WO2002056991A2

WO2002056991A2 - Systems and methods for performing electrokinetic chromatography

Info

Publication number: WO2002056991A2
Application number: PCT/US2002/001259
Authority: WO
Inventors: Wolfgang K. Goetzinger; Cai Hong
Original assignee: Arqule, Inc.
Priority date: 2001-01-16
Filing date: 2002-01-16
Publication date: 2002-07-25
Also published as: WO2002056991A3; AU2002239939A1

Abstract

Reagents and methods for performing electrokinetic chromatography are provided. The reagents of the invention comprise at least one organic acid and at least one organic base, and are substantially free of inorganic ions.

Description

SYSTEMS AND METHODS FOR PERFORMING ELECTROKINETIC CHROMATOGRAPHY

Background of the Invention

Analytical techniques for separating mixtures of chemical compounds have become increasingly powerful. Classical methods for separating compounds include gas chromatography (GC) and high pressure liquid chromatography (HPLC); these methods are widely used, but nevertheless are not sufficient to resolve mixtures of all types of compounds in a reasonable time. Recent developments in the field of separations science now permit the effective separation of complex mixtures of compounds through techniques such as capillary electrophoresis (CE) and electrokinetic chromatography (EKC). In both of these techniques, an applied electric potential results in differential migration of charged (ionic) compounds through a matrix or a bulk aqueous phase. In addition, the electric field causes the bulk aqueous phase to flow through the apparatus; this bulk flow, often termed electroosmotic flow or EOF, can carry analyte molecules, both charged and uncharged, through the analytical system.

In EKC, addition of a partitioning additive permits the analyte molecules to partition between the additive and the bulk aqueous phase; differential partitioning of analytes can result in changes in the relative elution times of analytes, which permits separation of compounds which might otherwise co-migrate through the system.

Unlike some electrophoretic techniques, EKC is capable of resolving both electrically charged (ionic) analytes and uncharged (neutral) analytes due to the combination of migration mechanisms which affect the analytes.

A common variety of EKC involves the use of a micelle-forming agent as the partitioning additive; in this variant, commonly known as MEKC, analyte molecules can partition between the bulk aqueous phase and the interior of the micelle. The micelle-forming agent can be anionic or cationic, although anionic micelle-forming agents are more common. A buffering agent is usually added to maintain a suitable pH range. For example, a typical reagent system may include sodium dodecyl sulfate (SDS) as the micelle-forming agent and an inorganic buffer salt such as sodium phosphate or sodium borate for pH control.

Unfortunately, typical MEKC buffers can suffer from disadvantages. For example, the presence in the buffer system of high concentrations of inorganic ions, such as sodium, results in high conductivity. The high concentration of inorganic ions tends to result in less EOF. Although lower rates of EOF can improve resolution of analytes under some circumstances, compounds will be eluted from the system more slowly as a result of the decreased bulk flow, resulting in longer analysis times. If a higher voltage is applied to reduce separation time, temperature gradients can form within the capillary tube, which can degrade the quality of the analytical separation. Zwitterionic buffers have been suggested for use in MEKC with SDS; such buffers avoid the addition of excessive amounts of inorganic ions due to the buffer, but the use of SDS will nevertheless result in the presence of a significant concentration of sodium ions in the aqueous phase.

U.S. Patent No. 6,083,372 (to Grover et al.) describes the use of a reagent for electrokinetic chromatography in which the buffer system includes a polycarboxylate for use with basic additives, or a polyamine base for use with acidic additives. This buffer system results in a lower conductivity for the aqueous phase, and therefore permits the use of a lower power level to perform the analysis. The '372 patent states that use of a diamine reagent results in a lower EOF and improved resolution. However, as noted above, lower EOF can result in longer analysis times.

Accordingly, improved buffer compositions for use in electrokinetic chromatography are needed.

Summary of the Invention

The present invention relates generally to improved reagents for performing electrokinetic chromatography.

It has now been discovered that the combination of an organic acid and an organic base, in proportions sufficient to provide a desired pH for the aqueous phase, results in an system suitable for electrokinetic chromatography. The systems of the invention can be used to provide rapid, high resolution separations of a variety of analytes, without requiring additional equipment. The low conductivity of the systems of the invention result in decreased power requirements; however, by selecting a suitable power level for use with a given system, the EOF can be adjusted to provide a rapid analysis time while maintaining acceptable resolution.

In one embodiment, the invention provides a reagent for analyzing a sample containing one or more analytes by electrokinetic chromatography. The reagent includes a buffer system having an organic acid and an organic base; the organic acid is preferably a carboxylic acid, more preferably a monocarboxylic acid, and the organic base is preferably an amine base, more preferably a monoamine.

In another embodiment, the invention provides methods for analyzing or purifying a sample containing one or more analytes by electrokinetic chromatography. The methods include the steps of providing an EKC apparatus which includes a reagent system of the invention; introducing a sample (which contains an analyte compound or a mixture of analyte compounds) into the EKC apparatus; and separating or purifying the compound (or compounds) by EKC, i.e., by application of an electric field to the EKC apparatus for a time sufficient to separate or purify the analyte(s).

