Patent Application
20140296485
Displacement chromatography (DC) in one of the three well-defined forms of column chromatography—elution, displacement, frontal. DC is principally a preparative method, but there are also analytical applications using “micropreparative” DC with packed “narrow-bore” or capillary columns.
Displacement chromatography may be carried out using any one of four general chromatographic methods when suitable, high purity displacer molecules are available. DC is used in (a) ion-exchange chromatography (cation-exchange, anion-exchange), (b) hydrophobic chromatography (reversed-phase, hydrophobic-interaction, hydrophobic charge-induction, thiophilic), (c) normal-phase chromatography including hydrophilic-interaction chromatography (HILIC) and (d) immobilized metal-ion affinity chromatography (IMAC).
With optimized DC, one may obtain, simultaneously, high purity (high resolution), high recovery (high yield) and high column loading (high capacity)—the latter much higher than overloaded preparative elution chromatography. In most cases, these advantages more than compensate for the disadvantages of DC (slower flow rates, longer run times, need for high purity displacers).
Displacement chromatography is carried out by choosing (a) an applicable chromatographic method, (b) a suitable chromatography column with proper dimensions, (c) proper mobile phase conditions, (d) a suitable displacer molecule and (e) suitable operation protocols with properly configured LC equipment. Initially, a suitable “weakly displacing mobile phase” (carrier) is chosen, and the column is equilibrated at a suitable flow rate. The carrier may contain a pH buffering compound adjusted to a useful pH value. Optimal displacement flow rates tend to be low, typically in the range of 35-105 cm/hr, though sometimes higher. A suitable amount of the sample solution is loaded onto the column at the sample-loading flow rate. The sample solution contains the material to be purified in the carrier along with the proper level of an ion-pairing agent or ion-pairing salt if the sample or displacer molecules are charged. Typical sample loadings are 50-80% of the operative breakthrough capacity. Next, a displacer mobile phase (displacer buffer), prepared from a suitable displacer compound at the proper concentration in the carrier solution, is pumped onto the column at the displacement flow rate until the displacer breakthrough is observed. The purified sample comes off the column before the displacer breakthrough front. Fractions from the column are collected and separately analyzed for content and purity. Finally, the displacer is removed from column using a “displacer removal solution”, and then the column is cleaned and regenerated to its original state for storage or for subsequent use.
Though different from elution chromatography, in some respects, displacement chromatography is easy to understand and easy to carry out. In DC, a sample is “displaced” from the column by the displacer, rather than “eluted” from the column by the mobile phase. When the output of the column is monitored online (e.g., via UV absorption, pH, or conductivity), a “displacement train” is obtained rather than an “elution chromatogram”. The displacement train is composed of side-by-side “displacement bands” rather than solvent-separated “elution peaks” in a chromatogram. When a displacement band is large enough to saturate the stationary phase, a trapezoidal “saturating band” Is formed. When a displacement band is not large enough to saturate the stationary phase, a small, triangular “non-saturating band” is formed. The height of a saturating band is determined by the binding isotherm at the point of operation; the area of a trapezoid-band or a triangle-band is proportional to the amount of the component.
Hydrophobic chromatography depends almost exclusively on the unique solvation properties of water that result from the highly structured, self-associated, hydrogen-bonded liquid. For conventional reversed-phase chromatography stationary phases (uncharged C18 column), binding is usually driven by entropy (+TΔS), which often must overcome unfavorable enthalpy (+ΔH). Thus, over the temperature ranges often used by chromatographers (10-70° C.), analyte-binding and displacer-binding often become stronger with increasing temperature. Another useful feature of hydrophobic chromatography is the use of additives that modify both the structure and strength of the self-hydrogen-bonding of the aqueous-based solvent. These additives include: salts (NaCl, K2HPO4, (NH4)2SO4), organic solvents (MeCN, MeOH, EtOH) and polar organic molecules (urea, oligo-ethyleneglycol) in chromatography buffers.
Hydrophobic displacement chromatography can be carried out using chiral analytes, chiral displacers and chiral chromatography matrices. Under these conditions, an achiral displacer may be used, but a racemic mixture of a chiral displacer cannot be used. Racemic chiral analytes can also be purified using an achiral chromatography column and an achiral displacer. In this case, impurities, including diastereomers, are removed from the racemic compound of interest, but there is no chiral resolution of the enantiomers. An anionic displacer may contain one or more chiral centers. With the proper choice of chiral chromatography matrix, mobile phase and achiral displacer, enantiomers are routinely resolved (separated) on a preparative scale. Depending on the specific circumstances, a good, enantiomerically pure, chiral displacer can have performance advantages over a good achiral displacer when carrying out a displacement separation of enantiomers on a chiral stationary phase.
Good hydrophobic displacer molecules should possess a unique combination of chemical and physical properties in order for them to function efficiently. Some soluble, hydrophobic molecules can function as displacers, but only a limited few function well. Many of the molecules described in this document fulfill the necessary requirements for well-functioning displacers.
Development of useful, reversed phase, preparative displacement chromatography has been hampered by the unavailability of suitable, high-purity displacer molecules. For example, U.S. Pat. No. 6,239,262 describes various reversed phase liquid chromatographic systems using low molecular weight surface-active compounds as displacers. U.S. Pat. No. 6,239,262 discloses an extremely wide range of possible charged moieties that may be coupled with hydrophobic moieties to form the disclosed surface active compounds used as displacers, but discloses that it is necessary to include a large proportion of organic solvent to mitigate the surface active properties of the disclosed displacers. The presence of such large proportions of organic solvents significantly alters the process, derogating from the benefits of reverse-phase hydrophobic displacement chromatography. In addition, the surface-active displacer compounds disclosed by U.S. Pat. No. 6,239,262 do not function well, resulting in relatively poor quality displacement trains in which a significant level of impurities may be present in the “purified” products.
We describe here new displacer molecules and methods to use them that have utility in various forms of hydrophobic displacement chromatography.
We have discovered and developed classes of negatively charged hydrophobic organic compounds, either salts or zwitterions, that uniquely possess that combination of chemical and physical properties necessary for good displacer behavior in hydrophobic displacement chromatography.
Accordingly, the present invention, in one embodiment, relates to a process for separating organic compounds from a mixture by reverse-phase displacement chromatography, comprising:
providing a hydrophobic stationary phase;
applying to the hydrophobic stationary phase a mixture comprising organic compounds to be separated;
displacing the organic compounds from the hydrophobic stationary phase by applying thereto an aqueous composition comprising a non-surface active hydrophobic anionic displacer molecule; and
collecting a plurality of fractions eluted from the hydrophobic stationary phase containing the separated organic compounds;
wherein the non-surface active hydrophobic anionic displacer molecule comprises a hydrophobic anion and a counterion, Cl, having the general formula A or B:
wherein in the general formulae A and B, each CM or CM′ is an independent hydrophobic chemical moiety with a negative formal charge selected from: carboxylate (XVI), N-acyl-α-amino acid (XVII), sulfonate (XVIII), sulfate monoester (XIX), phosphate monoester (XX), phosphate diester (XXI), phosphonate monoester (XXII), phosphonate (XXIII), tetraaryl borate (XXIV), boronate (XXV), boronate ester (XXVI); wherein the chemical moieties (XVI)-(XXVI) have the following chemical structures:
wherein in general formula B, CM and CM′ are independent charged chemical moieties having the same or opposite formal charge and are chemically attached to each other by a doubly connected chemical moiety, R*, which replaces one R1, R2 (if present), R3 (if present) or R4 (if present) chemical moiety on CM and replaces one R1, R2 (if present), R3′ (if present) or R4′ (if present) chemical moiety on CM′ (herein, an R group on CM′ is designated with a prime (′), e.g., R1′ is the R1 group on CM′);
wherein each of R1, R1′, R2, R2′, R3, R3′, R4 and R4′ is a linear or branched chemical moiety independently defined by the formula,
—CxX2x-2r-AR1—CuX2u-2s-AR2,
R* is a direct chemical bond or is a doubly connected, linear or branched chemical moiety defined by the formula,
—CxX2x-2rAR1—CuX2u-2s—,
and each R5 and R5′ independently are a linear or branched chemical moiety defined by the formula,
—CxX2x-2r-AR2;
wherein each AR1 independently is a doubly connected methylene moiety (—CX1X2—, from methane), a doubly connected phenylene moiety (—C6G4-, from benzene), a doubly connected naphthylene moiety (—C10G6-, from naphthalene) or a doubly connected biphenylene moiety (—C12G8-, from biphenyl);
wherein AR2 independently is hydrogen (—H), fluorine (—F), a phenyl group (—C6G5), a naphthyl group (—C10G7) or a biphenyl group (—C12G9);
wherein each X, X1 and X2 is individually and independently —H, —F, —Cl or —OH;
wherein any methylene moiety (—CX1X2—) within any —CxX2x-2r— or within any —CuX2u-2s— or within any —(CX1X2)p— may be individually and independently replaced with an independent ether-oxygen atom, —O—, an independent thioether-sulfur atom, —S—, or an independent ketone-carbonyl group, —C(O)—, in such a manner that each ether-oxygen atom, each thioether-sulfur atom or each ketone-carbonyl group is bonded on each side to an aliphatic carbon atom or an aromatic carbon atom;
wherein not more than two ether-oxygen atoms, not more than two thioether-sulfur atoms and not more than two ketone-carbonyl groups may be replaced into any —CxX2x-2r— or into any —CuX2u-2s—;
wherein mx is the total number of methylene groups in each —CxX2x-2r— that are replaced with ether-oxygen atoms, thioether-sulfur atoms and ketone-carbonyl groups, and mu is the total number of methylene groups in each —CuX2u-2s— that are replaced with ether-oxygen atoms, thioether-sulfur atoms and ketone-carbonyl groups;
wherein G is individually and independently any combination of —H, —F, —Cl, —CH3, —OH, —OCH3, —N(CH3)2, —CF3, —CO2Me, —CO2NH2; —CO2NHMe, —CO2NMe2;
wherein G* is individually and independently any combination of —F, —Cl, —R2, —OH, —OR2, —NR2R3, —CF3, —CO2Me, —CO2NH2; —CO2NHMe, —CO2NMe2;
wherein a pair of R2, R2′, R3, R3′, R4 and R4′ may comprise a single chemical moiety such that R2/R3, R2/R4, R3/R4, R2′/R3′, R2′/R4′ or R3′/R4′ is individually and independently —(CX1X2)p— with p=3, 4, 5 or 6;
wherein the integer values of each of x, r, u, s, mx, mu are independently selected for each R1, R1′, R2, R2′, R3, R3′, R4, R4′, R5, R5′ and R*, Integer values r and s are the total number of contained, isolated cis/trans olefinic (alkene) groups plus the total number of contained simple monocyclic structures and fall in the ranges 0≦r≦2 and 0≦a≦2, the numeric quantity x+u−mx−mu falls in the range 0≦x+u−mx−mu≦11;
wherein at least one aromatic chemical moiety, heterocyclic aromatic chemical moiety, imidazoline chemical moiety, amidine chemical moiety or guanidine chemical moiety is contained within CM or CM′ of A or B;
wherein a group-hydrophobic-index for each R-chemical-moiety (n) is numerically equal to the sum of the number of aliphatic carbon atoms plus the number of olefinic carbon atoms plus the number of thioether-sulfur atoms plus the number of chlorine atoms plus one-fifth the number of fluorine atoms plus one-half the number of ether-oxygen atoms plus one-half the number of ketone-carbon atoms plus one-half the number of aromatic carbon atoms beyond the number six minus the number of hydroxyl-oxygen atoms beyond the number one;
wherein an overall-hydrophobic-index (N) for each [CM] or [CM-R*—CM′] is numerically equal to the sum of the number of aliphatic carbon atoms plus the number of olefinic carbon atoms plus the number of thioether-sulfur atoms plus the number of chlorine atoms plus one-fifth the number of fluorine atoms plus one-half the number of ether-oxygen atoms plus one-half the number of ketone-carbon atoms plus one-half the number of aromatic carbon atoms beyond the number six minus the number of hydroxyl-oxygen atoms beyond the number one;
wherein the group-hydrophobic-indices (1n and 1′n) for R1 and R1′ fall in the range 4.0<1n,1′n<12.0, the group-hydrophobic-indices (2n, 2′n, 3n, 3′n, 5n, 5′n and *n) for R2, R2′, R3, R3′, R5, R5′, R*, when present, fall in the range 0.0≦2n, 2′n, 3n, 3′n 5n, 5′n, *n<12.0 and the group-hydrophobic-Indices (4n and 4′n) for R4 and R4′, when present, fall in the range 0.0≦4n, 4′n≦5.0;
wherein the overall-hydrophobic-index (N) divided by the value of g falls in the range 10.0≦N/g<24.0;
wherein in A, when the charged moiety, CM, has a formal negative charge, g=1, and in B, when both CM and CM′ have formal negative charges, g=2, and in B when CM and CM′ have opposite formal charges with the overall charge of [CM-R*—CM′] being negative, g=1;
wherein the numeric value of the group-hydrophobic-index calculated for a cyclic chemical moiety is divided equally between the two respective R-chemical-moleties;
wherein R1 or R1′ is identified as that R-chemical-moiety when only one such chemical moiety is attached to CM or CM′; wherein R1 or R1′ is identified as that R-chemical-moiety having the largest value of the group-hydrophobic-index when there are more than one such R-chemical-moieties attached to CM or CM′; wherein R4 or R4′ is identified as that R-chemical-moiety having the smallest value of the group-hydrophobic-index when there are more than three such chemical moieties attached to CM or CM′; and
wherein Cl is a non-interfering, oppositely-charged counter-ion or mixture of such counter-ions, and the value of d is zero, a positive whole number or a positive fraction such that electroneutrality of the overall hydrophobic compound is maintained.
