111111
`
`1111111111111111111111111111111111111111111111111111111111111111111111111111
`US 20050011836Al
`
`(19) United States
`(12) Patent Application Publication
`Bidlingmeyer et al.
`
`(10) Pub. No.: US 2005/0011836 A1
`Jan. 20, 2005
`( 43) Pub. Date:
`
`(54) ADDITIVES FOR REVERSED-PHASE HPLC
`MOBILE PHASES
`
`(52) U.S. Cl. ............................................ 210/656; 210/639
`
`(76)
`
`Inventors: Brian Bidlingmeyer, Frazer, PA (US);
`Qunjie Wang, Hock ssin (DE)
`
`(57)
`
`ABSTRACT
`
`Correspondence Address:
`AGILENT TECHNOLOGIES, INC.
`Legal Department, DL429
`Intellectual Property Administration
`P.O. Box 7599
`Loveland, CO 80537-0599 (US)
`
`(21) Appl. No.:
`
`10/621,953
`
`(22) Filed:
`
`Jul. 17, 2003
`
`Publication Classification
`
`(51)
`
`Int. Cl? ..................................................... BOlD 15/08
`
`The present invention provides silica-based reversed-phase
`HPLC methods that lead to higher retention of the analytes
`in the column and longer column lifetimes than usually
`observed under medium to high pH aqueous mobile phase
`conditions. The inventive methods comprise eluting the
`HPLC column using an aqueous mobile phase comprising at
`least one fluorinated additive. Preferred additives are poly(cid:173)
`fluorinated alcohols such as 2,2,2-trifluoroethanol and 1,1,
`1,3,3,3-hexafluoroisopropanol. The methods of the present
`invention may be used for analyzing, separating, purifying,
`and/or isolating small organic molecules, natural products,
`as well as biomolecules such as polypeptides, oligonucle(cid:173)
`otides and polynucleotides (e.g., DNA fragments).
`
`1
`
`MTX1011
`
`

`

`Patent Application Publication Jan. 20, 2005 Sheet 1 of 4
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`US 2005/0011836 Al
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`Patent Application Publication Jan.20,2005 Sheet 2 of 4
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`Patent Application Publication Jan. 20, 2005 Sheet 3 of 4
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`US 2005/0011836 Al
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`Patent Application Publication Jan. 20, 2005 Sheet 4 of 4
`
`US 2005/0011836 Al
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`
`

`

`US 2005/0011836 A1
`
`Jan.20,2005
`
`1
`
`ADDITIVES FOR REVERSED-PHASE HPLC
`MOBILE PHASES
`
`BACKGROUND OF THE INVENTION
`
`[0001] High-Performance
`Chromatography
`Liquid
`(HPLC) is one of the most widely used separation tech(cid:173)
`niques (1. G. Dorsey et al.,Anal. Chern. 1998,70: 591-644);
`and its applications range from industrial preparation to
`trace level detection. The popularity of HPLC stems from its
`ability to separate a large variety of analytes, including
`organics, inorganics and biomaterials with low to high
`molecular weights, and with different degrees of polarity,
`hydrophobicity, acidity, and ionization (1. Swadesh, HPLC.
`Practical and Industrial Applications, CRC Press, Boca
`Raton, 2001; 1. S. Fritz,Ion Chromatography, Wiley-VCH,
`Weinheim, 2000). The development of highly sophisticated
`HPLC procedures has provided a sensitivity that allows
`researchers to separate enantiomers (A. Satinder, Chiral
`Separations by Chromatography, Oxford University Press,
`Washington, D.C., 2000) and even isotopes; to detect muta(cid:173)
`tions in DNA fragments; and to achieve resolution of very
`similar polypeptides that differ by a single amino acid
`residue. The vast majority of biomedical and environmental
`analyses are currently performed by HPLC; and in the
`pharmaceutical industry, where it is the premier analytical
`technique, HPLC is used in all phases of drug discovery,
`development, and quality control.
