Mobile Phase Parameters Research Articles

Effect of the mobile phase on antibody-based enantiomer separations of amino acids in high-performance liquid chromatography

The effect of the mobile phase parameters flow rate, temperature, pH and ionic strength, as well as the addition of various organic modifiers on the enantiomer separation of various aromatic α-amino acids was investigated using two antibody-based chiral stationary phases that have opposing stereoselectivity. On both columns, a decrease in flow rate or temperature resulted in increased interaction with the retained enantiomer. It was found that the retention factor k 2 depends on the affinity between the analyte and the immobilized antibody and is not independent of the flow rate. Optimum separations of all amino acids investigated were obtained at pH 7.4 on both columns. While increased k 2 values were obtained at low ionic strength on the anti- d-amino acid antibody column, no such effect was observed on the anti- l-amino acid antibody column. The addition of organic modifiers did not improve separations. In all studies, the unretained enantiomer eluted with the void volume.

Effect of the mobile phase on antibody-based enantiomer separations of amino acids in high-performance liquid chromatography

The effect of the mobile phase parameters flow rate, temperature, pH and ionic strength, as well as the addition of various organic modifiers on the enantiomer separation of various aromatic alpha-amino acids was investigated using two antibody-based chiral stationary phases that have opposing stereoselectivity. On both columns, a decrease in flow rate or temperature resulted in increased interaction with the retained enantiomer. It was found that the retention factor k2 depends on the affinity between the analyte and the immobilized antibody and is not independent of the flow rate. Optimum separations of all amino acids investigated were obtained at pH 7.4 on both columns. While increased k2 values were obtained at low ionic strength on the anti-D-amino acid antibody column, no such effect was observed on the anti-L-amino acid antibody column. The addition of organic modifiers did not improve separations. In all studies, the unretained enantiomer eluted with the void volume.

Monolithic silica columns with chemically bonded tert-butylcarbamoylquinine chiral anion-exchanger selector as a stationary phase for enantiomer separations

An enantioselective silica rod type chiral stationary phase (CSP) is presented as a novel combination of the well-known enantiomer separation properties of immobilized tert-butyl-carbamoylquinine chiral anion-exchanger selector with the unique properties of monolithic silica material. The chromatographic behavior of the tert-butyl-carbamoylquinine silica rod was studied and compared with a similar prepared particulate material. Good selectivities were achieved for a spectrum of chiral test components like N-derivatized amino acids (DNB- Ac-, DNZ-, Bz-, Z-amino acids) and for Suprofen. The influence of mobile phase parameters, as well as the effect of serially coupling up to six 10 cm monolithic silica columns was studied and put in context to conventional columns of particulate 5 μm type CSP. Using that 60 cm long monolithic column it was possible to improve the enantiomer separation of Suprofen and achieve a baseline separation in less than 10 min of total separation time.

Retention modeling and simultaneous optimization of pH value and gradient steepness in RP‐HPLC using feed‐forward neural networks

AbstractA novel approach is proposed for the simultaneous optimization of mobile phase pH and gradient steepness in RP‐HPLC using artificial neural networks. By presetting the initial and final concentration of the organic solvent, a limited number of experiments with different gradient time and pH value of mobile phase are arranged in the two‐dimensional space of mobile phase parameters. The retention behavior of each solute is modeled using an individual artificial neural network. An “early stopping” strategy is adopted to ensure the predicting capability of neural networks. The trained neural networks can be used to predict the retention time of solutes under arbitrary mobile phase conditions in the optimization region. Finally, the optimal separation conditions can be found according to a global resolution function. The effectiveness of this method is validated by optimization of separation conditions for amino acids derivatised by a new fluorescent reagent.

