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 pi-pi 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: H2PO4- < CF3COO- < ClO4- < PF6-. 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.     

Effect of ionic liquid additives to mobile phase on separation and system efficiency for HPLC of selected alkaloids on different stationary phases

The silica-based stationary phases with favorable physical characteristics are the most popular in liquid chromatography. However, there are several problems with silica-based materials: severe peak tailing in the chromatography of basic compounds, non-reproducibility for the same chemistry columns, and limited pH stability. Ionic liquids (ILs) as mobile phase components can reduce peak tailing by masking residual free silanol groups. The chromatographic behavior of some alkaloids from different classes was studied on C18, phenyl, and pentafluorophenyl columns with different kinds and concentrations of ionic liquids as additives to aqueous mobile phases. Ionic liquids with different alkyl substituents on different cations or with different counterions as eluent additives were investigated. The addition of ionic liquids has great effects on the separation of alkaloids: decrease in band tailing, increase in system efficiency, and improved resolution. The retention, separation selectivity, and sequence of alkaloid elution were different when using eluents containing various ILs. The increase of IL concentration caused an increase in silanol blocking, thus conducted to decrease the interaction between alkaloid cations and free silanol groups, and caused a decrease of alkaloids retention, improvement of peak symmetry, and increase of theoretical plate number in most cases. The effect of ILs on stationary phases with different properties was also examined. The different properties of stationary phases resulted in differences in analyte retention, separation selectivity, peak shape, and system efficiency. The best shape of peaks and the highest theoretical plate number for most investigated alkaloids in mobile phases containing IL was obtained on pentafluorophenyl (PFP) phase.     

Effect of mobile phase anionic additives on selectivity,efficiency, and sample loading capacity of cationic drugs in reversed-phase liquid chromatography

In the present work, we study the effect of mobile phase anionic additive type and concentration on the selectivity, efficiency, and sample loading capacity of cationic drugs in reversed-phase liquid chromatography (RPLC). The type and concentration of an anionic additive are known to have a strong effect on the absolute retention of cations in RPLC; in contrast they have only a small effect on the selectivity of one cation relative to a second as seen here. This is mainly due to the similarity of the ion pair formation constants between the selected cations. The limiting retention factors of cations (i.e. the retention factor of the fully ion-paired analyte at very high additive concentration) are roughly proportional to their inherent hydrophobicities (i.e. the retention factor of the analyte in the absence of the anionic additive). With a given anion, differences in ion pairing strength between the solutes are required for effective selectivity adjustment. Based on the Wade–Lucy–Carr (W–L–C) kinetic model of overload peaks, the approach we developed in our previous work was used to study the effect of mobile phase anionic additives type and concentration on the limiting plate count (N₀) and sample loading capacity (ω_0.5) of various cationic drugs. Under linear chromatographic conditions, where the analyte exhibits its smallest peak width and thus maximum apparent plate count, the type and concentration of anionic additives have almost no effect on peak width. In comparison to neutral analytes the sorption isotherms of cationic species are very easily overloaded even when many fewer moles of cations as compared to neutrals are injected. We showed that different anionic additives profoundly affect the cations’ “overload profiles” (i.e. plots of plate count versus amount injected) by changing the sample loading capacities. The increase in sample loading capacities with different anions show the same order as the extent of ion pairing between the anions and the basic analytes. The detrimental effect of sample overloading on peak width can be greatly diminished by using either a stronger ion pairing agent or a higher concentration of a given ion pairing agent. Both effects operate by increasing the sample loading capacity, thereby allowing more solute to be injected. We believe that the increase in sample loading capacity described above is due in part to the increase in the number of ion-exchange sites as more anions sorb to the stationary phase. At the same time, the formation of a neutral ion-paired analyte also increases the amount of cation which can be loaded onto the stationary phase by allowing a greater fraction of the analyte to be present in the stationary phase as an electrically neutral (i.e. ion-paired) species.     

