Micro liquid chromatography coupled with evaporative light scattering detector at ambient and high temperature: optimization of the nebulization cell geometry

The recent developments in liquid chromatography (LC) are mainly dedicated to both system miniaturization (micro-, capillary-, and nano-LC) and analysis time decrease (fast-, and ultra-fast-LC). For the latter, several strategies can be used, and high temperature liquid chromatography (HTLC) seems very promising and easy to implement, especially in miniaturized system. In LC, the evaporative light scattering detector (ELSD) is considered an attractive alternative to conventional detector such as UV-vis due to its versatility and quasi-universality. Therefore, the compatibility of ELSD with micro-LC and micro-HTLC was investigated for several pharmaceutical compounds of interest. The nebulization process appeared to be the most critical parameter for performing the coupling and maintaining an efficient separation. Therefore, appropriate modifications in the nebulization cell geometry were brought to make ELSD fully compatible with micro-LC. The impact of optimized nebulization cell on chromatographic performance was evaluated in terms of efficiency and sensitivity. Finally, highly efficient, sensitive and fast separations of pharmaceutical drugs were performed with both techniques and the customized nebulization cell design.     

Combination of COFRADIC and high temperature-extended column length conventional liquid chromatography: a very efficient way to tackle complex protein samples, such as serum

The previously reported COmbined FRActional DIagonal Chromatography (COFRA-DIC) methodology, in which a subset of peptides representative for their parent proteins are sorted, is particularly powerful for whole proteome analysis. This peptide-centric technology is built around diagonal chromatography, where peptide separations are crucial. This paper presents high efficiency peptide separations, in which four 250 x 2.1 mm, 5 microm Zorbax 300SB-C18 columns (total length 1 m) were coupled at operating temperatures of 60'C using a dedicated LC oven and conventional LC equipment. The high efficiency separations were combined with the COFRADIC procedure. This extremely powerful combination resulted, for the analysis of serum, in an increase in the uniquely identified peptide sequences by a factor of 2.6, compared to the COFRADIC procedure on a 25 cm column. This is a reflection of the increased peak capacity obtained on the 1 m column, which was calculated to be a factor 2.7 higher than on the 25 cm column. Besides more efficient sorting, less ion suppression was noticed.     

Evaluation of the properties of a superficially porous silica stationary phase in hydrophilic interaction chromatography

The sample capacity, column efficiency (and its variation with flow) of a superficially porous unbonded silica phase (Halo) was investigated using hydrophilic interaction chromatography (HILIC), particularly for separation of basic compounds. Sample capacity compared with totally porous silica phases was somewhat reduced, broadly in line with the decreased surface area, but still favourable compared with reversed-phase separations of these solutes. Efficiencies in excess of 100,000 plates were obtained at room temperature in reasonable analysis times by using a 45 cm coupled column, while generating back pressures compatible with conventional HPLC. Shorter columns offered the possibility of fast analysis of bases, and the unfavourable mass transfer properties reported by others at high flow rate for similar reversed-phase columns, were not apparent. While excellent peak shapes were obtained for many bases on silica HILIC phases, problems may still occur for some solutes.     

Simultaneous, sequential quantitative achiral-chiral analysis by two-dimensional liquid chromatography

In general, chromatographic analysis of chiral compounds involves a minimum of two methods; a primary achiral method for assay and impurity analysis and a secondary chiral method for assessing chiral purity. Achiral method resolves main enantiomeric pairs of component from potential impurities and degradation products and chiral method resolves enantiomeric pairs of the main component and diastereomer pairs. Reversed-phase chromatographic methods are preferred for assay and impurity analysis (high efficiency and selectivity) whereas chiral separation is performed by reverse phase, normal phase, or polar organic mode. In this work, we have demonstrated the use of heart-cutting (LC-LC) and comprehensive two-dimensional liquid chromatography (LC × LC) in simultaneous, sequential achiral and chiral analysis and quantitation of minor, undesired enantiomer in the presence of major, desired enantiomer using phenylalanine as an example. The results were comparable between LC-LC and LC × LC with former offering better sensitivity and accuracy. The quantitation range was over three orders of magnitude with undesired D-phenylalanine detected at approximately 0.3% in the presence of predominant, desired L-phenylalanine (99.7%). The limit of quantitation was comparable to conventional high-performance liquid chromatography. A reversed-phase C18 achiral column in the primary and reversed-phase Chirobiotic Tag chiral column in the secondary dimension were used with a compatible mobile phase.     