Capillary electrochromatography of methylated benzo[ a]pyrene isomers : I. Effect of mobile phase tuning

It is of increasing importance that chromatographic methods be developed for the separation and identification of biological and environmentally harmful compounds such as methylated polycyclic aromatic hydrocarbons (PAH). Capillary electrochromatography (CEC) is fast becoming a useful technique for analysis of PAH, as it offers both high efficiency and superior resolution. The separation of 12 methylated benzo[ a]pyrene (MBAP) isomers is a challenge due to the extreme hydrophobicity and structural similarity of these compounds. In this work, we present Part I of our ongoing study, a method for the systematic mobile phase tuning for CEC separation of the 12 MBAP isomers. The CEC experiments were conducted utilizing a CEC-octadecylsilica (ODS) stationary phase and fused-silica capillary [(75 μm I.D., 363 μm O.D.) 36.5 cm total length, 25.0 cm effective length] which was slurry pressure packed in our laboratory. Several mobile phase parameters were manipulated to provide optimum separation. These included acetonitrile (ACN) concentration, tris(hydroxymethyl) aminomethane (Tris) concentration, pH, and addition of a tertiary buffer constituent such as tetrahydrofuran (THF) and isopropanol (IPA) to ACN–aqueous buffer mixtures. Optimum CEC separation conditions were achieved using 75% (v/v) ACN–25% (v/v) 12.5 m M Tris, pH 8.0, and 30 kV at 25 °C. These mobile phase conditions were then utilized for Part II of our study, the CEC stationary phase optimization for the separation of 12 MBAP isomers.

Prediction of the retention in reversed-phase liquid chromatography using solute–mobile phase–stationary phase polarity parameters

A previously reported algorithm, based on the equation: log k=( log k) 0+p(P N m −P N s ) , that relates the retention in reversed-phase liquid chromatography with solute ( p), mobile phase ( P m N ) and stationary phase ( P s N ) relative polarity parameters, is improved. The retention data reported by several authors for different sets of compounds, eluted with acetonitrile–water and methanol–water mixtures, are used to test the algorithm and elaborate a database of p values. The methodology is successfully applied to predict the retention using P m N values calculated as P m N =1.00−(2.13 ϕ)/(1+1.42 ϕ) for acetonitrile–water and P m N =1.00−(1.33 ϕ)/(1+0.47 ϕ) for methanol–water, ϕ being the organic solvent volumetric fraction. The polarity parameters are demonstrated to be useful to transfer retention data between solvent systems and between columns. Accordingly, the retention in a solvent system is predicted by characterising the working column with a small training set of compounds having diverse polarities, and using the p values known for another solvent system or column. The p polarity parameter is found to be a good descriptor of the retention, allowing the prediction of the expected elution order and peak overlaps.

Influence of mycophenolic acid on inosine 5′-monophosphate dehydrogenase activity in human peripheral blood mononuclear cells

Background: Inosine 5′-monophosphate dehydrogenase (IMPDH) catalyses the oxidation of inosine 5′-monophosphate (IMP) to xanthosine 5′-monophosphate (XMP). Thus, this enzyme plays an important role in the rate-limiting step of the de novo guanine nucleotide biosynthesis, making it a potent target for immunosuppressive drugs. Mycophenolic acid (MPA) is the most potent and specific inhibitor of IMPDH. Method: IMPDH activity is determined via evaluation of XMP formation and the inhibitory influence of MPA in human peripheral blood mononuclear cells (PBMCs) is assessed by means of high-performance liquid chromatography (HPLC). For this objective, we have optimised a method based on solvent-generated ion exchange chromatography by cautiously varying mobile phase parameters. Results: The optimised method renders it possible to separate 18 analytes in 54 min in a single isocratic experiment and to measure the IMPDH activity in the lysate of human PBMCs in dependence on incubation time, substrate, co-substrate and inhibitor concentrations. In this way, we have determined the Michaelis–Menten constants K M and V max for IMP and β-NAD + and the inhibitor constant K i for MPA. Conclusions: The chromatographic method presented in this report allows a rapid, reliable and reproducible quantification of IMPDH activity in PBMCs and therefore represents an attractive tool for the pharmacodynamic monitoring of the effects of MPA in patients under immunosuppressive therapy.

