Comparison of Reversed Stationary Phases for the Chromatographic Separation of Inorganic Analytes Using Hydrophobic Ion Mobile Phase Additives

Abstract

Alkyl-modified silica (RSi) and polystyrenedivinylbenzene (PRP-1) stationary phases are compared for the chromatographic separation of inorganic analyte anions and cations using hydro-phobic ions of opposite charge as mobile phase additives. Tetra-alkylammonium salts were used for anion separations and alkyl sulfonate salts for cation separations. Two major equilibria influence the retention of analyte ions on PRP-1. These are: retention of the hydrophobic ion on PRP-1 and an ion exchange selectivity between the hydrophobic counterion and the analyte ion. When using RSi retention is also influenced by ion exchange at residual silanol groups, which act as weak cation exchange sites. Mobile and stationary phase variables that influence analyte retention are identified. Optimization of these provides favorable eluting conditions for the separation of inorganic ionic analytes. Of particular interest is the potential use of PRP-1 and RSi columns for the separation of inorganic cations; conditions for the separation of alkali metals and alkaline earths are discussed.     

Capillary-electrochromatographic separations with copolymeric reversed-stationary phase and ion-exchanger-packed columns

A macroporous, spherical, 7 μm, polystyrene–divinylbenzene (PS–DVB), reversed-phase adsorbent (PRP-1) was evaluated as a stationary phase for the capillary electrochromatographic (CEC) separation of neutral, acidic, and basic analytes of pharmaceutical interest. Electroosmotic flow (EOF) for a PRP-1 packed capillary is nearly constant over the pH 2 to 10 range and is higher than for a silica-based C₁₈ packed capillary on the acidic side. EOF increases with an increase in buffer acetonitrile concentration or as applied potential increases. As analyte hydrophobicity increases, analyte retention and migration time increases. Increasing buffer acetonitrile concentration reduces analyte partitioning with the PS–DVB stationary phase and analyte retention and migration time decreases. When exchange sites are present on the PS–DVB copolymer, EOF (EOF is reversed for the anion-exchanger) increases as the exchange capacity increases. An increased exchange capacity also reduces partitioning of the analyte with the PS–DVB matrix and analyte retention and migration time decrease. Because of excellent stability in an acid environment, the PRP-1 packed capillary can be used in strong acid buffer solution and weak acid and base analytes depending on pK_a values can be separated as neutral species and cations, respectively. CEC separations on a PRP-1 capillary of neutral steroids, weak base pharmaceuticals (separation as cations), purines and pyrimidines (as cations), fatty acids (as undissociated species), and sulfa derivatives (as cations) are described. Efficiency for the PRP-1 packed capillary for acetone or thiourea as the analyte is about 6·10⁴ plates m⁻¹.     

Chromatographic behavior of ion pair enantiomers of dansyl leucine cyclohexylammonium salt on a beta-cyclodextrin stationary phase and the effect of a competitive-binding mobile phase additive

The separation of dansyl leucine enantiomers on a beta-cyclodextrin stationary phase is significantly complicated by the association of the amino acid with its cyclohexylammonium counter ion, in a mobile phase of 80:20 (v/v) methanol-water. This produces very unusual chromatography, with two partially superimposed peaks observed for each enantiomer at lower column temperatures. The peak shape is attributed to the irreversible, oncolumn conversion of the ion pair (I) to the free, protonated (neutral) dansyl amino acid (II+H). Increasing the ionic strength of the mobile phase greatly improves the chromatography by transforming the solute species to enantiomers of II (the anionic, free amino acid). Van't Hoff plots are constructed for both species I and II (under different mobile phase conditions) to provide thermodynamic insight into the major enantioselective driving forces of separation. The chiral discrimination of the stationary phase is found to be primarily enthalpically driven for both solutes. Finally, 1-adamantanecarboxylic acid (ACA) is investigated as a solute-competitive mobile phase additive to intentionally block the hydrophobic cyclodextrin cavities on the stationary phase. By varying the concentration of ACA additive in the mobile phase, control over the retention and chiral recognition of the stationary phase is demonstrated.     

