Mobile Phase Additives Research Articles

Enantioseparation of Seven Amino Alcohols on Teicoplanin Chiral Column

Under the polar organic mode, seven amino alcohols, propranolol, metoprolol, bisoprololfumarate, atenolol, salbutamol, isoprenaline, metoprolol and labetalol, were enantioseparated on teicoplanin chiral stationary phase (CSP), using methanol as mobile phase and acetic acid (HOAc) and triethylamine (TEA) as mobile phase additives. When the proportion of HOAc and TEA was 1:1, the retention factors and the resolutions of seven amino alcohols decreased with the increase of HOAc/TEA concentration, and the separation factors being kept constant. The excessive acid increased the polarity of mobile phase, and therefore the eluting ability was increased. Without HOAc additive, the retention factors of the amino alcohols decreased with the increase of the amount of TEA, with the separation factors being kept constant except labetalol. The polarity of mobile phase increased with the increasing of TEA concentration; therefore, the hydrogen bonding between solutes and CSPs decreased. All the results showed that the proportion of HOAc and TEA did not change the essentiality of chiral separation.

Retention factors and resolutions of amino benzoic acid isomers with some lonic liquids

Ionic liquids in the form of organic salts are being widely used as new solvent media. In this paper three positional isomers,o-amino benzoic acid,m-amino benzoic acid, andp-amino benzoic acids were separated with four different ionic liquids as mobile phase additives using high performance liquid chromatography (HPLC). The following ionic liquids were used: 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIm][BF4]), 1-ethyl-3-methylimidazolium tetrafluoroborate ([EMIm][BF4]), 1-ethyl-3-methylimidazolium methylsulfate ([EMIm][MS]), and 1-octyl-3-methylimidazolium methylsulfate ([OMIm][MS]). The effects of the alkyl group length on the imidazolium ring and its counterion, and the concentrations of the ionic liquids on the retention factors and resolutions of amino benzoic acid isomers were tested. The results of the separations with ionic liquids as the eluents were better than those without ionic liquids. Excellent separations of the three isomers were achieved using 2.0≈8.0 mM/L [OMIm][MS] and 1.0≈8.0 mM/L [EMIm][MS] as the eluent modifiers.

Separation of Periodate, Iodate and Iodide on a C-18 Stationary Phase. Dependence of the Retention on the Temperature and Solvent Composition. Monitoring of an Oxidative Cleavage Reaction

The separation of periodate, iodate and iodide can be easily achieved by HPLC on a C-18 column with a mobile phase consisting of acetonitrile and aqueous phosphoric acid, without the need of any mobile phase additives that are often employed. All three iodine species are well separated. Separation factors as high as 16 are observed, and retention factors k between ∼0.5 and 8. The retention time of all three ions increases with increasing acetonitrile concentration. The retention time of periodate and iodide decreases with increasing temperature, however, it increases slightly for iodate. An application of the HPLC method that was developed is the monitoring of an oxidative cleavage reaction, the cleavage of the enone-group of the steroid 4-androstene-3,17-dione. Periodate is used as oxidative reagent for this reaction. During the reaction periodate is reduced to iodate, and eventually both iodine species will be present in the reaction mixture, besides the starting material and product. In the final product the remaining iodate ions will be reduced to iodide which can be quantified accurately using the developed HPLC method.

Effect of Tertiary Alcohol Additives on Enantioselectivity of the Chiral‐AGP Column

The chiral‐AGP column is a protein based HPLC column widely used for analysis of chiral compounds in pharmaceutical and pharmacological applications. Organic solvents are frequently used as mobile phase additives to control analyte retention. In many cases, switching from one solvent additive to another can influence the enantioselectivity as well as retention. The group of solvents typically used as mobile phase additives includes methanol, ethanol, 1‐propanol, 2‐propanol, and acetonitrile. In this study, the column was used to resolve four different N‐substituted amino acid derivatives. The mobile phase consisted of a pH 7 phosphate buffer with the addition of organic solvent to control retention. During method optimization, nine different organic solvent additives were compared, including two tertiary alcohols. For three of the four analytes, the tertiary alcohol additives provided significantly higher enantioselectivity than any of the commonly recommended solvent additives, affording enantioselectivities in the range of 1.4 to 3.8.