Retention of ionizable compounds on high-performance liquid chromatography: XI. Global linear solvation energy relationships for neutral and ionizable compounds

A global linear solvation energy relationship (LSER) that simultaneously models retention in reversed-phase liquid chromatography as a function of solute LSER descriptors and mobile phase pH and composition has been derived from both the local LSER model and the linear solvent strength theory. At most only 13 mobile phase parameters and seven solute parameters are required to establish the global LSER model for neutral and ionizable solutes. This model implies only one mobile phase and two solute parameters more than the model previously set for neutral solutes. The additional mobile phase and solute parameters account for the ionization of the solute. The model has been successfully tested for 30 solutes of different type (acids, bases and non ionizable compounds) at 10 different pH values in three different acetonitrile–water mobile phases.

BUPROPION HYDROCHLORIDE: THE DEVELOPMENT OF A CHIRAL SEPARATION USING A CHIRAL AGP COLUMN

A chiral high-performance liquid chromatographic separation was developed for bupropion active ingredient. The mobile phase parameters that may influence the separation were studied and included: mobile phase pH, type and concentration of organic modifier, type and concentration of buffer, amount of analyte injected, and column temperature. The optimized chiral separation baseline resolved the two enantiomers in less than 10 minutes. Method validation was also performed for the separation. The method was found to be linear over a range of 0.5 μg/g to 99.8 μg/g with a correlation coefficient greater than 0.999. The limit of detection was determined to be 0.25 μg/g while the limit of quantitation was found to be 0.5 μg/g. Method precision was found to be 0.8% and 1.9% for the two enantiomers, while the system precision was found to be 0.4% and 0.5% for the two enantiomers.

Reversed-phase capillary electrochromatography of aloins and related constituents of aloe

The separation of some anthrone-C-glucosyl constituents of aloe was carried out by isocratic capillary electrochromatography using a 50 μm l.D. column packed with 3 μm ODS silica gel. In comparison to HPLC mode, CEC resolution of aloins was obtained in a very short time with a high selectivity factor. The influence of mobile phase parameters (pH and ionic strength of the buffer, acetonitrile amount in the eluent etc.) on the retention of aloins was examined in order to improve the separation conditions and to study the retention mechanism. Efficiency was found to increase with decreasing the EOF up to a reduced plate height of about 2 at a reduced linear velocity of 0.3 mm s−1. This effect was ascribed to the fact that, under the buffer conditions used (pH 8), aloins are partially anionically charged, with a consequent low mass transfer between the mobile and stationary phases. However, resolution remains almost the same in a wide pH range permitting baseline separations of aloins in short analysis times. Since aloins are known to undergo fast degradation reaction dependent on pH, a kinetic study was also carried out. The method was extended to the analysis of aloins in food and cosmetic products.

Enantioselective reversed-phase and non-aqueous capillary electrochromatography using a teicoplanin chiral stationary phase

Enantiomeric separation of chiral pharmaceuticals is carried out in aqueous and non-aqueous packed capillary electrochromatography (CEC) using a teicoplanin chiral stationary phase (CSP). Capillaries were slurry packed with 5 μm 100-Å porous silica particles modified with teicoplanin and initially evaluated using a non-aqueous polar organic mode system suitability test for the separation of metoprolol enantiomers ( R s=2.3 and 53 000 plates m −1). A number of pharmaceutical drugs were subsequently screened with enantioselectivity obtained for 25 racemic solutes including examples of neutral, acidic and basic molecules such as coumachlor ( R s=3.0 and 86 000 plates m −1) and alprenolol ( R s=3.3 and 135 000 plates m −1) in reversed-phase and polar organic mode, respectively. A statistical experimental design was used to investigate the effects of non-aqueous polar organic mobile phase parameters on the CEC electroosmotic flow, resolution and peak efficiency for two model solutes. Results primarily indicated that higher efficiency and resolution values could be attained at higher methanol contents which is similar to findings obtained on this phase in liquid chromatography.