Vacancy ion-exclusion chromatography of inorganic acids on a weakly acidic cation-exchange resin column

Vacancy ion-exclusion chromatography (VIEC) for inorganic acids such as H(2)SO(4), HCl, H(3)PO(4), HNO(3), HI and HF is tested on a polymethacrylate-based weakly acidic cation-exchange resin column in the H(+)-form. That is, mixture of inorganic acids in the mobile phase is adsorbed to the resin phase passing through the separation column, and each vacant peak induced by injecting water is determined. Retention times are dependent on the degrees of retention for each analyte in the resin phase. In VIEC, well-shaped peaks of inorganic acids are produced, leading to efficient separations. However, retention behaviors of inorganic acids were strongly affected by the concentrations of the acids in the mobile phase. Sulfosalicylic acid was mixed with inorganic acids in the mobile phase prior to the introduction of a separation column in order to obtain the well-resolutions in the lower concentrations of the acids. By using this method, the separations of inorganic acids could be achieved in the range of 0.01-1 mM, and the linear ranges could be extended over two-orders of magnitude. This is considered since the protonated carboxylic groups fixed on the resin phase were increased with increasing the acid concentrations in the mobile phase, and the penetration effects for the acids to the resin phase were thus enhanced. The detection limits (S/N=3) were below 1.0 microM for all analyte acids. Precision values for retention times were below 0.32% and for peak area were below 0.91%.     

Effects of aliphatic amines on capillary electrochromatographic performance of tricyclic antidepressants on octadecylsilica

Adding aliphatic amines to the mobile phase improves peak symmetry and efficiency in capillary electrochromatography of tricyclic antidepressants on octadecylsilica. The most hydrophobic aliphatic amine studied, dimethyloctylamine (DMOA), was the most efficient. Despite the fact that the amine additives substantially reduced the electroosmotic flow, the retention of the analytes decreased indicating a strong competitive effect of the additives. DMOA gave the largest retention decrease, and simultaneously reduced the resolution, indicating that silanophilic interaction is significant to the separation. Highest efficiencies were obtained at the lowest pH (2.8). Acetonitrile influenced both efficiency and peak symmetry, and best results were obtained at 60%.     

Effect of ionic additives on the elution of sodium aryl sulfonates in supercritical fluid chromatography

Addition of a small amount of polar solvent (i.e., modifier) to CO2 in packed column supercritical fluid chromatography (SFC) has shown major improvements in both polar analyte solubility and interaction of the polar analyte with the stationary phase. Recently, the addition of an ionic component (i.e., additive) to the primary modifier by one of us has been shown to extend even further the application of SFC to polar analytes. In this work, the effect of various ionic additives on the elution of ionic compounds, such as sodium 4-dodecylbenzene sulfonate and sodium 4-octylbenene sulfonate, has been studied. The additives were lithium acetate, ammonium acetate, tetramethylammonium acetate, tetrabutylammonium acetate, and ammonium chloride dissolved in methanol. Three stationary phases with different degrees of deactivation were considered: conventional cyanopropyl, deltabond cyanopropyl, and bare silica. The effect of additive concentration and additive functionality on analyte retention was investigated. Sodium 4-dodecylbenzene sulfonate was successfully eluted using all the additives with good peak shape under isocratic/isobaric/isothermal conditions. Different additives, however, yielded different retention times and in some cases different peak shapes.     

Low-conductivity background electrolytes in capillary zone electrophoresis--myth or reality?

The asymmetric triangle (fronting or tailing) concentration profiles and their broadening are the typical results of the electromigrational zone dispersion characterizing a system of the analyte in the background electrolyte (BGE). The present contribution suggests the parameter named the relative velocity slope, SBGE,X, which was introduced here as a quantity characterizing the peak broadening and the asymmetry. SBGE,X VS. analyte ionic mobility diagrams are suitable for the comparison of BGEs of given pH and the conductivity composed of electrolytes of different pKaS and ionic mobilities. The concept of SBGE,X diagrams is verified by capillary zone electrophoresis of the model analytes, which involve (i) the series of sulfobenzoylated poly(ethylene glycols) as examples of the strong electrolytes with different ionic mobilities and (ii) the series of monobasic phenols as weak electrolytes with different pKaS and similar ionic mobilities. It follows from both theoretical predictions of peak symmetry and their experimental verification that the optimum composition of BGEs is determined mostly by the suitable ionic mobility of the coion in dependence on the ionic mobility of the analyte. The low-conductivity BGEs based on low-molecular carrier ampholytes are at best only comparable with the properly chosen monobasic electrolytes.     