In one embodiment, the aqueous composition comprising a non-surface active hydrophobic anionic displacer molecule is free of added salt other than a pH buffer.
In one embodiment, CM has a general formula XXIV or XXV:
wherein in the general formula XXIV, R1 is phenyl, 4-EtC6H4—, 4-nPrC6H4—, 4-nBuCeH4—, 4-MeOC6H4—, 4-FC6H4—, 4-MeC6H4—, 4-MeOCsH4—, 4-EtC6H4—, 4-ClC6H4—, or C6F5—; and each of R2, R3 and R4 independently are phenyl, 4-FC6H4—, 4-MeCeH4—, 4-MeOC6H4—, 4-EtOH4—, 4-ClCeH4— or CeF5—; and
wherein in the general formula XXV, R1 is 4-(4-nBuCeH4)CsH4— or 4-(4-nBuCeH4)-3-ClC6H3—.
In one embodiment, CM has a general formula selected from 4-R1C6H4SO3H, 5-R6-2-HO—C6H3SO3H, 4-R1—C6H4—C6H3X-4′-SO3H, and 4-R1—C6H4—C6H3X-3′-SO3H, wherein R1 is CH3(CH2), wherein n=4-10 and X is H or OH.
In one embodiment, CM has a general formula XVIII or XXIII:
wherein in the general formula XVIII and in the general formula XXIII, R1 is C6Hs(CH2)n—, wherein n=5-11.
In one embodiment, CM has a general formula selected from 5-R1-2-HO—C6H3CO2H and R1C(O)NHCH(CeH5)CO2H, wherein R1 is CH3(CH2)n—, wherein n=4-10.
In one embodiment, CM has a general formula 4-R1C6H4PO3H2 wherein R1 is CH3(CH2)n—, wherein n=4-10.
In one embodiment, Cl is a non-interfering inorganic cation or mixture of such non-interfering cations selected from the groups: alkali metal ions (Li+, Na+, K+, Rb+, Cs+), alkaline earth metal ions (Mg2+, Ca2+, Sr2+, Ba2+), divalent transition metal ions (Mn2+, Zn2+) and NH4+; wherein Cl is a non-interfering organic cation or mixture of such non-interfering cations selected from the groups: protonated primary amines (1+), protonated secondary amines (1+), protonated tertiary amines (1+), protonated diamines (2+), quaternary ammonium ions (1+), sulfonium ions (1+), sulfoxonium ions (1+), phosphonium ions (1+), bis-quaternary ammonium ions (2+) that may contain C1-C6 alkyl groups and/or C2-C4 hydroxyalky groups.
As used herein, “non-surface-active”, with respect to a neutral non-surface-active anionic displacer compound employed in accordance with the present invention, means that the compound so described has a critical micelle concentration (“CMC”) greater than the concentration of the compound employed in a displacement chromatography process in accordance with the present invention. In one embodiment, the concentration of the non-surface-active displacer compound is less than about 80% of the CMC for that compound in water in the absence of organic solvent, salt or other agent that would affect the CMC. In one embodiment, the concentration of the non-surface-active displacer compound is less than about 60% of the CMC for that compound in water in the absence of organic solvent, salt or other agent that would affect the CMC. In one embodiment, the concentration of the non-surface-active displacer compound is less than about 50% of the CMC for that compound in water in the absence of organic solvent, salt or other agent that would affect the CMC.
In one embodiment, the aqueous composition comprising a non-surface-active hydrophobic anionic displacer molecule employed in accordance with the present invention does not exhibit adverse surface-active characteristics due to one or a combination of two or more of (1) the anionic non-surface-active displacer compound is present at a concentration lower than its CMC; (2) the overall-hydrophobic-index (N) for each [CM] or [CM-R*—CM′] divided by the value of g falls in the range 10≦N/g<24; (3) the group-hydrophobic-index (1n) for each R1 or R1′ falls in the range 4<1n<12, the group-hydrophobic-index (2n, 3n, 5n and *n) for each R2, R2′, R3, R3′, R5, R5′ and R*, when present, falls in the range 0≦2n, 3n, 5n, *n<12, and the group-hydrophobic-index (4n) for each R4 or R4′, when present, falls in the range 0≦4n≦5; (4) the composition contains greater than about 5 volume % or more of an organic solvent.
As used herein, “low organic solvent content” generally refers to an organic solvent content in, e.g., an aqueous “carrier” composition comprising a non-surface-active anionic displacer compound in accordance with the present invention, of less than about 25% by volume. In one embodiment, the organic solvent content of the aqueous “carrier” composition contains less than about 20% by volume of any organic solvent. In one embodiment, the organic solvent content of the aqueous “carrier” composition contains less than about 15% by volume of any organic solvent. In one embodiment, the organic solvent content of the aqueous “carrier” composition contains less than about 10% by volume of any organic solvent. In one embodiment, the organic solvent content of the aqueous “carrier” composition contains less than about 5% by volume of any organic solvent. In one embodiment, the aqueous “carrier” composition contains no organic solvent.
In one embodiment, the organic solvent is one or a mixture of two or more of methanol (CH3OH or MeOH), ethanol (C2H5OH or EtOH) or acetonitrile (CH3CN or MeCN). In one embodiment, the aqueous “carrier” composition contains a mixture of suitable organic solvents. In one embodiment, the aqueous “carrier” composition contains no organic solvent.
Hydrophobic displacement chromatography can be carried out using chiral analytes, chiral displacers and chiral chromatography matrices. Under these conditions, an achiral displacer may be used, but a racemic mixture of a chiral displacer cannot be used. Racemic chiral analytes can also be purified using an achiral chromatography column and an achiral displacer. In this case, impurities, including diastereomers, are removed from the racemic compound of interest, but there is no chiral resolution of the enantiomers.
An anionic displacer may contain one or more chiral centers; see for example, see chiral displacer compounds 689 and 698 (see Tables V-XIII below). Displacers 613 and 639 represent displacers compounds that are achiral. With the proper choice of chiral chromatography matrix, mobile phase and achiral displacer, enantiomers are routinely resolved (separated) on a preparative scale. Depending on the specific circumstances, a good, enantiomerically pure, chiral displacer can have performance advantages over a good achiral displacer when carrying out a displacement separation of enantiomers on a chiral stationary phase
Useful DIH Ranges—
Various classes of anionic hydrophobic displacers having the general formula A or B, have different useful pH ranges depending on the chemical nature of the charged moieties. Anionic hydrophobic displacers that contain protonatable anionic groups should be operated at a pH of 1-2 units or more above the actual pKa values.
Tetraarylborates—generally, these molecules are limited to the neutral pH range (6-8) owing to problems with hydrolytic instability outside that range. Some tetraarylborates have solubility limitations and can be used only with a narrow range of cationic ion-pairing agents.
Arylboronates—For most displacer applications, the pKa values of ordinary arylboronic acids (pKa˜8.9) are too high to be useful in the neutral pH range. Even with electron-withdrawing groups such as meta —NO2 (pKa˜7.4) or meta —Cl (pKa˜8.1), the pKa values are still high. However, the formation of boronate esters by the addition of certain polyols leads to compositions that have lower pKa values, by 2.0-2.5 pKa units in certain cases. The chemical nature of the polyol molecule also affects the hydrophobicity and solubility of the resulting boronate ester displacer. Useful polyols include fructose, ribose, sorbitol, mannitol, cis-1,2-dihydroxycyclopentane and cis-3,4-dihydroxytetrahydrofuran which lead to displacers that are useful in the pH range 7.1-10.0. A typical polyol concentration (mM) is about twice the concentration of the displacer or 25 mM greater than the concentration of the displacer, whichever is greater.
The displacer should bind to the column more strongly than all of the components of the sample or at least more strongly than all of the major components of interest. A good rule of thumb is that no more than 1-4% of the sample mass should bind more strongly than the displacer.