Facile separation of enantiomers,geometrical isomers,and routine compounds on stable cyclodextrin LC bonded phases

The effectiveness of employing stationary phases composed of chemically bonded cyclodextrin molecules in the high performance liquid chromatographic separation of a variety of different types of compounds is summarized. Over one hundred compounds, including optical, geometrical, and structural isomers, diastereomers, and epimers were successfully separated from each other via use of beta- or gamma-cyclodextrin bonded phases and aqueous methanolic mobile phases. The mechanism of separation is based upon inclusion complex formation between the compounds being separated and the cyclodextrin molecules bonded to the stationary phase. The effects of temperature, mobile phase composition and flow rate upon the chromatographic selectivity and resolution are described. The results indicate that the cyclodextrin columns may be more versatile, flexible, and effective compared to the conventional normal or reversed phase columns.     

Separation of chlamydosporol epimers by reversed-phase HPLC using commercial solvent optimisation software

Summary Two epimers of the mycotoxin chlamydosporol were separated by HPLC on an RP-18 column using a quaternary mobile phase consisting of water (79.1%), methanol (10.0%), acetonitrile (10.4%) and tetrahydrofuran (0.5%), with a flow rate of 1 ml min^–1. This optimal composition of mobile phase, with which the resolution value for the two epimers (1 and2) was 2.73 with retention times of 5.88 and 7.12 min, respectively, was achieved by the application of Philips Solvent Optimisation Software PU 6100. The presence of free silanols on the stationary phase was shown to be an essential requirement for the separation of the chlamydosporol epimers.     

Isolation by Means of Preparative Reversed-Phase Liquid Chromatography of Epimeric Alcohols Formed Upon Reduction of Pregnenolone and Progesterone

Abstract

A method is described which permits complete separation on a preparatory scale of the 20R and 20S epimeric alcohols obtained from lithium aluminium hydride and sodium borohydride reduction of pregnenolone and progesterone, respectively. The retention behaviour and resolution obtained on chromatography of the epimers on C-18 bonded phase material and elution with different acetonitrile/water and methanol/water mobile phases were studied. The order of retention is in both cases in accordance with ¹H-NMR chemical shift data which indicate a stable conformation with a more exposed 20-OH group in the 20S (=20α) epimer. Deviations from the elution order expected for true reversed-phase retention mechanisms were found on elution with mobile phase systems of reduced water content.     

Ion-Interaction Chromatographic Separation of Free Amino Acids

Abstract

An extensive study of the HPLC separation of 20 free amino acids by the addition of alkanesulfonate salts to the mobile phase was previously reported (1). This paper describes modifications in the procedure that improves the separation and resolution of the 20 free amino acids. Mobile phase variables (type and concentration of alkanesulfonate salt, organic modifier concentration, mobile phase pH, and mobile phase ionic strength), and stationary phase variables (particle size, type of packing) which can affect amino acid separation, resolution and selectivity were studied. Two stationary phases were compared, the 5 μm Hamilton PRP-1 and Phase Separations 3 μm, ODS-2. Longer chain alkanesulfonate salts (octane and decanesulfonate salts) were evaluated as mobile phase additives. A mobile phase gradient of increasing per cent organic modifier was necessary for separating complex mixtures of polar and nonpolar-basic amino acids. It is now possible to separate 19 of 20 free amino acids with this ion-interaction chromatographic procedure.     

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 CF3COO-, perchlorate ClO4-, and hexafluorophosphate PF6-). 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-, CF3COO-, ClO4-, PF6-) 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.     