`
`[0002] Chromatography may be defined as the separation
`of analyte molecules based on their differing affinities for
`distinct phases in relative motion. Essentially, in HPLC,
`components of a sample mixture are carried through a solid
`stationary phase by the flow of a liquid mobile phase, that is
`pumped under controlled conditions of high pressure. The
`stationary phase, which is composed of a packed bed of
`finely divided beads or particles contained within a chro(cid:173)
`matography column, acts as a retentive media and differen(cid:173)
`tially retards the migration of the analytes through the
`column so that the individual components of the mixture are
`gradually separated from one another and ultimately exit the
`column at different time points of the chromatographic run.
`
`[0003] Successful chromatography requires a proper bal(cid:173)
`ance of the intermolecular forces between the analyte, the
`mobile phase, and the stationary phase. In HPLC, both the
`mobile phase and stationary phase may be varied to alter the
`interaction mechanism(s). The important criteria to consider
`for HPLC method development are resolution, sensitivity,
`precision, accuracy, limit of detection, linearity, reproduc(cid:173)
`ibility, time of analysis and robustness of the method. The
`quality of the column itself, and its performance over time,
`contribute in important ways to each of these criteria.
`
`[0004]
`In spite of recent advances in the development of
`alternative stationary phases such as zirconia, alumina,
`titania, and polymer-based packings (K. K. Unger, Packings
`and Stationary Phases in Chromatographic Techniques,
`Marcel Dekker, New York, 1990), microparticulate silica is
`by far the most commonly employed chromatographic sup(cid:173)
`port in HPLC. This is mainly due to its versatility, high
`derivatization potential, high column efficiency, and easily
`controlled particle size and porosity. Most of the silica -based
`packings in use today are bonded phases that are formed by
`covalently attaching organic molecules with specific prop(cid:173)
`erties to silanol groups (Si-OH) present on the silica
`
`surface. Of these silica gel-based support materials,
`reversed-phase (i.e., weakly polar or non-polar) bonded
`packings are generally preferred because of their high reso(cid:173)
`lution power, separation efficiency, and mechanical stability.
`
`[0005] Although superior to most other available pack(cid:173)
`ings, silica-based bonded phases remain imperfect supports
`for reversed-phase HPLC, especially in the analysis of basic
`substances. The derivatization process that produces the
`bonded phase is rarely fully complete and generally leaves
`unreacted a significant number of silanol groups on the silica
`surface. The presence of these residual weakly acidic sil(cid:173)
`anols leads to irreversible adsorption of basic solutes, high
`peak tailing, and a strong dependence of retention times on
`solute concentrations (H. Engelhardt et al., 1. Chromatogr.
`1988, 458: 79-92). Separations at high pH (i.e., pH 9 or
`greater) appear, therefore, attractive for certain basic ana(cid:173)
`lytes since, under these conditions, (1) such analytes would
`be in their free-base form, and (2) the unreacted weakly
`acidic silanol groups would be totally ionized, a situation
`where irreversible and potentially deleterious electrostatic
`interactions between solute molecules and stationary phase
`would be minimized. Furthermore, operating at a pH well
`above the pi(, value of basic compounds should also allow
`more
`reproducible separations, since
`retention
`times
`changes due to the formation of ionized forms would not
`take place (1. 1. Kirkland et al., 1. Chromatogr. 1997, 762:
`97-112; 1. 1. Kirkland et al., 1. Chromatogr. A, 1998, 797:
`111-120). However, using reversed-phase HPLC columns
`under intermediate to high pH aqueous conditions, espe(cid:173)
`cially at elevated temperatures, is known to result in rapid
`loss of column performance and in reduced column lifetimes
`due to deterioration of the packing material, largely through
`solubilization of the silica support (1. 1. Kirkland et al., 1.
`Chromatogr. A, 1995, 691: 3-19; C. S. Horvath et al., Anal.
`Chern. 1977, 49: 142-154; A Wehrli et al., 1. Chromatogr.