An optimal displacer should bind to the stationary phase neither too strongly nor too weakly. The proper binding strength depends on the analyte of interest and the associated binding isotherms. Usually, a range of displacers with a range of binding strengths is needed for a variety of different columns and analytes to be purified. If a displacer binds too strongly, poor performance is obtained such as lower resolution, lower analyte binding capacity, difficulty in displacer removal and longer cycle times. If a displacer binds too weakly, a poor displacement train may result with too much “tailing” of the displaced analytes underneath the displacer, or there may be only partial displacement or no displacement at all.
A convenient, rule of thumb method that helps in choosing displacers with the proper binding strength is to carry out simple gradient elution chromatography of potential displacers and analytes using columns and mobile phases similar to those that are to be used in the displacement experiment. As a first screen, the displacer should elute 5-15 minutes later than the analytes of interest in a 60 minute gradient. Ideally one would measure the isotherms of the single analytes and mixtures of analytes but this is time consuming and often impractical. Because it operates early on the binding isotherm, this rule of thumb method is not perfect, but provides a convenient starting point for further DC optimization.
Apart from proper binding strength, useful hydrophobic displacers need to have binding isotherms with certain other useful characteristics.
(1) Monomodal, convex upward isotherms (Langmuir-type isotherm behavior) for displacer and analyte molecules facilitate the orderly formation of isotactic displacement trains and simplify the method optimization process. This is a useful property of many anionic displacer molecules in contrast to binding isotherms of many other uncharged hydrophobic displacer molecules such as aromatic alcohols (e.g., substituted phenols, naphthols, hydroxybiphenyls), fatty alcohols (e.g., 1-dodecanol, 1,2-dodecanediol) and uncharged fatty carboxylic acids (e.g., myristic acid) behave normally at lower concentrations and then become bimodal and rise again at higher concentrations (BET-type isotherm behavior). This binding behavior often arises from deposition of multiple layers of the hydrophobic displacer, each layer having different binding characteristics. This binding behavior greatly complicates the displacement process and its useful implementation.
(2) Chromatographic results in DC may be also complicated when displacer molecules undergo self-association in solution. As concentrations increase, problems with displacer self-association become worse. Again, the charged groups in anionic hydrophobic displacers decrease self-association problems in aqueous solution.
(3) Further complications may arise when product and/or impurity isotherms cross the displacer isotherm in the higher, non-linear binding region. This behavior leads to reversal of displacement order, broadening of overlap regions between displacement bands and problems with co-displacement. In this case, minor variations in displacer concentration can lead to large changes in the displacement train thereby making method optimization very difficult.
We have found that property designed anionic displacer molecules supplemented with the proper counter ions and, when needed, small amounts of useful organic solvents provide a family of effective hydrophobic displacers with Langmuir-type binding behavior and useful ranges of binding strengths.
With all of their many advantages, anionic hydrophobic displacer molecules have one extra requirement: an appropriate ion-pairing cation, Cl. The cation significantly affects the binding isotherm of the displacer and the functioning and utility of the displacer. The concentration of the cation is independently adjusted by adding appropriate amounts of Cl−/HCO2− salts of an ion-pairing cation. The properties of an ion-pairing cation for a charged hydrophobic displacer strongly affects its displacement properties. A few cations are involved in ion-pairing in solution, and all cations are involved in ion-pairing in the adsorbed state on the hydrophobic matrix. Furthermore, the cation can be involved in ion-pairing with the analyte (for example, anionic oligonucleotides at pH=7). The same ion-pairing cation for displacer and analyte should be used for good chromatographic resolution. Useful ion-pairing cations are usually singly charged. Owing to their higher solvation energies, divalent ions (Ca2+) and trivalent ions (La3+) are generally less useful but may be used in some specialized cases. Exceptions to the general rule are multiple, singly-charged moieties spaced apart in a single organic ion such as and Me3N+(CH2)4N+Me3. These ions can also be useful as counter-ions for di-anionic displacers.
Ion-pairing cations with greater hydrophobic character tend to increase binding strength and also decrease solubility. Furthermore, when using hydrophobic displacers, resolution of DC may decrease if the cation itself is either too hydrophobic or too hydrophilic. Typically, intermediate hydrophobic/hydrophilic character of the cation gives best results, but this varies depending on the molecule being purified. The optimal ion-pairing cation for each purification should be determined experimentally. Volatile ion-pairing agents are conveniently removed under reduced pressure, while nonvolatile ones are readily removed by other means: diafiltration, precipitation or crystallization. Table I gives a partial list of useful monovalent ion-pairing cations. When using protonated amine (pKa˜9.8-11.0), protonated amidine (pKa˜12.5) or protonated guanidine (pKa˜13.5) ion-pairing agents, the operating pH should be two or more pH units below the pKa of the respective amine.
nC6H13C(NH2)2+
nC5H11MeNC(NH2)2+ < nC6H13MeNC(NH2)2+
nC6H13NMeH2+
nC6H13NMe2H+
Mixed ion-pairing cations often lead to loss of chromatographic resolution and are generally to be avoided. However, there is one set of conditions when mixed cations may be used; that is, when both (a) the cation of interest has significantly stronger ion-pairing properties than the other counter-ions that are present and (b) the cation of interest is present in stoichiometric excess in the sample loading mixture and in the displacer buffer.
In order to simpllfy the potentially time consuming screening of lon-pairing counter-ions, the following recommendations serve as initial starting points for ion-pairing cations:
Peptides and small proteins (pH=6.0-9.0): Me4N+, Me3NH+, Me3NEt+, Me2EtNH+
Oligonucleotides (pH=6.5-8.0): nButyl-NH3+, nPentyl-NH3+, nHexyl-NH3+
DMT-On oligonucleotides (pH=7.0-8.0): EtNH3+, Et3NH+
Because many useful amines or amine salts are unavailable in HPLC-grade purity, a useful form is their protonated ammonium salts which are chemically more stable and are easily purified by repeated recrystallization. Hydrogenphosphate (2−), monomethylphosphate (2−), phosphonate (2−, aka phosphite), methylphosphonate (2−) and ethylphosphonate (2−) salts of suitable amines can be used with anionic displacers:
[MeNH3+]2[HOPO32−],[Me3NH+]2[HOPO32−],[nBuNH3+],[HOPO32−],[nC6H13NH3+]2[HOPO32−],
[MeNH3+]2[EtPO32−],[Me3NH+]2[EtPO32−],[nBuNH3+]2[EtPO32−],[nC6H13NH3+]2[EtPO32−].
In “hydrophobic chromatography” or, more properly, “solvophobic chromatography”, where the principal solvent component is water, potential hydrophobic displacer molecules often have limited solubility. Hydrophobic molecules usually do not dissolve in water to any appreciable extent unless there are “hydrophillic groups” attached to the hydrophobic molecule, such as charged ionic groups, hydrophillic counter-ions, polar groups or groups that function as hydrogen bond donors or acceptors. Aromatic molecules interact with water in a unique fashion owing to the unique manner in which the pi-electrons act as weak hydrogen bond acceptors. Furthermore, aromatic molecules can engage in face-to-face pi-stacking in aqueous solution. These small but important effects are reflected in the higher solubility in water of benzene (9 mM) and naphthalene (200 μM) compared with cyclohexane (˜10 μM) and trans-decalin (<1 μM) and in the higher solubility of phenol (960 mM) and β-naphthol (7 mM) compared with the unhydroxylated arenes. The molecular structure of a useful displacer molecule should facilitate a reasonable solubility (10-50 mM) in water or in water with low organic content yet at the same time be sufficiently hydrophobic that it binds strongly to the stationary phase. Generally, charged displacer molecules have better solubility properties than neutral ones owing to the increased solvation energies of charged species, especially counter-ions. It requires a unique balance of physical and chemical properties for anionic molecules to behave as good displacers.
It is important to note, generally speaking, that increasing the levels of the organic solvent in order to compensate for poor displacer solubility rarely leads to useful results. Best chromatographic results are obtained with 0-25% organic solvent, or more preferably, 2-15% organic solvent. Higher organic content (25-75%) of the mobile phase may be used in some cases but often capacity and resolution suffer badly.
One potential problem with hydrophobic displacement chromatography is the possible association of a hydrophobic displacer with a hydrophobic analyte in solution. This can lead to significant loss of resolution and contamination. Displacer-analyte association in the adsorbed state on the stationary phase also can occur but is less problematic with proper amounts of suitable ion-pairing agents present. A good method to deal with this problem is to use charged analytes and charged hydrophobic displacers with the same charge. These problems arise more frequently sometimes when neutral hydrophobic displacers and neutral hydrophobic analytes interact.
In some cases when the chemical structure and physical properties are conducive, anionic hydrophobic molecules can self-associate, forming micelles and micelle-like, self-associated structures in solution. This situation can lead to loss of resolution in DC as well as unwanted foaming of displacer solutions. The displacer in solution finds itself in various forms that are interrelated by various chemical equilibria. Furthermore, micelles can act as carriers for hydrophobic analyte molecules causing them to exist in solution in various forms. This unwanted phenomenon is concentration dependent and is effectively inhibited by the addition of small amounts of a suitable organic solvent such as methanol, ethanol or acetonitrile. Properly designed, anionic displacer molecules inhibit micelle formation and give better displacement results. Thus, keeping the group hydrophobic indices below 12.0 for R-groups, R1-R3, reduces the problem of unwanted detergency.
A displacer should have adequate purity. The object of preparative chromatography is to remove the impurities from a component of interest. Contamination of the desired compound by the displacer itself is rarely a problem, but contamination by “early displacing” impurities in the displacer solution may be problematic in some cases depending on the amounts of the impurities and their binding properties. Thus, a good displacer should contain little or no early displacing impurities.
In order to track the location and amounts of displacer throughout the DC experiment, to watch displacer breakthrough curves and to follow displacer removal during column regeneration procedures, it is useful to have a displacer with moderate ultraviolet absorption. High absorption is not needed nor is it preferred owing to the high concentrations of displacer and analyte. Generally, colorless displacers are preferred with a UV spectrum that has strategically located windows of low absorbance so that the analytes can be followed at some frequencies and the displacer monitored at other frequencies.
Convenient and cost-effective methods of chemical synthesis, production and manufacturing are important in order to produce useful displacers and reasonable costs. Furthermore, practical methods of purification, especially non-chromatographic purification, are needed in order to achieve the purity requirements in a cost-effective manner.
Among all its other desired chemical and physical properties, a useful displacer molecule should be chemically stable. It should be inert toward analyte molecules and chemically stable (non-reactive) toward water, common organic solvents, mild bases, mild acids and oxygen (air). It should be photo-stable and thermally stable under typical use and storage conditions and have a reasonable shelf life. It is greatly preferred that displacer molecules be visually colorless, yet have the requisite levels of UV absorbance. Dyes generally do not make useful displacers in DC. Useful displacer molecules also need to have low toxicity, not only to protect workers but to protect biological and drug samples that may come into contact with the displacer.