Effect of inorganic salts as mobile-phase additives on lipophilicity values determined by reversed-phase thin-layer chromatography for new 1,2,4-triazole derivatives

The chromatographic lipophilicity ( R M0 , ϕ 0 , S ) of eleven new 1,2,4-triazole derivatives has been determined by RP-TLC with methanol-water mobile phases containing sodium salts of the monovalent anions H 2 PO 4 − , Cl − , I − , NO 3 − , CF 3 COO − , ClO 4 − , and PF 6 − . Addition of iodide anions to the mobile phase proved a key factor in obtaining lipophilicity indexes which correlated better with the log P G scale for all the solutes investigated. Addition of neutral or strong chaotropic salts (chlorides, hexafluorophosphates), according to the Hofmeister classification, improved the correlations between R M0 and S and between R M0 and ϕ 0 . Experimental log P TLC values were also established on the basis of calibration plots. These values were also correlated with the calculated lipophilicity log P G . Highly significant improvement of this correlation was achieved for mobile phases modified with sodium iodide. The results obtained are discussed in connection with the known physicochemical proper...

Effect of chaotropic mobile phase additives on retention behaviour of beta-blockers on various reversed-phase high-performance liquid chromatography columns

In this study the effect of type and concentration of chaotropic counter anion in buffered mobile phase on retention behaviour of some beta-blockers was studied. Two types of anions (perchlorate, dihydrogen phosphate) were examined under different concentrations. Further different pH-values for each anion were used to investigate the effect of pH changes on chaotropic behaviour of these anions. The role of organic modifier (methanol, acetonitrile) type was also considered. All these parameters were studied for three different RP-columns (calixarene modified silica gel, monolithic RP-column and a conventional RP-column). The results indicate that all studied factors affected the chaotropic behaviour of the tested anions. Also the type of the used stationary phase has found to play an important role in retention behaviour of beta-blockers.

Evaluation of the silanol‐suppressing potency of ionic liquids

Recently, increasing attention has been paid to the use of ionic liquids for high-performance liquid chromatography (HPLC) and capillary electrophoresis. In the present study, the silanol-suppressing potency of ionic liquids was evaluated by HPLC using the two-retention site model proposed previously by Nahum and Horváth (J. Chromatogr. 1981, 203, 53-63). The binding constant, KA, in that approach has been demonstrated to reliably reflect the ability of the ionic liquids to block the silanols of the silica support material of the stationary phase. The determinations were carried out for ionic liquids of the 1-alkyl-3-methylimidazolium group with the use of a series of basic drugs as the test analytes. Comparison of ionic liquids with standard mobile phase additives such as triethylamine showed the former to possess advantages as silanol suppressors in HPLC. The main advantage of the method is that it provides a simple and fast determination of the silanol complex stability, which allowed comparison of the suppressing efficiency of several ionic liquids.

Contribution to the standardization of the chromatographic conditions for the lipophilicity assessment of neutral and basic drugs

The chromatographic conditions aiming to a better simulation of n-octanol–water partitioning using a base deactivated silica (BDS) column as stationary phase were investigated for structurally diverse basic and neutral drugs. Extrapolated retention factors log k w, determined using different methanol fractions as organic modifier, were considered as lipophilicity indices. The effect of n-decylamine and n-octanol as mobile phase additives was examined and the appropriateness of the final retention outcome to reproduce lipophilicity data was evaluated. Moreover, the influence of n-octanol on the linearity of the log k/methanol fraction relationship and on the uniformity of the retention mechanism was investigated. 1:1 correlation between log k w values and the logarithm of the distribution coefficient (log D) was established for basic drugs in presence of both n-decylamine and n-octanol as mobile phase additives. However, for neutral drugs n-decylamine proved to be a sufficient and more important factor than n-octanol.