Brief Description of the Drawings

Figure 1A is a chromatogram showing an MEKC separation of a mixture of three reference compounds using SDS in a borate buffer.

Figure IB is a chromatogram showing an MEKC separation of the same mixture of compounds as in Figure 1 A, using a lauric acid/Tris buffer.

Figure 1C is a chromatogram showing an MEKC separation of the same mixture of compounds as in Figure 1 A, using a cholic acid/ammonium hydroxide buffer. Figure 2 A is a chromatogram showing an MEKC separation of a mixture of five phenyl alcohols using SDS in a borate buffer.

Figure 2B is a chromatogram showing an MEKC separation of the same mixture of compounds as in Figure 2A, using a lauric acid/Tris buffer.

Figure 3 A is a chromatogram showing an MEKC separation of a mixture of four phthalate compounds using SDS in a borate buffer.

Figure 3B is a chromatogram showing an MEKC separation of the same mixture of compounds as in Figure 3 A, using a lauric acid/Tris buffer.

Figure 3C is a chromatogram showing an MEKC separation of the same mixture of compounds as in Figure 3 A, using a cholic acid/Tris buffer.

Figure 4 is a chromatogram showing an MEKC separation of a mixture of acidic compounds using a cholic acid/ammonium hydroxide buffer.

Figure 5 is a graph illustrating increase in current observed with increasing voltage for several buffer/reagent systems.

Figure 6 is a series of chromatograms showing MEKC separation under several conditions.

Figure 7 is a chromatogram showing the effect of different amine bases on an MEKC separation.

Figure 8 shows rapid MEKC separations using a lauric acid/Tris buffer.

Figure 9 is a chart illustrating detection efficiency with a mass spectrometer. Figure 10 shows tw.o MS scans from infusion of sample into the mass spectrometer.

Figure 11 shows single-ion MS detection of an MEKC separation.

Detailed Description of the Invention

Definitions For convenience, certain terms used in this specification are defined below.

The term "reagent system" or "buffer system" refers herein to a bulk aqueous phase which contains a buffer system including an organic anionic moiety and an organic cationic moiety, e.g., an organic acid and an organic base (or the deprotonated and protonated forms thereof, or mixtures thereof). The term "organic" is known to one of ordinary skill in the art, and, as used herein, refers to compounds or ions derived from carbon (except carbon dioxide and carbonic acid and its salts (carbonates)).

The "inorganic"is known to one of ordinary skill in the art, and, as used herein, refers to compounds or ions derived from elements other than carbon (although carbonates are considered to be inorganic for the purposes of this invention). Exemplary inorganic ions include cations such as sodium, potassium, lithium, barium, and the like; and anions such as chloride, bromide, iodide, sulfate, phosphate, borate, and the like.

The phrase "substantially free of inorganic ions", as used herein to refer to a reagent system, buffer or bulk aqueous phase, means a reagent, buffer or bulk aqueous phase which has a low concentration of inorganic ions, preferably such that the conductivity of the reagent, buffer, or bulk aqueous phase is less than about 5 mS/cm². In preferred embodiments, the concentration of any one inorganic ion (for example, sodium, potassium, borate, phosphate, chloride, and the like) is less than about 20 mM, more preferably less than about: 10 mM, 1 mM, 100 micromolar, 10 micromolar, or 1 micromolar. In other embodiments, the total concentration of inorganic ions is less than about 20 mM, more preferably less than about: 10 mM, 1 mM, 100 micromolar, 10 micromolar, or 1 micromolar. In certain embodiments, the concentration of (some or all) inorganic ions can be zero.

The term "bulk aqueous phase" or simply "aqueous phase" refers to a liquid phase (solvent) which contains at least some water, e.g., pure water or mixtures of water with other (preferably water-miscible) solvents, such as alcohols (e.g., methanol, ethanol, isopropanol, and the like), ethers (e.g., tetrahydrofuran, dioxane, and the like), dimethylformamide (DMF), N-methylpyrrolidone (NMP), acetone, 2- butanone, dimethylsulfoxide, and other solvents known in the art. The proportion of water to other solvents(s) in an aqueous phase can be varied and may range from 100% water (pure water) to mixtures in which water constitutes 90%, 80%, 70%>, 60%, 50%, 40%, 30%, 20% or 10% of the solvent mixture. The bulk aqueous phase is preferably chosen such that a micelle-forming agent can form micelles in the bulk phase, and such that analytes are able to partition between the bulk phase and the micelles. Choice of a suitable bulk aqueous phase and a micelle-forming agent is routine to one of ordinary skill in the art. Pure or substantially pure water is a particularly preferred bulk aqueous phase.

The term "micelle-forming agent", as used herein, refers to a compound that is capable of forming micelles in a bulk aqueous phase, or to a conjugate acid or base form of such a compound. For example, lauric acid is referred to herein as a micelle- forming agent, although micelles formed with lauric acid in a buffer system are primarily composed of laurate ions (with counterions for charge balance). Both laurate and lauric acid are referred to herein as micelle-forming agents. An "ionic micelle-forming agent" is a micelle-forming agent that bears a charge, e.g., a carboxylate anion.