While the most common type of reversed-phase column is octadecyl coated silica, many hydrophobic stationary phases find utility in DC. Examples of suitable stationary phases are listed in Table II. Ultimately, the best choice of stationary phase is experimentally determined for each system under study.
Better results in displacement chromatography are obtained with longer, well-packed columns that give better recovery and yield. Table III provides a guide for initial choices of column dimension and initial flow rates.
a500 mm or 2 × 250 mm
bInitial flow rate = 75 cm/hr (12.5 mm/min); needs to be optimized
Proper column length is important for good results. It should be long enough to fully sharpen the displacement train and give good resolution. Yet columns that are too long needlessly increase separation time and often lead to poorly packed beds and reduced resolution. In many cases, two well-packed shorter columns can be attached end-to-end with good chromatographic results. Considerable experimentation with small molecules (MW<3 KDa) indicates that optimal column length falls in the range 15-45 cm for 5 μm particles and 20-60 cm for 10 μm particles. Porous particles with pore sizes of 80-100 Å are suitable for traditional drugs and small peptides, pore sizes 120-150 Å are suitable for medium and large oligopeptides and oligonucleotides and pore sizes 300-500 Å are suitable for most proteins and DNA. Non-porous particles can be used, but loading capacity will significantly decrease.
In cylindrical columns, it is important that a planar flow front be established so that it is perpendicular to the axis of flow. Scaling up to purify larger amounts of sample is simple and straightforward in displacement chromatography once an optimized protocol has been developed on a smaller column. After the shortest acceptable column length is found, scale-up is simply accomplished by increasing column diameter while maintaining a constant linear flow rate. With proper modifications, displacement chromatography can be used with radial-flow columns and with axial-flow monolith columns. The principles of displacement chromatography also can be applied in analytical and preparative thin-layer chromatography.
Though displacement chromatography of organic compounds, traditional drugs and peptides has been carried out for many years, mediocre-to-poor results are often obtained. Good displacers, good columns and good operational protocols, as disclosed herein, lead to excellent reproduciblity and remarkably good chromatographic performance.
Initial evaluation is carried out using a good general purpose anionic displacer as the trimethylammonium salt with proper binding strength. At the operating pH, anionic analyte molecules require an anionic displacer. At the operating pH, anionic displacers can be used to purify anionic and neutral analytes. The displacer should bind to the column more strongly than the material to be purified, but the displacer should not bind too strongly. Typical displacer concentrations are in the range 10-50 mM. Initially, displacer concentration is set at 10-15 mM. As needed, pH buffer and ion-pairing agent are added to the displacer solution. The displacer solution and carrier solution should have identical compositions (including pH), except for the presence of displacer and the level of the ion-pairing salt. Displacers 579, 609 and 634 (see Tables V-XIII below) are examples of good general-purpose anionic displacers. During method optimization, it may be helpful to increase displacer concentration up to 20-30 mM or higher.
Table I contains a list of useful, monovalent, ion-pairing cations (Cl) that are useful for hydrophobic chromatography. They are needed when the displacer or analyte is charged. For anionic analytes and displacers, binding isotherms strongly depend on the chemical properties of the counter-ion and its concentration. Those ion-pairing cations with moderate to moderately strong binding properties are usually the best to use. Start with trimethylammonium formate or phosphate during initial experiments. When the analyte requires an on-pairing agent. It usually dictates the choice of ion-pairing agent during the DC experiment. The ion-pairing agent for the analyte and the displacer should be the same.
As noted earlier, using insufficient levels of a good ion-pairing agent is one of the major causes of poor chromatographic performance in displacement chromatography experiments. The formula for calculating the suitable concentration of the ion-pairing agent in the sample solution (CIPS, mM)) is given by,
C
IPS
=E
s
×C
s(mM)×Gs
where Es is the excess factor for the sample, Cs is the concentration of the sample (mM) and Gs is the absolute value of the net charge of the sample at the operative pH. The optimal value of Es is a parameter that needs to be determined experimentally. The formula for calculating the suitable concentration of the ion-pairing agent in the displacer solution (CIPD, mM) is given by,
C
IPD
=E
d
×C
d(mM)×Gd
where Ed is the excess factor for the displacer, Cd is the concentration of the displacer (mM) and Gd is the absolute value of the net charge of the displacer at the operative pH. The optimal value of Ed is a parameter that needs to be determined experimentally. It is essential that at least a stoichiometric amount of the ion-pairing agent be present in the solutions (Es≧1.0 and Ed≧1.0). In practice, it is our experience that Es should be in the range 1.1-10.0, more preferably in the range 1.2-6.0, more preferably yet in the range 1.5-4.5. Furthermore, it is our experience that Ed should be in the range 1.1-10.0, more preferably in the range 1.2-4.0. Serious deterioration in chromatographic performance results when the ion-pairing concentrations are unoptimized or too low, that is Es<1.0 and/or Ed<1.0.
For initial reversed-phase work, several good quality octadecyl on silica or phenylhexyl on silica columns should be evaluated (5 μm spherical particles with dimensions 4.6×250 mm). Scaleup to larger preparative columns can come later and is relatively straightforward. A critical issue is to choose a suitable pore size. Matrices with pores that are too large or too small often lead to reduced capacity and sometimes reduced resolution. See Tables II and III above.
Because displacement chromatography is a “quasi-equilibrium technique”, relatively slow flow rates are often needed. The optimal flow rate is the fastest flow rate possible without losing resolution. Sample loading flow rate and displacement flow rate should be about the same, both in the range of 35-105 cm/hr. Start at 75 cm/hr for traditional drugs, oligopeptides and oligonucleotides or 40 cm/hr for proteins and DNA. Regeneration flow rates should be 2-8 times the displacement flow rate. When purifying drugs, peptides or oligonucleotides at elevated temperatures on reversed-phase columns, faster flow rates might be used.
Because reversed-phase chromatography and other forms of hydrophobic chromatography are largely driven by +TΔS with +ΔH, higher temperature often leads to stronger binding, faster binding kinetics and distinctly different resolution. As a consequence, the temperature of the column and, to some extent, displacement buffers should be carefully regulated (+/−0.5° C.) in order to prevent band broadening. Initial work is often carried out at 25° C., and then elevated temperatures (45, 65° C.) are tried if the sample will tolerate it, and the boiling point of the organic solvent is suitable.
Although most water-miscible organic solvents will function, acetonitrile, methanol and ethanol are most commonly used. Some DC purifications are carried out with little or no organic solvent at all. This allows practical RPC and HIC purification of undenatured proteins with low salt and low organic solvent. Operating without organic solvent may also be helpful when there are safety issues associated with volatile, flammable solvents. When experimenting, first try acetonitrile for peptides, low molecular-weight organic drugs and small proteins or methanol for large proteins oligonucleotides and DNA. If solubility of the sample in water is acceptable, start with 3% v/v MeCN, 4% v/v EtOH or 5% v/v MeOH in the carrier buffer, the displacer buffer and sample loading solution; the organic content of these three solutions should be the same. Organic solvent content is an important parameter that needs to be optimized for each sample, column and displacer. For general purpose operation, organic solvent should be less than about 15 volume %, more preferably less than about 10 volume %, more preferably yet about 5 volume %. When Octadecyl columns are used, 2-3 volume % acetonitrile, 3-4 volume % ethanol or 4-5 volume % methanol is usually needed for optimal functioning of the matrix. Phenylhexyl and Octyl columns can usually tolerate the absence of organic solvent.
pH buffers are needed when there are ionizable protons in the sample, displacer, ion-pairing agent or on the stationary phase. Some analytes are only stable within certain pH ranges. For some analytes, chromatographic resolution is strongly pH-dependent. Anionic samples including anionic proteins (pH>pI) are purified using anionic displacers and anionic pH buffers. The cations associated with the anionic pH buffers should be the same as the ion-pairing cation but may be, in some cases, a cation that has significantly weaker ion-pairing properties. Furthermore, in some infrequent cases, cationic pH-buffers can be used but only when they possess weaker ion-pairing properties than the principal ion-pairing cation. Thus, N-methylmorpholine (7.4, NMM), triethanolamine (7.8, TEOA) and TRIS (8.1) can sometimes be used as pH buffers when trimethylammonium, triethylammonium or n-butylammonium is the ion-pairing cation. Anionic compounds with mid-range pKa values can be useful pH-buffers: Carbonate (9.9), Borate (9.0), TABS (8.9), TAPS (8.4), TAPSO (7.6), methylphosphonic acid (7.6, MPA), MOPS (7.2), MOPSO (6.9), phosphoric acid (6.8), monomethylphosphoric acid (6.3), phosphorous acid (6.3), MES (6.2), 3,3-dimethylglutaric acid (5.9, DMG), succinic acid (5.2, SUC), acetic acid (4.6, HOAc).
aAcid-amine buffer should be fully soluble in operating solvent, mixture of water and organic solvent.
bMore basic amine requires at higher pH (>7.8); displacer (D−) should have same counter-cation.
When working with samples that contain hundreds components and impurities, co-displacement is an almost unavoidable phenomenon because there are likely to be several minor components that co-displace with the major component of interest no matter where on the binding isotherms the DC experiments take place. Fortunately, co-displacement in displacement chromatography is a far less serious problem than co-elution in preparative elution chromatography. Co-displacement occurs under two conditions: (1) when binding isotherms are so similar that there is poor resolution and (2) when there is crossing of binding isotherms near the operating region of the binding isotherm. Fortunately, there are simple ways to deal with this issue: carry out a second DC experiment under different conditions by operating at a different point on the binding isotherms by,
a. changing the concentration of the displacer,
b. changing to a different displacer with different binding properties. Alternatively, the isotherms themselves can be changed by,
c. changing the chromatography matrix (stationary phase),
d. changing the concentration of the organic solvent,
e. changing to a different organic solvent,
f. changing to a different Ion-pairing agent,
g. changing the temperature.
A second “orthogonal” IP-RP DC step typically gives excellent purity (˜99.5%) with excellent yield (90-95%).
DC experiments are carried out at relatively high loading, typically in the range 60-80% of maximum loading capacity. The operative column loading capacity is not a fixed number; rather, it depends upon where on the binding isotherm the DC experiment operates.
Not all of the column capacity is available for use (see “Exception” below). In practice, only 90-98% of the column capacity is usable. Once the sample has been loaded onto the column, the displacer buffer is then pumped onto the column. There are three fronts that develop, each traveling at different velocities down the column: (1) the liquid front (T1, displacer buffer minus displacer), (2) the sample front (T2) and (3) the displacer saturation front itself (T3). The first front travels faster than the second and third fronts and limits the useable column capacity because the first front should exit the column before the displacement train (T2) begins to exit. The actual velocities of the fronts depend directly on the displacement flow rate. The ratio, α, of the front velocities, Vel1/Vel2, is given by the formula,
α=Km/(R×Cd)
where Km is the displacer binding capacity of the matrix (mg displacer per mL packed matrix) at displacer concentration of Cd, where Cd is the displacer concentration in the displacer buffer (mg displacer per mL displacer buffer), R is the ratio of the volume of the liquid in the column to the total volume of the column (mL liquid per mL bed volume). The maximum % usable column capacity is given by:
(100×(α−1))/α.
