Using strong injection solvents with 100% aqueous mobile phase in RP-LC

When samples are dissolved for HPLC analysis, organic solvents are often used to enhance the solubility of the sample components. However, when the diluent becomes significantly stronger than the mobile phase, peak shape distortions may develop during injection. This is a serious problem for weakly retained analytes under 100% aqueous mobile phase conditions. Under these extreme conditions, even small amounts of solvent in the diluent can cause serious broadening or distortion effects. However, if the organic solvent used in the diluent elutes significantly after the analyte, the peak shape of the analyte will be relatively unaffected, even when a relatively strong solvent such as THF is used. This phenomenon is counterintuitive considering the usual practice of keeping the sample diluent as weak as possible. This report describes the potential analytical utility and limitations of this approach for the analysis of weakly retained analytes.     

Mass overload in preparative liquid chromatography: Band broadening and maximum sample load

Summary Injection conditions in preparative liquid chromatography are optimized using the Haarhoff-Van der Linde model which was previously demonstrated as suitable for describing peak distortion in non-linear non-ideal chromatography. Ideally, the sample must be injected in a very narrow, concentrated plug form.     

Isolation and purification of flavonoid glycosides from Trollius ledebouri using high-speed counter-current chromatography by stepwise increasing the flow-rate of the mobile phase

Three flavonoid glycosides including orientin, vitexin, quercetin-3-O-neohesperidoside and one unknown compound were isolated and purified by high-speed counter-current chromatography (HSCCC) and semi-preparative HPLC from Trollius ledebouri Reichb., a traditional Chinese medicine. Preparative HSCCC with a two-phase solvent system composed of ethyl acetate-n-butanol-water (2:1:3, v/v/v) was successfully performed by increasing the flow-rate of the mobile phase from 1.5 to 2.5 ml/min after 190 min. Consequently, 95.8 mg orientin, 11.6 mg vitexin, 9.3 mg unknown compound with purities of over 97% and one partially purified peak fraction (contained quercetin-3-O-neohesperidoside at 85.1% purity) were obtained from 500 mg of the crude extract. Then the partially purified fraction was further purified by reversed-phase semi-preparative high-performance liquid chromatography. The structure identification of all pure fractions was carried out by UV, MS, 1H NMR and 13C NMR.     

Preparative isolation and purification of bergapten and imperatorin from the medicinal plant Cnidium monnieri using high-speed counter-current chromatography by stepwise increasing the flow-rate of the mobile phase

A high-speed counter-current chromatography (HSCCC) method was developed for the preparative separation and purification of bergapten and imperatorin from the Chinese medicinal plant Cnidium monnieri (L.) Cusson. The crude extract was obtained by extraction with ethanol from the dried fruits of Cnidium monnieri (L.) Cusson under sonication. Preparative HSCCC with a two-phase solvent system composed of n-hexane-ethyl acetate-ethanol-water (5:5:5:5, v/v/v/v) was successfully performed by increasing the flow-rate of the mobile phase stepwise from 1.0 to 2.0 ml min(-1) after 180 min. The components purified and collected were analyzed by high-performance liquid chromatography. The method yielded 45.8 mg of bergapten at 96.5% purity and 118.3 mg of imperatorin at 98.2% purity from 500 mg of the crude extract in a single run. The recoveries of bergapten and imperatorin were 92.1 and 93.7%, respectively.     

Evaluation of loop and pump injection systems in preparative liquid chromatography

The usefulness of pump or loop injectors in preparative liquid chromatography was evaluated. The concentration distribution of samples flowing through a UV detector cell, directly connected to the injector, was recorded at various flow velocities, at various diameters of loop tubing, and using various methods of injection [injection with sample volumes equal to a fraction or total loop volume, etc.]. The most advantageous methods were found to be either use of a loop injector to inject only a fraction of its total volume or use of a pump. Both of these methods ensure a almost rectangular concentration distribution.     

Optimization of throughput in preparative column luquid chromatography by column overloading and partial fractionation of the effluent

Summary The criteria for the optimization of preparative chromatography are sample throughput and product purity. It is shown for column liquid chromatography that the throughput has been maximized for a given purity by column overloading and appropriate fractionation. The size and position of the fraction must be determined by chemical analysis, since overlapping peaks under overloading conditions are usually distorted compared to the peaks of the same amounts of pure compounds. With overloading of the column larger particles can be used as column packings without reduction of the separation effect.     