I. Reagents and Buffer Systems

Abbreviations:

SDS: sodium dodecyl sulfate

Tris: tris(hydroxymethyl)aminomethane TEA: triethylamine DMSO: dimethylsulfoxide

In one aspect, the invention provides reagent systems (buffer systems) for use in EKC separations. The reagent systems comprise an ionic micelle-forming agent, together with a counterion-buffer, such that the micelle-forming agent and the counterion-buffer form a buffer. Preferred reagent systems are substantially free of inorganic ions, and have a low conductivity (preferably less than about 5 mS/cm²). In one embodiment, the micelle-forming agent is an organic acid, and the counterion- buffer is an organic base. In preferred embodiments, an organic micelle-forming agent is an organic monoacid, that is, a compound that has a single acid moiety. Although the discussion herein will refer for simplicity to carboxylic acids, one of ordinary skill in the art will recognize that other organic acids may be employed in the reagents and methods of the invention, including in addition to carboxylic acids, hydroxamic acids the like. In preferred embodiments, the acid moiety is a carboxylic acid (carboxylate).

Suitable carboxylic acids for use as micelle-forming reagents in the present invention include saturated, unsaturated, straight, branched, or cyclic acids, including, e.g., fatty acids preferably having from 8 to 20 carbons in the fatty acid chain, including lauric acid, myristic acid, palmitic acid, oleic acid, linoleic acid, arachidonic acid; cholic acid, and other carboxylic acids which are capable of forming micelles in aqueous phases. It will be understood that it may be possible to combine two or more carboxylic acids to create mixed micelles having characteristics suitable for use in the present invention, and such mixed micelles are within the scope of the invention. In certain embodiments, a carboxylic acid used as a component of the reagents or systems of the present invention is not an inner salt or zwitterion; for example, in certain preferred embodiments, the carboxylic acid is not an amino acid. For any carboxylic acid (or mixture of acids) used as a micelle-forming agent, the concentration of the acid(s) in the bulk phase should equal or exceed the critical micelle concentration (CMC) in that bulk phase, such that micelles are formed. The CMC can be determined, if desired, by methods well known in the art. Exemplary concentrations of micelle-forming carboxylic acids may be at least 5, 10, 25, or 50 mM, but are preferably not more than 200, 100, 75, or 50 mM. A preferred range is 25 - 50 mM.

Organic bases suitable for forming buffer systems (with an organic acid such as a carboxylic acid) include amine bases, such as (in preferred embodiments) monoamine bases (i.e., an amine having a single amino nitrogen atom) such as diisopropylamine, Tris, TEA, trimethylamine, diisopropylethylamine, and the like. The term "organic base", as used herein, also includes ammonia (or ammonium hydroxide). In certain embodiments, tertiary monoamine bases (i.e., an amine having a single amino nitrogen atom which has three alkyl or aryl groups attached) are particularly preferred. The organic base Tris is even more preferred. In certain embodiments, the amine base is not morpholine. Importantly, it has now been discovered that monoamine bases do not cause a reduction in EOF as large as the reduction caused by diamine bases. Thus, it is believed that monoamine bases can be used to obtain higher EOF and more rapid separations than are achieved under similar conditions with diamine (or other polyamine) bases.

It has been found that variation of the monoamine counterion with a micelle- forming organic acid can provide differing separations for mixtures of analytes. For example, a buffer system of lauric acid with Tris as the monoamine base gave somewhat different separation of a mixture of analytes than did a buffer system of lauric acid with TEA, or lauric acid with diisopropylamine. Thus, in some cases it may be possible to improve separation or resolution of compounds by changing the nature of the monoamine base.

In another embodiment, the micelle-forming agent can be an organic cationic moiety. Suitable organic cationic moieties for use as micelle-forming reagents include quaternary ammonium salts such as tetradecyltrimethylammonium, cetylpyridinium, hexadecyltrimethylammonium, and the like. In this case, an organic anion, such as a carboxylate ion (including, e.g., acetate, hexanoate, laurate, and the like, can be used as the counterion. In general, when an organic acid is used as a micelle-forming agent, an organic base can be used in varying amounts, depending upon the desired pH of the buffer system. In general, for each equivalent of carboxylic acid, at least one equivalent of an organic base will be present, to deprotonate the carboxylic acid. However, if a higher pH is desired (e.g., to ensure deprotonation of the acid or of analyte molecules), additional equivalents of base may be added. In a preferred embodiment, for each equivalent of organic acid present in the system, about 1 to about 2 equivalents of organic base (e.g., an amine) is present, with a 1:2 acid:base ratio being more preferred.

The concentration of the buffer system should be great enough to provide sufficient buffer capacity to maintain a suitable pH. It has been found that, for example, 50 mM lauric acid/ 100 mM Tris is a useful buffer system, pennitting the formation of micelles and also maintaining sufficient buffer capacity.