Supercritical fluid chromatography of metoprolol and analogues on aminopropyl and ethylpyridine silica without any additives

Metoprolol and a number of related amino alcohols and similar analytes have been chromatographed on aminopropyl (APS) and ethylpyridine (EPS) silica columns. The mobile phase was carbon dioxide with methanol as modifier and no amine additive was present. Optimal isocratic conditions for the selectivity were evaluated based on experiments using design of experiments. A central composite circumscribed model for each column was used. Factors were column temperature, back-pressure and % (v/v) of modifier. The responses were retention and selectivity versus metoprolol. The % of modifier mainly controlled the retention on both columns but pressure and temperature could also be important for optimizing the selectivity between the amino alcohols. The compounds could be divided into four and five groups on both columns, with respect to the selectivity. Furthermore, on the aminopropyl silica the analytes were more spread out whereas on the ethylpyridine silica, due to its aromaticity, retention and selectivity were closer. For optimal conditions the column temperature and back-pressure should be high and the modifier concentration low. A comparison of the selectivity using optimized conditions show a few switches of retention order between the two columns. On aminopropyl silica an aldehyde failed to be eluted owing to Schiff-base formation. Peak symmetry and column efficiency were briefly studied for some structurally close analogues. This revealed some activity from the columns that affected analytes that had less protected amino groups, a methyl group instead of isopropyl. The tailing was more marked with the ethylpyridine column even with the more bulky alkyl substituents. Plate number N was a better measure than the asymmetry factor since some analyte peaks broadened without serious deterioration of symmetry compared to homologues.     

Characterisation of reversed-phase stationary phases for the liquid chromatographic analysis of basic pharmaceuticals by thermodynamic data

This paper describes the characterisation of reversed-phase liquid chromatography (RPLC) columns using thermodynamic measurements. Retention versus 1/T data were used to construct Van't Hoff plots. The slope of these plots indicates the standard enthalpy of transfer of the analyte from the mobile to the stationary phase. The standard entropy can be calculated from the intercept. Van't Hoff plots were linear for the investigated RPLC columns, meaning that for basic analytes over the temperature range studied no changes in the retention mechanism occurred. Enthalpies and entropies of transfer of basic analytes from the mobile to the stationary phase revealed information about the types of interaction of protonated and neutral compounds with the stationary phases. However, a clear view using the present set of basic compounds on how these thermodynamic data may explain the observed substantial differences in peak symmetry cannot be given. It is considered that addition of N,N-dimethyloctylamine (DMOA) to the eluent will results in a dynamically coating of the stationary phase. Addition of DMOA to the eluent resulted for protonated basic compounds in a reduction of both enthalpy and entropy. In practice, with DMOA in the eluent symmetrical peaks were obtained. It is assumed that this is due to blocking residual silanols and/or ion exclusion effects.     

磷酸根离子在阴离子交换树脂上的保留行为及其机理探讨

首次发现磷酸根离子在阴离子交换柱上以两个色谱峰流出。在研究磷酸根离子的保留行为的基础上,提出了Ｈ２ＰＯ－４在固定相中进一步离解的保留机理,即Ｈ２ＰＯ－４在与阴离子交换树脂交换基进行离子交换的过程中,由于树脂交换基和淋洗离子的电荷相互作用促使一部分Ｈ２ＰＯ－４进行第２级离解。由于Ｈ２ＰＯ－４和ＨＰＯ２－４在阴离子交换树脂上的保留值不同,导致磷酸根离子出现“双峰”。     

Selective separation and purification of highly polar basic compounds using a silica-based strong cation exchange stationary phase

