is used to remove the CO.sub.2 after the separation, resulting in a detector background of 1.25 μS/cm. A 20 μL injection volume is used. Compare the total time for the separation and the response (noting that in the present method less than half the sample is injected).

[0028] FIG. 13B depicts the same separation as FIG. 13A with the CO.sub.2 eluent under identical conditions except the detector used is optical absorbance at 195 nm.

[0029] FIG. 14A compares conductivity detection to absorbance detection. CO.sub.2 is removed after the separation column by a carbonate removal device (CRD). Dionex™ IonPac™ ICE-AS6 column (9×250 mm); 1.0 mL/min; CO.sub.2 pressure=5 psia; 160 cm Engasser; 20 μL sample loop; 40° C.

[0030] FIG. 14B is a chromatogram showing the separation without carbon dioxide removal (without the CRD). Many of the ions are not identifiable with either absorbance or conductivity without the CRD, showing the merits of removing the eluent background. Dionex™ IonPac™ ICE-AS6 column (9×250 mm); 1.0 mL/min; CO.sub.2 pressure=5 psia; 160 cm Engasser; 20 μL injection volume; 40° C.

[0031] FIG. 15 is a graph illustrating a gradient delay time in ion exclusion chromatography. Because CO.sub.2 fully probes the occluded volume of the resin, it takes an extended time (˜32 min) for the gradient to reach the end of the column. Dionex™ IonPac™ ICE-AS6 column (9×250 mm), 0.5 mL/min, CO.sub.2 pressure actuated from 14.7-140 psia, 160 cm Engasser.

[0032] FIG. 16 is a chromatogram showing the measurement of low concentrations of organic acids in a high conductivity well water sample. The primary graph is the full scale separation while the inset contains a magnified view of just the organic acids of interest and shows how sensitive the method is using the engasser despite the overwhelming concentration of interfering strong acids. It was determined that formate and acetate were present due to contamination by the resin used to treat the sample. 1.0 mL of Sample treated with 0.5 mL of strong cation exchange resin; 100 μL Injection volume. Flow rate 0.5 mL/min; 160 cm long Engasser, 60 psi CO.sub.2; (1) Lactate, (2) Formate, (3) Acetate, (4) Propionate, (5) Isobutyrate, (6) Butyrate.

[0033] FIG. 17 is a plurality of chromatograms showing the separation of ions on a Dionex™ IonPac™ 4 mM i.d. AS-9HC column using electrodialytic KOH generation and CO.sub.2 Engasser. CO.sub.2 pressure was held constant at 145 psi (˜15.4 mM) CO.sub.2 and the KOH concentration were varied from 20 mM to 40 mM. The rectangular inset shows a region where the elution behavior is strongly dependent upon the eluent composition. Flow Rate 0.5 mL/min; Engasser length 5.5 cm; 20 μL injection volume.

[0034] FIG. 18 reproduces the top and bottom traces from FIG. 17. The middle trace utilizes a bicarbonate-carbonate gradient and results in a more attractive separation (i.e., faster analysis time) than either the top or the bottom chromatogram.

[0035] FIG. 19A shows the distribution of species (KHCO.sub.3, K.sub.2CO.sub.3, KOH) in the eluent as CO.sub.2 is held constant at a total dissolved concentration of 10 mM to maintain a stable constant background while the KOH concentration was altered from 0 to 25 mM. A large eluent pH range (˜4-12) is possible.

[0036] FIG. 19B similarly shows the distribution of species where a constant 10 mM KOH is used as the influent to the engasser and the total carbonate carbon content is varied from 0 to 25 mM. The total pH span possible is less than that in FIG. 19A.

[0037] FIG. 20 is a graph showing capacity factor (k′) dependence of ions based on the KOH concentration ranging from 20 to 40 mM while CO.sub.2 engasser (5.5 cm) pressure is held constant at 145 psi (˜15.4 mM total carbonate). Corresponding separations are shown in FIG. 17. Arsenate and phosphate retention increased as the pH is raised due to their increasing charge. The other ions showed a decreasing capacity factor with increasing KOH concentration. Both axes have logarithmic scaling. The curvature is caused due to the transition from bicarbonate to carbonate.

[0038] FIG. 21 is a graph showing the effect of the carbonate gradient on the background conductance. 5.5 cm Engasser pressure is maintained constant at 145 psi CO.sub.2 (˜15.4 mM) while the KOH concentration is changed with time as follows: 0-5 min 18 mM KOH; 5-35 min 18-23 mM KOH; 35-55 min 23-40 mM KOH; 55-80 min 40 mM KOH; 80-80.5 min 40-18 mM KOH; 80.5-95 min 18 mM KOH. A rise can be seen where HCO.sub.3.sup.− is displaced by CO.sub.3.sup.2−. Conversely, a dip is caused as CO.sub.2 is resorbed back onto the column to make HCO.sub.3.sup.−. Use of higher concentrations of CO.sub.2 in the eluent is advantageous for faster re-equilibration of the column. Flow rate 0.5 mL/min; Dionex™ IonPac™ AG/AS-9HC 4mm Φ column set.

[0039] FIG. 22 shows separations of 3 different racemic mixtures of chiral amines using an eluent containing 60:40 acetonitrile:MeOH, 0.05% triethylamine that goes through a 160 cm Engasser operating with 60 psi CO.sub.2. A chiral stationary phase (LARIHC CF6-P (4.6×150 mm) column with 5 μm fully porous particles (a chiral stationary phase (CSP) with an alkyl derivatized cyclofructan 6 chiral stationary phase, which is commericially available from AZYP, LLC) was used. The injection volume was a 20 μL injection loop and optical absorbance detection at 254 nm was used. Note that these drugs/drug precursors can be obtained in pure form after separation as all eluent components are volatile.

[0040] FIG. 23 is a series of chromatograms showing different separation conditions tested for the separation of the chiral amine 1-(1-naphthyl)ethylamine. Effective separation was not achieved in more heavily aqueous media operating in a reverse phase mode and use of even 10% water resulted in no chiral selectivity. HILIC using polar organic solvents was found to separate the compounds. Triethylamine was required and no elution occurred without it. Other amines such as ammonia and trimethylamine were too strong, and were ineffective. According to a method of the invention, these amines could be separated under 60:40 ACN:MeOH and 0.05-0.2% triethylamine. Use of an eluent formed by introduction of gaseous CO.sub.2 into the eluent precursor/eluent is critical for elution. Other conditions were similar to those described for FIG. 22, except detection at 225 nm. Sample contained 25 mg/L racemic 1-(1-naphthyl)ethylamine.

[0041] FIG. 24A and FIG. 24B are illustrative derivations for programmable pH and ionic strength control. Engasser permeation constants are assumed to be known. Addition of other acid or base species in the influent solution to the Engasser is readily incorporated in this algorithm to calculate gas pressures necessary to achieve a certain pH and/or ionic strength.

[0042] FIG. 25A shows a chromatogram of 23 amino acids separated by cation exchange on Dionex™ IonPac™ CG/CS-3 4 mmΦ column set. The top trace is the total ion chromatogram from a mass spectrometer for all the amino acids. The lower traces are extracted ion chromatograms at the appropriate mass to charge ratio of the individual amino acids. All amino acids are 100 μM in concentration. The NH.sub.3 Engasser was 25 cm in length and the CO.sub.2 Engasser was 40 cm long. The Engasser influent was 20 mM formic acid flowing at 1 mL/min.

[0043] FIG. 25B shows the pressure sensor readout out for the gradient amino acid separation performed in FIG. 25A. An air purge was used to remove NH.sub.3 from the engasser to achieve a lower concentration of NH.sub.4.sup.+ in the eluent. During the air purge, the formic acid converts the column from NH.sub.4.sup.+ to H.sup.+. H.sup.+ is a weaker eluent than NH.sub.4.sup.+. The initial separation is performed isocratically using formic acid. A brief NH.sub.3 pulse is applied to fill the Engasser with a low pressure of NH.sub.3 and then the vent solenoid is opened. The column then is converted back to NH.sub.4.sup.+. This creates a transition zone. Some amino acids are eluted easily by the NH.sub.4.sup.+ and not H.sup.+ and therefore concentrate in the transition zone. To prevent this, a vacuum can be applied to the gas outlet to allow lower concentrations of NH.sub.3 without removing it entirely as occurs in the air purge.

[0044] FIG. 26A is similar to FIG. 25A, except the separation is conducted on an anion exchange column and the mixture contains 18 amino acids. Obviously the cation exchange mode provides for a better separation. The top trace is the total ion chromatogram from a mass spectrometer for all the amino acids. The lower traces are extracted ion chromatograms at the appropriate mass to charge ratio of the individual amino acids. All amino acids are 100 μM in concentration. The NH.sub.3 Engasser was 25 cm in length and the CO.sub.2 Engasser was 40 cm long. The eluent contained 20 mM Formic Acid flowing at 1 mL/min.