In Example 2 below, the respective α-value is 21.98, and the respective maximum usable capacity is 95.4%. Note that as Cd increases, Km will also increase, but not as much if operating high on the nonlinear part of the isotherm. Thus, α will decrease and maximum % usable column capacity will decrease.
There is an exception to the foregoing. If significant levels of unwanted, early-displacing impurities are present in the sample, one can increase the usable capacity of the column, even beyond 100% by overloading the column and spilling out these impurities during sample loading before the displacer flow is started. Thus, the column loading could be 105% of maximum based on the whole sample, but the column loading would be only 80% based on the amount of main product plus late-displacing impurities.
A sample is loaded onto the column through a sample injection valve using one of two methods. The sample should be loaded under frontal chromatography conditions at the same point on the binding isotherm at which the DC experiment takes place. The carrier is not passed through the column after the sample is loaded. Method 1: A sample loading pump is used; Method 2: An injection loop is used. Usually, only partial loop injection is used. The sample in the loop should be driven out of the loop onto the column first by the carrier and then the displacer solution. Not more that 85-95% of the loop volume should be loaded onto the column so that sample diluted by carrier is not loaded.
The concentration of the load sample is an important operating parameter. The optimal sample loading concentration (mg/mL) is the same as the output concentration of the purified product from the displacement experiment—the plateau region of the displacement train. Binding-isotherms, the column binding capacities and the output concentrations are initially unknown. Simply carry out the first displacement experiment with the sample solution loaded onto the column using initial estimates as shown below:
(1) Pick an initial column loading percentage at which the one wishes to work, say 75%.
(2) Pick an initial concentration for the sample by one of two methods:
Initial sample conc.(mg/mL)=(col. binding capacity(mg/mLm)×col. volume(mLm)/((T2−T1)×sample flow-rate(mL/min))=(50 mg/mLm×4.155 mLm)/((736 min-312 min)×0.208 mL/min)=2.36 mg/mL (for Example 1)
If the first DC experiment with loaded sample leads to overloaded conditions (>100% loading), rerun the experiment at one-half the sample concentration. From the results of the first successful DC experiment while using a sample, actual loading concentration and actual column loading capacity are readily calculated, and those values are then used in adjusting sample concentration and loading for the second DC experiment.
The loading sample solution is prepared at the concentration and amount described above. Enough excess solution is needed for overfilling the loop or filling the dead volume of a sample loading pump and delivery lines. The pH, amount of pH buffer and amount of organic solvent are the same as the carier and displacer buffer. Dissolving the sample in the carrier changes its pH, so the pH of the sample solution will have to be re-adjusted after dissolution. However, the amount of ion-pairing agent may be different. The ion-pairing agent used in the sample solution must be the same one used in the displacer buffer. In this regard, the ion-pairing requirements of the sample dictate which ion-pairing agent is used in the sample solution and in the displacer solution. Based on the formal chemical charge at the operating pH and the concentration of the main analyte, the concentration of the concentration is the ion-pairing agent or ion-pairing salt is calculated. See “Concentration of ion-Pairing Agent” above.
The composition and history of the sample should be known. If the sample contains a cation, its chemical nature and amount (concentration) should also be known. (a) Obviously, if no cation is present, then no adjustment is made in sample preparation. (b) If the cation in the sample is the same as the ion-pairing agent used in the DC, then the amount of added ion-pairing agent to the sample solution is reduced accordingly. (c) If the cation in the sample has significantly weaker ion-pairing properties than the ion-pairing cation used in the DC, then its presence is ignored. (d) If the cation in the sample has stronger ion-pairing properties than the ion-pairing cation used in the DC, then the cation should be exchanged or removed before proceeding.
Displacement chromatography gives excellent chromatographic resolution, especially with optimized protocols using a good C18-reversed-phase column. However, the resolution is difficult to see because all of the bands come off the column together as back-to-back bands in the displacement train. Many of the small impurity triangle-bands are less than 30 seconds wide (<100 μL). Thus, an experiment with a displacer breakthrough time of 250 minutes and 80% sample loading, the displacement train would be about 200 minutes wide, and more that 400 fractions would have to be taken so that chromatographic resolution is not lost during the fraction-collection process. Analyzing 400 fractions is truly enlightening and interesting but also a daunting task. This is when online real-time fraction analysis would be useful. In practice, one may somewhat disregard resolution and collect only 100-130 larger fractions. Analysis of even this reduced number of fractions represents a substantial amount of work.
In the circumstance in which a preparative DC experiment is conducted and only the purified main component is of interest, the fraction collecting process is greatly simplified. Based on the shape of the displacement train observed at various frequencies (UV), the beginning and ending of main band of interest is judged and then about 10 fractions are analyzed in both regions in order to determine which fractions to pool. Analyzing 20 fractions instead of 100-130 fractions is an easier task.
In Example 3 below, analysis is even easier. Based on the method above, the beginning and ending of the main band of Interest is judged, a conservative pooling is made without any analysis and only one analysis is carried out on the final pool.
The displacer is removed using 5-10 column volumes of 95/5 (v/v) ethanol-water or 80/10/10 (v/v/v) acetonitrile-npropenol-water without any pH buffer or ion-pairing agent. The object is to efficiently remove >99.9% or more of the displacer from the column in the shortest amount of time. The flow rate is increased (100-400 cm/hr) in order to speed up the column regeneration process if the matrix will tolerate the increased back-pressure. Observing the displacer removal near the absorption maximum of the displacer (see displacer instructions) allows the regeneration process to be carefully monitored and optimized by UV detection.
Salts in aqueous solvents lead to solvents that are less hospitable to dissolved hydrophobic analytes and hydrophobic displacers resulting in stronger binding to hydrophobic chromatographic matrices. This is the principle behind hydrophobic-interaction chromatography (HIC). So long as solubility of the analyte is sufficient in the salt solution, the addition of salt is a good way to modulate analyte binding and selectivity to a hydrophobic matrix.
In some cases, analyte binding to a hydrophobic matrix is so weak that added salt is needed in order to obtain sufficient analyte binding. Commonly used salt solutions include one of 0.5-2.5M (NH4)2SO4, K2SO4, Na2SO4, NaCl and KCl. With the help of many different salts at various concentrations, HIC in displacement mode offers many options for useful chromatographic separations of proteins.
See the example protocol for Example 2 (dual pump operation).
Because residual displacer from previous experiments is a potential problem, the protocol has line purging operations, a quick column regeneration and equilibration operations in order to make sure that the HPLC system and column are completely clean and properly equilibrated just before sample loading. These steps are simply precautionary and not always necessary. The protocol includes the (a) a pre-equilibration sequence, (b) an equilibration sequence, (c) a sample loading sequence (d) a displacement sequence and (e) a regeneration sequence in a single protocol. In order to overcome problems with dead-volume in the system, all loading buffers, displacer buffers and sample solutions are purged through the system to waste just prior to pumping onto the column. This way, the column sees a sharp front of undiluted solutions immediately upon valve switching. The sample solutions should be degassed so that gas bubbles do not form in them. When injection loops are used, they need to be overfilled by about 10%. The overfill can be collected for further use. Full loop injections should not be used, only partial loop injections. Experience dictates that only 85-95% of the loop volume can be used depending on the inner diameter of the loop tubing because the sample solution mixes with the driver solution and dilutes it. The sample in the loop is driven onto the column by the loading buffer, but toward the end of the sample loading process, the driving solution is changed to the displacer buffer. This allows the displacer buffer to be purged through the system just prior to the displacer buffer itself being pumped directly onto the column. During the initial part of the regeneration process, slower flow rates are used Thus, problems with high backpressure rarely occur. Once most of the displacer has been removed, higher flow rates can be used.
As with all forms of preparative chromatography, optimization of the chromatographic methods and procedures is important, but it requires some experimentation. The benefits of displacement chromatography often come with a price—time. The time-consuming factors can be minimized during method optimization.
Existing protocols provide a useful starting point for method optimization, but they will need modification for the specific sample under study. A sample protocol (Example 2) is shown below that has been optimized for purity without regard to time. It is important to carry out method optimization adapted for the specific physical properties and chromatographic properties of the sample of interest. Upon optimization, longer process times (600-800 min) often can be reduced to 200-300 minutes and in some cases reduced to 100-150 minutes.
Hydrophobic chromatography used in displacement mode has (a) high matrix productivity (gram of product per liter matrix over the lifetime of the matrix), (b) high volume productivity (gram of product per liter of column volume), (c) high solvent productivity (gram of product per liter of solvent used) yet (d) may have mediocre time productivity (gram of product per unit of time). Proper method optimization mitigates the time factor.
A typical instrumental configuration for a small preparative HPLC system is given below.
Starting Peptide: Crude 20-mer Peptide, 65.8% purity, FW˜2.300 mg/pmole, charge=−4
Column: Waters Xbridge BEH130, 5 μm, 135 Å, 4.6×250 mm SS, —C18 on silica
Flow Rates Loading=208 μL/min; Displacement=208 μL/min
Ion-Pairing Agent: Trimethylammonium (Me3NH+);
Temperature=23° C.; pH=7.1;
Displacer Buffer: 10.0 mM Displacer 579+15 mM H3PO4 (HPLC grade) in DI water w/5% (v/v) MeOH, pH=7.1, w/37% Me3N(HPLC grade), final Me3NH+ concentration ˜25 mM.
Loading Buffer: 15 mM H3PO4 (HPLC grade) in water w/5% (v/v) MeOH, pH=7.1 w/37% Me3N(HPLC grade), Me3NH+ concentration ˜25 mM.
Sample Solution: 3.02 mg/mL peptide in water with 5% (v/v) MeOH, 15 mM H3PO4; pH=7.1 w/37% Me3N, Me3NHconcentration ˜25 mM.
Load Amount: 157.2 mg from loading pump (pump 2); Loading Time=250.0 min.
Fraction Size: 520 μL; 5 mL formic acid added to each fraction, pH reduced to 3.5; samples immediately frozen (−20° C.) until analysis or pooling.
Fraction Analysis: Undiluted fractions analyzed (17 μL injection volume) by analytical elution HPLC at 215 nm; calculations based on area %.