Compared to moderately and weakly hydrophilic bases, highly polar basic compounds are even more difficult to separate due to their poor retention in reversed phase (RP) mode. This study described the successful applications of a strong cation exchange (SCX) stationary phase to achieve symmetric peak shape, adequate retention and selectivity in the separation of very polar basic compounds. Salt and acetonitrile concentrations were adjusted to optimize the separation. Good correlations (R² = 0.998–1.000) between the logarithm of the retention factor and the logarithm of salt or acetonitrile concentration were obtained. Gradients generated by changing salt or acetonitrile concentration were compared for the analysis of different highly polar bases. Although all of the analytes were eluted more quickly with an acetonitrile gradient, the effect of the gradients tested on peak width and peak shape varied with respect to analyte. In addition, the effects of different types of cation and anion additives were also investigated. After separation parameters were acquired, the SCX-based method was utilized to analyze highly hydrophilic alkaloids from Scopolia tangutica Maxim with high separation efficiency (plate numbers > 32,000 m⁻¹). Concurrently, one very polar alkaloid fraction was purified with symmetric peak shape using the current method. Our results suggest that SCX stationary phase can be used as an alternative to RP stationary phase in the analysis and purification of highly hydrophilic basic compounds.     

High-performance liquid chromatographic analysis of basic drugs on silica columns using non-aqueous ionic eluents. I. Factors influencing retention, peak shape and detector response

The use of silica columns together with non-aqueous ionic eluents provides a stable yet flexible system for the high-performance liquid chromatographic analysis of basic drugs. At constant ionic strength, eluent pH influences retention via ionisation of surface silanols and protonation of basic analytes, pKa values indicating the pH of maximum retention. At constant pH, retention is proportional to the reciprocal of the eluent ionic strength for fully protonated analytes and quaternary ammonium compounds. The addition of water up to 10% (v/v) has little effect on retention if the protonation of the analytes is unaffected. Thus, it is likely that retention is mediated primarily via cation exchange with surface silanols. However, additional factors must play a part with compounds such as morphine which give tailing peaks at acidic or neutral eluent pHs.     

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.     

Characterization of a microparticulate strong anion-exchanger in the HPLC of acidic drugs

The use of Waters Spherisorb S5SAX for the HPLC of acidic compounds, including a number of non-steroidal anti-inflammatory drugs (NSAIDs), has been investigated. Adequate retention, separation, and peak efficiency and symmetry were obtained for most analytes on a 250 x 4.6 mm i.d. column using methanol containing ammonium perchlorate (10 mmol L(-1), pH 6.7 or pH 8.3) as eluent. The results of changes in (i) eluent pH (constant ionic strength); (ii) eluent ionic strength (constant pH); and (iii) adding water to the eluent (constant pH) were consistent with a retention mechanism dominated by ion-exchange with the bonded strong anion-exchange (SAX) moieties. However, there were some unexpected observations, including (i) a general decrease in retention at eluent pH values above 7.7; (ii) a marked increase in retention on adding 1% (v/v) water to the eluent; (iii) a subsequent marked decrease in retention on adding 5% (v/v) or more water; and (iv) decreased column activity with time. These observations may be due to (i) interaction between the charged SAX moieties and ionised surface silanols (with ionization increasing at higher eluent pH values) and (ii) influence of the solvation of silanols, analytes, SAX moiety, and counter-ion varying with both pH and water content. Nevertheless, the factors influencing separation of individual NSAIDs remain unclear especially as no relation between log k and pKa exists for these compounds. Hydrophobic interactions are unlikely to be important since basic and neutral compounds were hardly retained. Ease of accessibility of the counter-ion to the SAX moiety for analyte displacement may be a factor.     

An ion/molecule reaction for the identification of analytes with two basic functional groups

A mass spectrometric method is presented for the identification of analytes with two basic functionalities and PA between 222 and 245 kcal/mol, including diamines. This method utilizes gas-phase ion-molecule reactions of protonated analytes with neutral 1,1-diethoxyethene (DEE) in a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR). A variety of protonated mono-, bi-, and trifunctional analytes containing different functional groups, namely, amido, amino, N-oxide, hydroxy, carboxylic acid, keto, thio, thioether, alkene, phosphite, and phosphonate, were tested in the FT-ICR. The results demonstrate that basic protonated bifunctional compounds (PA between 222 and 245 kcal/mol) react selectively with DEE by forming a specific addition/elimination product ion (adduct - EtOH) (this product was also observed for lysine with three functionalities). The diagnostic reaction sequence involves proton transfer from the protonated analyte to the basic vinyl group in DEE, followed by addition of one of the functional groups of the analyte to the electrophilic α-carbon in protonated DEE. The next step involves proton transfer from this functionality to the other analyte functionality, followed by proton transfer to DEE and elimination of ethanol. Since the mechanism involves proton transfer between two functional groups of the analyte, the reaction does not occur for analytes where the two functionalities cannot be in close proximity (i.e., meta-phenylenediamine), and where no proton is available (i.e., dimethylaminoketone).     