[0045] FIG. 26B is a graph showing the pressure sensor readouts of the NH.sub.3 and CO.sub.2 gradient used in FIG. 26A. The indicated pressure is in pounds per square inch absolute (psi).

[0046] FIG. 27 shows a chromatogram for the separation of 23 amino acids, Dionex™ IonPac™ CG/CS-3 4 mmΦ column set. Separation is identical to that performed in FIG. 25A using the gradient shown in FIG. 25B except that no CO.sub.2 was used during the separation. The top trace is the total ion chromatogram from a mass spectrometer for all the amino acids. The lower traces are extracted ion chromatograms at the appropriate mass to charge ratio of the individual amino acids. In FIG. 25A, CO.sub.2 allows buffering at higher pH and higher attainable ionic strength. This makes the separation of tyrosine, phenylalanine, and tryptophan easier than in the present case since these amino acids are highly retained at low pH but rapid transition to higher pH may cause them to elute all at once. While a higher pH is attainable when CO.sub.2 is not used and histidine, lysine, and arginine can be eluted earlier, the resolution between histidine and lysine is reduced.

[0047] FIG. 28 shows a chromatogram for the separation of 23 amino acids, Dionex™ IonPac™ CG/CS-3 4 mmΦ column set. Separation is identical to that performed in FIG. 25A using the gradient shown in FIG. 25B except that no formic acid was used during the separation. Without formic acid to lower the pH and render the amino acids cationic, all but the most basic amino acids are poorly retained and poorly resolved.

[0048] FIG. 29 shows a chromatogram for the separation of 23 amino acids Dionex™ IonPac™ CG/CS-3 4 mmΦ column set. Separation is identical to that performed in FIG. 25A using the gradient shown in FIG. 25B except that no air purge was used to lower the NH.sub.3 concentration in the engasser prior to the separation. The top trace is the total ion chromatogram from a mass spectrometer for all the amino acids. The lower traces are extracted ion chromatograms at the appropriate mass to charge ratio of the individual amino acids. While many of the acids are now unresolved and elute in 5 minutes, it can be see that isoleucine, leucine, and norleucine are well resolved and elute in a reasonable time unlike in FIG. 25A. Tyrosine and phenylalanine however are significantly less resolved than before. A greater dynamic range in the NH.sub.3 pressure, especially at the low end, can be achieved with a vacuum on the engasser outlet.

[0049] FIG. 30 is a series of chromatograms showing a comparison of ion chromatograms of various gradient methods tested. The CO.sub.2—NH.sub.3-formic acid system allowed the best separation. The additional CO.sub.2 created a greater buffered region and allowed higher ionic strengths so that elution of late ions provided more efficient (e.g., sharper, narrower) peaks.

[0050] FIG. 31 is a chromatogram showing separation of monoclonal antibody variants using 2-(N-morpholino)ethanesulfonic acid (MES) eluent. Column: Thermo Scientific™ Dionex™ MAbPac™ SCX-10, 10 μm, 4×250 mm; Eluent: A; 20 mM MES+60 mM NaCl pH 5.6; B: 20 mM MES+300 mM NaCl, pH 5.6; Gradient: 15-36.44% B in 50 min; Flow Rate: 1 mL/min; Temp: 30° C.; Sample: MAb, 5 mg/mL; 1) Sample 1: 25 μg; 2) Sample 2: 50 μg; Detection: 280 nm; Peak: 1-5: Acidic variants; Peaks 6, 8, and 11: C-terminal Lys variants.

[0051] FIG. 32 is a chromatogram of the same sample and on the same column as in FIG. 31 except a gradient eluent system of NH.sub.3—CO.sub.2 is used for separation; this will allow the antibodies to be recovered in pure form.

[0052] FIG. 33 is the gradient pressure readout used for the separation shown in FIG. 32. Separation starts with a low concentration of NH.sub.3 and a high concentration of CO.sub.2 to produce a low pH low ionic strength eluent. NH.sub.3 pressure is first increased to increase the ionic strength followed by a reduction of CO.sub.2 pressure to raise the pH. Spikes observed in the signal were due to electronic noise caused by valve actuation.

[0053] FIG. 34 is a table with information relevant to the 5 proteins separated in FIG. 35 and includes the protein mass and isoelectric point (pI).

[0054] FIG. 35 shows separation of five proteins—Total Ion Chromatogram in the m/z 500-1500 range the protein peaks are indicated with an asterisk. Flow Rate 0.2 mL/min; Injection volume=10 μL; 1 mg/mL each Lactalbumin, Ubiquitine, Myoglobin, Cytochrome C (Bovine), Cytochrome C (Equine),; 20 mM Formic Acid; Thermo Scientific™ Dionex™ MAbPac™ SCX-10. Proteins are eluted in their listed order. In the subsequent figures the mass spectra acquired for the asterisked peaks are provided. The elution order is governed by the pH of the solution except for the 2 cytochrome C proteins which are separated based on affinity for the column.

[0055] FIG. 36 shows the gradient pressure readout for the separation of 5 proteins shown in FIG. 35.

[0056] FIG. 37 shows the mass spectrum for the Lactalbumin peak eluting around 18 minutes. Thermo Scientific™ Dionex™ MAbPac™ SCX-10; Flow Rate 0.2 mL/min; Injection Volume Volume 10 μL; sample concentration 1 mg/mL; Engasser influent 20 mM Formic Acid.

[0057] FIG. 38 shows the mass spectrum for the ubiquitin peak eluting around 25 minutes. Thermo Scientific™ Dionex™ MAbPac™ SCX-10; Flow Rate 0.2 mL/min; Injection Volume 10 μL; sample concentration 1 mg/mL; Engasser influent 20 mM Formic Acid;

[0058] FIG. 39 shows the mass spectrum of the myoglobin peak eluting around 48 minutes. Thermo Scientific™ Dionex™ MAbPac™ SCX-10; Flow Rate 0.2 mL/min; Injection Volume 10 μL; sample concentration 1 mg/mL; Engasser influent 20 mM Formic Acid;

[0059] FIG. 40 shows the mass spectrum of the cytochrome c (bovine) peak eluting around 63 minutes. Thermo Scientific™ Dionex™ MAbPac™ SCX-10; Flow Rate 0.2 mL/min; Injection Volume 10 μL; sample concentration 1 mg/mL; Engasser influent 20 mM Formic Acid.

[0060] FIG. 41 shows the mass spectrum of the cytochrome c peak (equine) peak eluting around 67 minutes. Thermo Scientific™ Dionex™ MAbPac™ SCX-10; Flow Rate 0.2 mL/min; Injection Volume 10 μL; sample concentration 1 mg/mL; Engasser influent 20 mM Formic Acid.

DETAILED DESCRIPTION OF THE INVENTION

I. Introduction

[0061] Eluents used in high-performance liquid chromatography (HPLC) are produced using a gradient pump and each individual eluent component must be prepared separately before being combined into the gradient.

[0062] Gases are typically available in very high purity (99.999+%). A gas permeable membrane defining an eluent fluid flow channel can serve as an efficient barrier to any nonvolatile material/adventitious particles present while allowing the gas to penetrate the membrane and contact an eluent precursor fluid and/or an eluent disposed within the eluent fluid flow channel. Because the rate of gas introduction (permeation) into the eluent fluid flow channel is dependent on the differential partial pressure of the gas across the membrane, the dissolved gas concentration in the eluent generators of the invention may be altered simply by altering the external pressure of the gas. Using this principle, the present invention provides eluent generators producing eluents including gas-derived components of pre-determined and reproducible concentration(s).

[0063] Described herein are the construction, and principles of operation of exemplary eluent generators of the invention, systems incorporating such eluent generators and methods of using such eluent generators and systems in various chromatographic separations relying on diverse separation modalities.

[0064] The present invention provides devices and methods of eluent generation using gas permeable membranes for the introduction of neutral, acidic and basic gases to an eluent precursor/eluent form eluents consisting of acids, bases, salts buffers, and/or organic solvents. The present invention is of particular use in forming eluents that are also volatile and hence easily removed under vacuum.

[0065] In accordance with the present invention, devices, systems, and methods are provided for generating a high purity aqueous, polar organic, or hydroorganic stream with selected acidic or basic species and suitable for use as a chromatography eluent. In one form, an eluent generator includes an eluent flow channel defined by a gas permeable membrane. The membrane is disposed within a housing, which is substantially impermeable to the gas. The housing is charged with the gas while an eluent precursor solution flows through the eluent flow channel. The gas crosses the membrane in to the eluent precursor or eluent solution, thereby forming the eluent, e.g., an eluent of a predetermined composition. Exemplary gases include CO.sub.2, NH.sub.3, gases derived from volatile amines, and those derived from volatile acids.