The effect of chaotropic mobile phase additives on the separation of selected alkaloids in reversed-phase high-performance liquid chromatography

The retention behavior of selected alkaloids from different classes was studied. The effect of chaotropic salts additives to the mobile phase on chromatographic parameters of protonated basic analytes was investigated on Zorbax Extend-C18 column. The influence of the type of salts and their concentration on retention, efficiency, peak symmetry and separation selectivity of investigated alkaloids was established. Buffered acetonitrile–water mobile phase was chosen because of significant retention of added liophilic ions due to strong dispersive π–π interactions. These conditions are responsible for great contribution of electrostatic forces in the retention of protonated bases. The addition of salt, such as hexafluorophosphate, perchlorate, trifluoroacetate leads to the increase in retention, efficiency and separation selectivity of examined analytes. The influence of added salts on increase in retention parameters could be expressed as follows: H 2PO 4 − < CF 3COO − < ClO 4 − < PF 6 −. This order is in agreement with ability of salts to “salting-in” effect according to Hofmeister series. Obtained chromatograms of alkaloids mixture illustrate suitability of chaotropic effect to improve their separation selectivity.

Influence of Zinc and Nickel Sulfates as Mobile Phase Additives on the Retention, Efficiency, and Selectivity of Organic Hydrocarbons with Polar Groups in RP‐HPLC

Inorganic eluent additives affect the retention and selectivity of chosen hydrocarbons with polar proton‐acceptor groups in reversed phase HPLC. Following that, nickel and zinc sulfates have been used in organic‐aqueous mobile phase at the concentration ranging from 0.001 to 0.01 M. The influence of salts is attributed to the desolvation of the analytes mainly due to the sulfate anions in most cases, or selective complexation with particular entities by metal cathions. The effect of salts concentrations on efficiency on a RP‐18‐bonded silica column has also been studied. All investigated compounds displayed significant changes in efficiency with the increase of salt concentration.

The effect of acidic and basic additives on the enantioseparation of basic drugs using polysaccharide‐based chiral stationary phases

The enantioseparation of nine commercially available basic drugs was achieved on polysaccharide-based chiral stationary phases with the acidic additive ethanesulfonic acid and the basic additive butylamine. Seven different commercially available CSPs were used for the study (AD, AS, OD, OJ, OG, OB, and OC). Mobile phase additives have been proven to be essential in obtaining satisfactory enantio-resolution in terms of both efficiency and selectivity. Significantly improved selectivities were obtained for the basic probe drugs with the acidic additive, ethanesulfonic acid, rather than the basic additive, butylamine. This is best seen with Chiralpak AS CSP. No enantioseparation for the nine drugs was observed when butylamine was used as an additive; however, satisfactory enantioseparation for the nine drugs was achieved using ethanesulfonic acid. Higher column efficiencies were observed with the acidic additive, especially when isopropanol was used as a modifier. Higher sensitivity was also achieved with ethanesulfonic acid because of the significantly lower background at the UV detection wavelength. The acidic additive was demonstrated to be superior to the basic additive for the enantioseparation of basic drugs using seven different polysaccharide-based CSPs. These results are counterintuitive to the common "rule of thumb" in enantioseparation that states acidic additives work best for acidic analytes and basic additives work best for basic analytes. The beneficial effects of acidic additive in enantioseparations observed in this study could significantly improve the applicability of polysaccharide-based CSPs for the enantioseparation of basic analytes.

Ionic liquids versus triethylamine as mobile phase additives in the analysis of β-blockers

A comparative study of peak shape, elution behavior, elution strength and resolution of seven β-blockers (acebutolol, alprenolol, labetalol, metoprolol, nadolol, pindolol and propranolol) chromatographed with aqueous-organic mobile phases containing additives such as the ionic liquid (IL) 1-butyl-3-methylimidazolium (BMIM BF 4) or triethylamine (TEA) is performed using a conventional reversed-phase Kromasil C 18 column and isocratic elution. The efficiencies and asymmetry factors achieved for the group of β-blockers in the Kromasil C 18 column improve when the cationic modifiers are added to the aqueous-organic mobile phase as competing additives for the silanol active sites. BMIM BF 4 is a significantly better additive compared to TEA. The improvement is more notably for the asymmetry factor, BMIM BF 4 allowing to obtain higher plate numbers than TEA at the same concentration. The effects of both modifiers on elution strength and retention factors are, however, different. TEA decreases the solute retention factors when BMIM BF 4 does not change them significantly. Using other additives taken in the IL family such as 1-butyl-3-methylimidazolium hexafluorophosphate and 1-octyl-3-methylimidazolium tetrafluoroborate (OMIM BF 4), it is shown that the silanol screening effect is always observed, due to the IL cation, when it is possible to increase or to decrease the solute retention factors playing with the hydrophobic nature or chaotropic character of its anion.