Total Run Time: 12.3 hr
Output Concentration: 2.77 mg/mL
Column Loading: 88.2% of maximum capacity
Column Capacity:
˜42.9 mg peptide/mL matrix 2.79 mg peptide/mL solution based on whole sample
˜58.6 mg peptide/mL matrix 2.79 mg peptide/mL solution based on pure product
˜212 μmole displacer/mL matrix 10.0 μmole displacer/mL solution
Sample Conc./Output Conc.=1.1
Amount of Me3NH+ in sample=4.7 times stoichiometric amount. Good results are obtained with reasonable loading (42.9 g/L), excellent purity and excellent yield (99.3% purity 90% yield) using a small “analytical-type” column in one step from crude peptide. This example is designed to show the purification of a crude synthetic anionic peptide at neutral pH (7.1) using an anionic displacer with a cationic ion-pairing agent. Rather than using a primary or secondary amine, a protonated tertiary amine was chosen as IP agent in order to prevent any possibility of trans-amidation reactions. Screening of a range of tertiary amines showed that trimethyl amine gave the best resolution, purity and yield in this example. Under preparative conditions using the same column, the principal impurities are better removed at pH=7.1 (99.3% purity@90% recovery; 5% MeOH with trimethylammonium phosphate) than at pH=2.0 (98.6% purity@90% yield; 3% MeCN with trifluoroacetic acid; data not shown). The displacer band and the peptide bands are quite sharp leading to nearly rectangular displacement bands and nearly uniform codisplacement (total of 0.7% composed of 5 minor impurities). As a result, the recovered purity is nearly invariant at 99.3% throughout the range of yields from 80-95%. Only one impurity codisplaces under both pH conditions (2.0, 7.1) suggesting that a two-step purification of this peptide on the same column at two different pH values would lead to a final purity of 99.8-99.9% at an overall, two-step yield of 85-90%.
Main Pump(1) with 4 buffer lines, Sample Loading Pump(2) with 1 solvent line, Pump Selector Valve, Column Bypass Valve Pump Selector Valve: 6-port valve controlled by single-channel toggle logic (S3=0, Pump1 to column-Pump2 to waste, S3=1 Pump1 to waste-Pump2 to column)
Column Valve: 6-port valve controlled by single-channel toggle logic (S6=0, liquid flow through column, S6=1, liquid flow bypasses column)
UV photodiode array detector after column (flow cell: 0.5 mm pathlength, 9 μL volume) followed by conductivity detector (flow cell: 170 μL volume); conductivity
flow cell is removed when fractions are being collected for analysis.
Loading Buffer=A-Line on Pump1 (81=1-flow on, S1=0-flow off); Displacer Buffer=B-Line on Pump 1 (S2=1-flow on, S2=0-flow off); Displacer Removal Buffer=C-Line on Pump1 (S4=1-flow on, S4=0-flow off); Column Storage Buffer=D-Line on Pump1 (85=1-flow on, 85=0-low off); Sample Solution=A-line on Pump2 (S7=1, sample flow on, S7=0 sample flow off). Sample solution is filtered (0.211) and degassed.
Before sequence begins, cleaned column is briefly purged with A-buffer to remove column storage buffer. See Example 3 for description of other details.
Displacer Removal Buffer (C-Buffer)=10% (v/v) 1-propanol, 10% (v/v) DI water in acetonitrile.
Column Storage Buffer (D-Buffer)=70/30 (v/v) methanol/water with formic acid (15 mM) and ammonium formate (15 mM).
Starting Peptide: Crude synthetic oligonucleotide (20-mer, A2G6T8C4, ammonium salt, monothiophosphate backbone), 82.2% purity, FW=6.7397 mg/pmole, charge=−19.
Column: Waters Xbridge BEH130, 5 μm, 135 Å, 4.6×250 mm SS, —C18 on slica
Flow Rates Loading=208 μL/min; Displacement=208 μL/min
Ion-Pairing Agent: nButylammonium (nBuNH3+)
Temperature=23° C.
pH=7.0
Displacer Buffer: 15.0 mM Displacer 607b+20 mM H3PO4 (HPLC grade)+50 mM purified nBuNH2 in DI water w/5% (v/v) MeOH, pH=7.0 w/50% HCO2H (HPLC grade).
Loading Buffer: 20 mM H3PO4 (HPLC grade)+50 mM purified nBuNH2 in water w/5% (v/v) MeOH, pH=7.0 w/50% HCO2H(HPLC grade).
Sample Solution: 3.78 mg/mL (561 μM) oligo in water with 5% (v/v) MeOH, 20 mM H3PO4+100 mM purified nBuNH2; pH=7.0 w/50% HCO2H(HPLC grade).
Load Amount: 124.2 mg from loading pump (pump 2); Load Time=158.0 min. (2.63 hr)
Fraction Size: 624 μL
Sample Preparation Crude synthetic 20-mer oligonucleotide in ammonia water (25.1 mg/mL) is placed under vacuum on a rotary evaporator (30° C.) until almost all of the NH3 and water is removed forming a colorless sticky residue. Using a combination of Dilution Buffer-1 and Loading Buffer, the sample is diluted back to the same original volume (before solvent removal on rotary evaporator) so that the sample contains 100 mM nBuNH3+. Only slight pH adjustment is needed. A portion of concentrate is diluted from 25.1 mg/mL to 3.78 mglmL using Dilution Buffer-2. The diluted sample solution is filtered, degassed and stored at 4° C. until use. Dilution Buffer-1 is the same as the Loading Buffer except with 120 mM nBuNHB3+. Dilution Buffer-2 is the same as the Loading Buffer except with 100 mM nBuNH3+.
Fraction Analysis: Fraction analysis is not carried out. Purified fractions are conservatively pooled based on the shape of the displacement trace.
Analysis of pooled product is carried out using analytical elution IP-RP chromatography and anion-exchange chromatography.
Fractions are monitored at 260 nm; calculations are based on area %.
Total Run Time: 7.5 hr
Output Concentration: 2.40 mg/mL
Column Loading: 100.3% of maximum capacity based on total sample; 85.2% of maximum capacity based on main band
Column Capacity: ˜30.0 mg oligo/mL matrix 2.40 mg oligo/mL based on whole sample ˜35.3 mg oligo/mL matrix@2.40 mg oligo/mL based on main band ˜221 μmole displacer/mL matrix@15.0 mole displacer/mL solution
Purity %:99.0%
Yield %:75%; data based on conservative fraction-pool without fraction analysis; actual usable yield is likely to be higher (80-85%).
Comments: Sample ConcJOutput Conc.=1.57
Amount nBuNH3+ in sample=9.4 times stoichiometric amount.
Even though the loading concentration is a little high (3.78 vs 2.40 mg/mL) and loading amount is a little high (100% vs 80%), good results are obtained with reasonable loading (30.0 g/L) and good purity (99.0% purity 75% yield) using a small “analytical-type” column in one step from crude oligonucleotide. This example is designed to show the displacement purification of a crude synthetic oligonucleotide using an anionic displacer with a cationic ion-pairing agent. This experiment also shows that successful sample pooling can be carried out without extensive analysis of fractions. Screening of primary, secondary and tertiary amines as well as quaternary ammonium salts shows three ion-pairing agents that give reasonable binding strength and resolution: nC4H9NH3+, nC5H11NH3+, and nC6H13NH3+. Preparative IP-RP displacement chromatography is both a complement to and replacement for preparative anion-exchange displacement chromatography of oligonucleotides. Furthermore, this method that operates near neutral pH (pH=7-8) and is compatible with purification of oligo-RNA's.
Method 4a, 4b—Reversed-Phase for Anions (Sulfonates, Carboxylates):
Method 4c—Reversed-Phase for Anions (Phosphates, Phosphonates):
Method 4d—Reversed-Phase for Anions (Boronates):
Method 4e—Reversed-Phase for Long-Chain Alkyl Halides:
97.37 g freshly distilled n-hexylbenzene (0.60 mole, fw=162.276, ˜113 mL) is added to 300 mL stirring, anhydrous 1,2-dichloroethane in a 1 L, 3-neck round-bottom flask that is equipped with a reflux condenser, a magnetic stirrer, 500 mL addition funnel and Teflon®-coated thermocouple. The glassware and other equipment are dried in an oven to remove surface moisture. The reflux condenser is really not needed except as a safety precaution. The reaction is carried out under a dry nitrogen atmosphere with a slow N2 purge. At ambient temperature, 69.92 g chlorosulfonic (0.60 mole, fw=−1 16.525, ˜40 mL) in 300 mL anhydrous 1,2-dichloroethane is added in a dropwise fashion to the stirring reaction mixture at such a rate (˜5-6 mL/min) that the entire addition is complete in about 60 minutes. A mild reaction exotherm is observed (˜10° C.). After all the chlorosulfonic acid is added, the mixture is stirred at ambient temperature for about 20 hours. The reaction mixture is periodically monitored by HPLC. When, after 20-24 hours, the amount of residual n-hexylbenzene is 5-10% of the original amount, the reaction is considered complete. If not, additional chlorosulfonic acid in dichloroethane is added while the mixture is stirred for an additional 12 hours. At the end of the reaction, small mounts of residual, unreacted hexylbenzene are observed and there is no indication of di-sulfonation. Some monosulfonation isomers are observed (0.5% 3-isomer, 3.5% 2-isomer) that are removed by subsequent purification. At this point, the reaction solution is a clear yellow solution. If, however, the reaction solution is cloudy or hazy, this indicates that small amounts of water have entered the system. Without exposing the mixture to humid air, the solvent and HCl byproduct are removed under vacuum affording a light brown oily product. The gaseous HCl is removed by using a trap containing KOH pellets prior to using a low temperature trap to retain the solvent and other volatiles. Finally, the product is placed under high vacuum (0.01 torr) for 24 hours at ambient temperature in order to complete the removal of volatiles. This preparation of 4-nhexylbenzenesulfonic acid (free acid) is also used in subsequent preparations (product a). Alternatively, the product is dissolved in a minimum amount of dry n-pentane (˜1.5 L) and then used as a pentane solution (product b); the concentration of the sulfonic acid in pentane is estimated by HPLC. Again, without exposing the mixture to humid air, the n-pentane is removed under vacuum affording a purified, light brown oily product (product c). Note that when making the pentane solution, most of the sulfonic acid product (90-95%) dissolves; the pentane insoluble fraction is saved for other uses. If, however, the sulfonic acid product is exposed to a little atmospheric moisture, the oxonium salt forms which is insoluble in pentane and a lesser amount of the sulfonic acid is taken up during the pentane extraction. nHexylbenzenesulfonic acid (free acid) is a unique molecule being appreciably soluble in virtually all solvents: n-pentane, cyclopentane, methylene chloride, 1,2-dichloroethane, diethyl ether, tbutyl methyl ether, acetonitrile, toluene, dioxane, isopropanol, absolute ethanol, methanol and water.
Method A.
Exactly one equivalent of purified water (ca 10.8 g) is carefully added in a dropwise fashion with agitation to the entire nhexylbenzenesulfonic acid (free acid) product from Example 5 (product a). The reaction with water is noticeably exothermic. The cooled reaction mixture is extracted twice with 200 mL n-pentane in order to remove nhexylbenzene and other organic materials. The combined pentane fraction is discarded while residual pentane is removed under vacuum from sulfonic acid monohydrate. The reaction is nearly quantitative yielding a viscous light brown liquid.