II. Definitions

[0066] A “weakly dissociated acid” is an acid analyte with a pK.sub.a of from about 5 to about 10.

[0067] A “weakly dissociated base” is a base analyte with a pK.sub.b of from about 5 to about 10.

[0068] A “volatile acid” or a “volatile base” refers to the respective species in vapor, neat liquid or solution form.

[0069] A “gas permeable membrane” refers to a membrane at least partially permeable to an uncharged molecule. The membrane can be of any useful configuration, including flat, tubular, multi-annular, etc. Exemplary permeable membranes include Teflon AF, and ion exchange membranes. The permeable membrane can be a gas permeable and liquid impermeable membrane (at a given pressure).

[0070] An “eluite” refers to a chromatographic solute or analyte.

[0071] An “eluent precursor” refers to any liquid capable of dissolving the gas in the Engasser. Exemplary eluent precursors are those liquids generally recognized as chromatographic eluents and includes water, polar organic solvents, hydroorganic mixtures, with or without acids, bases, or salts further added.

[0072] The terms “Engasser” and “eluent generator” are used interchangeably herein to refer to an eluent generator of the invention.

[0073] An “amorphous fluoropolymer” refers to a family of polymers based on copolymers of 2,2-bistrifluromethyl-4,5-difluro-1,3-dioxole. An exemplary amorphous fluoropolymer is Teflon AF®.

[0074] “Dionex IonPac ICE-AS6” is an ion exclusion chromatography column where the resin has two types of ion-exchange groups (sulfonic and carboxylic acid) and hydrophobic groups. The substrate is a 8 micron microporous bead with 8% divinylbenzene crosslinker.

[0075] “Dionex MAbPac SCX-10” is a strong cation exchange chromatography column where the resin is a highly crosslinked divinylbenzene media with sulfonic acid groups.

[0076] “PSIA” refers to pounds per square inch absolute.

III. Exemplary Embodiments

[0077] In various embodiments, the invention provides a system for performing a chromatographic separation of an analyte. The system comprises, (a) an eluent generator, which comprises, (i) a housing configured to be pressurizable by gas. The housing includes an annular void defined by the housing. The housing includes a gas inlet for the gas and a gas outlet for the gas in fluid communication with the annular void. The eluent generator also includes (ii) a membrane permeable to the gas. The membrane defines an eluent flow channel disposed within the annular void. The eluent flow channel has an eluent precursor fluid inlet and an eluent outlet. The eluent generator further includes (b) a source of gas in fluidic communication with the gas inlet; and (iii) a source of the eluent precursor fluid. Downstream of the eluent generator and in fluidic communication with the eluent outlet is a sample interjector and a chromatography column configured to perform the chromatographic separation of the analyte. Exemplary embodiments include a valve, a vacuum, a trap, or a reservoir of inert gas (e.g., N.sub.2) at the gas outlet.

[0078] Exemplary systems of the invention further include a member selected from a gas input solenoid in fluidic communication with said source of gas and the gas inlet and interposed therebetween, a gas exhaust solenoid in fluidic communication with the gas outlet, and a combination thereof. According to these embodiments, the eluent generator can be pressurized with the gas to a pre-selected pressure, providing the operator the ability to control the amount of gas traversing the gas permeable membrane and entering the eluent flow channel, thereby controlling the concentration of gas (or reaction products between the gas and the eluent precursor) in the eluent flow channel.

[0079] The system and its components, e.g., the eluent generator, can be controlled manually or electronically. In an exemplary embodiment, at least a component or substructure of the system is controlled by a microprocessor or similar device. For example, to control the pressure of the gas in the eluent generator, the gas input solenoid, the gas exhaust solenoid and a combination thereof is controlled by a controller such that pressure of the gas within said eluent generator is controlled to a pre-selected pressure by the controller. Thus, in one embodiment, the gas input solenoid is open and the gas exhaust solenoid is closed, allowing the eluent generator to be pressurized with the gas. In various embodiments, the gas exhaust solenoid is open and the gas input solenoid is closed, allowing the device to be depressurized. In yet another embodiment, the gas input solenoid is open and the gas exhaust solenoid is open, allowing the device to operate at essentially atmospheric pressure with respect to the gas.

[0080] In a still further embodiment, the gas exhaust is fluidically connected to a vacuum, which promotes the passage of the gas across the external surface of the gas permeable membrane. In an exemplary embodiment, the vacuum allows the gas to be present in the eluent generator at a pressure that is less than atmospheric pressure. Thus, the device provides multiple configurations for adjusting the pressure of the gas and, therefore, the concentration of the gas or reaction components derived from the gas, in the eluent.

[0081] In various embodiments, the system further comprises a pump in fluidic communication with the eluent flow channel inlet. The pump is configured to supply the eluent precursor fluid to the eluent flow channel. The Engasser can be on the high pressure side or on the low pressure side of the pump depending on whether it is desirable for a particular application to drive the eluent precursor and eluent through the eluent flow channel or to pull the fluids through the flow channel.

[0082] The system is configured for the use of any gas permeable membrane in any format of eluent flow channel. In an exemplary embodiment, the gas permeable membrane is configured to permit passage of the gas from the annular void into the eluent flow channel and prevent or retard passage of the eluent from the eluent flow channel into the annular void. An exemplary gas permeable membrane is Teflon AF®.

[0083] The system can be used with acidic gases, basic gases and gases which are neutral but which form acids or bases upon contacting the eluent precursor or the eluent. Exemplary gases include CO.sub.2, a gas derived from a volatile acid, a gas derived from a volatile base, and ammonia. In some embodiments, it is desirable to use two or more gases. In some embodiments, the gases have different characteristics. Thus, in various embodiments, the system is configured to run in a gradient mode in which two or more eluents containing different gases or products derived from gases are mixed.

[0084] The system of the invention is versatile and is of use in the formation of organic eluents, aqueous eluents and combinations thereof in both isocratic and gradient formats. In an exemplary embodiment, the eluent is an aqueous eluent and the eluent precursor fluid is an aqueous eluent precursor fluid.

[0085] An exemplary device of the invention is shown in FIG. 1. Eluent precursor fluid enters the eluent flow channel 4 contained in housing 3 through eluent precursor fluid inlet 1. The exemplary device includes a coupling union 2 that includes three fluid ports. Coupling union 2 fluidically couples eluent precursor inlet 1 to eluent flow channel 4 and a downstream coupling union fluidically couples eluent precursor outlet 7 to eluent flow channel 4. Coupling union 2 also fluidically couples gas inlet 5 to the annular void within the housing 3 and the downstream coupling union fluidically couples gas outlet 6 to the annular void within the housing 3. The gas enters the annular void within the housing 3 through gas inlet 5, contacting the exterior surface of gas permeable membrane 8 of the eluent flow channel before existing the housing through gas outlet 6. Spacer inlet/outlet tubing 9 helps form a seal between coupling union 2 and gas permeable membrane tube 8. The resulting eluent exits the eluent flow channel through eluent outlet 7.

[0086] In various embodiments, the system of the invention further comprises a delay device in fluidic communication with the eluent outlet and the chromatography column and interposed therebetween. The delay device provides for a finite delay, of the order of several to 100 seconds, between the gas dissolving in the eluent at the generator and the resulting liquid reaching the chromatography column. This may be of particular relevance with CO.sub.2 as the half life for its conversion to H.sub.2CO.sub.3 is ˜20 s.

[0087] An exemplary embodiment of the invention is shown in FIG. 2. The system is controlled by microprocessor 10 (e.g., FreeSoC), which is operatively connected to comparators 17 and 29 through lines 14 (12 bit Aout) and 13 (12 bit Ain1), respectively. Comparator 29 is operatively linked to transistor 30, and comparator 17 is operatively linked to transistor 18. An exemplary transistor is a MOSFET. The microprocessor is also attached to pressure sensor 27 through line 12 (12 bit Ain2) and to conductivity detector CD1. Resistor 15 connects line 13 and comparator 17. Gas inlet solenoid 20 controls the flow of gas from gas inlet 19 into capillary 33 and then into eluent generator 22. Eluent precursor enters the eluent generator through eluent precursor inlet 21. Gas outlet solenoid 31 is operatively coupled to the gas outlet by means of a T-junction, which also connects the gas outlet to pressure sensor 28. Solenoid 31 is also coupled to capillary 32. In an embodiment, a vacuum pump (not shown) can be coupled to solenoid 31 or capillary 32 so that the pressure of the annular void of the housing of eluent generator 22 can be lowered to less than atmospheric pressure. The eluent outlet of eluent generator 22 is fluidically coupled to coil 23, which is of use to further dissolve the gas in the eluent or react the components of the gas with the eluent. Outlet from this coil is routed to high pressure conductivity cell 24 and exits from this cell to the chromatographic system, e.g., to the chromatography column and other downstream components. Conductivity detector CD-1 receives data from conductivity cell 24 via lines 25.