Simultaneous correlation of hydrophobic interactions in HIC and protein solubility in aqueous salt solutions and mixed solvents

The chromatographic retention in hydrophobic and reversed phase chromatography and the solubility of proteins display some common features. The chromatographic retention, as well as the solubility, is modulated by the thermodynamic properties of the solute in the fluid phase. The retention measurements at linear conditions provide information of the solution properties of the protein at infinite dilution, and the solubility measurements produce the supplementary information about the solution properties at the saturation limit. This provides a useful approach to simultaneous correlation of the chromatographic retention and the solubility.The experimental data, used for the correlation, comprise retention measurements of lysozyme on different HIC adsorbents using an aqueous ammonium sulphate eluant, an aqueous ammonium sulphate eluant with an admixture of ethanol, as well as published solubility data.The chromatographic retention data and the corresponding solubility data have been correlated using a chemical potential model derived from Kirkwood's theory of solutions of charged macro-ions and zwitterions in electrolyte solutions. The model correlated the chromatographic retention factor and the solubility data within the precision of the measurements. The model was applied in a pH range from 4 to 11. It was demonstrated experimentally, as well as theoretically, that an admixture of ethanol to the aqueous eluant changes the thermodynamic retention factor on various adsorbents identically when compared to the thermodynamic retention factor in an ethanol free eluant.     

Improvement in the solvent evaporation injection technique for preparative supercritical fluid chromatography

Summary In supercritical fluid chromatography, the sample load that can be injected using the sample solvent evaporation technique is limited by the volume of the loading precolumn. A multiple sample loading procedure is investigated in order to increase the injectable amount and to optimize injection conditions. This work also deals with optimization of solvent removal conditions.     

Formation and migration of zones in overloaded preparative liquid-solid chromatography

Summary An equilibrium sandwich chamber for continuous thinlayer chromatography was used to study overloaded systems of the heptane + methylene chloride-silica type. Mixtures of two or three dyes were used as the model samples. Wide starting zones were formed using a glass distributor (frontal chromatography stage), then the movement of the zones was recorded during continuous elution. The effect of sample concentration and volume on the maximum separation yield was investigated. Band compression effects are illustrated for samples dissolved in solvents having a low eluent strength. Satisfactory analogy was found to separations in preparative column chromatography. Good separation yield was obtained for frontal + elution TLC: depending on the differences in the R_F values of the components, 6–16 mg samples were completely separated on 0.5×100×75 mm layers containing ca. 1 g of silica.Presented at the Eighth International Symposium on Liquid Chromatography, Baden-Baden, 3–7 May 1983.     

Determination of the solubility, dissolution enthalpy and entropy of Deflazacort in different solvents

The solubility of Deflazacort in four solvents, acetone, ethyl acetate, ethanol and 2-propanol, was measured at temperatures ranging from 293.15 K to 348.15 K at atmospheric pressure using Laser Monitoring Technique. The solubility data were correlated by the modified Apelblat model. Then the dissolution enthalpy and entropy of Deflazacort were predicted from the solubility data using van’t Hoff equation. In this study, it should be concluded that the viscosity and surface tension of solvents affect the solubility behavior, dissolution enthalpy and entropy of Deflazacort in different solvents.     

Optimization of the mobile phase conditions for the purification ofTaql endonuclease

Summary Taql endonuclease was purified by high performance ion exchange liquid chromatography with linear gradient elution. Of the chromatographic media tested as mobile phases for the HPLC purification ofTaql endonuclease, sodium acetate (pH 5.0) and phosphate (pH 7.0) buffers were appropriate for use with cation exchange columns, and L-histidine (pH 6.0) and Tris (pH 8.0) buffers with anion exchange columns.     

Generic method for systematic phase selection and method development of biochromatographic processes. Part I. Selection of a suitable cation-exchanger for the purification of a pharmaceutical protein

Even if the first protein therapeutics are now for more than 20 years on the market the selection of suitable adsorbents for the preparative downstream processing (DSP) of these biomolecules as well as the method development towards process conditions are still based mainly on 'trial and error'. Therefore, theses processes are not perfectly efficient, but indeed very time consuming and laborious. In this study a novel systematic method is introduced to find a suitable adsorbent (not necessarily the best one) with appropriate separation parameters for a specific separation with reduced effort. Following this strategy, the adsorbents must first be packed into columns under preparative conditions and then characterized completely with regard to, e.g. pressure drop, k'-values, plate heights (HETP curves), selectivity and capacity by using test substances, which are similar in their characteristics (molecular mass, size, charge distribution, hydrophobicity) to the target proteins. With the database once determined, a preselection of most suitable adsorbents including separation parameters is made regarding chromatographic and also economical properties. After this, preparative experiments must be conducted with a reduced number of adsorbents to figure out the individual influence of side components. This approach is demonstrated for the separation of an exemplary industrial protein mixture using cation-exchange chromatography (CEX). Characterization of different weak CEX-adsorbents is illustrated. After comparing these phases with each other, a first preselection and a prediction of suitable adsorbents is made. In the following preparative separation conditions (load, velocity, gradient) are determined for the preparative separations using the database and results of some additional experiments. The final comparison of separation performance in preparative scale confirms this selection and so the applicability of the new method.     