Chiral anion exchangers applied to capillary electrochromatography enantioseparation of oppositely charged chiral analytes: investigation of stationary and mobile phase parameters

Weak anion-exchange (WAX) type chiral stationary phases (CSPs) based on tert.-butyl carbamoyl quinine as chiral selector (SO) and different types of silica particles (porous and non-porous) as chromatographic support are evaluated in packed capillary electrochromatography (CEC). Their ability to resolve the enantiomers of negatively charged chiral analytes, e.g., N-derivatized amino acids, in the anion-exchange mode and their electrochromatographic characteristics are described in dependence of several mobile phase parameters (pH, buffer type and concentration, organic modifier type and concentration) and other experimental variables (electric field strength, capillary temperature). The inherent “zwitterionic” surface character of such silica-based WAX type CSPs (positively charged SO and negatively charged residual silanols) allows the reversal of the electroosmotic flow (EOF) towards the anode at pH values below the isoelectric point (p I) of the modified surface, whereas a cathodic EOF results at pH values above the p I. Since for negatively charged analytes also an electrophoretic transport increment has to be considered, which can be either in or against the EOF direction, several distinct modes of elution have been observed under different stationary phase and mobile phase conditions: (i) co-electrophoretic elution of the negatively charged solutes with the anodic EOF in the negative polarity mode, (ii) counter-electrophoretic elution with the cathodic EOF in the positive polarity mode, and (iii) electrophoretically dominated elution in the negative polarity mode with a cathodic EOF directed to the injection end of the capillary. Useful enantioseparations of chiral acids have been obtained with all three modes. Enantioselectivity values as high as under pressure-driven conditions and theoretical plate numbers up to 120 000 per meter could be achieved under electrically driven conditions. A repeatability study yielded RSD values below 2% for retention times and RSD values in the range of 5–10% for theoretical plate numbers and resolution, thus clearly establishing the reliability of the investigated anion-exchange type CEC enantioseparation methods.

Separation and quantification of ropinirole and some impurities using capillary liquid chromatography

Ropinirole, 4-[2-(dipropylamino)ethyl]-1,3-dihydro-2H-indol-2-one, is a potent anti-Parkinson’s disease drug developed by SmithKline Beecham Pharmaceuticals. Capillary liquid chromatography (CLC) was used for the separation and quantification of ropinirole and its five related impurities, potentially formed during its synthesis. A simultaneous optimization of three mobile phase parameters, i.e., pH, buffer concentration and acetonitrile content was performed employing an experimental design approach which proved a powerful tool in method development. The retention factors of the investigated substances in different mobile phases were determined. Baseline resolution of the six substances on a C 18 reversed stationary phase was attained using a mobile phase with an optimized composition [acetonitrile–8.7 m M 2-( N-morpholino)ethanesulfonic acid adjusted to pH 6.0 (55:45, v/v)]. It was shown that CLC, operated in the isocratic mode under the mobile phase flow-rate of 4 μl/min, can determine the level of these impurities, down to a level of 0.06% of the main component within 25 min.

Use of immobilized amyloglucosidase as chiral selector in chromatography. Control of enantioselective retention and resolution in liquid chromatography

Several mobile phase parameters were investigated for controlling enantioselective retention and resolution on a chiral stationary phase made in-house. The chiral selector was the enzyme amyloglucosidase, which was immobilized onto a silica support via reductive amination. The influences of the mobile phase pH, concentration and type of uncharged organic modifier, ionic strength and column temperature on enantios-electivity were studied. The analysis time for resolving enantiomers could be adjusted with only a minor decrease in enantioselectivity by using a high ionic strength mobile phase buffer. This indicated a retention mechanism involving ion-exchange interactions. It was further confirmed by the decreasing enantioselectivity of amines when using a mobile phase pH below the isoelectric point of the native protein. Interesting effects were observed when the organic modifier concentration was increased and also when the column temperature was raised. Both retention and enantioselectivity increased with increasing concentration of 2-propanol in the mobile phase. Examples are given where both enantioselectivity and retention increased with increasing column temperature. Thermodynamic studies were performed to calculate the entropy and enthalpy constants. The results showed that, depending on mobile phase composition, the enantioselective retention may be caused by differences in entropy or enthalpy.