Advantageously, the reagents (buffer systems) of the present invention can also be used when it is desired to interface an EKC apparatus with a mass spectrometer. While it is possible to port an eluent stream into the inlet of a mass spectrum, the presence of non- volatile compounds or elements in the eluent will tend to cause the spectrometer inlet to become contaminated, and can cause significant degradation of the analyte signal in the MS. These difficulties can be reduced or eliminated by using only volatile reagents in the formulation of a bulk phase for use in EKC (or by using reagents which when combined yield volatile products). Thus, in a preferred embodiment, the components of the reagents of the invention are substantially volatile under MS conditions; that is, the components are all capable of being volatilized in an MS instrument. For example, ammonia and ammonium salts are often quite volatile; the low molecular weight and boiling point of ammonia result in high volatility. In selecting reagents for use in a buffer system which is to used with an MS interface, volatile carboxylic acids or amine bases are preferred. In addition to ammonia (NH₃), trimethylamine and triethylamine are relatively low- boiling amines that may be useful for MS applications. Similarly, shorter-chain carboxylic acids generally have a lower boiling point (and a higher vapor pressure at a given temperature and pressure) than do longer chain acids. Thus, a carboxylic acid having a short carbon chain may be preferable to a longer-chain acid (subject to the requirement, in MEKC systems in which the carboxylic acid is the micelle-forming agent, that the acid be capable of forming micelles in an aqueous phase).

One of ordinary skill in the art will appreciate that the reagent systems of the invention can be prepared in several ways. For example, a free carboxylic acid can be suspended in water, and an amine (as the free base) can be added. The resulting preparation can be said to include a carboxylic acid (such as lauric acid) and an amine base (such as ammonia or ammonium hydroxide), although at a pH greater than 7, substantially all of the carboxylic acid exists as the deprotonated carboxylate, while at least some fraction of the amine exists as the (protonated) conjugate acid of the amine base. A similar result could be obtained by dissolving an ammonium salt of a carboxylic acid in water; an example would be the use of ammonium laurate to form the aqueous phase; again, the carboxylic acid will be deprotonated at alkaline pH, and the amine will be at least partly protonated.

The reagents of the invention generally will have relatively low conductivity when compared to conventional MEKC reagents which include inorganic ions. For example, in preferred embodiments, the reagent systems of the present invention have a conductivity of less than about 5 mS/cm², more preferably less than about 3 mS/cm², still more preferably less than about 2 mS/cm² or less than about 1 mS/cm². The pH of a reagent system is preferably selected to permit efficient separation of analytes, while avoiding significant degradation of the analytes. The pH of the buffer should be selected to allow the appropriate form of an acid or base to exist in the reagent system. For example, lauric acid (protonated form) is not highly soluble in water and does not efficiently form micelles. However, the (deprotonated) laurate anion is soluble and can form micelles. Thus, when lauric acid is used as the micelle-forming agent, it is necessary to select a pH range at which significant amounts of laurate anion exist. At pH greater than about 7, the majority of the lauric acid is present in the form of laurate; thus, in this illustrative example, a buffer system should be selected to ensure that the pH is greater than 7. However, an excessively alkaline pH could cause degradation of analytes. In general, when a carboxylic acid is used as a micelle-forming reagent, a preferred pH range is from about 7 to about 12, preferably about 7 to about 10, more preferably from about 7.5 to about 9.5, and still more preferably from about 8.0 to about 9.0. If a cationic ammonium compound is used as a micelle-forming agent, a preferred pH range is from about 2 to about 8, more preferably about 2 to about 7.

In another aspect, the invention provides a kit for preparing a buffer system for use in EKC. The kits of the invention comprise (i) a container of an organic acid and a container of an organic base, or (ii) a container of an ammonium salt of an organic acid or (iii) a container containing a solid organic acid and a solid organic base. The organic acid and base, or the ammonium salt of the organic acid, are provided in an amount sufficient such that, when reconstituted in an aqueous phase, a buffer system is fonned and micelles are formed.

II. Methods

The present invention also provides methods for separating or purifying compounds by electrokinetic chromatography, and methods for preparing reagents useful for performing electrokinetic chromatography.

In one embodiment, the invention provides methods for separating or purifying compounds by EKC. The methods include the steps of providing an EKC apparatus which includes a reagent system of the invention; introducing a sample (which contains an analyte compound or a mixture of analyte compounds) into the EKC apparatus; and separating or purifying the analyte compound (or compounds) by EKC, i.e., by application of an electric field to the EKC apparatus (or capillary) for a time and under conditions (e.g., electric potential) sufficient to separate or purify the analyte(s). In a preferred embodiment, the analyte compound or compounds (or any impurity present) is detected by mass spectrometry. The reagents of the invention are suited for use in a variety of apparatuses for performing EKC separations. EKC equipment will generally include a power source for applying an electric potential to a capillary which contains the reagent system. EKC or capillary electrophoresis equipment is available commercially, for example, from Beckman, Inc. EKC is usually performed in a silica capillary, commonly a fused silica capillary; these are conventional and can be obtained from commercial sources (e.g., Polymicro Technologies). In preferred embodiments, EKC separations according to this invention can be performed at temperatures near ambient, e.g., at temperatures in the range between 15-35°C, more preferably about 25°C. The electrical potential applied to a capillary- can vary according to the type of capillary, the bulk phase,a nd other considerations known to the skilled artisan; exemplary electrical potentials are in the range of 10 - 50 kN for a 10 cm capillary.