[0088] Another exemplary embodiment is shown in FIG. 6. Eluent precursor fluid is pulled from reservoir 40 through line 41 by pump 42, exiting the pump through line 43, which joins T-junction 44. In the upper branch, the eluent precursor flows through line 45 into Engasser 57, where it is contacted with CO.sub.2, entering the Engasser through line 55. CO.sub.2 flow into the eluent generator is controlled by solenoid 56. Excess CO.sub.2 exits the Engasser through line 57, where the pressure is determined by pressure transducer 58. The flow of CO.sub.2 out of the eluent generator is controlled by solenoid 59. In the lower branch, eluent precursor fluid enters line 46 and passes into Engasser 51. NH.sub.3 enters the system through line 47, and is controlled by solenoid 48. NH.sub.3 pressure is measured by pressure transducer 49. The NH.sub.3 passes through buffer coil 50 and into the Engasser. Excess NH.sub.3 exits the Engasser through line 64 and the flow through this line is controlled by solenoid 52. The eluent formed in the upper branch exits Engasser 57 through line 61. The eluent formed in the lower branch exits Engasser 51 through line 63. The eluents formed in the upper and lower branches are combined in T-junction 63 from which they are passed to gradient mixer 53. Conductivity of the eluent exiting the gradient mixer is optionally measured by means of high pressure conductivity cell 54. The signal from this cell is transmitted to conductivity detector 60. In an exemplary embodiment, the chromatography system includes one or more injection valves 66, chromatography columns or detectors after the gradient mixer 53 or high pressure conductivity cell 54.

[0089] In various embodiments, it is desirable to maintain the gas at a temperature higher than ambient temperature. For example, it may be desirable to prevent the gas from condensing from the gaseous state to the liquid state or to increase the pressure of the gas within the eluent generator. Gas permeation through a membrane is also temperature dependent, thus in various embodiments the device includes a thermostated enclosure to generate reproducible eluents containing the dissolved gas. Thus, in various embodiments, the system of the invention, e.g., the eluent generator, further comprises a heating device configured to heat at least a portion of the eluent generator, thereby maintaining the gas in a gaseous state with consistent membrane permeation.

[0090] When the heater is connected to the eluent generator or a portion thereof, it may be used to heat the entire eluent generator or a portion thereof. In an exemplary embodiment, the heater heats at least the gas input or a component thereof. In an exemplary embodiment, the heater heats the gas input and the gas input solenoid. The heater may be of any useful design, including resistance heaters, air heaters and jackets, liquid-based heaters and jackets, etc.

[0091] The system of the invention is of use in ion chromatography in both suppressed and non-suppressed modes. In an exemplary embodiment, the system further comprises a suppressor configured to suppress ions of said eluent, said suppressor disposed downstream of said chromatography column and in fluidic communication therewith.

[0092] In an exemplary embodiment, the system of the invention further comprises a suppressor downstream of and in fluidic communication with the chromatography column. Suppressed conductometric anion chromatography (SCAC) applications range from trace analysis in semiconductor manufacturing (Vanatta, L. E. TrAC, Trends Anal. Chem. 2001, 20, 336-345) to pharmaceutical analysis (Michalski, R. Mini-Rev. Med. Chem. 2014, 14, 862-872) to name a few. While SCAC excels in measuring strong acid anions, weak acid anions respond poorly or not at all. Even though acids with pK.sub.a<7 show some response, they do so in a nonlinear manner. Anions from very weak acids (pK.sub.a>7.0), e.g., silicate or cyanide are essentially not measurable.

[0093] Suppressed ion chromatography is a known technique for the analysis of ions. Suppression or stripping of the electrolyte is described in U.S. Pat. Nos. 3,897,213; 3,920,397; 3,925,019; and 3,926,559 by an ion exchange resin bed. A different form of suppressor column is described and published in U.S. Pat. No. 4,474,664, in which a charged ion exchange membrane in the form of a fiber or sheet is used in place of the resin bed. In this form of suppressor, the sample and eluent are passed on one side of the membrane with a flowing regenerant on the other side, the membrane partitioning the regenerant from the effluent of chromatographic separation. The membrane passes ions of the same charge as the exchangeable ions of the membrane to convert the electrolyte of the eluent to weakly ionized form, followed by detection of the ions. Other useful suppressors are disclosed in U.S. Pat. Nos. 4,751,004, 4,459,357, 4,403,039, 4,500,430 and 4,647,380, and 4,999,098.

[0094] In various embodiments, the system of the invention further comprises a detector configured to detect at least one component of the analyte. The detector is disposed downstream of said chromatography column and in fluidic communication therewith. A particular detection scheme is chosen based on the properties of the analytes. For example, in ion chromatography, analysis of nitrate, bromide or iodide can be pursued by ultraviolet detection (UV) since these analytes absorb in UV. However other common ions such as fluoride, sulfate, and phosphate do not absorb UV and so will not respond to direct UV detection. Those of ordinary skill in the art understand how to select the proper detector or detectors for a particular analysis.

[0095] Another exemplary system of the invention includes one or more evaporative type of detector. In this technique, the eluent along with the analyte are outputted (e.g., nebulized) from the chromatography column and then evaporated where the analyte remains as a residual solid. The residual solid particles can then be detected with an evaporative light scattering detector, a charged aerosol detectors, and/or an electrospray ionization mass spectrometer. The charged aerosol detector (CAD) nebulizes the effluent flow and creates charged particles that can be measured as a current proportional to the analyte concentration. Details regarding the charged aerosol detector can be found in U.S. Pat. Nos. 6,544,484; and 6,568,245, which are hereby fully incorporated by reference herein. Evaporative light scattering detector (ELSD) includes a nebulizer receiving a solution eluting from a separation column, then atomizing and spraying the solution as droplets, which dry to form residue aerosol particles. An air stream carries the residue particles past a beam of light, each particle scattering (reflecting or refracting) the light as it intersects the beam. One or more photodetectors sense the scattered light. The scattered light intensity increases with the size of the particle. Accordingly, the amplitude of the photodetector output signal is used to measure particle size. Details regarding an example of an ELSD can be found in International PCT Publication WO 2010/068272, which is hereby fully incorporated by reference herein. In various embodiments, the evaporative step removes the volatile and ionically charged eluent that can interfere with mass spectrometry.

[0096] In various embodiments, the system includes one or more conductivity detectors. Conductivity detection is a bulk property detection and the total conductance depends on the nature of the ions via the charge on the ion and the mobility and the concentration in a sample. The specific conductance of a solution is the sum of the concentration-mobility product of the different ions present. It is well known that equal concentrations of specific different compounds, e.g. NaCl and HCl, have vastly different specific conductance.

[0097] According to the present invention, an exemplary volatile amine is an amine capable of permeating through the permeable membrane of the permeant membrane device of the invention from a solution of the volatile amine in which the membrane is in contact while an eluent stream containing one or more weakly ionizable acid flows within the annulus of the permeable membrane. Exemplary volatile amines of use in the present invention include those having a general structure according to Formula I:

##STR00001##

in which R.sup.1, R.sup.2 and R.sup.3 are selected from H and substituted or unsubstituted alkyl. Exemplary alkyl moieties include C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6, C.sub.7, and C.sub.8 straight, branched chain and cyclic substituted or unsubstituted alkyl moieties. In various embodiments, two or more of R.sup.1, R.sup.2 and R.sup.3, together with the nitrogen to which they are bonded are joined to form a ring structure. The amines can be used individually or in combination.

[0098] Exemplary volatile amines include, without limitation, (methyl).sub.namine, (ethyl).sub.n, (propyl).sub.namine, and (butyl).sub.namine, in which n is 1, 2, or 3; alkanolamines (including, but not necessarily limited to, monoethanolamine (MEA), methyldiethanolamine (MDEA), diethanolamine (DEA)); ethylenediamine (EDA), methoxypropylamine (MOPA), diethylaminoethanol (DEAE) and the like and mixtures thereof. Although ammonia is not strictly speaking an amine, in the context herein ammonia is included in the same group of nitrogen compounds as amines.

