Influence of the eluent composition on column efficiency in reversed-phase high-performance liquid chromatography

Summary The dependence of column efficiency on the eluent (MeOH/H₂O) composition in a reversed-phase liquid chromatography system within a wide concentration range has been systematically examined. It is shown that when the intracolumn effect of mass transfer and diffusion is the main factor controlling band broadening the column efficiency decreases with the increase of the viscosity of the MeOH/H₂O mixture; on the other hand, when the extra-column effect is the main factor an increase in the viscosity of the eluents will help in improving column efficiency. The column efficiency is also related to the properties of the sample.     

Column-induced selectivity in separation of polynuclear aromatic hydrocarbons by reversed-phase,high-performance liquid chromatography

Summary Differences in retention time and relative elution order were observed when a standard mixture of 11 PAH was injected on three C-18 columns from different manufacturers under equivalent conditions.Working on a grant from the Swedish Natural Science Research Council.     

High performance liquid chromatography analysis of wine anthocyanins revisited: Effect of particle size and temperature

The complex anthocyanin fraction of red wines poses a demanding analytical challenge. We have found that anthocyanins are characterised by extremely low optimal chromatographic velocities, and as a consequence generic HPLC methods suffer from limited resolving power. Slow on-column inter-conversion reactions, particularly between carbinol and flavylium species, are shown to occur on the same time scale as chromatographic separation, leading to increased plate heights at normal chromatographic velocities. In order to improve current routine HPLC separations, the use of small (1.7 μm) particles and high temperature liquid chromatography (HTLC) were investigated. 1.7 μm particles provide better efficiency and higher optimal linear velocities, although column lengths of ∼20 cm should be used to avoid the detrimental effects of conversion reactions. More importantly, operation at temperatures up to 50 °C increases the kinetics of inter-conversion reactions, and implies significantly improved efficiency under relatively mild analysis conditions. It is further demonstrated using relevant kinetic data that no on-column thermal degradation of these thermally labile compounds is observed at 50 °C and analysis times of <2 h.     

Faster analysis by reversed-phase high-performance liquid chromatography

Summary Many analysts are not taking full advantage of the high speed possibilities of modern LC. Some analytical procedures reported in the literature, and many in regular use in control laboratories, could be achieved in less time without loss in precision. Some factors which affect retention times are discussed and the advantages and disadvantages of employing shorter column lengths and finer packing materials in reversed-phase HPLC are examined. The effect on efficiency of increased flow rates with 10,5 and 3 m ODS materials is shown. The ability to couple shorter column lengths without loss of efficiency is also demonstrated. This allows a minimum length to be selected that gives adequate resolution. Examples of high speed separations are shown and limitations in state of the art HPLC equipment and chromatographic data systems are discussed briefly.Presented at the 14th International Symposium on Chromatography London, September, 1982     

Determination of fungicides in wine by mixed-mode solid phase extraction and liquid chromatography coupled to tandem mass spectrometry

A novel procedure for the determination of nine selected fungicides (metalaxyl-M, azoxystrobin, myclobutanil, flusilazole, penconazole, tebuconazole, propiconazole, diniconazole and difenoconazole) in wine samples is presented. Sample enrichment and purification is simultaneously performed using mixed-mode, anion exchange and reversed-phase, OASIS MAX solid-phase extraction (SPE) cartridges. Analytes were determined by liquid chromatography coupled to tandem mass spectrometry using atmospheric pressure electrospray ionization (LC-ESI-MS/MS). Parameters affecting the chromatographic determination and the extraction-purification processes were thoroughly investigated. Under optimized conditions, 10 mL of wine were firstly diluted 1:1 with ultrapure water and then passed through the mixed-mode SPE cartridge at a flow of ca. 5 mLmin(-1). After a washing step with 5 mL of an aqueous NH(4)OH solution (5%, w:v), analytes were recovered with just 1 mL of methanol and injected in the LC-MS/MS system without any additional purification. The selective extraction process avoided significant changes in the ionization efficiency for red and white wine extracts in comparison with pure standards in methanol. Performance of the method was good in terms of precision (RSDs<11%) and accuracy (absolute recoveries>72%, determined against pure standards in methanol) reporting method LOQs in the range of 0.01-0.79 ngmL(-1) for target compounds, which are far below the EU maxima residue levels (MRLs) for fungicides in vinification grapes and wine. Several commercial wines from different geographic areas in Spain were analyzed. In most samples, metalaxyl-M and azoxystrobin were found at concentrations up to several ngmL(-1).     