Method B.
Exactly one equivalent of purified water (6-10 g) is carefully added in a dropwise fashion with vigorous stirring to the entire nhexylbenzenesulfonic acid (free acid) product in n-pentane from Example 5 (product b). The reaction with water is noticeably exothermic. The cold reaction mixture is cooled to about 0° C., the upper pentane layer is removed and the lower product layer is extracted once with 200 mL n-pentane. The combined pentane fractions are discarded while residual pentane is removed from sulfonic acid monohydrate. The reaction is nearly quantitative yielding a viscous yellow liquid.
Method C.
While the foregoing method is simple and straightforward, the alkylsulfonic acid produced has mediocre purity and is difficult to purify. Another method involves the isolation and purification of the sodium salt which in turn is converted back into the free acid. 26.4 g Purified and recrystallized, sodium 4-nhexylbenzenesulfonate (100 mmole, fw=264.32) is suspended in 300 mL HPLC-grade 2-propanol. The stirring mixture is heated to 70° C. for about 10 minutes and then cooled to 0-5° C. The cold mixture is weighed. Gaseous HCl is slowly and carefully bubbled into the cold, stirring mixture while the temperature is maintained below 10° C. (caution, strongly exothermic heat of solution). The HCl addition is terminated after 18-20 g HCl is added. The stirring mixture is carefully warmed to about 50° C. and maintained at that temperature for about 6 hours. The solvent and excess HCl are removed under vacuum (rotary evaporator). 300 mL 2-propanol is added to the mixture and again the solvent is removed under vacuum. The residue is taken up in 150 mL diethyl ether, the solution is filtered and the solvent and other volatiles are removed under vacuum using a rotary evaporator. Finally, the product is placed under high vacuum (0.01 torr) for 24 hours at ambient temperature in order to complete the removal of all volatiles. This procedure affords 22-23 g of a pure, colorless, viscous oil which is stored in a desiccator over P2O5 at 5° C. in the dark. Because the starting sodium salt is pure, the purity of the resulting sulfonic acid monohydrate is >99.7% (HPLC). Residual chloride content is <0.5 mole %. Karl-Fisher titrations indicate that the sulfonic acid exist as the monohydrate (0.9-1.1 equiv. H2O per sulfonic acid).
Method D.
To the final reaction solution from Example 5 before 1,2-dichloroethane or HCl is removed, 10.8 g purified water is carefully added dropwise with stirring. The solution of the monohydrates of alkylbenzene sulfonic acids are soluble in dichloroethane at room temperature. However, at low temperature (−20° C.), the monohydrates of the higher alkylsulfonic acids (noctyl, nnonyl, ndecyl, nundecyl) nicely crystallize from solution as colorless, waxy crystals. The oxonium salts of the higher alkylsulfonic acids are isolated using low-temperature filtration through sintered glass in a moisture-free atmosphere (dry N2). The crystals are washed with cold, anhydrous solvent: dichloroethane/pentane (50/50) and then n-pentane. Residual pentane is removed under vacuum. When warmed to room temperature, solid monohydrates melt and then resolidify again when cooled to −20° C. In contrast, the monohydrates of the intermediate alkylsulfonic acids (npentyl, nhexyl, nheptyl) do not crystallize from dichloroethane at −20° C.
At ambient temperature, the entire product, 4-nhexylbenzenesulfonic acid, from the above preparation (Example 5, product a) is added in a dropwise fashion over a period of about 30 minutes to 800 mL of a vigorously stirred NaBr solution in water (3.0 M, filtered) that is contained in a 2 L beaker. During the addition, an off-white solid forms, and after the addition is complete, the mixture is vigorously stirred at ambient temperature for about 60 minutes. Enough NaOH (50% aqueous) is added dropwise to the stirring solution until the pH is near neutral (pH=6-8 by pH paper). The solid is recovered by filtration through sintered glass and washed twice with absolute ethanol and washed twice again using acetonitrile. The solid product is dried by sucking dry air through it overnight. Recrystallization is accomplished by dissolving the product in 4× its weight of hot ethanol-water (50/50 w/w) followed by quick filtering through sintered glass into a clean flask. If needed, small amounts of absolute ethanol are added to the filtered solution in order to make up for that which lost during vacuum filtration. The filtered solution is heated to about 65-70° C., and then an equal amount (4×) of warm (65-70° C.) absolute ethanol is rapidly added with stirring. The warm mixture is allowed to stand at room temperature for about 4 hours during which time crystals (white platelets) of the desired product begin to form. The crystallization mixture is then allowed to stand at −20° C. for about 4 hours to complete the crystallization. The cold mixture is rapidly vacuum-filtered using a cold fritted glass filter, and then the mixture is allowed to warm up to ambient temperature. At room temperature, the product is washed once using small amounts of acetonitrile and then dried by passing dry air through the filter. This procedure produces about 82 g (57% yield based on amount of reacted nhexylbenzene) of a white crystalline product with a HPLC purity of >99.8% (HPLC). By adjusting the amount of solvent and the amount of water in the ethanol-water mixture, other high-purity sodium salts of alkylbenzenesulfonic acids are similarly prepared: npentyl, nheptyl, noctyl, nnonyl, ndecyl and nundecyl.
Method A.
At ambient temperature, the entire product, 4-nhexylbenzenesulfonic acid (free acid), from the above preparation (Example 5, product a) is dissolved in 1000 mL of peroxide-free diethyl ether in a 2 L flask. Gaseous trimethylamine is slowly bubbled into the stirring ether solution until no more reaction is observed. The hygroscopic, pale yellow crystalline solid is filtered, washed with diethyl ether and dried by passing dry nitrogen through the filter cake. The product is dissolved in 900 mL of warm (40° C.) 2-propanol and then cooled to room temperature. The mixture filtered to remove insolubles, 900 mL more 2-propanol is added and then diethyl ether (˜5 L) is slowly added to induce initial crystallization of the product. The mixture is cooled to −20° C. and allowed to stand overnight at this temperature. Filtration of the cold mixture using sintered glass produces off-white crystals that are subsequently washed with diethyl ether and dried. This product is recrystallized again from 2-propanol/diethyl ether yielding 113 g (69% yield based on reacted nhexylbenzene) of white hygroscopic crystals with a purity of >99.5% (HPLC). The product is dried in a vacuum oven (50° C., 25 torr) for about 6 hours and stored in a desiccator over P2O5. Many other alkylammonium salts of alkylbenzenesulfonic acids may be similarly prepared. All are soluble in water and simple alcohols. Some are insoluble in diethyl ether and n-pentane (such as trimethylammonium and tetramethylammonium salts) while others are soluble in diethyl ether yet insoluble in n-pentane (such as nbutylammonium and nhexylammonium salts).
Method B.
In 1000 mL of a cooled (10° C.), stirring n-pentane solution of 4-nhexylbenzenesulfonic Acid (0.33 mmole free acid per mL, Example 5, product b) is slowly bubbled gaseous trimethylamine as such a rate that the temperature is maintained below 20° C. until the supernatant solution is basic (damp pH paper). The solution is stirred at ambient temperature for about 15 minutes to complete crystallization of the product. The white crystals are filtered using fritted glass, washed with diethyl ether and dried by passing dry nitrogen through the filter cake. The white solid is dissolved 2-propanol at ambient temperature, filtered and then recrystallized from 2-propanoVdiethyl ether according to Example 20, Method A above. This procedure yields about 84 g (84% yield) of a white, crystalline product with HPLC purity of >99%.
Method C.
26.4 g Purified and recrystallized, Sodium Salt (100 mmole, fw=264.32) is suspended in 300 mL HPLC-grade 2-propanol. 11.0 g Trimethylammonium chloride crystals (115 mmole, fw=95.57) is added, several drops of freshly prepared HCl (−8% gaseous HCl in HPLC-grade 2-propanol) are added until the pH is distinctly acidic (damp pH paper) and then the mixture is stirred and briefly heated to 75° C. for 10 minutes. The stirring mixture is slowly allowed to cool to ambient temperature over a period of 6 hours. The mixture is gently heated to about 40° C. and then filtered through sintered glass in order to remove the sodium chloride. The solvent is removed from the filtrate under vacuum (rotary evaporator), 300 mL additional 2-propanol is added and then also removed under vacuum. More 2-propenol is added so that the total volume of the mixture is about 270 mL, and then 730 mL peroxide-free, inhibitor-free diethyl ether is added and mixed. This mixture is cooled to −20° C. overnight in order to complete crystallization of the product. The cold mixture is quickly filtered through sintered glass, washed with diethyl ether, dried by passing dry nitrogen through the filter-cake, further dried in a vacuum oven (50° C., 25 torr) for about 6 hours and stored in a desiccator over P2O5. This procedure yields about 26 g (86% yield) of a pure, white, crystalline product. This material is suitable for use a displacer. HPLC purity of the arene sulfonate anion is essentially identical to that of the starting material (>99.8%). Residual chloride content is <0.2 mole % and residual sodium is <0.05 mole %.
117.8 g freshly distilled 4-npropylbiphenyl (0.60 mole, fw=196.29, ˜120 mL) is added to 300 mL stirring, anhydrous 1,2-dichloroethane in a 1 L 3-neck round-bottom flask that is equipped with a reflux condenser, a magnetic stirrer, 500 mL addition funnel and Teflon®-coated thermocouple. The glassware and other equipment are dried in an oven to remove all surface moisture. The reflux condenser is really not needed except as a safety precaution. The reaction is carried out under a dry nitrogen atmosphere with a slow N2 purge. At ambient temperature, 69.92 g chlorosulfonic (0.60 mole, fw=116.53, ˜40 mL) in 300 mL anhydrous 1,2-dichloroethane is added in a dropwise fashion to the stirring reaction mixture at such a rate (˜5-6 mL/min) that the entire addition is complete in about 60 minutes. A mild reaction exotherm is observed. After the entire chlorosulfonic acid is added, the mixture is stirred at ambient temperature for about 20 hours. The reaction mixture is periodically monitored by HPLC. When, after 20 hours, the amount of residual npropylbiphenyl is 5-10% of the original amount, the reaction is considered complete. If not, additional chlorosulfonic acid in dichloroethane is added while the mixture is stirred for an additional 12 hours. Besides the main product at the end of the reaction, some unreacted butylbiphenyl (˜5%) is observed along with a monosulfonation isomer (˜0.2%) and some disulfonation (˜2%) which are all subsequently removed by extraction or recrystallization. At this point, the reaction solution is a clear yellow solution. Without exposing the mixture to humid air, the solvent and HCl byproduct are removed under vacuum affording a light brown oily product. The gaseous HCl is removed by using a trap containing KOH pellets prior to using a low temperature trap to retain the solvent and other volatiles. Finally, the product is placed under high vacuum (0.01 torr) for 24 hours at ambient temperature in order to complete the removal of volaties. This preparation of 4-npropylbiphenyl-4′-sulfonic acid (free acid) is used in subsequent displacer preparations.