`1978, 149: 199-210).
`
`[0006] Several studies have been carried out to devise
`ways to extend the lifetime of reversed-phase HPLC col(cid:173)
`umns. Thus, the use of untreated silica pre-columns that
`partially saturate the mobile phase with dissolved silica,
`thereby reducing or precluding solubilization of the packing
`material in the analytical column, has been found to signifi(cid:173)
`cantly improve the longevity of a column (1. G. Atwood et
`al., 1. Chromatogr. 1979, 171: 109-115). Addition to the
`mobile phase of a large variety of certain additives and
`modifiers, such as organics, salts and detergents (L. C.
`Sander and S. A Wise, CRC Crit. Rev. Anal. Chern. 1987,
`18: 299), that interact with the analytes and/or the stationary
`phase, has been reported to increase the lifetime of HPLC
`columns by, for example, preventing irreversible adsorption
`of basic solutes to the reversed-phase support and/or by
`decreasing the solubility of microparticulate silica at inter(cid:173)
`mediate to high pH.
`
`[0007] Chemical approaches have also been developed to
`extend the lifetime of reversed-phase HPLC columns by
`shielding, eliminating, or reducing the number of residual
`silanol groups on the silica surface (K. K. Unger, Porous
`Silica, Elsevier, Amsterdam, N.Y., 1976; 1. Kohler and 1. 1.
`Kirkland, 1. Chromatogr. 1987, 385: 125-150; D. B. Mar(cid:173)
`shall et al., 1. Chromatogr. 1986, 361: 71-82; 1. G. Dorsey
`and W. T. Cooper, Anal Chern. 1994, 66: 857 A-867 A). Work
`has, for example, been directed at increasing the efficiency
`of the derivatization reaction in order to produce more
`
`6
`
`

`

`US 2005/0011836 Al
`
`Jan.20,2005
`
`2
`
`densely bonded silica supports with a smaller number of
`unreacted silanols. Silanol groups have also been partially
`removed by curing, a condensation reaction of adjacent
`silanols (T. G. Waddell et al., 1. Am. Chern. Soc. 1981, 103:
`5303-5307), or by end-capping, a chemical process in which
`relatively small reagents (such as activated silanes) are
`reacted with the remaining silanols (G. B. Cox, 1. Chro(cid:173)
`matogr. A, 1993, 656: 353-367; 1. 1. Kirkland et al., 1.
`Chromatogr. A, 1997, 762: 97-112). Bonding of bidendate
`silanes or bulky silanes (1. 1. Kirkland et al., Anal. Chern.
`1989, 61: 2-11) with tentacle-like chains (M. Ashri-Khoras(cid:173)
`sani, et al., Anal. Chern. 1988, 60: 1529-1533) has been
`shown to produce stationary phases with improved stability
`properties as compared with the commonly used monofunc(cid:173)
`tional packings. Similarly, polymeric (T. Darling et al., 1.
`Chromatogr. 1977, 131: 383-390; A 1. Alpert and F. E.
`Regnier, 1. Chromatogr. 1979, 185: 375-392) and horizon(cid:173)
`tally polymerized silanes (G. Schomburg et al., 1. Chro(cid:173)
`matogr. 1983, 282: 27-39; M. 1. Wirth and H. 0. Fatunmbi,
`Anal. Chern. 1993, 65: 822-826) have been found to exhibit
`some stability in high pH mobile phases. In a slightly
`different approach, treatment of silica particles with metal
`oxides or hydroxides, which results in the formation of a
`protective layer over the silica, has also been demonstrated
`to increase the lifetime of the stationary phase thus obtained
`(see, for example, U.S. Pat. No. 4,600,646).
`
`[0008] Although most of the modifications of the silica
`surface generate chromatographic supports with improved
`stability properties at medium to high pH, several of the
`chemical reactions cannot easily be applied to many of the
`currently commercially available columns.