On-column endcapping and derivatization in reversed-phase high-performance liquid chromatography

Summary On-column endcapping and derivatization or regeneration of C₈ and C₁₈ reversed-phase HPLC columns with newly introduced reagents were studied. These treatments can increase column life expectancy by restoring retention times and original chromatographic characteristics of the columns. This is illustrated by examples. Presented at the 15th International Symposium on Chromatography, Nürnberg, October 1984     

High-efficiency high performance liquid chromatographic analysis of red wine anthocyanins

The analysis of anthocyanins in natural products is of significant relevance in recent times due to the recognised health benefits associated with their consumption. In red grapes and wines in particular, anthocyanins are known to contribute important properties to the sensory (colour and taste), anti-oxidant- and ageing characteristics. However, the detailed investigation of the alteration of these compounds during wine ageing is hampered by the challenges associated with the separation of grape-derived anthocyanins and their derived products. High performance liquid chromatography (HPLC) is primarily used for this purpose, often in combination with mass spectrometric (MS) detection, although conventional HPLC methods provide incomplete resolution. We have previously demonstrated how on-column inter-conversion reactions are responsible for poor chromatographic efficiency in the HPLC analysis of anthocyanins, and how an increase in temperature and decrease in particle size may improve the chromatographic performance. In the current contribution an experimental configuration for the high efficiency analysis of anthocyanins is derived using the kinetic plot method (KPM). Further, it is shown how analysis under optimal conditions, in combination with MS detection, delivers much improved separation and identification of red wine anthocyanins and their derived products. This improved analytical performance holds promise for the in-depth investigation of these influential compounds in wine during ageing.     

Liquid chromatography and characterization of ether-functionalized imidazolium ionic liquids on mixed-mode reversed-phase/cation exchange stationary phase

A new series [C_nO_mmim]Cl of imidazolium cation-based ionic liquids (ILs), with an ether functional group on the alkyl side-chain, has been prepared. The possibility of analyzing the ionic liquids by high performance liquid chromatography (HPLC) was investigated on mixed-mode reversed/cation exchange stationary phase with the aqueous-acetonitrile mobile phase. Elution parameters, such as retention factor, selectivity and column efficiency, were studied as functions of mobile phase composition and pH. The ILs were characterized by elemental analysis, and infrared, UV and ¹H, ¹³C NMR spectroscopy.     

Effect of the alkyl chain length of the bonded stationary phase on solute retention in reversed-phase high-performance liquid chromatography

Summary A comparison of the chromatographic retention characteristics of nonpolar bonded stationary phases was investigated. The results show that the interactions between the solutes and the stationary phases having C₂, C₈ and C₁₈ alkyl groups are almost similar in the range of the mobile phase investigated. This interaction is considered as the solvophobic effect.     

Comparison of reversed-phase column materials for high-performance liquid chromatography of proteins

Nine reversed-phase materials with various bonded phases from different suppliers were studied for the separation of hydrophilic proteins with two solvent systems. Protein retention, resolution and recovery were not correlated with the nature of the hydrocarbonaceous ligand. Peak volumes increased with molecular weight, which led to broad, irregular peaks for the larger proteins on some columns. Four columns that performed equally well were selected for the purification of hydrophobic Sendai virus membrane proteins. In this case, more distinct differences were found between columns. Recovery of the membrane proteins strongly depended on the combination of column and solvent systems.     

Separation of positional isomers of mono-poly(ethylene glycol)-modified octreotides by reversed-phase high-performance liquid chromatography

The purpose of this study was to develop a reversed-phase high-performance liquid chromatographic method (RP-HPLC) for separating each positional isomer from low- to high-molecular-weight mono-PEGylated octreotides prepared by polyethylene glycol (PEG) derivatives with various molecular weights (2, 5, or 20 kDa). In the gradient elution using acetonitrile and 10 mM phosphate buffer at pH 7.0 on a Phenomenex Gemini C-18 column (250 mm × 4.6 mm id, 5 μm), each positional isomer of the mono-PEGylated octreotides was completely resolved with good resolution (PEG-2K: 7.6, PEG-5K: 6.6, and PEG-20K: 3.1). The optimal RP-HPLC condition also resolved the degradation products of mono-PEG-octreotide isomers in thermal stability studies at 55 °C and enzymatic stability studies with trypsin. In conclusion, the developed RP-HPLC method will be valuable for studying the effect of PEGylation site and the attached PEG size on the physicochemical and pharmacological properties of PEGylated octreotides.     