Preconcentration and separation of haloacetic acids by ion chromatography

A comparative study was made of the chromatographic behaviour of five haloacetic acids (mono-, dibromoacetic and mono-, di-, trichloroacetic acids). The techniques investigated included reversed-phase ion interaction chromatography with UV detection, suppressed and non-suppressed anion-exchange chromatography. The systems are discussed studying the retention as a function of the mobile phase parameters and the stationary phases used (LiChrospher 100 RP-18, IonPac AS9, AS10 and AS11). A preconcentration step, performed on different substrates, namely LiChrolut-EN and activated vegetal carbon, has been optimized in order to reduce the method detection limits. Results obtained for drinking water samples are shown.

Sulphonated azoligand for metal ion determination in ion interaction chromatography

An azosulphonated ligand, namely cyclo-tris-7-(1-azo-8-hydroxynaphthalene-3,6-disulphonic acid), Calcion, which is generally used as a metallochromic indicator for metal ion titrations, has been studied in order to verify its suitability as a new ligand for metal ion separation in a liquid chromatography technique based on the ion interaction mechanism. Before chromatographic method development, studies on the complexation capabilities of Calcion with Ni 2+, Cu 2+, Fe 3+ and Al 3+ metal ions were performed, determining the stability constants of the complexes between such metal ions and the ligand. The procedure is based on ion-exchange chromatography and involves the retention of the negatively charged complexes on an anion-exchange resin and the determination of free and complexed metal by inductively coupled plasma atomic emission spectrometry. Chromatographic separation of Ni 2+–Cu 2+and Fe 3+–Al 3+ was reached, using a silica-based octyl stationary phase, through pre-complexation with Calcion, and UV–visible detection of the resulting anionic complexes. The effect of the main mobile phase parameters (concentration of the ion interaction reagent tetrabutylammonium chloride, organic modifier methanol and pH) on k′ of analytes was studied and optimization of the metal ion separation was achieved. In order to obtain the simultaneous determination of metal ions, the concentration of CH 3OH and pH gradient elution were also considered. The method developed has been applied to tap water analysis.

Enantioseparation of semisynthetic ergot alkaloids on vancomycin and teicoplanin stationary phases

The macrocyclic antibiotics, vancomycin and teicoplanin, were used as chiral stationary phase selectors for the enantioselective separation of semisynthetic ergot alkaloids in reversed-phase high-performance liquid chromatography (RP-HPLC). The chromatographic behavior of the ergot preparations was investigated in order to obtain a deeper insight into the enantiodiscriminative process. A variety of factors, including mobile phase parameters such as the nature and concentration of the organic modifier, buffer concentration and pH, were examined. Conditions for the enantioseparation of real pharmaceutical preparations, i.e. lisuride, terguride and nicergoline, were found. Differences in the chiral stationary phases are presented and the interaction mechanism is discussed.