High throughput screening of active pharmaceutical ingredients by UPLC

Ultra performance LC (UPLC) was evaluated as an efficient screening approach to facilitate method development for drug candidates. Three stationary phases were screened: C-18, phenyl, and Shield RP 18 with column dimensions of 150 mm x 2.1 mm, 1.7 microm, which should theoretically generate 35,000 plates or 175% of the typical column plate count of a conventional 250 mm x 4.6 mm, 5 microm particle column. Thirteen different active pharmaceutical ingredients (APIs) were screened using this column set with a standardized mobile-phase gradient. The UPLC method selectivity results were compared to those obtained for these compounds via methods developed through laborious trial and error screening experiments using numerous conventional HPLC mobile and stationary phases. Peak capacity was compared for columns packed with 5 microm particles and columns packed with 1.7 microm particles. The impurities screened by UPLC were confirmed by LC/MS. The results demonstrate that simple, high efficiency UPLC gradients are a feasible and productive alternative to more conventional multiparametric chromatographic screening approaches for many compounds in the early stages of drug development.     

Peak shape improvement of basic analytes in capillary liquid chromatography

The analysis of bases is of special interest in pharmaceutical research because numerous active substances contain basic functional groups. Capillary and conventional size LC separations of drug substances spiked with potential impurities were compared. In the case of the nonpolar drug levonorgestrel equal separation efficiency was readily attained by both techniques. The peaks of basic substances, however, showed extensive tailing when separated by capillary LC. The peak deformation was attributable to interactions of the basic substances with the polar inner surface of the fused silica capillaries employed in capillary LC and does not appear with the steel tubing generally used in conventional size LC. This drawback of capillary LC was overcome by use of deactivated fused silica capillaries for column hardware and transfer lines.     

The impact of extra-column band broadening on the chromatographic efficiency of 5 cm long narrow-bore very efficient columns

Small columns packed with core-shell and sub-2 μm totally porous particles and monolith columns are very popular to conduct fast and efficient chromatographic separations. In order to carry out fast separations, short (2-5 cm) and narrow-bore (2-2.1 mm) columns are used to decrease the analyte retention volume. Beside the column efficiency, another significant issue is the extra-column band-spreading. The extra-column dispersion of a given LC system can dramatically decrease the performance of a small very efficient column. The aim of this study was to compare the extra-column peak variance contribution of several commercially available LC systems. The efficiency loss of three different type 5 cm long narrow bore, very efficient columns (monolith, sub-2 μm fully porous and sub-2 μm core-shell packing) as a function of extra-column peak variance, and as a function of flow rate and also kinetic plots (analysis time versus apparent column efficiency) are presented.     

Enantiomeric separations by means of nano‐LC

Enantiomers represent a class of compounds extensively investigated since they can show totally different behaviors when they interact with a chiral environment. Because of their identical chemical structure (they differ only in the spatial arrangement of the atoms in the molecule), the separation of optical isomers is a challenging task of analytical chemistry. So far employed methods for the separation of enantiomers are mainly based on chromatography. CE as well was considered as an analytical technique suitable for chiral separations, characterized by high efficiency and low consumption of reagent. Recently, miniaturization was introduced in LC to answer the needs to perform analyses in the minimum time, to use the smallest amount of samples and to reduce environmental pollution. Nano‐LC represents nowadays a valid alternative to the abovementioned conventional analytical techniques, and can be advantageously exploited for enantiomeric separation especially because it needs minute amounts of the chiral material necessary to carry out enantiomeric separations. This review describes the development and applications of nano‐LC in the field of chiral separations. The data reported in literature show its relevance for the study enantiomers‐chiral selectors interaction, as well as for application in pharmaceutical and clinical research.     

Robust isocratic liquid chromatographic separation of functional poly(methyl methacrylate)

The separation of telechelic poly(methyl methacrylate) (PMMA) prepolymers based on the number of end-groups under critical liquid chromatography (LC) conditions has been studied using a bare-silica column, which can interact with polar functional groups. The critical solvent compositions for non-functional, mono-functional and bi-functional PMMAs were determined in normal-phase LC using mixtures of acetonitrile and dichloromethane (DCM) of varying composition as the mobile phase. The telechelic prepolymers were successfully separated according to hydroxyl (OH) functionality (with zero, one, or two OH groups, respectively) under the critical conditions, in which fast (5 min), base-line separations were obtained independent of molecular weight. Changing the column temperature, flow rate, and mobile-phase composition within a certain range did not affect the functionality separation. Therefore this isocratic LC separation method is quite robust. Evaporative light-scattering detector (ELSD) calibration curves were used for the quantitative analysis of functional PMMA prepolymers.     