[0099] In various embodiments, the useful amines include relative stronger amines having a pKa between about 10.5 to about 12. In one non-limiting embodiment, the amine does not contain oxygen. In another non-restrictive embodiment, the amines are di-alkylamines which have a pKa range of between about 10.7 to about 11.4. In various embodiments, the amine has a normal boiling point less than about 95° C., e.g., less than about 70° C., less than about 50° C., or less than about 40° C. Suitable amines include, but are not limited to, dimethylamine, diethylamine, dipropylamine, di-isopropylamine, di-n-butylamine, diisobutylamine, di-sec-butylamine, di-tert-butylamine, pyrrolidine, piperidine, and combinations (e.g., mixtures) thereof.

[0100] Exemplary volatile amines of use in the invention have sufficiently low conductance at the concentration used such that they do not excessively interfere with detection of the analytes.

[0101] According to the present invention, an exemplary volatile acid is an acid capable of generating a gas, which can permeate through the permeable membrane of the eluent flow channel of the eluent generator of the invention from a solution of the neat or diluted volatile acid while an eluent precursor/eluent stream flows within the lumen of the permeable membrane. Useful volatile acids include those acids which have a pKa range from <0 to about 4. Another definition of volatile acid is on that is relatively readily removed from a solution under vacuum. Exemplary volatile acids include all haloacids (e.g., HF, HCl, HBr, and HI) as well as methanesulfonic acid, toluenesulfonic acid, carboxylic acids such as formic acid, etc., and other acids that at least 99% of which can be removed under vacuum (e.g., <10 mm Hg pressure) within 24 hours.

[0102] Any useful concentration of gas can be incorporated into the eluent flow channel within the permeable membrane of the eluent flow channel. In exemplary embodiments, the gas concentration in the eluent solution will vary for the specific chromatographic mode used. For the separation of weak acids by a CO.sub.2 eluent, typically useful CO.sub.2 concentrations range from 5 to 500 mM CO.sub.2. In carbonate bicarbonate eluent preparation for suppressed ion chromatography, the useful range is from very low (<˜1 mM) to 30 mM total carbonate carbon. In NH.sub.3—CO.sub.2 gradient applications, one or the other eluent component will typically start at zero concentration and range up to 250 and 500 mM, respectively, for NH.sub.3 and CO.sub.2.

[0103] In an exemplary embodiment, the invention uses a high concentration of gas, e.g., CO.sub.2, NH.sub.3 or a gas derived from a volatile base or volatile acid external to the membrane, and the gas is at least moderately permeable through the membrane. In this manner, the desired concentration of the gas within the eluent flow channel is reached. A high external concentration of gas coupled with a modest permeability of these components across the membrane ensures that, in exemplary embodiments, the external gas needs be only infrequently replenished. By “high external concentration” it is intended that the concentration of the gas be sufficiently high so as to not require constant changing, replacement or replenishment.

[0104] In an exemplary embodiment, the device of the invention includes a pressure readout and gas is added to or removed from the eluent generator automatically based upon the measured gas pressure and desired concentration of the gas or its derived species in the eluent.

[0105] An exemplary gas source is a compressed gas cylinder, e.g., for NH.sub.3 and CO.sub.2.

[0106] In various embodiments, a small external gas volume is desirable since it results in less wasted gas, particularly when a gradient is run.

[0107] Though recited in terms of certain ranges, it will be understood that all ranges from the lowest of the lower limits to the highest of the upper limits are included, including all intermediate ranges or specific values, within this full range or any specifically recited range. It is within the ability of one of ordinary skill to select membrane permeability (e.g., molecular weight cutoff, length, composition, thickness, etc.) and the concentration and identity of the gas to achieve a desired concentration of base or acid within the eluent flow channel. In an exemplary embodiment, the device is capable of prolonged use without replacement or replenishment of the gas source or eluent precursor source.

[0108] The present invention also provides methods of using the systems and devices disclosed herein. In an exemplary embodiment, the invention provides a method of performing a chromatographic separation using a system of the invention. The system comprises: (a) an eluent generator, comprising: (i) a housing configured to be pressurizable by gas, comprising an annular void defined by the housing, a gas inlet for the gas and a gas outlet for the gas in fluid communication with the annular void. The eluent generator also comprises: (ii) a membrane permeable to the gas defining an eluent flow channel disposed within the annular void and having an eluent precursor fluid inlet and an eluent outlet; (ii) a source of gas in fluidic communication with said gas inlet; (iii) a source of the eluent precursor fluid; and (b) a chromatography column disposed downstream of and in fluidic communication with the eluent outlet. An exemplary method comprises: (a1) flowing the eluent precursor fluid through the eluent flow channel while the annular void is maintained under positive pressure of the gas. The positive pressure is selected to be of a magnitude sufficient to cause the gas to cross the gas permeable membrane, dissolving it in said eluent precursor fluid, thereby forming said eluent. Step (b1) includes, passing the eluent from the eluent outlet into the chromatography column. Step (c1) includes contacting the chromatography column with the analyte, thereby performing the chromatographic separation of the analyte.

[0109] As will be apparent to those of skill in the art, an analyte is injected onto the chromatography column, and at least one component of the analyte is eluted from the chromatography column by eluent generated by the eluent generator flowing through the chromatography column. In various embodiments, the at least one component of the analyte is detected by a detector following its elution from the chromatography column.

[0110] The eluent precursor can be any useful liquid including, without limitation, e.g., water, aqueous bases, e.g., NaOH(aq), NH.sub.4OH(aq), aqueous acids, e.g., HC1(aq), H.sub.2SO.sub.4 (aq), organic solvents, e.g., polar organic solvents, e.g., acetonitrile, methanol, ethanol, etc. and mixtures of water and aqueous acids, bases and/or salts with organic solvents. In various embodiments, acids, bases, and salts do not exceed about 200 mM in the eluent precursor. In various embodiments, appropriate selection of membrane geometries/type and gas pressure is deliberately chosen to generate gas-derived eluent component concentrations as high as 1 M.

[0111] In various embodiments, the eluent precursor is selected from aqueous NaOH or KOH. In an exemplary embodiment, NaOH or KOH can be present in the eluent precursor at a concentration of from about 0.001M to about 200 mM.

[0112] In various embodiments, the gas is a member selected from CO.sub.2, ammonia, a gas derived from a volatile amine and a gas derived from a volatile acid.

[0113] The system of the invention is highly versatile and, because a wide range of eluents of different formats can be generated, the invention provides methods in which the eluent format is an isocratic format or a gradient format.

[0114] Because of this versatility, a wide range of different chromatographic procedures can be practiced using the system of the invention. Thus, the invention provides methods of chromatographic separation as varied as ion exclusion, ion exchange, chiral amine separation, amino acid separation, and protein separation, including separation of monoclonal antibodies.

[0115] One of the sources of the versatility of the system and the methods of use in conjunction with the system is the ability to predictably and reproducibly control the content and composition of the eluent produced with the eluent generator of the instant invention. Thus, in various embodiments, the invention provides a method in which, prior to performing a separation, the system is controlled to produce an eluent having a pre-determined composition. In an exemplary embodiment, the controlling comprises, (a) selecting the gas, and pre-determining; (i) pressure of the gas in the annular void; (ii) composition of the eluent precursor solution in the eluent flow channel; and (iii) flow rate of the eluent precursor solution through the eluent flow channel. The controlling further comprises, (b) controlling the eluent generator such that; (i) the pressure of said gas in the annular void is maintained at the pre-determined pressure; (ii) the composition of the eluent precursor fluid is maintained at the predetermined composition; and (iii) the flow rate of the eluent precursor fluid in the eluent flow channel is maintained at the predetermined flow rate, thereby producing said eluent having said pre-determined composition.

[0116] The system of the invention includes eluent generators in which a gas is dissolved in the eluent precursor/eluent. Also provided are systems in which the gas reacts with the eluent precursor/eluent in the eluent flow channel, forming new species for use as components of the eluent. Thus, it is desirable in certain embodiments to predetermine the rate of dissolution of the gas in the eluent precursor/eluent and/or the rate of reaction of the gas with the eluent precursor/eluent in the eluent flow channel. The pre-determined values of gas solubility and/or dissolution rate and/or gas reaction with the eluent precursor/eluent allow determining a concentration of the gas and/or a reaction product between the gas and the eluent precursor/eluent in the eluent precursor/eluent in the flow channel. In various embodiments, the rate of reaction of the gas with the eluent precursor is determined, thereby determining concentration of a reaction product resulting therefrom in the eluent.