`
`[0009] Therefore, a need continues to exist for improved
`strategies for extending the lifetime of silica gel-based
`reversed-phase HPLC columns. There is a particular need
`for approaches that are widely applicable, do not involve
`chemical modifications of the stationary phase, increase the
`retention of analytes in the column, and yet preserve the
`integrity of the HPLC column.
`
`[0010] Such strategies would for example be useful in the
`case of a particular superficially porous silica-based packing
`material that has been reported to show great performance
`for the separation of large molecules. This packing material,
`which is composed of particles made of a solid silica core
`and a macroporous shell, cannot practically be used at
`medium pH (i.e., 6-8) and at high temperature (i.e., >50° C.)
`since these conditions cause a dramatic shortening of its
`lifetime. Methods to increase the longevity of this packing
`material under the above conditions, which are typical for
`the analysis of some large biomolecules such as DNA,
`oligonucleotides, and some proteins, would considerably
`widen the range of applications of this chromatographic
`support. Furthermore, in the case of DNA fragments, it may
`be desired that the retention of the fragments take place in
`the order of the fragments' length regardless of their com(cid:173)
`position. A reverse order is sometimes observed due to
`interactions with silanol residues on the silica surface.
`Methods that would reduce or eliminate this problem and
`thus allow DNA fragment sizing are therefore highly desir(cid:173)
`able.
`
`SUMMARY OF THE INVENTION
`[0011] The present invention provides systems for per(cid:173)
`forming reversed-phase high-performance liquid chroma-
`
`tography analyses. In particular, the present invention
`encompasses reagents and strategies for efficiently separat(cid:173)
`ing components of a sample mixture by reversed-phase
`HPLC while avoiding problems linked to column instability
`such as those generally observed under intermediate to high
`pH aqueous conditions. More specifically, the present inven(cid:173)
`tion provides additives for aqueous mobile phases, and silica
`gel-based reversed-phase HPLC methods that lead to high
`retention of the analytes in the column and to extended
`column lifetimes.
`
`[0012]
`In one aspect, the present invention provides meth(cid:173)
`ods for analyzing, separating, isolating, and/or purifying
`components of a sample mixture by silica-based reversed(cid:173)
`phase HPLC comprising the step of eluting the HPLC
`column, which is packed with a superficially porous silica(cid:173)
`based reversed-phase support and loaded with the sample,
`mixture using an aqueous mobile phase comprising less than
`10% by volume of at least one additive. In these methods,
`the presence of the additive in the mobile phase leads to an
`increased column lifetime as compared with the lifetime
`observed in the absence of the additive, all other conditions
`being equal. Additionally or alternatively, the presence of
`the additive in the mobile phase leads to a higher retention
`in the column of at least one component of the mixture as
`compared with the retention observed for the same compo(cid:173)
`nent in the absence of the additive, all other conditions being
`equal.
`
`[0013]
`In certain preferred embodiments, the additive is a
`neutral, polar fluorinated organic modifier. Preferably, the
`fluorinated organic modifier is a polyfluorinated alcohol.
`Polyfluorinated alcohols for use in the present invention
`include, but are not limited to, 2,2-difluoroethanol; 2,2,2-
`trifluoroethanol; 3,3,3-trifluoropropanol; 1H,1H -dihydro(cid:173)
`penta-fluoropropanol;
`1,1,1,3,3,3-hexafluoroisopropanol;
`perfluoropropanol; 2-methy 1-1, 1,1 ,3,3,3-hexafluoro-2-pro(cid:173)
`panol; 4,4,4-trifluoro-1-butanol; 3,3,4,4,4-penta-fluoro-2-
`butanol; 1H,1H -dihydroheptafluoro-1-butanol; 2,2,3,3,4,4,
`4-heptafluoro-1-butanol; and perfluoro-1-butanol. Preferred
`polyfluorinated alcohols for use in the inventive HPLC
`methods include 2,2,2-trifluoroethanol; 1,1,1,3,3,3-hexa(cid:173)
`fluoroisopropanol; and combinations thereof.