Chiral separation of ketoprofen on an achiral C8 column by HPLC using norvancomycin as chiral mobile phase additives

A high-performance liquid chromatographic method for chiral separation of ketoprofen racemate was developed. ( R)- and ( S)-ketoprofen enantiomers were separated on a Hypersil BDS C8 column (150 mm × 4.6 mm i.d., 5 μm) at 25 °C, using acetonitrile–triethylamine acetate (TEAA) buffer (pH 5.2, 20 mM) (35:65, v/v) containing 2.0 mM norvancomycin as the mobile phase. Effects of norvancomycin concentration, content of acetonitrile and TEAA buffer pH on the enantioseparation were investigated. The method was validated for linearity, repeatability, limits of detection (LOD) and limits of quantification (LOQ). Calibration curves ( r 2 = 0.999) were constructed in the range of 2.01–200.8 μg ml −1 for ( S)-ketoprofen and 2.04–152.4 μg ml −1 for ( R)-ketoprofen, respectively. Repeatability ( n = 5) showed less than 2% relative standard deviation (R.S.D.). LOD and LOQ for the two enantiomers were found to be 0.20 and 0.78 ng for ( S)-ketoprofen, 0.20 and 0.86 ng for ( R)-ketoprofen, respectively. Norvancomycin and vancomycin as chiral mobile phase additives (CMPAs) in the chiral separation showed similar abilities of enantioseparation. However, to obtain the optimum enantioseparation, a lower concentration of norvancomycin than that of vancomycin is required.

Determination and characterization of organic explosives using porous graphitic carbon and liquid chromatography–atmospheric pressure chemical ionization mass spectrometry

A new LC–MS method for the determination and characterization of three groups of commonly used organic explosives (nitroaromatic compounds, cyclic nitroamines and nitrate esters) was developed using a porous graphitic carbon (PGC) (Hypercarb) column. Twenty-one different explosive-related compounds – including 2,4,6-trinitrotoluene, its by-products and its degradation products – were chromatographically separated in a single analysis. This efficient separation facilitates the identification of the manufacturer of the explosive using the identified analytes as a fingerprint. A final, conclusive identification of the analytes can be obtained using LC–MS equipped with an atmospheric pressure chemical ionization (APCI) interface. Solvent effects on chromatographic behaviour were investigated, as were the effects of solvent mixtures and mobile phase additives. The number and the relative positions of the nitro groups within analyte molecules influence their order of elution; these effects were investigated. The data thus generated can be interpreted to support a hypothesis concerning the retention mechanism of nitro-containing compounds when using PGC. Limits of detection ranged from 0.5 to 41.2 ng. The new methodology described herein improves the sensitivity and selectivity of explosive detection. The effectiveness of the method is demonstrated by the analysis of soil samples containing explosives residue from test fields in Sweden and Afghanistan.

Sensitivity enhancement in liquid chromatography/atmospheric pressure ionization mass spectrometry using derivatization and mobile phase additives