[0117] As noted above, the system of the invention provides eluents in both isocratic and gradient formats. When a gradient format is desired, a parameter selected from: (i) the pressure of the gas in the annular void; (ii) the composition of the eluent precursor fluid; (iii) the flow rate of the eluent precursor fluid in the eluent flow channel; and (iv) a combination thereof is maintained for a pre-determined time after which it is varied, thereby producing the eluent in a gradient format. As those of skill in the art are aware, the gradient may include two or more eluent components, which vary in concentration, pH, composition, etc. The gradient can be selected to have any convenient profile and duration.

[0118] A key component of the systems and methods set forth above is the eluent generator of the invention. In an exemplary embodiment, the eluent generator is configured for generating an eluent of a pre-determined composition and for incorporation into a system for chromatographic separation of an analyte. An exemplary eluent generator includes: (a) a housing configured to be pressurizable by gas. An exemplary housing includes an annular void defined by the housing, a gas inlet for the gas and a gas outlet for the gas. Both the gas inlet and gas outlet are in fluid communication with the annular void within the housing. The eluent generator also includes, (b) a membrane permeable to the gas. The membrane defines an eluent flow channel disposed within the annular void. The eluent flow channel has an eluent precursor fluid inlet and an eluent outlet. The outlet is configured for coupling to a chromatography column and fluidically communicating therewith. To control the gas flow and pressure within the eluent generator, a first solenoid is coupled to and communicates fluidically with the gas inlet. The solenoid is optionally controlled by a first controller. Similarly, the eluent generator includes a gas inlet coupled to and communicating fluidically with a second solenoid. The second solenoid is optionally controlled by a second controller. The first and second controller can be the same controller. In an exemplary eluent generator and system of the invention, the first controller and the second controller are programmed to operate cooperatively to control pressure of the gas in the annular void at a predetermined value, thereby generating the eluent of said pre-determined composition.

[0119] The foregoing descriptions of specific embodiments of the present invention have been presented for purposes of illustration and description. They are not intended to be exhaustive or to limit the invention to the precise forms disclosed, and obviously many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and its practical application, to thereby enable others skilled in the art to best utilize the invention and various embodiments with various modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the Claims appended hereto and their equivalents. All references are incorporated herein by reference in their entirety.

EXAMPLES

Example 1

Eluent Generator Design and Control

[0120] The construction of the Engasser is shown in FIG. 1. While the permeative introduction NH.sub.3 and CO.sub.2 are largely discussed here, it should be readily understood that any permeable fluid can be introduced in this fashion. What is formed in the liquid in the lumen will depend on the composition of the influent liquid. With CO.sub.2 as the Engasser gas, a solution of pure carbonic acid will be the effluent with pure water as the influent, with a base solution as the influent, a variable composition that can vary all the way from carbonic acid to bicarbonate to carbonate can be generated.

[0121] An illustrative example for an engasser device was presently constructed by first inserting the end of a piece of Teflon AF® tubing (0.28 mm i.d.×0.68 mm o.d.) into a 1/16″ o.d. tubing (0.762 mm i.d.) that is not particularly permeable to CO.sub.2. Teflon AF is tradename for an amorphous fluoropolymers based on copolymers of 2,2-bistrifluromethyl-4,5-difluro-1,3-dioxole. This tube is then swaged down upon the Teflon AF® using standard polyetheretherketone (PEEK) chromatography fittings (e.g., a coupling union). The Teflon AF® then is fed through a PEEK tee (1.27 mm thru hole), ⅛″ o.d., 1/16″ i.d. PTFE Teflon® or PEEK tubing, (very low CO.sub.2 permeability), another PEEK tee, and finally into another length of CO.sub.2 impermeable tube which again is swaged down on the Teflon AF®. This connecting impermeable tubing is then connected to the rest of the chromatography system. The length used depends on the eluent flow rate and concentration desired.

[0122] Application of the gas pressure to the device may be accomplished most simply using a pressure regulator, but this results in only a static pressure that is not programmable by the user. Experiments carried out in this report used a solenoid valve setup that allowed analog control of the pressure (FIG. 2). Digital pressure controllers are available for inert gases and may be more convenient to use if compatible with the gas to be introduced (Mini Pressure Controller).

[0123] The solenoid setup was controlled using a FreeSoC board which contains a Cypress Semiconductor P5 programmable system on a Chip. The device was configured to allow for data acquisition of the pressure sensors and a high pressure conductivity cell as well as analog outputs to control the pressure. The pressure was controlled using 2 LM311 comparators and, 2 MOSFETS, and 2 solenoid valves (SV). The analog out from the FreeSoC is directly coupled to both comparators but on opposite inputs. The high pressure sensor output (this sensor only provides gauge pressure and not absolute) is wired to the other terminals of the comparator and is also recorded using the FreeSoC. It was necessary to step the voltage down to the input of the comparator that controls the source valve to prevent continuous venting and filling. When a particular comparator has a high output, (the +input terminal is less than the −input terminal), the MOSFET energizes the solenoid valve SV connected to it. Silica capillaries (150 μm i.d., ˜20 cm length) are used to reduce the gas introduction rate into the jacket to prevent immediate over pressurization of the device that will result in immediate venting. Similarly, silica outlet capillaries are used to limit the venting rate. An ambient pressure sensor is used to correct for any changes in atmospheric pressure. This is generally not an issue at a given location but can be important in translating elution/eluent conditions exactly if a method from a reference laboratory is to be accurately implemented in another varying considerably in altitude (and hence ambient pressure).

[0124] Permeation is governed by the partial pressure of the gas across the membrane. This means that at zero gauge pressure there is still ˜1 atm partial pressure of the gas to be introduced in the jacket and thus there will still be permeation through the membrane. The dynamic range can be improved by adding a vacuum system, such as that commonly used with current degassers used in electrodialytic eluent generation or by a He/N.sub.2 flush system, as these gases are already often available for pressurizing eluent reservoirs. A hydration or delay coil is shown after the Engasser. This is relatively unique to CO.sub.2 introduction and is needed because the hydration of CO.sub.2 (CO.sub.2+H.sub.2O.fwdarw.H.sub.2CO.sub.3), especially into pure water or media of low [OH.sup.−] is relatively slow (the dehydration step is fast in comparison) (Soil and Byrne, Mar.Chem. 2002, 78 (2) 65-73). A hydration coil or catalyst is therefore particularly beneficial when a pure carbonic acid eluent is desired as used in Ion Exclusion Chromatography. The conductivity of the solution was measured after the coil for evaluating the CO.sub.2 introduction rate. Note that the conductivity was always higher after the hydration coil because of slow hydration kinetics. The conductivity measurement cell was simply two stainless steel HPLC tubes used as electrodes connected and electrically separated by a PEEK union. The conductivity was measured by a Dionex CDM-1 detector, the analog output of which was acquired by the FreeSoC.

[0125] An embodiment of the eluent generator can use two gases, e.g., NH.sub.3 and CO.sub.2. Both of these gases liquefy at modest pressures (relative to HPLC) and ambient temperature. At 25° C., for example, CO.sub.2 liquefies at ˜930 psi while NH.sub.3 becomes a liquid at ˜145 psi. As ambient pressure is ˜14 psi, this only allows an order of magnitude of control for NH.sub.3 pressure compared to almost 2 orders of magnitude for CO.sub.2 if vacuum or inert gas flush is not used at the low end. Especially with NH.sub.3, it was also found necessary to keep the gas introduction valve heated; decompression of NH.sub.3, commonly used as a refrigerant gas, through the valve causes the temperature to drop and leads to NH.sub.3 condensation. Additional complications caused by this behavior include liquid NH.sub.3 flowing through the valve and accumulating in the Engasser; even after the inlet valve is closed, the pressure continues to increase as the liquid warms up and evaporates. NH.sub.3 is not vented directly into the laboratory air but through an acid trap; while a bubbler containing a dilute sulfuric acid solution can be used, a cartridge packed with acid impregnated silica gel or ammonium or sodium bisulfate (into which an exhaustion indicator like bromthymol blue can be incorporated is convenient. A solid trap prevents problems associated with potential back aspiration of a solution phase acid into the Engasser.

[0126] Note also that while CO.sub.2 has to be introduced in a high pressure format because of its much lower solubility, NH.sub.3 has a very high Henry's law constant (highly soluble in water), 28% w/w or 14.5 mol/L NH.sub.3 has a vapor pressure of only 1 atm. Ammonia can thus also be introduced through a low pressure Engasser before the pump if desired. Even with introduction on the high pressure side, pressures in excess of 100 psi NH.sub.3 were not needed when the proper length of tubing was chosen.

