Evaluation of the potential of nonlinear gradients for separating a ternary mixture

The potential of applying nonlinear gradients in preparative chromatography is evaluated theoretically and experimentally. The second eluting component of a ternary model mixture is considered as the target component. Systematic gradient resolution experiments were conducted for this mixture using reversed phase chromatography. In preliminary experimental investigations the effect of the mobile phase composition on the specific distribution equilibria was quantified. Concave and convex solvent strength gradients were analysed and compared with conventional linear gradients. Main result of the study is the fact that significant improvement in productivity can be achieved if the gradient shapes are designed carefully.     

Improvement of separation in zonal preparative thin-layer chromatography by gradient elution

Summary Complex extracts of the plants Azulan and Hemorigen were separated by zonal micropreparative thin-layer chromatography in sandwich chambers of the ES and DS type which permitted zonal application of large volumes of sample, without auxiliary equipment. Application from the edge of the layer, in the frontal chromatography mode, markedly improved the separation efficiency and capacity owing to displacement effects which narrow the initially broad zones. Further improvement of separation efficiency and purity of fractions, revealed by densitometry, was observed using stepwise gradient elution. This was confirmed by extraction of some of the separated fractions from the layer and rechromatography; the composition of these fractions were generally simpler than for the corresponding isocratic chromatograms.     

A practical approach to the preparative purification of peptides using analytical instrumentation with analytical and semipreparative columns

Summary We have examined the effect of particle size of silica-based reversed-phase packings and column packing techniques on the reversed-phase analytical separation of a peptide mixture. A C₁₈ packing of 15–20 μm average particle size produced satisfactory peptide resolution, allowing a relatively inexpensive scale up to the preparative purification of peptides. A shallow gradient (0.1% acetonitrile/min) elution procedure was developed for the preparative purification of closely related decapeptides (differing by one methyl group) on analytical (250×4.1 mm I.D.) and semipreparative (250×10 mm I.D.) columns. Up to 30 mg and 225 mg of the two-peptide mixture was efficiently resolved, with high yields of homogeneous peptides, on analytical and semipreparative columns, respectively, containing the 15–20 μm packing. We have also demonstrated the potential of our purification procedure for resolving more complex multicomponent mixtures by efficiently separating a total of 22 mg of three closely-related peptides on analytical columns containing 7 μm or 15–20 μm particle size reversed-phase packings. The use of the inexpensive 15–20 μm packing, coupled with the ability to pack efficient columns with analytical HPLC instrumentation, offers great cost saving potential.     

Theoretical background of short chromatographic layers. Optimization of gradient elution in short columns

Although linear salt gradient elution ion-exchange chromatography (IEC) of proteins is commonly carried out with relatively short columns, it is still not clear how the column length affects the separation performance and the economics of the process. The separation performance can be adjusted by changing a combination of the column length, the gradient slope and the flow velocity. The same resolution can be obtained with a given column length with different combinations of the gradient slope and the flow velocity. This results in different separation time and elution volume at the same resolution. Based on our previous model, a method for determining the separation time and the elution volume relationship for the same resolution (iso-resolution curve) was developed. The effect of the column length and the mass transfer rate on the iso-resolution curve was examined. A long column and/or high mass transfer rate results in lesser elution volume. The resolution data with porous bead packed columns and monolithic columns were in good agreement with the calculated iso-resolution curves. Although the elution volume can be reduced with increasing column length, the pressure drop limits govern the optimum conditions.     

HPLC Analysis of complex mixtures of triglycerides using gradient elutions and an ultraviolet detector

Summary A method of reserved-phase HPLC analysis for mixtures of triglycerides (TG's) that provides good resolution at acceptable analysis times for high-ECN TG's has been developed. An elution gradient of methyltert-butyl-ether (MTBE) in acetonitrile (ACN) was used with an ultraviolet detector operated at 215nm. The effect of the proportion of MTBE in the mobile phase, gradient time, temperature and sample solvent on TG retention and resolution was studied. Linear relationships were derived between the logarithm of the capacity factor (log k'), and the logarithm of selectivity (log α) and the above-mentioned chromatographic factors. The conditions selected were: an elution gradient of from 23 to 30% MTBE, an elution gradient time of 25 minutes, and a temperature of 30°C, which provided good resolution of soybean oil TG's in less than 30 minutes.     

Simple automated generation of gradient elution conditions in sequential injection chromatography using monolithic column

The paper deals with the concept of simple automated creation of gradient profile of the mobile phase for gradient-elution sequential injection chromatography (GE-SIC). The feasibility and merits of this concept are demonstrated on the separation and simultaneous assay of indomethacin as active principle and of its two degradation products (5-methoxy-2-methylindoleacetic acid and 4-chloro-benzoic acid) in a topical pharmaceutical formulation.The GE-SIC separation was performed with a FIAlab^® 3000 SIC set-up (USA) equipped with an Onyx™ Monolithic C18 (25 mm × 4.6 mm, Phenomenex^®) column, a six-port selection valve, a 5-mL syringe pump and a fiber-optics UV CCD detector. Ketoprofen was used as an internal standard (IS). The gradient elution was achieved by automated reproducible mixing of acetonitrile and aqueous 0.2% phosphoric acid in the holding coil of the SIC system. Different profiles of the gradient elution were tested. The optimal gradient using two mobile phases 30:70 and 50:50 of acetonitrile/0.2% phosphoric acid (v/v) was achieved under the optimum flow rate 1.2 mL min⁻¹. The chromatographic resolution R between the peaks of all solutes (including the IS) was >2.00. The repeatability of retention times was characterized by the RSD values 0.18-0.30% (n = 6). Net separation time was 3.5 min and the mobile phase consumption was 4.5 mL for a single GE-SIC assay. The figures of merit of the novel GE-SIC method compared well with those of conventional HPLC.     