`
`[0014]
`In some embodiments, the pH of the aqueous
`mobile phase is between 2 and 11. Preferably, the pH of the
`aqueous mobile phase is between 6 and 8. In addition to the
`fluorinated additive, the mobile phase may further comprise
`one, or more than one, modifier selected from the group
`consisting of a buffering agent, an ion-pairing agent, a
`multivalent cation binding agent, a surfactant, and a water(cid:173)
`soluble organic solvent. The mobile phase may be run
`through the HPLC column using an isocratic elution or a
`gradient elution.
`
`[0015] Preferably,
`is
`the silica -based bonded phase
`selected from the group consisting of C4 (butyl), C8 ( octyl),
`C18 ( octadecyl), CN ( cyano ), and phenyl.
`
`[0016] The inventive methods may be used to analyze,
`detect, separate, isolate, and/or purify any compound, agent,
`or molecule that can be loaded on and eluted through a silica
`gel-based reversed-phase HPLC column. In certain embodi(cid:173)
`ments, at least one of the components of the mixture is
`detected as it elutes from the column as a solution in the
`mobile phase. In other embodiments, the HPLC method
`allows the analysis of at least one component of the mixture.
`
`7
`
`

`

`US 2005/0011836 Al
`
`Jan.20,2005
`
`3
`
`In still other embodiments, at least one of the components of
`the mixture is collected in a distinct fraction as it emerges
`from the column as a solution in the mobile phase. In yet
`other embodiments, the HPLC method allows the prepara(cid:173)
`tive isolation of at least one component of the mixture.
`
`[0017] The inventive methods may, in particular, be used
`to analyze, detect, separate, isolate, and/or purify small
`organic molecules, natural products, as well as biomolecules
`such as polypeptides, polynucleotides, and oligonucleotides.
`
`In certain preferred embodiments, the inventive
`[0018]
`HPLC methods are used for analyzing, detecting, separating,
`isolating, and/or purifying DNA fragments. The additive in
`the aqueous mobile phase is preferably a polyfiuorinated
`alcohol selected from the group consisting of 2,2,2-trifiuo(cid:173)
`roethanol; 1,1,1,3,3,3-hexafiuoroisopropanol; and combina(cid:173)
`tions thereof. The mobile phase may further comprise at
`least one ion-pairing agent, at least one multivalent cation
`binding agent, and at least one water-soluble organic sol(cid:173)
`vent. In preferred embodiments, the ion-pairing agent is a
`trisubstituted ammonium salt; the multivalent cation binding
`agent is ethylenediaminetetraacetic acid (EDTA); and the
`water-soluble organic solvent is acetonitrile. The mobile
`phase may be run through the HPLC column using an
`isocratic elution or a gradient elution. Preferably, a gradient
`is used.
`
`[0019] Other aspects, features and advantages of the
`present invention will become apparent from the following
`detailed description. It should be understood, however, that
`the detailed description and specific examples, while indi(cid:173)
`eating preferred embodiments of the invention, are given by
`way of illustration only.
`
`BRIEF DESCRIPTION OF THE DRAWING
`
`[0020] FIG. 1 illustrates the effects of column aging on the
`separation of DNA fragments. Part A of FIG. 1 shows a
`chromatogram obtained by injecting a pbr 322 Hae III DNA
`digest sample (2 ,uL) on a Poroshell C18 HPLC column. The
`conditions of the chromatographic run were as follows:
`Mobile phase, solvent A: 0.1 M TEAA/0.1 mM EDTA,
`pH=7, solvent B: A in 25% acetonitrile; Gradient: 40-80% B
`in 30 minutes; Flow rate: 0.25 mL/mn; Column temperature:
`50° C. Part B of FIG. 1 shows a chromatogram obtained
`under the same experimental conditions 62 hours after that
`displayed in part A Between the two injections of DNA
`digest, the column was "aged" in a controlled manner as
`described in Example 1.