Any suitable detector may be used to detect the presence of a compound in a sample. UN detectors can be used for compounds which are UN active. However, as mentioned above, the low conductivity reagent systems of the invention can be used to provide a mass spectrometer-compatible EKC system. Thus, in a preferred embodiment, the EKC system, including the reagent system of the invention, is connected to or interfaced with a mass spectrometer (MS), and an analyte (or analytes) is detected by MS. Interfaces suitable for connection between a capillary and a mass spectrometer are known in the art and are commercially available. The methods of the invention provide rapid, efficient separation or purification of analytes (compounds). In preferred embodiments, the separation or purification is accomplished in less than 10 minutes, more preferably less than 5 minutes, more preferably in less than 2 minutes, and even more preferably in less than about one minute. Such rapid separations can be used in high-throughput applications, for example, in laboratories in which hundreds or thousands of samples must be processed per day. One example of such an application is in the analysis or purification of the products of combinatorial synthesis (for another method of analyzing or purifying combinatorial libraries, see, e.g., U.S. Patent No. 5,968,361, which is incorporated herein by reference). Thus, in one embodiment, the analyte or analytes are components of a combinatorial synthesis or library.

The present methods provide good separation efficiency. In preferred embodiments, the separation efficiency is at least about 300,000 N/m (N is the number of theoretical plates), more preferably at least about 500,000, 700,000, 800,000, 900,000, or one million N/m. In another embodiment, the invention provides a methods for preparing a reagent system useful for performing electrokinetic chromatography. The method comprises the steps of providing an ionic micelle-forming agent; providing a counterion-buffer; and combining the ionic micelle-forming agent and the counterion- buffer under conditions such that the micelle-forming agent and the counterion-buffer form a buffer. In certain embodiments, the ionic micelle-forming agent and the counterion-buffer are combined in an aqueous phase. In other embodiments, it may be desirable to combine the micelle-forming agent and counterion-buffer in non- aqueous solvent systems (or no solvent at all); this reagent system could then be dissolved in an aqueous solvent system to form a buffer system suitable for performing EKC. Such pre-mixed systems may be provided in a container, as part of kit for use in EKC systems. Preferred reagent systems are substantially free of inorganic ions, and have a low conductivity (preferably less than about 5 mS/cm²). In one embodiment, the micelle-forming agent is an organic acid, and the counterion- buffer is an organic base.

The following non-limiting examples are provided to illustrate the present invention.

Examples

General Experimental

All MEKC separations were completed on a Beckman P/ACE system MDQ (Beckman Instruments, Fullerton, CA). The system was run using P/ACE workstation software version 1.6. UN detection was performed with a UN detector operated at 214 nm; it was found that a data rate of 32 Hz gave the sharpest peaks, especially for rapid separations. The cartridge coolant was thermostated at 25 °C. Fused silica capillaries of 50 mm and 75 mm ID. were purchased from Polymicro Technologies, Phoenix, AZ. Phenethylalcohol, 3-phenyl-l-propanol, 4-phenyl-l- butanol and 5-phenyl-l-pentanol were purchased from Aldrich (Milwaukee, WI). Lauric acid, cholic acid, sodium laurate, sodium cholate and Tris were purchased from Fluka (Milwaukee, WI). 100 mM SDS buffer was purchased from Hewlett Packard Company (Santa Clarita, CA), then diluted to 50 mM. The lauric acid/Tris buffer was prepared by adding 0.025 mole of lauric acid and 0.05 mole of Tris to 0.5L water. A similar procedure was used to prepare cholic acid/Tris buffer. Sodium laurate buffer was prepared by adding 0.005 mole of sodium laurate to 100 ml 25 mM sodium tetraborate stock. The same procedure was followed for preparing sodium cholate buffer. All the buffers were prepared fresh before use. The buffer pH was measured using an Accumet Research AR15 pH meter (Fisher Scientific, Pittsburgh, PA).

Example 1

The following instrument settings were used for this example: The voltage was 30kN; a fused silica capillary with 50 μm I.D. was used. Total capillary length was 60.2 cm; length from inlet to detector was 50 cm. Separation temperature was 25 °C. Pressure injection 1.5 psi second, with a data rate of 32 Hz.

Figure 1A shows an MEKC separation of a mixture of three neutral compounds (quality control standard mixture or QCSTD): 2-acetamidophenol, 2- hydroxydibenzofuran, and 3-(4-tert-butylphenoxy)benzaldehyde. The micelle- forming reagent was SDS (50 mM) in tetraborate buffer (25 mM), pH 9.25. It can be seen that the three compounds are well separated (the peak at about 3.4 minutes is a result of the injection solvent); the overall time for the analysis is more than thirteen minutes. Figure IB shows an MEKC separation of the same three compounds using lauric acid as the micelle-forming agent (50 mM) and Tris as the counterion-buffer (100 mM), pH 8.38. The compounds are fully resolved; the total time for analysis was less than seven minutes.

Figure 1C shows an MEKC separation of the same three compounds using cholic acid as the micelle-forming agent (50 mM) and ammonium hydroxide as the counterion-buffer (100 mM), pH 9.40. The compounds are fully resolved; the total time for analysis was less than seven minutes.