High performance liquid chromatography with atmospheric pressure ionization (API) mass spectrometry has been essential to a large number of quantitative analytical applications for a variety of compounds. Poor detection sensitivity however is a problem observed for a number of analytes because detection sensitivity can be affected by many factors. The two most critical factors are the chemical and physical properties of the analyte and the composition of the mobile phase. In order to address these critical factors which may lead to poor sensitivity, either the structure of the analyte must be modified or the mobile phase composition optimized. The introduction of permanently charged moieties or readily ionized species may dramatically improve the ionization efficiency for electrospray ionization (ESI), and thus the sensitivity of detection. Detection sensitivity may also be enhanced via introduction of moieties with high proton affinity or electron affinity. Mobile phase component modification is an alternative way to enhance sensitivity by changing the form of the analytes in solution thereby improving ionization efficiency. pH adjustment and adduct formation have been commonly used to optimize detection conditions. The sensitivity of detection for analytes in bio-matrices could also be enhanced by decreasing ion-suppression from the matrix through derivatization or mobile phase addition. In this review, we will discuss detection-oriented derivatization as well as the application of mobile phase additives to enhance the sensitivity of detection in liquid chromatograph/atmospheric ionization/mass spectrometry (LC/API/MS), focusing in particular on the applications involving small molecules in bio-matrices.

The effect of the mobile phase additives on sensitivity in the analysis of peptides and proteins by high-performance liquid chromatography–electrospray mass spectrometry

The study of the effect of mobile phases on sensitivity in the analysis of peptides and proteins by high-performance liquid chromatography (HPLC)-electrospray mass spectrometry (ESI-MS) has been the aim of this review. Reversed-phase chromatography (RPLC) is the chromatographic mode most suitable for coupling with ESI-MS since mobile phases containing organic modifiers are used. The analysis of proteins and peptides by RPLC mostly involves the use of trifluoroacetic acid (TFA) as an ion-pairing agent despite its being a strong suppressor of the MS signal. Different studies reporting the effects of using other ion-pairing agents (other perfluorinated acids, acetic acid, formic acid, etc.) and buffers (ammonium acetate, ammonium formate, ammonium bicarbonate, morpholine, etc.) in RPLC-ESI-MS of proteins and peptides did not yield a single strong candidate that could generally replace TFA. The enhancement in sensitivity with other reagents observed in some cases strongly depended on the analyte, the experimental conditions used, and the mass spectrometer and, usually, it did not compensate for the loss in separation resolution related to TFA. The examples of direct coupling of affinity, size-exclusion, or ion-exchange chromatography (IEC) to ESI-MS are very limited because of incompatibilities related to the use of mobile phases containing high salt concentrations. To overcome this problem, an intermediate desalting step is needed. Multidimensional chromatography, microdialysis, and ion-capture modules can be used to couple these chromatographic modes with ESI-MS. Multidimensional chromatography with RPLC as a second dimension has most often been used. Although most examples involve the trap and analysis in the second dimension of a certain part of the first separation, some comprehensive analyses of the entire sample in the second dimension have also appeared.

Role of ion pairing in anionic additive effects on the separation of cationic drugs in reversed-phase liquid chromatography

Mobile phase additives can significantly affect the separation of cationic drugs in reversed-phase liquid chromatography (RPLC). Although there are many applications for anionic additives in RPLC separations, the retention mechanism of basic drugs in the presence of inorganic and highly hydrophilic anionic species in the mobile phase is not at all well understood. Two major retention mechanisms by which anionic additives can influence the retention of cations are: (1) ion pair formation in the mobile phase with subsequent retention of the neutral ion pair; (2) pre-sorption of anionic additives on the stationary phase followed by “dynamic ion-exchange” or “electrostatic interaction” with the analytes. Because the use of ion pair chromatography in the separation of proteins, peptides, and basic drugs is rapidly increasing, understanding the retention mechanism involved is becoming more important, especially for the smaller commonly used hydrophilic anionic additives (e.g., formate HCOO −, chloride Cl −, trifluoroacetate CF 3COO −, perchlorate ClO 4 −, and hexafluorophosphate PF 6 −). In this work, we compared various anionic additives in light of their effects on the retention of basic drugs. As did many others we found that the addition of anionic additives (Cl −, CF 3COO −, ClO 4 −, PF 6 −) profoundly influences the retention of basic drugs. In order to explain the data and differentiate the mechanisms by which the anionic additives perturb the chromatography, we used ion pair formation constants independently measured by capillary electrophoresis (CE) under the mobile phase conditions (pH, solvent composition) identical to those used in chromatography. Agreement between the predicted and experimental chromatographic data under various conditions was evaluated. Under specific circumstances (e.g., pH, stationary phase, and nature of anionic additive), we conclude that the ion pair mechanism is more important than the dynamic ion-exchange and at other conditions it remains a significant contribution.