Example 2

Permeation Rate

[0127] The rate of permeation of CO.sub.2 or NH.sub.3 into pure water was measured using conductivity as shown in FIG. 2. A backpressure coil was used to generate 1000 psi at 0.5 mL/min. The applied CO.sub.2 or NH.sub.3 pressure was changed gradually over time and the conductivity and the applied pressure values were recorded. FIG. 3 shows illustrative data for CO.sub.2 permeating through a 160 cm long Engasser. Based on the known limiting equivalent conductance of HCO.sub.3.sup.− and H.sup.+, the conductivity data can be used to calculate their concentrations and hence pH. The known dissociation constants relevant for the system then permit the estimation of the total amount of permeated CO.sub.2 as well as a permeation rate per unit pressure of CO.sub.2. FIG. 4 shows the relationship between pressure, conductivity, pH, and permeation rate for CO.sub.2 using 2 Engasser lengths differing by a factor of 30). Because H.sub.2CO.sub.3 is a weak acid, the conductance is nonlinear with pressure. The total dissolved concentration is linear however with pressure (R.sup.2≧0.9996). We found that the 5.5 cm Engasser had a permeation rate per unit length (9.7 nmol/cm/min/psi) almost twice that of the 160 cm Engasser (compared to 4.9 nmol/cm/min/psi). The present systems are non-equilibrium systems—the solution composition in the lumen will never reach equilibrium with the external gas pressure. The introduction rate does not increase linearly with increasing length because the internal CO.sub.2 concentration/ pressure increases along the length of the Engasser, resulting in greater differential pressure across the membrane in the entrance region that continually decreases with the downstream length. Using the Henry's law constant, the solution partial pressure can be calculated. For the 5.5 cm engasser, at a water flow rate of 0.5 mL/min, the solution partial pressure of CO.sub.2 reaches 4.6% of the external pCO.sub.2, while under the same conditions the 160 cm engasser reaches 68% of the equilibrium pCO.sub.2. Obviously the overall rate of permeation will also be lumen flow dependent, reducing the flow will increase the degree to which equilibrium is approached by increasing the residence time in the Engasser, the effective permeation rate per unit time will decrease.

[0128] In a situation where the introduced gas reacts immediately with the lumen fluid (i.e., eluent precursor/eluent), for example when CO.sub.2 is introduced into a NaOH solution in the lumen, the gas partial pressure in the lumen effectively remains near zero until the reactive consumption stops. In this case the permeation rate remains independent of flow rate. Consider an online titration of NaOH flowing into the lumen with CO.sub.2 (FIG. 5) using the 160 cm Engasser and varying the external CO.sub.2 pressure while holding the influent NaOH concentration and flow rate constant. The CO.sub.2 first reacts with the NaOH to form Na.sub.2CO.sub.3. Additional CO.sub.2 is then consumed reactively to form NaHCO.sub.3. Tangent based determination of the first equivalence point was determined at two different NaOH flow rates from which the permeation rate of the CO.sub.2 could be calculated. Given the known input flux of NaOH, the permeation rate at the two different flow rates were calculated to be within 0.5% of each other, 1.94±0.01 μmol/min/psi. For this particular tubing this would be the maximum attainable permeation rate. Note that this value is 1.26× that of what the 5.5 cm Engasser exhibits with a water influent. Given that in this very short device the approach to equilibrium is <5% complete, the difference should have been less—suggesting that concentration polarization occurs within the lumen, meaning that the actual pCO.sub.2 at the interior boundary layer of the wall is higher than in the bulk solution, thus reducing the permeation rate more than what will be estimated of the degree of attainment of equilibrium of the bulk solution. Predictably the average permeation rate for the much longer 160 cm engasser with a water influent was much lower (by 2.5× compared to the NaOH influent case).

[0129] Summary of permeation rate data for the various experimental devices are presented in Table 1. Note that the permeation rate/length is nearly constant for NH.sub.3 regardless of flow rate or device length because of its much greater Henry's law solubility compared to CO.sub.2 and the fact that NH.sub.4.sup.+ is a considerably stronger acid than H.sub.2CO.sub.3. From a practical standpoint, the greater extent of ionization also makes the slope of conductance vs. total dissolved amount steeper than in the case of CO.sub.2, facilitating the measurement of the total permeated amount or the permeation rate (for which measurement at only one flow rate in this case is sufficient).

TABLE-US-00001 TABLE 1 Permeation Rate Constants for CO.sub.2 and NH.sub.3 of various lengths and eluent flow rates measured by titration or conductivity. Tubing Permeation Length Titration Flow Rate Permeation Rate Rate/Length (cm) (Y/N) (mL/min) (umol/min/psi) (nmol/min/psi/cm CO.sub.2 160 N 0.5 0.774 4.840 5.5 N 0.5 0.053 9.631 160 Y 2 1.946 12.163 160 Y 3 1.946 12.163 40 N 0.2 0.169 4.228 NH.sub.3 160 N 0.5 1.300 8.125 5.5 N 0.5 0.055 9.949 160 Y 1 1.322 8.265 160 Y 1.25 1.377 8.604 40 N 0.2 0.345 8.630 40 N 0.2 0.382 9.562 40 N 0.2 0.355 8.879 25 N 0.2 0.231 9.231 25 N 0.2 0.233 9.311

[0130] Once the NH.sub.3 permeation rate for a system is known, a dynamic titration of CO.sub.2 using NH.sub.3 can be used to determine the CO.sub.2 permeation rate under the given experimental conditions. The setup for performing this titration as well as generating an ammonium carbonate gradient is shown in FIG. 6. The bulk of the flow is sent through the CO.sub.2 engasser since it is more sensitive to lower flow rates than NH.sub.3. The NH.sub.3 compatible valves readily available to us were relatively large resulting in significant system hysteresis. This was mitigated through the use of a long (2 m) buffer coil of PTFE tubing (3 mm od 1.5 mm id). FIG. 7 shows the results for titration of a fixed pressure of NH.sub.3 with varying CO.sub.2 pressures with both ascending and descending pCO.sub.2. CO.sub.2 reacts with NH.sub.3 in H.sub.2O at first to form (NH.sub.4).sub.2CO.sub.3. The increase in conductivity that results from CO.sub.2 introduction into ammonia is due both to the formation of NH.sub.4.sup.+ and CO.sub.3.sup.2−. Once (NH.sub.4).sub.2CO.sub.3 formation is complete there is only a minor decrease in conductance as the conversion of CO.sub.3.sup.2− to less mobile HCO.sub.3.sup.− is partly offset by the increase in [NH.sub.4.sup.+] due to the decrease in pH. The net result is a virtual plateau in conductance. As the rate of ammonia introduction is known and will be equal to twice the CO.sub.2 introduction rate on a mole basis at the (NH.sub.4).sub.2CO.sub.3 equivalence point. The ratio of the CO.sub.2 to NH.sub.3 pressures at the equivalence point can be multiplied by the NH.sub.3 permeation rate to compute the CO.sub.2 rate. Similarly a fixed CO.sub.2 pressure can be titrated by varying the ammonia introduction rate; all of the resulting CO.sub.2 permeation rate data are listed in Table 2 and are remarkably constant, no matter how it has been determined.

Example 3

3.1 Ion Exclusion

[0131] In ion exclusion chromatography, the separation of weak acids is carried out based upon their diffusion into an occluded volume of a like charged resin and adsorption to the resin matrix. For efficient chromatography, typically a strong mineral acid is used to lower the pH of the separation environment and suppress weak acid ionization. However, this separation design results in poor sensitivity for suppressed conductometric detection compared to ion exchange chromatography because the eluent cannot be converted to water or to a weakly conducting acid like carbonic acid.

[0132] If the carbonic acid concentration is high enough in the system, H.sub.2CO.sub.3 is capable of lowering the pH sufficiently to conduct efficient chromatography. Just as importantly, it can then be removed by a degasser device like the carbonate removal device (CRD) (U.S. Pat. No. 7,306,720), widely used in suppressed carbonate eluent anion exchange chromatography. In the present example, the membrane in a commercial CRD was modified by coiling it around a ⅛″ rod and using a vacuum pump that drew air through a needle into a NaOH reservoir to trap any ambient CO.sub.2. Chromatograms obtained in the isocratic eluent mode (constant CO.sub.2 pressure on the engasser) are shown in FIG. 8. The background was only 1.644 μS/cm using up to 250 mM CO.sub.2. The CO.sub.2 pressure also perceptibly affects the separation (FIG. 9). Glutaric and propionic acids may have their positions reversed while fumaric acid may be eluted over a rather wide range depending on the CO.sub.2 concentration. Early eluters have their resolution improved at higher applied pressures, but this comes at a loss of resolutions between peaks 7-10. This indicated a gradient in pCO.sub.2 may be beneficial. Because there is a large occluded volume that CO.sub.2 itself can probe, there is a very large gradient delay time (FIG. 15) (>30 minutes) suggesting that the gradient should commence even prior to sample injection (FIG. 10). The gradient separation (FIG. 11) increased the resolution (FIG. 12) for some of the early eluting ions and allowed control over where fumarate elutes as well as the retention order for glutaric and propionic acids. Early eluters were also further separated from the column void. The applicability of the method towards measuring some key weak acids in groundwater that have been identified as signatures for hydraulic fracking induced contamination has also been successfully demonstrated (FIG. 16).