It can be seen that the separation is slower for the SDS-based system at the same applied voltage. The above results demonstrate that carboxylic acids can be used as micelle-forming agents, together with organic base counterions, to provide reagent systems suitable for rapid separation of compounds by MEKC.

Example 2

Figure 2 A shows an MEKC separation of a mixture of five neutral compounds: benzyl alcohol, phenethyl alcohol, 3-phenyl-l-propanol, 4-phenyl-l- butanol, and 5-phenyl-l-pentanol. The micelle-forming reagent was SDS (50 mM) in tetraborate buffer (25 mM), pH 9.25. The voltage was 30kN. It can be seen that the five compounds are well separated; the overall time for the analysis is more than ten minutes. The separation efficiency for the third peak (at about 7 minutes) is calculated to be about 380,000 Ν/m.

Figure 2B shows an MEKC separation of the same five compounds using lauric acid as the micelle-forming agent (50 mM) and Tris as the counterion-buffer (100 mM), pH 8.38. The compounds are fully resolved; the total time for analysis was less than six minutes. The separation efficiency for the third peak (at about 4 minutes) is calculated to be about 485,000 Ν/m.

It can be seen that the separation is slower for the SDS-based system at the same applied voltage. In addition, the separation efficiency of the lauric acid-bsed buffer system is higher than the SDS-based system. The above results demonstrate that carboxylic acids can be used as micelle-forming agents, together with organic base counterions, to provide reagent systems suitable for rapid separation of neutral, uncharged compounds by MEKC.

Example 3

Figure 3 A shows an MEKC chromatographic separation of a mixture of four phthalates: dimethylphthalate, diethylphthalate, dipropylphthalate, and dibutylphthalate. The micelle-forming reagent was SDS (50 mM) in tetraborate buffer (25 mM), pH 9.25. The voltage was 3 OkN. It can be seen that the four compounds are well separated (the peak at about 3.4 minutes is a result of the injection solvent); the overall time for the analysis is more than thirteen minutes. Figure 3B shows an MEKC separation of the same four compounds as in Figure 3A, using lauric acid as the micelle-forming agent (50 mM) and Tris as the counterion-buffer (100 mM), pH 8.38. The voltage was 30kN. The compounds are fully resolved; the total time for analysis was less than seven minutes. Figure 3C shows an MEKC separation of the same three compounds using cholic acid as the micelle-forming agent (50 mM) and Tris as the counterion-buffer (100 mM), pH 8.36. The voltage was 30kN. The compounds are fully resolved; the total time for analysis was less than nine minutes.

It can be seen that the separation is slower for the SDS-based system at the same applied voltage. The above results demonstrate that carboxylic acids can be used as micelle-forming agents, together with organic base counterions, to provide reagent systems suitable for rapid separation of neutral aromatic esters.

Example 4

For this example, a sample mixture containing several acidic compounds was separated: ibuprofen, naproxen, nicotinic acid, and phthalic acid.

The following instrument settings were used for this example: The voltage was 30kN; a fused silica capillary with 50 μm I.D. was used. Total capillary length was 60.2 cm; length from inlet to detector was 50 cm. Separation temperature was 25

°C. A 1.5 psi second injection was used, with a detector data rate of 32 Hz. The buffer system was 25 mM cholic acid with 50 mM ammonium hydroxide, pH 9.51.

The analytes were prepared as a mixture (0.1 mg/ml) in the buffer system.

The results are shown in Figure 4. It can be seen that all four acids were separated in a total analysis time of about 11 minutes. The elution order is ibuprofen

(first), naproxen, nicotinic acid, and phthalic acid (last). This example demonstrates that acidic compounds can be separated using a buffer system according to the invention.

Example 5

The relationship between current and field strength was investigated for several reagent systems. For this Example, five buffer systems were prepared: SDS: 50 mM SDS with 25 mM sodium setraborate, pH 9.25. Cholic Acid Sodium Salt: 50 mM sodium salt of cholic acid with 25 mM sodium tetraborate, pH 9.67. Lauric Acid Sodium Salt: 50 mM sodium salt of lauric acid with 25 mM sodium tetraborate, pH 9.23. Cholic Acid with Tris: 50 mM cholic acid with 100 mM Tris, pH 8.36. Lauric Acid w/ Tris: 50 mM lauric acid with 100 mM Tris, pH 8.38. All data were measured by using a 50 micron I.D. fused silica capillary at 25 °C.

Figure 5 illustrates the change in current as the separation voltage is increased in different buffer systems. A significant reduction of the current was observed by changing the buffer from traditional sodium salt buffers to lauric acid/Tris or cholic acid/Tris buffers. At 500 N/cm field strength using a 50 mM sodium salt lauric acid with 25 mM sodium tetraborate buffer, a current above 50 μA was observed. However, using 50 mM lauric acid with 100 mM Tris, a current less than 10 μA was detected under the same field strength. It is well known that separation efficiency increases with increasing field strength within the liner range of Ohm's law. By using low conductivity buffers, the analysis can be performed in a relatively high voltage range to improve separation efficiency and increase separation speed.

Example 6

The separation of a mixture of compounds was performed with SDS buffer (50 mM SDS / 25 mM borate) and with a buffer system of the invention (lauric acid 50 mM / 100 mM Tris) to compare the power levels and separation efficiency.