Solubility and limiting activity coefficient of simvastatin in different organic solvents

Equilibrium mole fraction solubility of Zocor^® (simvastatin) a pharmaceutically important compound, was measured between 279 and 315 K, in fifteen different industrial-relevant organic solvents including: methyl acetate, ethyl acetate, propyl acetate, iso-propyl acetate, butyl acetate, iso-butyl acetate, sec-butyl acetate, tert-butyl acetate, and ethanol, propanol, 1-butanol, 2-butanol, 1-pentanol, 1-hexanol, and 1-octanol. Fusion enthalpy, Δ_fusH, melting point temperature, T_m, were measured to be 32,169 J/mol, 412.6 K, respectively; and the difference in the molar heat capacity (at constant pressure) of the liquid, and solid form of simvastatin, ΔC_P, was approximated (by extrapolation) to be 230 J/mol K. Dissolution of simvastatin was found to be endothermic, and entropically favorable. The activity coefficient at infinite dilution of simvastatin in each solvent was calculated from the experimental data, then fitted to Gibbs–Helmholtz equation to estimate the limiting partial molar excess enthalpies, , and the limiting partial molar excess entropies, . The data was also fitted to the non-random-two-liquid (NRTL) activity coefficient equation to generate the model interaction parameters for dissolution of simvastatin in the organic solvents studied here.     

Efficient protocol for isolation and purification of different soyasaponins from soy hypocotyls

Soyasaponins are naturally occurring triterpenoid glycosides associated with many biological activities. The aim of the present study was to develop an effective method for isolation and purification of differently glycosylated, acetylated, and 2,3‐dihydro‐2,5‐dihydroxy‐6‐methyl‐4H‐pyran‐4‐one (DDMP)‐conjugated soyasaponins from soy hypocotyls. Both gel filtration using Sephadex LH‐20 chromatography (Amersham Pharmacia Biotech AB; elution phase: methanol, flow rate: 3.0 mL/min, sample loading: 60 mg) and high‐speed countercurrent chromatography (stationary phase: n‐butanol–acetic acid (5.0%, v/v), mobile phase: water flow rate: 3.0 mL/min, sample loading: 100 mg) could effectively fractionate isoflavones and soyasaponins from the crude extract with yield of soyasaponin complexes 20.5 mg and 22.3 mg, respectively. After fractionation, the soyasaponin complexes could be purified further using preparative HPLC to separate individuals. A total of nine soyasaponins, triacetyl soyasaponin Ab (yield 1.55%, HPLC purity >98%), Aa (2.68%, >99%), Ab (18.53%, >98%), Ae (0.85%, >98%), Ba (0.63%, >91%), Af (1.12%, >85%), Bb (3.45%, >98%) and Be (0.59%, >76.8%) were obtained. DDMP‐conjugated groups, αg (2.06%, >85%), βg (7.59%, >85%), and γg (0.29%, >85%) that were very labile even in mild conditions, were also collected. The method described here can be used as an effective protocol to separate different soyasaponins occurring in the original sample.     

Large-scale isolation and purification of geniposide from the fruit of Gardenia jasminoides Ellis by high-speed counter-current chromatography

High-speed counter-current chromatography (HSCCC) was applied to the isolation and purification of geniposide from Gardenia jasminoides Ellis. Analytical HSCCC was used for the preliminary selection of a suitable solvent system composed of ethyl acetate-n-butanol-water (2:1:3, v/v/v). According to the above solvent system, preparative HSCCC was successfully performed with the optimal solvent system composed of ethyl acetate-n-butanol-water (2:1.5:3, v/v/v) yielding 389 mg of geniposide at over 98% purity from 1g of the partially purified extract with 38.9% recovery in a one-step separation.     

Solubility of daidzin in different organic solvents and (ethyl alcohol + water) mixed solvents

The solubility of daidzin in different organic solvents and (ethyl alcohol + water) mixed solvents was measured by high performance liquid chromatography (HPLC) analysis method from T = (283.2 to 323.2) K at atmosphere pressure. The results show that at higher temperature more daidzin dissolves, and moreover, the solubility increases with the ethyl alcohol mole fraction increase in the (ethyl alcohol + water) mixed solvents. The experimental solubility values were correlated by a simplified thermodynamic equation, λh equation and modified Apelblat equation. Based on the solubility of daidzin, the enthalpy and entropy of solution were also evaluated by van’t Hoff equation. The results illustrated that the dissolution process of daidzin is endothermic and entropy driven.     

Analysis of herbicides in water using temperature-responsive chromatography and an aqueous mobile phase

A simple and rapid method has been developed for herbicides in water using temperature-responsive liquid chromatography (LC) and a column packed with poly(N-isopropylacrylamide) (PNIPAAm), a polymer anchored on the stationary-phase surface of modified silica. PNIPAAm reversibly changes its hydrophilic/hydrophobic properties in water in response to temperature. The method was used to determine five sulfonylurea and three urea herbicides. Separation was achieved with a 10 mM ammonium acetate (pH 3.0) isocratic aqueous mobile phase, and by changing the column temperature. The analytes were extracted from water by off-line solid-phase extraction (SPE) with an N-vinyl-pyrrolidone polymer cartridge. The average recoveries of the eight herbicides from spiked pure water, tap water and river water were 70-130% with relative standard deviations (RSDs) of <10%. The limits of quantitation (LOQ) of the eight herbicides were between 1 and 4 microg l(-1).