Computerized optimization of gradient elution conditions with ternary solvent mobile phases in RPLC

Summary In this work, an optimization procedure for gradient RPLC separation, using ternary mobile phases, is described. This procedure requires eight preliminary experiments in gradient elution mode to predict the retention surface for each solute over the whole triangular space. This is followed by computerized calculations to determine the best ternary gradient elution profile with respect to both selectivity and analysis time. The efficiency of this procedure from the point of view of rapidity and of accuracy, is illustrated for the specific separation of twelve phenyl urea herbicides.     

Application of a new capsule-type silica gel coated with hydrophobic polymer to preparative liquid chromatography on an industrial scale

Summary A new type of column packing material designed for preparative liquid chromatography, silicone polymer-coated silica gel modified with octadecyl group (S/S-C₁₈), was applied to chromatographic purification of a lipophilic biotechnological product. Triglycerides containing γ-linolenic acid were separated from the curde oil that consisted of triglycerides, diglycerides, free fatty acids, sterols and other polar substances, using a S/S-C₁₈ packed column (150 mm I.D. × 1000 mm). No column deterioration was observed after more than 1500 times of sample introductions.     

Development of an efficient method for the preparative isolation and purification of chlorophyll a from a marine dinoflagellate Amphidinium carterae by high-speed counter-current chromatography coupled with reversed-phase high-performance liquid chromatography

In our research into chlorophylls of marine dinoflagellates, chlorophyll a was separated rapidly from the hexane extract of Amphidinium carterae in three steps. The first step was silica gel column chromatography, where elution was performed with 0–50% ethyl acetate in n-hexane. The second was high-speed counter-current chromatography using a two-phase solvent system consisting of n-hexane–ethyl acetate–methanol–water (5:5:5:1, v/v), and the third step was preparative reversed-phase high-performance liquid chromatography using a solvent system of acetone–water (89:11, v/v). HPLC analysis showed that the purity of chlorophyll a from the second step was over 83%, and after the third it was over 99%. Thirty milligrams of chlorophyll a was isolated from a crude sample of 250 mg of chlorophylls, and its structure was identified by analyzing its MS, ¹H NMR and ¹³C NMR spectra.     

Determination of chlorophenoxy acid herbicides and their esters in soil by capillary high performance liquid chromatography with ultraviolet detection, using large volume injection and temperature gradient

A simple method for the simultaneous determination of chlorophenoxy acid herbicides and their esters in soil is presented. Compounds studied are: 2,4-dichlorophenoxyacetic acid (2,4-D), 4-(2,4-dichlorophenoxy)butanoic acid (2,4-DB), 2,4-dichlorophenoxyacetic-1-butyl ester (2,4-D-1-butyl ester), and 2,4-dichlorophenoxyacetic-1-methyl ester (2,4-D-1-methyl ester).

The chromatographic analysis was carried out by HPLC, after ultrasonic extraction, on a C₁₈ packed capillary column with temperature gradient, large injection volumes and UV detection at 232 nm. Samples were spiked with amounts between 2.5 and 6.0 μg g⁻¹ of each herbicide; recoveries obtained were between 72 and 97% (n=3 for each spiked level) and detection limits were between 0.3 and 0.5 μg g⁻¹.

Application of this procedure to the analysis of herbicides in ester and acid forms showed the effectiveness of the methodology proposed.     

Determination of flavonoids from Gynostemma pentaphyllum using ultra-performance liquid chromatography with triple quadrupole tandem mass spectrometry and an evaluation of their antioxidant activity in vitro

Gynostemma pentaphyllum, a traditional herbal tea, and its flavonoids possess antioxidant activities. This study was carried out to isolate and identify these flavonoids from G. pentaphyllum and determinate the contents of the main flavonoids and their antioxidant activities. Nine flavonoids were determinated rapidly from G. pentaphyllum using medium-pressure liquid chromatography combined with other simple chromatographic methods. And nine flavonoids were identified as rutin (1), 4′-O-methyl-kaempferol-3-O-rutinoside (2), ombuoside (3), kaempferol-3-β-D-O-rutinoside (4), isorhamnetin-3-O-β-D-rutinoside (5), quercetin-3-O-β-D-glucoside (6), isorhamnetin (7), kaempferol (8), and quercetin (9) by liquid chromatography mass spectrometry-ion trap time of flight and ¹³C nuclear magnetic resonance spectroscopy, respectively. All of them displayed various potent antioxidant effects against the DPPH radical and A549 cell injury by using H₂O₂-generate peroxyl radicals in vitro . A sensitive and specific ultra-performance liquid chromatography coupled with multiple-reaction monitoring mode was utilized to rapidly analyze the main six flavonoids of the ethanol extract of G. pentaphyllum within less than 8?min using a Waters Acquity UPLC BEH C₁₈ column. The contents of them were from 57.11 to 12907.74?µg/g in G. pentaphyllum.