A 50 micron I.D. fused silica capillary was used for the separation. Total capillary length was 31.2 cm. The length from injection end to detector was 21 cm. Separation was performed at 25 ° C. Injection was at 0.6 psi second, detection was with a UN detector set at 214 nm, and a data rate of 32 Hz. The results are shown in Figure 6. It can be seen that both analysis time and separation efficiency are improved by switching from the SDS buffer system (bottom trace) to the lauric acid/Tris system (middle and top traces). Instrumental specifications suggest that the power applied to P/ACE_MDQ instruments during the separation should be less than 3 Watts per meter. When 16 kN was applied to the SDS buffer system, it generated 2.92 watts per meter, close to the instrument's limitation. On the other hand, when 16 kN was applied to the lauric acid/Tris system (middle trace), the power was only 0.55 watts per meter. Thus, the lauric acid/Tris system allowed for higher voltages to be used. For example, when 30 kN was applied to the lauric acid/Tris system (top trace), the power was only 2.1 watts, still below the manufacturer's specifications. The lauric acid system provides a faster separation at similar field strength than the SDS system. The top trace shows that the last peak was observed at 1.22 minutes using 30 kN and lauric acid/Tris buffer, as compared to 4.56 minutes with 16 kN and the SDS, nearly a four-fold improvement. Furthermore, comparing the separation efficiency using the last peak, the lauric acid system gave better performance (plate count is close to 1 million / meter in lauric acid system at 16 kN, 480,352 / meter in SDS system at the same voltage). Furthermore, the efficiency for the first peak in the separation using the SDS system is greater than the efficiency for the last peak in the separation with SDS, possibly due to the generation of excessive heat. However, the separation efficiency for the last peak in the separation with the lauric acid is greater than the efficiency for the first peak in the lauric acid- based separations (at both power levels), suggesting that excess heat buildup is not a problem for this system.

The EOF (units of 10^"4 cm²/N*s) was 5.0 for the SDS system, and 5.7 for both power levels with the lauric acid/Tris buffer. Separation efficiencies (Ν/m) for benzylalcohol were: SDS, 621,000; lauric acid/Tris at 16 kN applied, 810,000; lauric acid/tris at 30 kN applied: 708,000. Ν/second was 1090 (SDS), 1930 (lauric acid/Tris at 16 kN); 3080 (lauric acid/Tris at 30 kN).

It can be seen that the lauric acid system performed well compared to SDS in this case; in fact, the lauric acid system appeared to provide greater efficiency per time unit (more plates per second).

Example 7

Several monoamine bases were compared for use in creating a buffer system with lauric acid. Diisopropylamine, TEA, and Tris were compared a bases by separating a mixture of five compounds chosen to represent pharmaceuticals: guaiacol glyceryl ether, procainamide, caffeine, acetophenetidine, and tetracaine. The results are shown in Figure 7. It can be seen that not all bases resulted in a buffer system that separated all the analytes. In addition, separations with diisopropylamine and TEA as the base both showed some peak tailing. For this set of analytes, Tris was the best amine base for use in the buffer system.

Example 8

In this example, conductivity was calculated for several buffer systems. In the table below, the following abbreviations are used: LA is lauric acid; CA is cholic acid; Cap ID is capillary inside diameter; cap cm is capillary length. Conductivity was calculated according to the following formula: k=I*L/N*A

Table

Buffer pH Cap Cap Voltage CE Current Conductivity

ID (mS/cm) mm cm kN mA

1 50mMSDS/25mMΝa 9.25 50 60.2 30 54.5846 5.58 tetraborate

2 50mMLA/100mMTris 8.38 50 60.2 30 10.533 1.08

3 50mMLA/100mMTEA 11 50 60.2 30 17.6379 1.80

4 50mMLA/100mM 11.4 50 60.2 30 16.4331 1.68 Diisopropylamine

5 50mMLA/ 100 mM 9.65 50 60.2 30 28.423 2.91 NH₄OH

6 50mMCA/ 100 mM Tris 8.36 50 60.2 30 17.7111 1.81

7 50mMCA/100mMTEA 10.8 50 60.2 30 21.1994 2.17

8 50mMCA/100mM 11.2 50 60.2 30 21.268 2.17 Diisopropylamine

9 50mMCA/100mM 9.4 50 60.2 30 42.4582 4.34

NH₄OH

It was found that the calculated conductivity was lower for all of the buffer systems of the invention than for the SDS/borate system. Buffer systems using Tris as the organic base had the lowest calculated conductivity of the these buffer systems. Example 9 ^•

In this example, rapid separation of analytes was achieved by use of a short silica capillary. Four standard mixtures (describes above) were separated using a lauric acid/Tris buffer system. The samples were injected by "short end" injection on the capillary, so the effective length of the capillary was about 10 cm. It can be seen from Figure 8 that the separation for all four mixtures was rapid, with all separations being complete in under 0.8 minutes.

Example 10 For this example, a buffer system of the invention was tested to determine compatibility with MS detection.