On-fibre solid-phase microextraction coupled to conventional liquid chromatography versus in-tube solid-phase microextraction coupled to capillary liquid chromatography for the screening analysis of triazines in water samples

This paper compares the advantages and disadvantages of two different configurations for the extraction of triazines from water samples: (1) on-fibre solid-phase microextraction (SPME) coupled to conventional liquid chromatography (LC); and (2) in-tube SPME coupled to capillary LC. In-tube SPME has been effected either with a packed column or with an open capillary column. A critical evaluation of the main parameters affecting the performance of each method has been carried out in order to select the most suitable approach according to the requirements of the analysis. In the on-fibre SPME configuration the fibre coating was polydimethylsiloxane (PDMS)-divinylbenzene (DVB). The limits of detection (LODs) obtained with this approach under the optimized extraction and desorption conditions were between 25 and 125 microg/L. The in-tube SPME approach with a C18 packed column (35 mm x 0.5 mm I.D., 5 microm particle size) connected to a switching micro-valve provided the best sensitivity; under such configuration the LODs were between 0.025 and 0.5 microg/L. The in-tube SPME approach with an open capillary column coated with PDMS (30 cm x 0.25 mm I.D., 0.25 microm of thickness coating) connected to the injection valve provided LODs between 0.1 and 0.5 microg/L. In all configurations UV detection at 230 nm was used. Atrazine, simazine, propazine, ametryn, prometryn and terbutryn were selected as model compounds.     

Physically adsorbed chiral stationary phase of avidin on monolithic silica column for capillary electrochromatography and capillary liquid chromatography

The physical adsorption method proposed previously has been successfully applied to a monolithic silica column. By virtue of the physical adsorption, a chiral stationary phase of avidin was prepared onto the silica monolith. The phase ratio of resulting stationary phase was evaluated with frontal analysis. The method proved to be comparable in phase ratio to the chemical bonding methods used in high-performance liquid chromatography (HPLC). Enantiomer separations were carried out in capillary electrochromatography (CEC) and capillary liquid chromatography (CLC) modes. Due to its larger phase ratio, the resulting column showed more powerful separation capability as compared to open-tubular CEC (OTCEC). Twelve chiral compounds were baseline-resolved. The resulting column showed high separation efficiency, with average theoretical plate numbers of 66 000/m for CLC and 122 000/m for CEC. Good reproducibility was observed, with RSD value less than 1.3% for retention time, retention factor and separation factor, and less than 6.6% for plate counts and resolution (n = 40). Fast separations were achieved with a short column. The test enantiomers were baseline-resolved within 4 min under CLC and CEC modes. In addition, field-enhanced sample injection (FESI) was coupled to CLC as well as CEC to improve the detection sensitivity.     

Elevated temperature-extended column length conventional liquid chromatography to increase peak capacity for the analysis of tryptic digests

High efficiency separations (200 000 plates) were obtained on conventional LC equipment by coupling 8 x 25 cm x 2.1 (or 4.6) mm id x 5 microm d(p) ODS columns (total length 2 m) and operation at 60 degrees C using a dedicated LC oven. The peak capacity in this 1-D set-up was 900 for the separation of human serum tryptic peptides analyzed after depletion of six highly abundant proteins. The chromatographic performance of an elevated temperature-extended column length conventional LC is highlighted.     

Fast hydrophilic interaction liquid chromatographic separations on bonded zwitterionic stationary phase

Separation science is an art of obtaining adequate resolution of the desired compounds in minimum time, and with minimum effort in terms of sample preparation and data evaluation. In LC, where selectivity is a main driving force for separation, the availability of different separation modes capable of operating at high flow rates is a way to make combined optimal use of selectivity, efficiency, and speed. The separation of polar and hydrophilic compounds is problematic in RP LC due to the poor retention. Hydrophilic interaction liquid chromatography (HILIC) is a more straightforward separation mode to address this problem. Herein, it is shown that separations in HILIC mode are equally efficient as for RP, providing a potential for very fast separations on short columns. This is not only facilitated by the low viscosity of the mobile phase compositions used, compared to typical RP eluents, but also due to higher column permeability. To exemplify this, baseline separations of uracil and cytosine are shown in less than 4 s and of Tamiflu and its main metabolite in less than 40 s, both under isocratic conditions. HILIC must therefore be considered having potential for high throughput purposes, and being an attractive candidate as the second separation dimension in 2-D HPLC.     