Development of an immunochromatographic method to determine β-lactoglobulin at trace levels

β-Lactoglobulin (βLG) is one of the main components of whey proteins. Among other reasons, its allergenic character makes necessary its determination in hypoallergenic foods and biopharmaceutical products at very low concentration making use of very sensitive methods. In this paper, the development of an enzyme linked immunoaffinity chromatographic (ELIAC) method in sandwich format is described. The optimized method includes the use of a minicolumn (4.9 mm × 1 mm) packed with Nucleosil ® 4000-7OH derivatized with high density of antibody coverage, the employment of rabbit serum and Zwittergent ® 3–12 as mobile phase additives, and the incubation of the sample containing βLG with anti βLG labeled with horseradish peroxidase prior to their injection into the immunochromatographic system. The method allows determination of βLG with a practical detection limit of 20.7 pM and a sensitivity of 1.05 × 10 9 AU mol −1 L. The application of this ELIAC method to determine βLG in a commercial hypoallergenic formula based on caseins is shown. The amount of allergen, 0.32 pmol βLG per gram of sample, is about 5 million times lower than that naturally occurring in cow's milk.

Influence of inorganic mobile phase additives on the retention, efficiency and peak symmetry of protonated basic compounds in reversed-phase liquid chromatography

Inorganic eluent additives affect the retention of protonated basic analytes in reversed-phase HPLC. This influence is attributed to the disruption of the analyte solvation-desolvation equilibria in the mobile phase, also known as "chaotropic effect". With an increase of counteranion concentration analyte retention increases with concomitant decrease in the tailing factor. Different inorganic counteranions at equimolar concentrations affect protonated basic analyte retention and peak symmetry to varying degrees. The effect of the concentrations of four different inorganic mobile phase additives (KPF6, NaClO4, NaBF4, NaH2PO4) on the analyte retention, peak symmetry, and efficiency on a C8-bonded silica column has been studied. The analytes used in this study included phenols, toluene, benzyl amines, beta-blockers and ophthalmic drugs. The following trend in increase of basic analyte retention factor and decrease of tailing factor was found: PF6- > ClO4- approximately BF4- > H2PO4-. With the increase of the counteranion concentration greater analyte loading could be achieved and consequently an increase in the apparent efficiency was observed until the maximum plate number for the column was achieved. At the highest concentration of counteranions, the peak efficiency for most of the basic compounds studied was similar to that of the neutral markers. In contrast, the neutral markers, such as phenols, showed no significant changes in retention, efficiency or loading capacity as counteranion concentration was increased.

Influence of inorganic mobile phase additives on the retention, efficiency and peak symmetry of protonated basic compounds in reversed-phase liquid chromatography

Inorganic eluent additives affect the retention of protonated basic analytes in reversed-phase HPLC. This influence is attributed to the disruption of the analyte solvation–desolvation equilibria in the mobile phase, also known as “chaotropic effect”. With an increase of counteranion concentration analyte retention increases with concomitant decrease in the tailing factor. Different inorganic counteranions at equimolar concentrations affect protonated basic analyte retention and peak symmetry to varying degrees. The effect of the concentrations of four different inorganic mobile phase additives (KPF 6, NaClO 4, NaBF 4, NaH 2PO 4) on the analyte retention, peak symmetry, and efficiency on a C8-bonded silica column has been studied. The analytes used in this study included phenols, toluene, benzyl amines, beta-blockers and ophthalmic drugs. The following trend in increase of basic analyte retention factor and decrease of tailing factor was found: PF 6 − > ClO 4 − ∼ BF 4 − > H 2PO 4 −. With the increase of the counteranion concentration greater analyte loading could be achieved and consequently an increase in the apparent efficiency was observed until the maximum plate number for the column was achieved. At the highest concentration of counteranions, the peak efficiency for most of the basic compounds studied was similar to that of the neutral markers. In contrast, the neutral markers, such as phenols, showed no significant changes in retention, efficiency or loading capacity as counteranion concentration was increased.