Ruthenium (II) 1,10-Phenanthroline Salts as Mobile Phase Additives for the Separation and Indirect Detection of Free Amino Acids

Abstract

Ruthenium (II) 1,10-phenanthroline, Ru(phen)²⁺ ₃, salts are used as ion interaction reagents in a basic mobile phase for the retention, resolution, and indirect photometric detection (IPD) of free amino acids on a polystyrene divinylbenzene (Hamilton PRP-1) column. Mobile phase Ru(phen)²⁺ ₃ concentration and pH and type and concentration of organic modifier and counteranion affect retention and IPD. Underivatized amino acid elution order is influenced by side chain structure typical of ion exchange processes. Detection limits for the separation and detection of free amino acids using an isocratic elution condition are about 0.1 nmole for lower retained amino acids and 0.25 nmole for higher retained amino acids for a 3:1 signal:noise ratio. Gradient elution is possible but at higher detection limits.     

Isolation and Identification of Drugs of Forensic Interest by High Performance Reverse Phase Ion Pair Partition Chromatography

Abstract

A method is presented for the isolation and identification of milligram or microgram quantities of drugs of forensic interest. High performance reverse phase ion pair partition chromatography is performed on a 9.4 millimeter internal diameter microparticulate octadecylsilane column employing a water, methanol, acetic acid, alkylsulfonate mobile phase. Subsequent to collection from the liquid chromatograph, a simple extraction is performed followed by Infrared (IR) analysis and/or solid probe Mass Spectrometry (MS). A study is presented using phenylpropanolamine hydrochloride and ephedrine hydrochloride as a test model for the determination of the optimum concentration of counter ion required for a semi-preparative separation. The method is applied to an LSD seizure from a clandestine laboratory, methamphetamine in a street exhibit, and an amobarbital - secobarbital mixture in a multi-barbiturate capsule.     

Ion-pair LC of carbohydrates at alkaline pH. Separation and retention behaviour of glycoconjugates

Summary A new technique for the separation of carbohydrates as ion-pairs in strongly alkaline solution is presented. Carbohydrates are weakly acidic and partly present as anions at pH12 [1]. They are retained as ion-pairs on polymeric solid phases (PRP-1 and PLRP-S) with a hydrophobic quaternary ammonium counter ion present in the mobile phase. The effects of nature and concentration of mobile phase components on the retention of carbohydrates have been investigated and an ion-pair distribution model is proposed. The influence of temperature indicated no changes in retention mechanism with high counter ion concentration, but the resolution decreased with increasing temperature. Saccharides added to the mobile phase were shown to increase the retention and the selectivity.     

Simultaneous determination of epimers (+)-catechin and (‒)-epicatechin in Onosma bracteatum Wall. using high-performance thin-layer chromatography‒mass spectrometry method

High-performance thin-layer chromatography‒mass spectrometry (HPTLC‒MS) method was developed for the estimation of epimers (+)-catechin (CA) and (‒)-epicatechin (ECA) in Onosma bracteatum Wall. Resolving these epimers is challenging and so method optimization was done for the selection of the stationary phase and the mobile phase to achieve their coherent separation. To further increase the reliability of the obtained densitometric results, HPTLC–MS analysis was performed. The genus Onosma L. is a species-rich genus that exhibits complex patterns of morphological and karyological diversity, and highly debatable taxonomic approaches. Thus, many similar species are described based on morphological differences and often quite ambiguous. To facilitate the identification of O. bracteatum, separation was achieved using pre-coated silica gel 60 F₂₅₄ HPTLC plate as the stationary phase and a mixture of diisopropyl ether–ethyl acetate–formic acid (9.0:0.2:0.7, V/V) as the mobile phase for the separation of epimers CA and ECA. Sample preparation, mobile phase selection and optimization were given importance to manage good resolution (R_F) of these markers. Flavan-3-ols CA and ECA were identified and confirmed on the basis of R_F and in situ UV and MS overlaid spectra with respective standards. The method was validated for linearity, inter-day precision, intra-day precision, repeatability, accuracy, specificity, limit of detection, and limit of quantification. The average recoveries for epimers CA and ECA from ethyl acetate extract fraction (MEF) were found 98.86 and 99.03% indicating the good reproducibility for each marker. The proposed validated HPTLC method is simple, accurate and reproducible and is the first report on the separation and quantification of the epimers CA and ECA in O. bracteatum using HPTLC–MS.

