Control mobile phase additives

The chiral resolution using cyclodextrins as mobile phase additives is also controlled by a number of chromatographic parameters as in case of CSPs. The chiral resolution occurred by the formation of diastereoisomeric inclusion complex formation and, hence, the composition of the mobile phase, pH, concentration of cyclodextrins, and temperature are the most important controlling parameters. 

Mobile-phase additives are used in HPLC to control the pH and ensure efficient and reliable separations. They also have to be compatible with ESI or APCI conditions. If the pH of the mobile phase needs to be reduced for better LC separations, the most suitable additives in LC/MS are acetic acid and formic acid with typical concentrations ranging from 0.1% to 1%. Note that addition of acids will suppress ionization in negative ion mode. Weakly acidic compounds may not form deprotonated ions under acidic conditions. If the pH of the mobile phase needs to be increased to enhance LC separations, ammonium hydroxide (0.1% to 1%) is suitable. Weakly acidic compounds can be ionized effectively in negative ion mode. Triethylamine is another additive that may be useful to enhance ionization of other compounds in negative ion mode because it is basic. It should be cautioned that the presence of triethylamine might suppress ionization of other compounds in the positive ion mode. A commonly used volatile salt in LC/MS to buffer mobile phases is ammonium acetate (<0.1 M). It is used to replace nonvolatile salts such as phosphates because these nonvolatile salts tend to crystalUze in the ion source and block the source, suppressing ionization of analytes. 

With respect to positive-ion ESI-MS, it shoidd be noted that simvastatin and lovastatin do not contain A -atoms. In the negative-ion mode, the deprotonated molecule is observed for the simvastatin lactone and its P-hydroxy acid [46-47]. In positive-ion ESI-MS, strong sodium adducts are observed [46]. Control of the sodium content and thereby of the relative abundance of [M+Na]" relative to other adducts was not completely possible during sample pretieatment and analysis [47]. In the presence of AmOAc, the formation of [M+Na] and [M+K]" is somewhat suppressed in favour of [M+H]" and [M+NHJ" [47]. Various mobile-phase additives were investigated to improve the sensitivity, to direct the adduct formation, and/or to direct the fragmentation of simvastatin in ESI-MS [48]. The best performance was achieved by the addition of 2 mmoEl of methylamine to the aqueous phase (adjusted to pH 4.5 by means of acetic acid). This resulted in a fourfold enhancement in the... 

Chiral mobile phase additives provide a more versatile and cost-effective approach for enantiomer separations in thin-layer chromatography. Typically, chemically bonded layers with cyclodextrin and its derivatives, bovine serum albumin, or macrocyclic glycopeptides are used as chiral additives in the reversed-phase mode [59,60,172-178]. For [5- and y-cyclodextrins and their derivatives, a 0.1 to 0.5 M aqueous methanol or acetonitrile solution of the chiral selector is used as the mobile phase. Bovine serum albumin is generally used at concentrations of 1-8 % (w/v) in an aqueous acetate buffer of pH 5 to 7 or in a 0.5 M acetic acid solution, in either case with from 3-40 % (v/v) propan-2-ol (or another aliphatic alcohol), added to control retention. Enantioselectivity usually increases with an increase in concentration of the chiral selector, and may be non existent at low concentrations of the chiral selector. 

The effect of the mobile-phase composition on the ESI-MS mass spectra of the chemotherapeutic agent paclitaxel has been investigated [64]. The use of different mobile-phase additives was evaluated. Paclitaxel is prone to formation of [M-i-Na] - and [M+K]+-ions and for quantitative bioanalysis of this drag, control over the adduct formation is important to obtain reliable results. The use of either docedylamine (Cj2N)/acetic acid, providing predominantly [M-i-C,2N+H] -ions,... 

We have already mentioned some of the commonly used mobile phase additives for MS-compatible pH control. The most important attribute is the volatility of all buffer components. The most frequently used mobile phase additives are formic acid and acetic acid, together vhth the true buffers formic acid/ammonium formate and acetic acid/ammonium acetate. In the alkaline pH range, the preferred additive is ammonia, and the buffer of choice is ammonium hydrogencarbonate. One can also use the ammonium ion with volatile counterions such as formate or acetate to establish a true buffer at the pfQ of the ammonium ion (pKj = 9.24). One finds sometimes in the literature the reported use of ammonium formate or acetate at neutral pH. It needs to be pointed out that this has little to do with pH control, since these salt solutions have no buffer capacity at pH 7 ... 

Retention and stereoselectivity on the BSA columns can be changed by the use of additives to the aqueous mobile phase (30). Hydrophobic compounds generally are highly retained on the BSA, and a mobile-phase modifier such as 1-propanol can be added to obtain reasonable retention times. The retention and optical resolution of charged solutes such as carboxyUc acids or amines can be controlled by pH and ionic strength of the mobile phase. 

Controlling for these forces requires variation in the amount of salt, organic solvent, and the pFI of the mobile phase. It is impractical to perform such experiments with 50 mM formic acid an alternative additive must be used that maintains its chaotropic properties independent of salt content or pFI. Fortunately, mobile phases containing 50 mM hexafluoro-2-propanol (HFIP) afford a fractionation range comparable to that of the formic acid (Fig. 8.6), permitting the effects of these variables to be studied systematically. 

Commercial grades of PVP, K-15, K-30, K-90, and K-120 and the quaternized copolymer of vinylpyrrolidone and dimthylaminoethylmethacrylate (poly-VP/ DMAEMA) made by International Specialty Products (ISP) were used in this study. PEO standard calibration kits were purchased from Polymer Laboratories Ltd. (PL), American Polymer Standards Corporation (APSC), Polymer Standards Service (PSS), and Tosoh Corporation (TSK). In addition, two narrow NIST standards, 1923 and 1924, were used to evaluate commercial PEO standards. Deionized, filtered water, and high-performance liquid chromatography grade methanol purchased from Aldrich or Fischer Scientific were used in this study. Lithium nitrate (LiN03) from Aldrich was the salt added to the mobile phases to control for polyelectrolyte effects. 

Valko et al. [37] developed a fast-gradient RP-HPLC method for the determination of a chromatographic hydrophobicity index (CHI). An octadecylsilane (ODS) column and 50 mM aqueous ammonium acetate (pH 7.4) mobile phase with acetonitrile as an organic modifier (0-100%) were used. The system calibration and quality control were performed periodically by measuring retention for 10 standards unionized at pH 7.4. The CHI could then be used as an independent measure of hydrophobicity. In addition, its correlation with linear free-energy parameters explained some molecular descriptors, including H-bond basicity/ acidity and dipolarity/polarizability. It is noted [27] that there are significant differences between CHI values and octanol-water log D values. 

Retention and selectivity in LSC are dramatically influenced by the presence of even low concentrations of polar additives in the mobile phase, particularly water [20,22,253-255]. Their influence is most pronounced when the mobile phase is nonpolar. However, when used in controlled amounts (in which case they are... 

In addition to water, virtually any organic polar modifier may be used to control solute retention in liquid-solid chromatography. Alcohols, alkyl2aiines, acetonitrile, tetrahydrofuran and ethyl acetate in volumes of less than one percent can be incorporated into nonpolar mobile phases to control adsorbent activity. In general, column efficiency declines for alcohol-moderated eluents cogqpared to water-moderated eluent systems. Many of the problems discussed above for water-moderated eluents are true for organic-moderated eluents as well. 

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