Vitamin U-bonded stationary phase in capillary ion chromatography

A vitamin U-bonded stationary phase was prepared and the retention behavior of inorganic anions was examined using ion chromatography. Inorganic anions were retained on the vitamin U-bonded stationary phase under acidic as well as neutral eluent conditions in the ion-exchange mode. The elution order of the examined anions under neutral eluent conditions was nearly the same as that observed in common ion exchange mode, while the elution order observed under acidic eluent conditions was completely different from that observed in common ion exchange mode. The retention of the analyte anions under the neutral eluent conditions was due to the sulfonium groups of the vitamin U, while protonated primary amino groups caused retention of the analyte anions with different selectivity under acidic conditions. The retention factor of the analyte anions increased with decreasing eluent concentration under both eluent conditions. The present system was applied to the determination of bromide and nitrate contained in seawater.     

Evaluation of analyte protectants to improve gas chromatographic analysis of pesticides

A common problem in gas chromatography (GC) applications is the analyte losses and/or peak tailing due to undesired interactions with active sites in the inlet and column. Analytes that give poor peak shapes or degrade have higher detection limits, are more difficult to identify and integrate, and are more prone to interferences than stable analytes that give narrow peaks. For susceptible analytes, significant peak quality improvements are obtained when matrix components are present because they fill active sites, thus reducing analyte interactions. This phenomenon is called "matrix-induced chromatographic response enhancement." Several approaches have been proposed to minimize peak distortion phenomena and compensate for matrix-induced effects, which is especially important for accurate quantitation, but each approach has serious limitations for routine multi-pesticide analysis. In this study, we demonstrate the feasibility of using "analyte protectants" to provide a more convenient and effective solution to the problem than other approaches developed thus far. The protecting agents are added to extracts and matrix-free standards alike to provide the chromatographic enhancement effect even for the most susceptible analytes in a very dirty GC system. In this study, we evaluated 93 different compounds to find the most suitable ones for improving chromatographic quality of the signal. Because hydrogen bonding has been shown to be an important factor in analyte interactions with active sites, we mainly focused on additives with strong hydrogen bonding capabilities. Dramatic peak enhancements were achieved using compounds containing multiple hydroxy groups, such as sugars and sugar derivatives, and gulonolactone appears to be the most effective protecting agent for the most pesticides that we tested. The benefits of using analyte protectants versus alternative procedures for overcoming matrix-induced effects in quantitation include: (a) simpler procedure; (b) easier integration of peaks; (c) lower detection limits; (d) better quantitation; (e) less maintenance of the GC inlet; and (e) lower cost. However, long-term influences on the performance of the chromatographic system have yet to be established.     

Modification of silica surfaces for CZE by adsorption of non-ionic hydrophilic polymers or use of radial electric fields

Polyvinylalcohols (PVA) and hydroxyethylcelluloses (HEC) have been used as additives in cyclodextrin-modified capillary zone electrophoretic chiral separations of aromatic amines such as tocainide and its analogs in unpretreated 50 μm i.d. fused silica capillaries. The additives were used at low concentrations (<0.05%) in common buffers, together with the γ-cyclodextrin as chiral selector. They reduce the electroosmotic flow, i.e. increase the migration times of the analytes in these chiral separations, and, moreover, considerably improve both peak symmetry and the widths of the peaks relative to migration time. In terms of the chromatographic theory of efficiency, more than 200000 theoretical plates can be achieved with unpretreated fused silica capillaries. This enhancement of efficiency arises because adsorptive “dynamic” coating with the hydroxylic modifier molecules suppresses adsorption of the analyte molecules by the capillary walls. The influence of field strength and buffer composition on the separation efficiency attainable with and without modifier in the buffers has also been investigated. Alternative experiments on the influence of analyte adsorption on efficiency have been performed by superimposing radial electric fields on the capillary to modify the ζ potentials. Although the EOF could be freely adjusted, it was not possible to obtain an improvement in efficiency comparable with that furnished by coating the adsorptive surface with PVA or HEC.