`[0021] FIG. 2 illustrates the effects of column aging on the
`separation of DNA fragments when 1,1,1,3,3,3-hexafiuor(cid:173)
`oisopropanol is present in the mobile phase. Part A of FIG.
`2 shows a chromatogram obtained by injecting a pbr 322
`Hae III DNA digest sample (2 ,uL) on a Poroshell C18 HPLC
`column. The conditions of the chromatographic run were as
`follows: Mobile phase, solvent A: 0.1 M TEAA/0.1 mM
`EDTN0.08 M hexafiuoroisopropanol, pH=7, solvent B: A in
`25% acetonitrile; Gradient: 60-100% Bin 30 minutes; Flow
`rate: 0.25 mL/mn; Column temperature: 50° C. Part B of
`FIG. 2 shows a chromatogram obtained under the same
`experimental conditions 62 hours after that displayed in part
`A Between the two injections of DNA digest, the column
`was "aged" in a controlled manner in the presence of
`1,1,1,3,3,3-hexafiuoroisopropanol as described in Example
`2.
`
`[0022] FIG. 3 illustrates the effects of column aging on the
`separation of DNA fragments when 2,2,2-trifiuoroethanol is
`present in the mobile phase. Part A of FIG. 3 shows a
`chromatogram obtained by injecting a pbr 322 Hae III DNA
`digest sample (2 ,uL) on a Poroshell C18 HPLC column. The
`conditions of the chromatographic run were as follows:
`Mobile phase, solvent A: 0.1 M TEAA/0.1 mM EDTN0.08
`M trifiuoroethanol, pH=7, solvent B: A in 25% acetonitrile;
`Gradient: 60-100% Bin 30 minutes; Flow rate: 0.25 mL/mn;
`Column temperature: 50° C. Part B of FIG. 3 shows a
`chromatogram obtained under the same experimental con(cid:173)
`ditions 62 hours after that displayed in part A Between the
`two injections of DNA digest, the column was "aged" in a
`controlled manner in the presence of trifiuoroethanol as
`described in Example 3.
`
`[0023] FIG. 4 illustrates the effects of the presence of
`1,1,1,3,3,3-trifiuoroisopropanol in the mobile phase on the
`separation of two single strain oligonucleotides (a 23-mer
`and a 25-mer). Part A of FIG. 4 shows a chromatogram
`obtained by injecting a mixture of the two oligonucleotides
`on a Poroshell C18 HPLC column. The conditions of the
`chromatographic run were as follows: Mobile phase, solvent
`A: 0.1 M TEAA/0.1 mM EDTA, pH=7, solvent B: A in 25%
`acetonitrile; Gradient: 20-55% Bin 5 minutes; Flow rate: 0.5
`mL/mn; Column temperature: 20° C. The chromatogram
`presented in part B of FIG. 4 was obtained under the same
`experimental conditions with the exception that the column
`temperature was 50° C. instead of 20° C.; and the gradient
`was: 20-55% B in 8 minutes. Part C of FIG. 4 shows a
`chromatogram obtained as in part B except that solvent A
`was 0.1 M TEAA/0.1 mM EDTN0.08 M hexafiuoroisopro(cid:173)
`panol.
`
`DEFINITIONS
`
`[0024] Throughout the specification, several terms are
`employed, that are defined in the following paragraphs.
`
`[0025] The term "HPLC" has herein its art understood
`meaning and refers to a particular type of liquid chroma(cid:173)
`tography that characteristically operates under high-pressure
`conditions. In a liquid chromatography system, the mol(cid:173)
`ecules of "analytes" (i.e., compounds or agents under inves(cid:173)
`tigation) interact with two non-miscible phases: a "station(cid:173)
`ary phase" (the solid support contained within a column
`chromatography), and a "mobile phase" (the liquid media
`that is run through the column and acts as a carrier for the
`analytes ). Preferred stationary supports for use in the present
`invention are silica gel-based reversed-phase packings. In
`the context of the present invention, analytes may be any
`chemical or biological molecule, compound, agent, or moi(cid:173)
`ety that can be analyzed by silica-based reversed-phase
`HPLC. Analytes (also called solutes) include, but are not
`limited to, small organic molecules, natural products, and
`biomolecules such as polypeptides, polynucleotides and
`oligonucleotides. Particularly preferred analytes are DNA
`molecules.