To the final reaction solution from Example 9 before 1,2-dichloroethane or HCl is removed, 10.8 g purified water is carefully added in a dropwise fashion with stirring over a period of 30 minutes. During water addition, white crystals of the monohydrate deposit from solution. The mixture is stirred for 60 minutes at room temperature and then cooled to about 0° C. and allowed to stand at this temperature for 2 hours. The cold mixture is filtered using sintered glass in a dry N2 atmosphere, washed with dichloroethane, cyclopentane and finally n-pentane. The product is dried by passing dry N2 through the filter-cake and finally dried in a vacuum oven (45° C., 25 torr) for about 6 hours. The white solid is stored in a sealed container that is kept in a desiccator over P2O5. This procedure produces about 128 g (79% yield based on amount of reacted propylbiphenyl) of a hygroscopic, white crystalline solid that is 98-99% pure by HPLC. Generally, the monohydrates of alkylbiphenyl sulfonic acids crystallize nicely as white solids from dichloroethane at room temperature.
58.8 g 4-nPropylbiphenyl-4′-sulfonic Acid Monohydrate (Example 10, 200 mmole, fw=294.37) is dissolved in 175 mL absolute ethanol. If particulates are present, the solution is filtered through sintered glass. Through this stirring solution at ambient temperature, gaseous trimethylamine is carefully bubbled into solution until it is basic (pH paper). The solution becomes noticeably warm as the acid is neutralized by the amine, and the temperature rises to 50-55° C. Upon cooling and standing at room temperature for about 1 hour, a mass of white crystals separate from solution. About 450 mL of diethyl ether is added and the mixture is again stirred at ambient temperature for about 1 hour. The mixture is filtered using sintered glass under a dry N2 atmosphere, washed with twice with diethyl ether and dried by passing dry N2 through the filter cake. The product is recrystallized a second time from warm absolute ethanol and washed/dried as before. Finally the product is dried in a vacuum oven (45° C., 25 torr) for about 6 hours. The white solid is stored in a sealed container that is kept in a desiccator over P2O5. This procedure produces about 65 g (92% yield) of a white crystalline solid that is >99.5% pure by HPLC. Na+, NH4+ and other alkylammonium salts are similarly prepared.
25.5 g Freshly distilled 1-bromo-7-phenylheptane (100 mmole, fw=255.20) and 190 mL acetonitrile are placed in a in a one-liter, 3-neck round-bottom flask that is equipped with a heating mantle, mechanical stirrer, reflux condenser and Teflon®-coated thermocouple. The reaction is carried out under a nitrogen atmosphere with a slow N2 purge. 50.4 g Sodium sulfite (400 mmole, fw=126.04) is dissolved in 150 mL warm, purified water, and the solution is filtered in order to remove particulates. The aqueous sulfite solution is added all at once to the reaction flask, and the reaction mixture is heated at 76° C. for about 20 hours with vigorous mechanical stirring. The reaction mixture is allowed to stand overnight at 15-20° C. in order to allow the mixture to fully separate into two layers. The layers were separated, and the upper layer (product plus acetonitrile) is dried with anhydrous magnesium sulfate. The solvent is then removed under vacuum, and a white crystalline product forms. The mixture is filtered using a sintered glass filter, washed several times with peroxide-free diethyl ether and dried on the filter by passing dry N2 through the filter cake. The product is further dried in a vacuum oven (50° C., 15 torr) overnight. This procedure yields about 23.9 g (86% yield) of a white crystalline solid that is >99% pure by HPLC.
28.42 g L-2-Phenylglycine (188 mmole, >98% ee, fw=151.17, S-enantiomer) is suspended in 200 mL purified water in an open 750 mL Erlenmeyer flask that is equipped with a thermometer, magnetic stirring and external cooling. 13.60 g 50% NaOH (170 mmole, fw=40.00) solution is added dropwise over a period of 15 minutes. The mixture is stirred at room temperature for 30 minutes and then cooled to 10-15° C. 13.83 g Freshly distilled octanoyl chloride (85 mmole, fw=162.66) is added in a dropwise fashion over a period of 15 minutes, and then the mixture is stirred at 10-15° C. for an additional 60 minutes. Two more additions of NaOH solution followed by octanoyl chloride with stirring are carried out: 8.96 g 50% NaOH, 9.16 g octanoyl chloride, stirring for 1 hour at 10-15° C., 7.52 g 50% NaOH, 7.59 g octanoyl chloride, stirring for three hours at room temperature. The mixture is filtered through sintered glass, the filtrate is cooled to 10-15° C., and conc. aqueous HCl (reagent, 37%) is added dropwise until the pH is about 1 (pH paper). During acid addition, a clear oily layer or sticky solid forms as the crude product comes out of solution. 250 mL peroxide-free diethyl ether is added with stirring in order to take up the product in ether. The layers are separated, the upper ether layer is dried over anhydrous magnesium sulfate which is subsequently removed by filtration, and the ether is removed under vacuum leaving behind a colorless oil or sticky white crystalline mass. This material is taken up in a minimum amount of slightly warm (30° C.) diethyl ether, and then allowed to crystallize at −20° C. overnight. The cold mixture is quickly filtered using sintered glass, then washed with n-pentane and finally dried under vacuum (0.01 torr) at ambient temperature to yield a pure white crystalline solid (˜42 g, 81% yield).
A dry, filtered diethyl ether solution of L-N-octanoyl-2-phenylglycine, free acid (5 mL ether per g) is placed in a suitable Erlenmeyer flask and magnetically stirred at ambient temperature. Gaseous trimethylamine (>99%) is slowly bubbled into the stirring ether solution while maintaining the temperature below 30° C. Immediately, white crystals of the trimethylammonium salt crystallize from solution. Once the supernatant liquid is sufficiently basic (pH>8, damp pH paper), the addition of the amine is terminated, and the mixture is allowed to stand at 5° C. for two hours. The cold mixture is quickly filtered through sintered glass, washed with ether and then dried under vacuum producing a near quantitative yield (>95%) of the trimethylammonium salt. The product is recrystallized once from absolute ethanol/diethyl ether yielding an HPLC purity of >99.5%. No octanoic acid or its salt is detected. The white solid is stored in a sealed container that is kept at room temperature in a desiccator over P2O5.
In order to avoid contamination, carefully cleaned boroslicate glassware, beakers, flasks, fritted glass filters and pipettes are used; organic extractable plastic labware is avoided. 32.62 g Freshly distilled n-butylamine (440 mmole, fw=74.138) is added in a dropwise fashion over a period of about 15 minutes to a stirring solution of 19.21 g freshly recrystallized methanephosphonic acid (200 mmole, fw=96.023) dissolved in 110 mL HPLC-grade denatured ethanol (90/5/5 w/w ethanol/methanol/2-propanol). During the course of the amine addition, there is a noticeable exotherm. The warm solution is briefly heated to 70° C. and then allowed to cool to room temperature for about 6 hours in order to begin crystallization. Then 110 mL peroxide-free, inhibitor-free diethyl ether is added, the mixture is stirred and then allowed to stand overnight at −20° C. The di-ammonium salt is filtered using sintered glass, washed twice with diethyl ether, washed once with HPLC-grade n-pentane and dried by sucking dry nitrogen through the filter cake. The recrystallization process is repeated a second time by dissolving the white crystalline product in an appropriate of hot denatured ethanol. After the final recrystallization, the product is further dried in a vacuum oven (45° C., 25 torr) for about 6 hours and then stored in a desiccator over P2O5. This procedure yields about 43 g (88% yield) of a stable, solvent-free, mildly hygroscopic, white crystalline salt that is pure enough to add to HPLC buffers in the range of 10-100 mM.
Generally, HPLC-grade amines and acids for use as HPLC buffers are difficult to obtain except for a very limited range of reagents that are commonly used such as triethylamine and phosphoric acid. For this reason, it is useful to obtain stable, high-purity ammonium salts of buffering anions for HPLC uses. The ammonium salts are chemically more stable than the corresponding free amines, and they are easier to handle than pure amines themselves. This general procedure with minor modifications is used to make purified di-ammonium salts of methanephosphonic acid, phosphoric acid and phosphorous acid. Good quality, HPLC-grade phosphoric acid, good quality, freshly recrystallized, solid phosphorous acid or methanephosphonic acid are used. Liquid amines are distilled prior to use in order to remove many of the impurities. High purity (>99%) gaseous amines, Me3N, Me2NH, MeNH2, EtNH2, are delivered in the gaseous form from pressurized cylinders. Generally, commercially available aqueous solutions of these amines are not pure enough to be used. High purity, quaternary ammonium hydroxide solutions in water (−35% w/w) are also used. Either of two solvents, HPLC-grade methanol or HPLC-grade denatured ethanol, are used for crystallization at a usage rate of 2-20 mL solvent per gram of salt. A wide range of crystalline di-ammonium salts are made using this method.
bQ1 = 4-(4-nBuC6H4)C6H4—
cQ2 = 4-(4-nBuC6H4)-3-ClC6H3—; Q2I prepared via coupling 4-nBuC6H4Br and 4-AcNH-2-Cl-C6H3B(OH)2 followed by deacylation, diazotization, iodination.
aMethod: R. Shintani, M. Takeda, T. Tsuji, T. Hayashi, J. Amer. Chem. Soc., 132 (2010) 13168-13169
dS. Yamada, S. Ikukawa, S. Nakayama, M. Yudasaka, Japanese Patent JP04266836 (1992)
nBuNH3+ salt
nC5H11NH3+
nC6H13NH3+
nC6H13NH3+
aMinor 3-isomer (meta) obtained in low yield by multiple recrystallization.
nBuNH3+ salt
d Method: N. A. Bumagin, E. V. Luzikova, J. Organomet. Chem., 532 (1997) 271-273; H. X. Sun, S. H. Sun, B. Wang, Tetr. Lett., 50 (2009) 1596-1599.
g Method: the Arbusov method is used as described by N. D. Dawason and A. Burger, J. Amer. Chem. Soc., 74 (1952) 5312-5314 followed by acid hydrolysis (HCl) of the diethyl ester.
zmeasured at pH = 6.1
h Method: L. J. Goossen, M. K. Dezfuli, Synlett, 3 (2005) 445-448, followed by acid hydrolysis (HCl) of the diethyl ester.
zmeasured at pH = 6.1
i See V. Caplar, L. Frkanec, N. S. Vujicic, M. Zinic, Chem.-Eur. J., 16 (2010) 3066-3082
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US2012/058544 | 10/3/2012 | WO | 00 | 4/2/2014 |
| Number | Date | Country | |
|---|---|---|---|
| 61542424 | Oct 2011 | US |