The experiment was done with Quattro I mass spectrometer (Micromass, UK) with a positive electrospray ionization (ESI) interface. The ESI voltage was maintained at 3.5 kN. Cone voltage was set at 20 N. Sample was infused to the mass spectrometer through an infusion pump (Harvard Apparatus, Νatick, MA). The sample was premixed tetracaine in micellar buffer; the sheath liquid was MeOH/H₂O/CH₃COOH 80/20/1. The flow ratio of sample (in buffer) to sheath liquid was 1 :2. Infusion flow rate was 3 microliters per minute. The mass spectrometer was run at positive full scan from 175 - 300 amu. The scan rate was (0.5 sec / scan). Nebulizer gas flow was maintained at 0.5 L per min.

A buffer of 50 mM cholic acid A 00 mM ammonium hydroxide was compared to a buffer of 50 mM SDS/25 mM sodium tetraborate. The results are shown in Figures 9 and 10. When the sample was mixed with cholic acid buffer and then infused into the MS, the detection sensitivity was almost 10-fold greater than when the SDS buffer was used. Figure 10 shows the MS scan from 150 -300 m z for the SDS and cholic acid systems.

Example 11

In this example, a sample mixture was separated by MEKC using a buffer of the invention, and the compounds were detected by MS.

The experiment was performed with a Fimiigan TSQ 700 mass spectrometer. ESI positive voltage was maintained at 4.2 kN. Single ion monitoring was set at masses 180.0-181.0, 194.5-195.5, and 264.5-265.5. The scan rate was 0.2 second per scan. The sheath flow rate was 5 ul per minute. Auxiliary gas was set at 3-4 psi. The CE high voltage supply was a Spellman CZE 1000R (Hauppauge, ΝY). A 50 mm I.D. 27 cm long fused silica capillary was used for the separation. The separation voltage was 12 kN. Samples were introduced by manual pressure injection 5 second at 10 cm high. Separation buffer was 50mM cholic acid with 100 mM ΝH₄OH, pH 9.35. Sample concentration was 0.1 mg per ml each of acetophenitidin, caffeine and tetracaine in separation buffer. The results are shown in Figure 11. The single ion peaks corresponding to all three of the sample analytes were clearly detected (Figure 11) using the mass spectrometer.

The reagents and methods of the invention are useful for EKC in conjunction with MS detection.

Example 12

By allowing the use of reduced current for EKC separations (compared to standard SDS buffers), the buffers of this invention permit the use of capillaries having larger inner diameters. This can be advantageous for use with detectors such as ultraviolet (or visible-light) detectors; the wider capillary increases detection sensitivity.

For this example, the same relative amount of sample (a mixture of five phenyl alcohols) was injected into two capillaries containing a buffer system of the invention, one having an I.D. of 50 microns; the other having a 75 micron I.D. Fused silica capillaries were used, total length of the capillary 31.2 cm. The length from injector inlet to detector was 21 cm. Temperature was at 25 °, and the acquisition data rate was 32 Hz. Separation voltage was 15 kN; the samples were injected by electrokinetic Injection at 5 kN, 3 seconds. Separation buffer was 50 mM Lauric acid with 100 mM Tris, pH at 8.38. The voltage was 15kN; the power was 0.51 Watts/m for the 50 micron capillary and 1.09 Watts/m for the 75 micron capillary. Retention times for the phenyl alcohols were similar in the two capillaries under these conditions. As the capillary size was increased from 50 to 75 microns, the signal was observed to increase almost 2.5 times.

This example demonstrates that the sensitivity of detection can be improved by using lower current and a larger diameter capillary.

The contents of all references (including patents) cited herein are hereby incorporated by reference.

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not to limit the scope of the invention. Other aspects, advantages, and modifications are within the following claims.

Claims

What is claimed is:

1. A reagent system for separating or purifying by electrokinetic chromatography a sample containing at least one analyte compound, the reagent system comprising: an aqueous phase; an ionic organic micelle-forming agent in an amount effective to form micelles, such that an analyte compound can partition between the micelles and the aqueous phase; and an organic counterion present in an amount sufficient to provide charge balance for the ionic micelle-fonning agent; wherein the ionic organic micelle-forming agent and the organic counterion together comprise a buffer; and further wherein the reagent system is substantially free of inorganic ions.

2. The reagent system of claim 1, wherein the ionic organic micelle-fonning agent is a carboxylic acid and the organic counterion is a monoamine base.

3. The reagent system of claim 2, wherein the carboxylic acid is selected from the group consisting of lauric acid and cholic acid.

4. The reagent system of claim 4, wherein the monoamine base is selected from the group consisting of ammonia, Tris, triethylamine, and diisopropylamine.

5. A method for separating or purifying a compound or compounds by electrokinetic chromatography, the method comprising the steps of: providing an electrokinetic chromatography apparatus which includes a reagent system according to claim 1; introducing a sample which contains the compound or compounds into the electrokinetic chromatography apparatus; and separating or purifying the compound or compounds by electrokinetic chromatography.

6. The method of claim 5, wherein the ionic organic micelle-forming agent is a carboxylic acid and the organic counterion is a monoamine base.

7. The method of claim 5, wherein the presence of the compound or compounds is detected by mass spectrometry.