Regioselective separation of isomeric triacylglycerols by reversed-phase high-performance liquid chromatography: stationary phase and mobile phase effects

Complete regioselective separation of five pairs of isomeric dipalmitoyl polyalkenoyl glycerols with two to six double bonds in the unsaturated acyl residues has been achieved by RP-HPLC on a single ODS column. Four ODS columns with stationary phases containing different percentages of free silanol groups have been tested. Binary mobile phases of ACN admixed with dichloromethane, tetrahydrofuran, 1-propanol, 2-propanol, ethanol, or acetone have been examined. The choice of modifier depended on the nature of the stationary phase. The more polar solvents were better suited for stationary phases with higher percentage of free silanol groups. Isomeric species were eluted according to chain length, number of double bonds, and the position of the unsaturated acyl chain in the glycerol molecule. Retention increases in the order 20:5 < 22:6 < 18:3 < 20:4 < 18:2. Within each isomeric pair, the species with unsaturated acyl chain occupying either the sn-1- or the 3-position were retained preferentially. Complete simultaneous regioselective separation of 10 isomeric triacylglycerols in a single isocratic run on a single ODS column was demonstrated.     

Stability study and quantitative determination of mebeverine hydrochloride in tablets by means of reversed-phase high-performance liquid chromatography

Summary A rapid and specific reversed-phase high-performance liquid chromatographic method (RPHPLC) is described for the determination of mebeverine hydrochloride in tablets. Elution was performed on an octyl silane column with a methanol-water mixture (75-25), containing 0.05% hexylamine as silanol-blocking agent, adjusted to pH 5.0 with phosphoric acid. The method gave accurate, precise and reproducible results. The mean recovery of the drug from six synthetic tablet mixtures was 100.0% with a relative standard deviation (RSD) of 0.94%. In order to test the specificity of the method, the interference of the degradation compounds of mebeverine hydrochloride and of the intermediates from the synthesis was investigated. None of them did interfere. By means of mass spectrometry and UV-spectrophotometry, the degradation compounds of mebeverine were identified as veratric acid and as 4-|ethyl-[2-(4-methoxyphenyl)-1-methylethyl]amino| 1-butanol. The stability study proved that mebeverine hydrochloride is very stable in tablets; the tablets still contain more than 95% of the declared drug potency after storage for more than one year at 50°C.Colofac; Duspatal; Duspatalin     

Resolution of triacylglycerol positional isomers by reversed-phase high-performance liquid chromatography

The ability of reversed-phase high-performance liquid chromatography (RP-HPLC) to separate some positionally isomeric disaturated and monounsaturated triacylglycerols (TAGs) as intact species is demonstrated for the first time. Mobile phases of acetonitrile modified with methanol, ethanol, 2-propanol, 1-propanol, 1-butanol, acetone, or dichloromethane were tested for the separation of POP-PPO, PLP-PPL, PEP-PPE, and PDP-PPD (P-palmitic, O-oleic, L-linoleic, E-eicosapentaenoic, D-docosahexaenoic acid residue) on a single RP-HPLC column. The resolution improved with increasing number of double bonds in the acyl residues. While POP and PPO were only partially resolved, PDP and PPD were fully separated with all tested mobile phases, except those containing methanol. Also separated were the four TAGs having the same equivalent carbon number (ECN = 42), PEP, PPE, PDP, and PPD, on a single RP-HPLC column with mobile phase acetonitrile-2-propanol (70:30, v/v) at 0.8 mL/min. In all cases the isomer with the unsaturated acyl residue in either 1- or 3-position was retained more strongly than the respective 2-isomer.     