High-efficiency enantioseparations of N-derivatized amino acids by packed capillary electrochromatography using ODS silica and a quinine-derived chiral selector as ion-pair agent

The enantiomers of N-derivatized amino acids, e.g., 3,5-dinitrobenzoyl, 3,5-dinitrobenzyloxycarbonyl, 2,4-dinitrophenyl and 9-fluorenylmethoxycarbonyl amino acids, have been separated by enantioselective ion-pair formation and packed capillary electrochromatography (CEC) using RP18 silica particles. Thus, a CEC-Hypersil ODS-3 μm packed capillary column, 335 mm (effective length 250 mm)×0.1 mm I.D., was used in combination with a quinine carbamate type chiral ion-pair agent (selector, SO) which was added to aqueous and non-aqueous buffered mobile phases, respectively. The negatively charged analyte enantiomers interact with the positively charged chiral SO by multiple intermolecular interactions to form a pair of transient diastereomeric ion-pairs which may differentially adsorb to the ODS-stationary phase. Enantioseparation is achieved due to different observed mobilities of the analyte enantiomers originating from different ion-pair formation rates of the enantiomers and/or differential adsorption of the diastereomeric ion-pairs to the ODS-stationary phase. Countercurrent-like electrophoretic migration of oppositely charged ion-pair agent and solute enantiomers may give rise to enhanced enantioselectivity. At high electrolyte concentrations (>10 m M), e.g. if the chiral ion-pair agent is added to the mobile phase that contains, compared to typical RP-CEC conditions (<10 m M), relatively high amounts of background electrolyte, electrophoretic transport of the analytes is dominating (negative polarity mode); this method is characterized by high efficiency with theoretical plate numbers up to 170 000 per meter, e.g. for DNP-Val, but with moderate enantioselectivity. In contrast, at low electrolyte concentrations (<10 m M), e.g. if the chiral ion-pair agent itself is used as electrolyte without any other background electrolyte, the analytes are driven through the capillary column with the electroosmotic flow (positive polarity mode); this operation mode is characterized by relatively poor efficiency, but high enantioselectivity. The influence of several mobile phase parameters (aqueous versus non-aqueous, selector concentration, type of background electrolyte) on observed mobility, enantioselectivity and efficiency was evaluated.

Behavior of Cation-Exchange Materials in Capillary Electrochromatography

The behavior of a strong, cation-exchange material (propanesulfonic acid, SCX) has been studied in capillary electrophoresis (CE) and capillary electrochromatography (CEC) by the use of coated and packed capillaries. In aqueous electrolytes, the SCX-coated capillary showed a far more consistent electroosmotic flow over the pH range 3.6-10.5, compared to untreated fused silica. However, in similar electrolytes containing 80% (v/v) acetonitrile, both coated and untreated capillaries performed similarly, casting doubts upon the stability of the SCX coating. The effect of voltage and mobile-phase parameters such as pH, ionic strength, and organic content was studied in CEC for both 3-μm SCX and C(18) packing materials, and the results were compared in terms of linear velocities, currents, and conductivities. Only at pH 5 and below was a higher EOF velocity than expected observed for the SCX column. In accordance with theory, the EOF was seen to increase with decreasing ionic strength for the C(18) column. However, for the SCX column, this was not the case: the EOF showed a general reduction as the ionic strength was decreased. The greatest anomaly was observed on changing the acetonitrile composition: the EOF showed a consistent decline with increasing organic, whereas the EOF in both the open capillary and C(18) column decreased and then started to rise with acetonitrile contents above 70% (v/v).

Evaluation of the macrocyclic antibiotic LY333328 as a chiral selector when used as a mobile phase additive in narrow bore HPLC

The macrocyclic antibiotic LY333328 has been evaluated as a chiral selector for the enantioseparation of nine dansylated amino acids. This macrocyclic glycopeptide was used as a chiral mobile phase additive (CMPA) in conjunction with narrow bore high-performance liquid chromatography (HPLC). The key mobile phase parameters of LY333328 concentration and buffer pH were varied, along with variations in stationary phases consisting of C8, phenyl, cyano, and silica. After observing and plotting changes in retention and resolution based on corresponding variation in these parameters, a better understanding of the behavior of this chiral selector was obtained. The pKa values of the dansyl amino acid analytes and LY333328 were measured and used to gain a better understanding of the microenvironment in which these enantioseparations occur. Optimized conditions resulted in the baseline separation of eight of nine dansyl amino acids.