`
`[0026] The term "reversed-phase", as used herein to char(cid:173)
`acterize a mode of chromatography, refers to a method or a
`process in which the mobile phase is more polar than the
`stationary phase. In reversed-phase chromatography, hydro(cid:173)
`phobic compounds that exhibit more affinity for the hydro(cid:173)
`phobic, less polar stationary phase than for the mostly
`aqueous mobile phase, elute less quickly than do hydrophilic
`
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`compounds. Reversed-phase chromatography is generally
`used for the study of non-polar or weakly polar water(cid:173)
`soluble molecules; however, more polar molecules can also
`be separated by using a more polar bonded phase or other
`techniques such as ion-pair chromatography. In contrast, the
`term "normal phase", refers to a method or a process in
`which the mobile phase is less polar than the stationary
`phase. In normal phase chromatography, hydrophobic ana(cid:173)
`lytes that exhibit more affinity for the mobile phase than for
`the stationary phase, elute more quickly than do hydrophilic
`compounds. Normal phase chromatography is generally
`used for the study of organic molecules that are not soluble
`in water or aqueous solutions.
`
`[0027] The term "superficially porous stationary support",
`as used herein, refers to a solid support composed of a
`packing material having surface pores. More specifically, the
`particles of solid support have a solid core and a thin porous
`shell.
`
`[0028] The term "aqueous mobile phase", as used herein,
`refers to a liquid that is mainly aqueous (i.e., non-organic) in
`character. In the context of the present invention, an aqueous
`mobile phase may comprise a certain amount of organic
`solvent, as well as other additives and modifiers. The amount
`of organic solvent may vary (for example, from 0 to 100%)
`during the chromatographic run. The term "organic solvent",
`as used herein, refers to any organic (i.e., non-aqueous)
`liquid that is suitable for use in reversed-phase chromato(cid:173)
`graphic separations. Preferably, the organic solvents for use
`in the present invention are polar (e.g., more polar than the
`reversed-phase stationary support material) and water
`soluble.
`
`[0029] The mobile phase may be eluted through the chro(cid:173)
`matography column using an isocratic elution or a gradient.
`The terms "gradient" and "gradient elution" are used herein
`interchangeably. They refer to an elution process that
`involves changes in the mobile phase composition during
`the chromatographic run. The mobile phase composition
`may be changed continuously or stepwise during the elution
`process. The composition may be varied to modify the
`character or properties (e.g., elution strength, polarity or pH)
`of the mobile phase. A gradient elution is generally used to
`decrease the time necessary to separate components of a
`mixture. In an "isocratic elution", the composition of the
`mobile phase remains essentially constant for any part or all
`of the duration of the chromatographic separation process,
`however, the concentrations of the mobile phase compo(cid:173)
`nents may vary. Thus, the term "isocratic" refers to an
`elution process in which the concentrations of the compo(cid:173)
`nents of the mobile phase are maintained constant (i.e.,
`within ±0.1 %, preferably within ±0.05%) throughout the
`separation.
`
`[0030]
`In a HPLC system, a detector is generally placed
`downstream from the analytical column, and is used to
`continuously monitor whole or part of the eluant exiting the
`column. A "detector" typically responds to a property of the
`analyte (e.g., absorbance, fluorescence, chemiluminescence,
`optical activity, radiochemical or electrochemical property,
`mass) or to a property of the mobile phase (e.g., index of
`refraction, thermal conductivity).