     

Adsorption of an Anionic Surfactant on an Octadecyl Bonded Phase from a Pure Aqueous Mobile Phase

Abstract

Difficulties encountered when using pure aqueous mobile phases in ion pair reversed phase liquid chromatography are described.

The results presented in this paper show the influence of the structure and wettability of the stationary phase surface on the adsorption of an anionic surfactant (sodium octyl sulfate).     

Effect of anionic additive type on ion pair formation constants of basic pharmaceuticals

Due to their beneficial effect on selectivity, peak shape, and sample loading, the use of mobile phase anionic additives, such as formate (HCOO-), chloride (Cl-), and trifluoroacetate (CF3COO-), is increasing in both reversed-phase chromatography (RPLC) and liquid chromatography-mass spectrometry (LC/MS). Similarly, perchlorate is a common "ion pair" agent in reversed-phase separation of peptides. Although many studies have suggested that anions effect in chromatography is due to the formation of ion pairs in the mobile phase between the anions and cationic analytes, there has been no independent verification that ion pairs are, in fact, responsible for these observations. In order to understand the mechanisms by which anionic additives influence retention in chromatography and ionization efficiency in electrospray mass spectrometry, we studied the formation of ion pairs between a number of prototypical basic drugs and various additives by measuring the effect of anionic additives on the electrophoretic mobility of the probe drugs under solvent conditions commonly used in chromatography. For the first time, ion pair formation between basic drugs and anionic additives under conditions commonly used in reversed-phase liquid chromatography has been confirmed independently with all anions (i.e. hexafluorophosphate, perchlorate, trifluoroacetate, and chloride) used in this study. We measured ion pair formation constants (Kip) for different anionic additives using capillary electrophoresis (CE) and obtained quantitative estimates for the extent of ion pairing in buffered acetonitrile-water. The data clearly indicate that different anionic additives ion pair with cationic drugs to quite different extents. The ion pair formation constants show a clear trend with the order being: PF6- > ClO4- > CF3COO- > Cl-. However, the extent of ion pairing is not large. At a typical RPLC mobile phase additive concentration of 20mM, the percentages of the analytes that are present as ion pairs are about 15%, 6%, and 3% for hexafluorophosphate, perchlorate, and trifluoroacetate, respectively. The fraction of the analytes present as a chloride pair is even smaller.     

Industrial application of green chromatography--I. Separation and analysis of niacinamide in skincare creams using pure water as the mobile phase

In this work, chromatographic separation of niacin and niacinamide using pure water as the sole component in the mobile phase has been investigated. The separation and analysis of niacinamide have been optimized using three columns at different temperatures and various flow rates. Our results clearly demonstrate that separation and analysis of niacinamide from skincare products can be achieved using pure water as the eluent at 60 °C on a Waters XTerra MS C18 column, a Waters XBridge C18 column, or at 80 °C on a Hamilton PRP-1 column. The separation efficiency, quantification quality, and analysis time of this new method are at least comparable with those of the traditional HPLC methods. Compared with traditional HPLC, the major advantage of this newly developed green chromatography technique is the elimination of organic solvents required in the HPLC mobile phase. In addition, the pure water chromatography separations described in this work can be directly applied in industrial plant settings without further modification of the existing HPLC equipment.     

Quantitation of trace betamethasone or dexamethasone in dexamethasone or betamethasone active pharmaceutical ingredients by reversed-phase high-performance liquid chromatography

Adequate separation is essential for the quantitation of trace amounts of dexamethasone that are typically found in betamethasone active pharmaceutical ingredients and vice versa. In this paper, we describe three simple and efficient high-performance liquid chromatography methods from which true baseline separations between betamethasone and dexamethasone are achieved even when the concentration ratios between these two epimers are larger than 2000:1. One method is developed on a 5 cm ACE C8 column that uses water and acetonitrile as the mobile phase and 20 mM beta-cyclodextrin as the mobile phase additive. The resolution factor between betamethasone and dexamethasone is 3.3. The second method is developed on a 10 cm ACE C8 column that uses water and acetonitrile as the mobile phase, in which the resolution factor between the epimers is 2.7. The third method is developed on a 10 cm ACE C8 column using water and tetrahydrofuran as the mobile phase, in which the resolution factor between the epimers is 3.1. Preliminary validation studies are carried out for the second and third methods.     