[0133] FIG. 13A and FIG. 13B show conductivity and absorbance chromatograms for a standard mixture of 13 organic acids. The current commercially standard separation is provided in the inset. The same concentrations are used, but with different temperature and injection volumes for the separation. The dip around 17.5 minutes is the CO.sub.2 vacancy peak. Fumaric acid has much greater absorbance than the other weak acids which highlights the advantage of measuring using conductivity where ions will have similar response relative to their molar concentration.

[0134] Conductivity provided better results at low concentrations compared to absorbance. Some residual CO.sub.2 or other dissolved gas caused air bubbles to develop to which the absorbance detector was very sensitive. Subsequently more efficient CO.sub.2 removal was accomplished by using narrower bore CRD tubing with the tubing coiled around a ⅛″ rod. FIG. 14B shows the separation without the CRD. In neither absorbance nor conductivity are any of the ions identifiable without CO.sub.2 removal.

3.2 Ion Exchange Chromatography

[0135] Very high purity carbonate eluents with low background conductance can be easily generated without the need to prepare solutions or manually adjust the pH. By placing an eluent generator (and degasser) prior to the engasser it is possible to make solutions of HCO.sub.3.sup.−/CO.sub.3.sup.2−/OH.sup.− of varying strength and pH. Separation was performed on a Dionex™ IonPac™ AS-9HC column prior to suppression and removal of CO.sub.2 through the aforementioned modified CRD. Speciation models are provided in FIG. 19A and FIG. 19B that illustrate solution composition and pH. Because of the pH control and difference in selectivity between HCO.sub.3.sup.− and CO.sub.3.sup.2−, carbonate based elution still offers some advantages over the hydroxide eluent ion chromatography and will likely see increasing use as a new low noise carbonate suppressor has been developed (U.S. Patent Application Publication No. U.S.20160041133A1). Isocratic separation with carbonate eluents made in line can be seen in FIG. 17 for 19 different anions. FIG. 20 shows the capacity factor dependence upon the hydroxide concentration. As the concentration increases, so does the pH which increases the charge on phosphate and arsenate and thus their retention. Such selectivity choices for polyvalent weak ions are lost when using a pure hydroxide gradient. A hydroxide gradient using the eluent generator may be produced to improve the separation (FIG. 18). The gradient consisted of isocratic elution using 18 mM KOH from 0-5 minutes followed by a linear ramp from 18-23 mM KOH during 5-35 min, then another linear ramp from 35-55 min in which the KOH was raised from 23-40 mM, then the KOH was held constant at 40 mM from 55-80 minutes, then the system was allowed to re-equilibrate with 18 mM KOH for 15 minutes. Of note is the low background and the lack of baseline drift/shift (FIG. 21). There is a slight increase in conductivity during the start of the gradient as HCO.sub.3 is displaced by C0.sub.3.sup.2−. Then during the re-equilibration stage the conductivity decreases as CO.sub.2 is readsorbed back onto the stationary phase as HCO.sub.3.sup.−.

3.4 Chiral Amine Separation

[0136] Chiral amines are most often separated by an ion pairing effect using an acid as the pairing agent. Here, chiral amines were separated using CO.sub.2 as the pairing agent. This may be particularly advantageous for large scale separations where cleanup from the pairing agent (HClO.sub.4.sup.−) can be difficult or costly. FIG. 22 shows the separation of 3 different chiral amines on a LARIHC CF6-P (4.6 mm i.d.×15 cm, 5 μm fully porous particles, AZYP, LLC). No separation was found to occur without the presence of CO.sub.2 or a base such as triethylamine (FIG. 23).

3.5 Amino Acid Separation

[0137] The separation and determination of amino acids have been and continue to be important. Amino acids have both cationic and anionic functionalities. The net charge is dependent on the pH. This allows the use of cationic or anionic separation columns. If an anion exchange column is used, elution is first carried out using HCO.sub.3.sup.− which is a weak eluent before switching to CO.sub.3.sup.2−, a much stronger eluent. Analytes tend to concentrate in the zone between the two as is observed with phenylalanine, glutamine and arginine in FIG. 26A. (It is possible to operate with ammonium carbonate entirely, just go from very low to higher concentrations.) A cation exchange column on the other hand allows a single eluent ion: NH.sub.4.sup.+. In addition there are other volatile weak acids that can extend the pH range even lower (e.g. formic acid). Similar volatile bases are not as readily available. A mass spectrogram shows a separation of a number of amino acids on a Dionex™ IonPac™ 4 mm inner diameter column set are shown in FIG. 25A. NH.sub.4.sup.+ was found to be a fairly strong eluent ion on this particular column. To reduce the concentration at the beginning of a gradient separation, the Engasser was first purged with air. 20 mM formic acid in the eluent then converted the column into the weaker H.sup.+ form and served as the primary eluent at the start of the separation. The gradient program pressure readback can be seen in FIG. 25B. This however creates the same problem observed with the anion column where there is a transition zone in which analytes may concentrate, though the effect was less pronounced in going from H.sup.+ to NH.sub.4.sup.+ than from HCO.sub.3.sup.− to CO.sub.3.sup.2−. Leucine and norleucine were found to elute in the transition zone as seen in FIG. 25A. The transition was created using a controlled brief pulse of NH.sub.3 to the Engasser shortly after the start of the run. The top trace is the total ion chromatogram of all the monitored mass to charge (m/z) signals. Extracted signals at the relevant m/z for each amino acid are provided in the lower traces to identify the amino acid. Because NH.sub.3 and CO.sub.2 are both volatile and of high purity, coupling to mass spectrometry is simple.

[0138] When using an anion exchange column for amino acid separation as shown in FIG. 26A, phenylalanine was observed to elute where the gradient is transitioning from bicarbonate to carbonate so it is focused at the transition front. The same is true of arginine. The column was an IonPac PA-10 2 mm. The flow was split 15× to dilute the concentration of the eluent but still most of the amino acids elute very early. The pressure gradient is shown in FIG. 26B. The top trace in the chromatogram is the total ion chromatogram obtained on a mass spectrometer. The lower traces are marked with symbols and are the extracted ion chromatograms at the relevant m/z ratio to identify the eluting amino acids.

[0139] The role of CO.sub.2, formic acid, and air purge were explored and are provided in FIG. 27, FIG. 28, and FIG. 29 respectively. While the separation without CO.sub.2 is still of some use, its absence reduces the upper ionic strength limit and thus elution strength at a given pH, as well as buffering in neutral to alkaline pH. This is expected to play a more crucial role with larger peptides and biomolecules. Formic acid was found to be necessary to lower the pH below that obtained using only CO.sub.2 in order to increase retention of amino acids. The air purge as described previously lowers the NH.sub.3 concentration and allows elution by H.sup.+, a weaker eluent. FIG. 30 compares all 4 separation conditions.

3.6 Monoclonal Antibody Separation

[0140] A pH separation of a monoclonal antibody lysate is shown in (FIG. 32) using a Thermo Scientific™ Dionex™ MAbPac™ SCX-10 2 mm column. Column effluent was monitored by UV absorbance detection. The separation is in good agreement with that provided as reference (FIG. 31). The gradient is shown in FIG. 33. A large peak eluting at 60 minutes is caused by the gradient transition to CO.sub.3.sup.2− from HCO.sub.3.sup.−. Longer monitoring wavelengths don't show this peak as prominently but are less sensitive to the monoclonal antibody. The concentration used was 0.5 mg/mL.

3.7 Protein Separation

[0141] A separation of 5 proteins was also performed using a pH gradient on the Thermo Scientific™ Dionex™ MAbPac™ SCX-10 column. The proteins were Myoglobin, Cytochrome C, Bovine, Ubiquitin, Lactalbumin, and Cytochrome C (Equine). FIG. 34 provides reference molecular weights and isoelectric point (pI) values. The proteins eluted from the column (FIG. 35) in order of ascending pI values as the gradient (FIG. 36) pH was increased. The total ion chromatogram of scans of m/z 500-1500 is shown in FIG. 35. Spectra of the identified peaks are provided in FIG. 37-41. Protein identification based on molecular weight shows that the proteins are eluted in order of increasing pI with the exception of the cytochrome c proteins which have nearly identical pI but different affinities for the stationary phase.