`
`[0031] The term "chtromatogram" has herein its art under(cid:173)
`stood meaning. It refers to a plot corresponding to the
`detector response as a function of time. Ideally (i.e., in the
`
`absence of co-elution) each peak in a chromatogram corre(cid:173)
`sponds to a single analyte. The elution time of the analyte (or
`retention time of the peak) provides evidence for the identity
`of the analyte (qualitative analysis) and the height or area of
`the peak can be related to the concentration of the analyte
`(quantitative analysis).
`
`[0032] The term "retention time" refers to a measure of the
`elution time of an analyte (i.e., the time needed, after
`injection, for an individual solute to reach the detector). The
`retention time reflects the extent of retention (or retardation)
`undergone by a particular analyte in a specific HPLC column
`under given mobile phase and elution conditions (the higher
`the retention time, the stronger the interactions between the
`chromatographic support and the solute). When comparing
`the retention times of a given compound or molecule using
`different chromatographic systems (e.g., different stationary
`phases and/or different mobile phases), it is necessary to
`correct the retention time values for the time it takes the
`mobile phase alone to reach the detector.
`
`[0033] The terms, "durability", "longevity" and "lifetime"
`are used herein interchangeably. They refer to a time period
`during which a HPLC column can be used without under(cid:173)
`going significant loss of performance. These terms also refer
`to the number of analytical injections that can be performed
`before observing significant loss in column performance.
`Column performance is a function of selectivity, specificity,
`stability and reproducibility.
`
`[0034] As used herein, the term "selectivity" refers to a
`parameter that measures the relative retention of two par(cid:173)
`ticular compounds on a given chromatography column (the
`higher the selectivity, the better the separation). When
`operating under constant experimental conditions, shifts in
`selectivity are an indication of problems with loss of bonded
`phase or column fouling.
`
`[0035] The term "specificity" refers to the ability of a
`chromatographic method to measure accurately and specifi(cid:173)
`cally an analyte of interest in the presence of other compo(cid:173)
`nents that may be expected to be present in the sample. It is
`a measure of the degree of interference from other ingredi(cid:173)
`ents, excipients,
`impurities, and degradation products,
`ensuring that a peak response is due to a single component
`only (i.e., that co-elution does not take place). In a separa(cid:173)
`tion, specificity is measured and documented by the reso(cid:173)
`lution, efficiency (i.e., plate count), and peak tailing.
`
`[0036] As used herein, the term "resolution" refers to the
`difference in retention of adjacent peaks divided by their
`average band width. Sufficient resolution between peaks is
`required for proper quantitation and efficient separation of
`different analytes.
`
`[0037] The terms "column efficiency" or "theoretical
`plates" are used herein interchangeably. They have their art
`understood meaning and refer to a measure of the efficiency,
`or resolving power, of a chromatography column. The
`efficiency of a column can be measured by several methods.
`A description and an evaluation of such methods have been
`reported by B. A Bidlingmeyer and F. V. Jr. Warren (in:
`Anal. Chern. 1984, 56: 1583-1596). The most common
`reasons for loss of column efficiency (or theoretical plates)
`are column voiding and column fouling. Decreasing effi(cid:173)
`ciency leads to broadening of the peaks in the chromato(cid:173)
`gram.
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`[0038] The term "tailing", as used herein, refers to a peak
`that does not exhibit a Gaussian (symmetric) shape, but in
`which the front part is steeper than the rear. Tailing may be
`caused by column voiding or by the presence of sites on the
`packing material exhibiting a stronger than average retention
`for the solute.
`[0039] The term "stability", as used herein, refers to the
`ability of a chromatography column to retain its specificity,
`selectivity, and reproducibility properties over a long period
`of time.
`[0040] The term "reproducibility", as used herein, is a
`measure of the degree of repeatability of a HPLC method
`under normal operation. It is usually expressed as the
`percent relative standard deviation for a statistically signifi(cid:173)
`cant number of samples.
`[0041] The term "column voiding", has herein its art
`