Examination of some reversed-phase high-performance liquid chromatography systems for the determination of lipophilicity

Three reversed-phase systems [based on the divinylbenzene-styrene copolymer (PRP-1), the C₁₈-derivatized divinylbenzene-styrene copolymer (ACT-1), and the Nucleosil C₈ columns] were studied for their suitability in lipophilicity determination. Acetonitrile-water was selected as the mobile phase. Correlation between log k′ and log P_cyc for both the PRP-1 and Nucleosil C₈ systems was superior to the correlation between log k′ and either log P_oct or log P_cyc (oct = octanol; CYC = cyclohexane) on the ACT-1 column. On the PRP-1 and Nucleosil columns, correlation between log k′ and log P_oct was much improved when test compounds were grouped into classifications of non-H bonding, single amphiprotics (alcohols, phenols, amides) or double amphiproties. Although the PRP-1 system gave broad peaks with lipophilic substrates, there was good correlation between log k′ values on the Nucleosil silica-based reversed-phase system and the polymer PRP-1 system, indicating that either is suitable for the determination of lipophilicity.     

Chromatographic Separation of Cresol Isomers by a β‐Cyclodextrin: Application for the Determination of Volatile Phenols in Alcoholic Beverages

Abstract

The chromatographic separation of o‐cresol, m‐cresol, and p‐cresol by using β‐cyclodextrin as a chiral reagent has been studied. Conditions for the chromatographic separation of these isomers by using the cyclodextrin in the mobile phase or bonded in the stationary phase were optimized, and both procedures provided good results for the resolution of the chromatographic peaks. The use of fluorimetric detection (λ_exc 275 nm λ_em 300 nm) allows detection and quantification limits of the µg/L for eight studied phenols by using both procedures. The determination of volatile phenols in alcoholic beverages must be carried out using the cyclodextrin in the mobile phase because of the co‐elution of phenol and ethylguaiacol with other compounds of some studied matrix.     

Separation of epimeric aromatic acid (−)‐menthol esters by countercurrent chromatography using hydroxypropyl‐β‐cyclodextrin as an additive

The separation of ten epimeric aromatic acid (−)‐menthol esters by countercurrent chromatography with hydroxypropyl‐β‐cyclodextrin as the mobile phase additive was investigated, and methods for the analysis of all the epimeric esters by reversed‐phase high‐performance liquid chromatography were established. A biphasic solvent system composed of n‐hexane/20–70% methanol containing 50 mmol/L of hydroxypropyl‐β‐cyclodextrin (1:1, v/v) was selected, which provided high separation factors for five of the epimeric esters, and successful separations by countercurrent chromatography were achieved. The complete separation of five pairs of epimeric ester was obtained with the purity being over 98% for each peak fractions, as determined by high‐performance liquid chromatography. The recovery of each analyte from the eluted fractions reached around 80–88%.     

High-performance liquid chromatographic analysis of a new beta-lactam antibiotic, 6059-S (moxalactam)

Reversed-phase high-performance liquid chromatography was applied to the quantitative determination of a new beta-lactam antibiotic, 6059-S, ant its R- and S-epimers were resolved. The procedure was also applied to pharmaceuticals and human urine samples. Chromatographic separation was effected on a bonded hydrophobic stationary phase with two mobile phases: methanol-phosphate butter for the resolution of the epimers and methanol-tetra-n-butylammonium phosphate for the quantitation of 6059-S. For the determination of 6059-S in human urine, the latter mobile phase was used successfully without interference by the other urine components. An in vivo experiment was conducted by administering intravenously 1 g of 6059-S to seven volunteers and analysing their urine by chromatographic and microbiological assays, and a comparison of the results gave a correlation coefficient of 0.9954. One-compartment model analysis of the time-course data revealed that 6059-S was excreted in urine intact with a rate constant of 0.433h-1.