Separation of pyranoanthocyanins from red wine by column chromatography

With the aim of monitoring the formation of anthocyanin-derived pigments and contributing to the study of their chromatic properties, stability and relative contribution to the colour of red wines, a method for fractionation of the colouring material was set up. The method was based on the distinct reactivity of the different pigment families towards bisulfite (hydrogen sulfite). The wine, acidified and bleached with NaHSO₃, was placed in a Toyopearl^® HW-40(s) gel column and submitted to elution with ethanol. Two fractions with different pigment compositions were collected and analysed by liquid chromatographay diode array detection-mass spectrometry. Compounds present in each fraction were identified according to their UV-visible and MSⁿ mass spectra, showing that the first one was mostly constituted of pyranoanthocyanins, whereas the second basically contained anthocyanins and anthocyanin-flavanol condensation products. A large variety of new pigments were detected, some of which had not been previously reported in red wines, as far as we know. Characteristic MS² and MS³ fragmentation patterns were observed within each family of compounds, which could be further applied for characterisation of unknown pigments in other wines.     

Direct aqueous determination of glyphosate and related compounds by liquid chromatography/tandem mass spectrometry using reversed-phase and weak anion-exchange mixed-mode column

Analysis of the broad-spectrum herbicide glyphosate and its related compounds is quite challenging. Tedious and time-consuming derivatization is often required for these substances due to their high polarity, high water solubility, low volatility and molecular structure which lacks either a chromophore or fluorophore. A novel liquid chromatography/tandem mass spectrometry (LC/MS–MS) method has been developed for the determination of glyphosate, aminomethylphosphonic acid (AMPA) and glufosinate using a reversed-phase and weak anion-exchange mixed-mode Acclaim^® WAX-1 column. Aqueous environmental samples are directly injected and analyzed in 12 min with no sample concentration or derivatization steps. Two multiple reaction monitoring (MRM) channels are monitored in the method for each target compound to achieve true positive identification, and ¹³C,¹⁵N-glyphosate is used as an internal standard to carry out isotope dilution mass spectrometric (IDMS) measurement for glyphosate. The instrument detection limits (IDLs) for glyphosate, AMPA and glufosinate are 1, 2 and 0.9 μg/L, respectively. Linearity of the detector response with a minimum coefficient of determination (R²) value (R² > 0.995) was demonstrated in the range of ∼10 to 10³ μg/L for each analytes. Spiked drinking water, surface water and groundwater samples were analyzed using this method and the average recoveries of analytes in three matrices ranged from 77.0 to 102%, 62.1 to 101%, 66.1 to 93.7% while relative standard deviation ranged from 6.3 to 10.2%, 2.7 to 14.8%, 2.9 to 10.7%, respectively. Factors that may affect method performance, such as metal ions, sample preservation, and storage time, are also discussed.     

Development of a mixed-mode solid phase extraction method and further gas chromatography mass spectrometry for the analysis of 3-alkyl-2-methoxypyrazines in wine

A new method for analysing 3-isopropyl-2-methoxypyrazine, 3-sec-butyl-2-methoxypyrazine and 3-isobutyl-2-methoxypyrazine in wine has been developed and applied to wine. The analytes are extracted from 25 mL of wine in a solid-phase extraction cartridge filled with 60 mg of cation-exchange mixed-mode sorbent. Analytes are recovered with triethylamine in dichloromethane and the organic extract is analysed by GC-SIM-MS using 3-isopropyl-2-ethoxypyrazine as internal standard. The detection limits of the method are in all cases under 1 ng/L, below the olfactory thresholds of the compounds in wine. The repeatability of the method is around 15% for levels in wine of 2 ng/L. Linearity is satisfactory and recoveries are in all cases close to 100% with RSD between 13% and 20%. The method has been applied to the analysis of 12 Chilean white and 8 Spanish red wines. The levels found suggest that 3-alkyl-2-methoxypyrazines can exert a significant sensory contribution to the aroma of Chilean Sauvignon Blanc wines, while most likely they play a nearly negligible role on traditional Ribera and Rioja Spanish red wines.     

Comparison of ion-exchange chromatography, isoelectric precipitation and reversed-phase high-performance liquid chromatography for the separation of individual cardiac myosin light chains.

Three modified procedures for the separation of cardiac myosin light chains are carefully compared. Ion-exchange chromatography gives a purified cardiac myosin light chain 1, whereas light chain 2 is always contaminated by light chain 1. Reversed-phase high-performance liquid chromatography gives the best resolution of these light chains and needs only 20 min for each run. However, it requires pure preparation of myosin light chains before separation. Isoelectric precipitation is the simplest procedure and suitable for large quantities of material. Although it gives the highest yield the separation is not adequate. A modified and rapid procedure for the isolation of cardiac and skeletal total myosin light chains is also presented.