Implementations of two-dimensional liquid chromatography

Today scientists must deal with complex samples that either cannot be adequately separated using one-dimensional chromatography or that require an inordinate amount of time for separation. For these cases we need two-dimensional chromatography because it takes far less time to generate a peak capacity n(c) twice in a row than to generate a peak capacity n(c)(2) once. Liquid chromatography has been carried out successfully on thin layers of adsorbents and along tubes filled with various adsorbents. The first type of separation sorts out the sample components in a physical separation space that is the layer of packing material. The analysis time is the same for all the components of the sample while their migration distance increases with decreasing retention. The resolution between two components having a certain separation factor (alpha) increases with increasing migration distance, i.e., from the strongly to the weakly retained compounds. In the second type of separation, the sample components are eluted from the column and separated in the time space, their migration distances are all the same while their retention times increase from the unretained to the strongly retained compounds. Separation efficiency varies little with retention, as long as the components are eluted from the column. We call these two types of separation the chromatographic separations in space (LC(x)) and the chromatographic separations in time (LC(t)), respectively. In principle, there are four ways to combine these two modes and do two-dimensional chromatographic separations, LC(t)xLC(t), LC(x)xLC(t), LC(t)xLC(x), and LC(x)xLC(x). We review, discuss and compare the potential performance of these combinations, their advantages, drawbacks, problems, perspectives and results. Currently, column-based combinations (LC(t)xLC(t)) are the most actively pursued. We suggest that the combination LC(x)xLC(t) shows exceptional promise because it permits the simultaneous second-dimension separations of all the fractions separated in the first-dimension, thus providing remarkable time saving.     

Interference of chitosan in glucose analysis by high-performance liquid chromatography with evaporative light scattering detection

The purpose of this work was to quantify glucose in aqueous solutions containing chitosan by high-performance liquid chromatography (HPLC) with evaporative light scattering detection (ELSD). Chitosan is a natural compound that is used alone or as an additive in several formulations. Microencapsulation of bioactive compounds such as glucose, by means of chitosan, is being explored, but difficulties arise when glucose needs to be determined in the presence of chitosan. HPLC is the technique most commonly used for glucose analysis, and ELSD may offer advantages (e.g. sensitivity and the possibility of operating in gradient mode) compared with other detectors. The influence of chitosan in the analysis of glucose by HPLC with ELSD was investigated at different pH values of the aqueous solutions. Isocratic elution with an acetonitrile/water mixture (80:20, v/v) and water washing between runs were the best options to avoid the mucoadhesive properties of chitosan, which are responsible for column degradation and variability of the retention time of glucose. The developed methodology was considered completely adequate for rapid glucose analysis in aqueous solutions with low pH (< 3), in the presence of chitosan.     

Comprehensive two-dimensional liquid chromatography to quantify polyphenols in red wines

A comprehensive two-dimensional liquid chromatography method has been applied for the quantification of polyphenols in red wines and compared to the most commonly employed conventional LC approach. Such methodology comprised the use of a microbore conventional HPLC column packed with totally porous particles in the first dimension and a partially porous column of conventional diameter in the second dimension. Even though a good number of applications in comprehensive LC have been reported, quantification experiments have been rarely described. To this regard, the advantages of comprehensive LC together with the employment of dedicated software capable of detecting and quantifying each peak from the 2D plot, have been taken into account for quantifying the most representative polyphenols in three different commercial Sicilian red wine samples. The optimized method has been validated in terms of linearity, sensitivity, detection and quantification limits. LODs as low as 0.02 ppm were obtained using the one-dimensional HPLC-DAD method, whereas values lower than 0.10 ppm were obtained by comprehensive LC. However, comprehensive LC allowed the quantification of a higher number of compounds with RSD lower than 10% thanks to its improved resolving power. The separation capabilities of comprehensive LC allowed the analysis of complex natural samples without any pre-treatment to effectively reduce the interferences coming from the matrix.     

Increased productivity in quantitative bioanalysis using a monolithic column coupled with high-flow direct-injection liquid chromatography/tandem mass spectrometry

The feasibility of using a monolithic column as the analytical column in conjunction with high-flow direct-injection liquid chromatography/tandem mass spectrometry (LC/MS/MS) to increase productivity for quantitative bioanalysis has been investigated using plasma samples containing a drug and its epimer metabolite. Since the chosen drug and its epimer metabolite have the same selected reaction monitoring (SRM) transitions, chromatographic baseline separation of these two compounds was required. The results obtained from this monolithic column system were directly compared with the results obtained from a previously validated assay using a conventional C18 column as the analytical column. Both systems have the same sample preparation, mobile phases and MS conditions. The eluting flow rate for the monolithic column system was 3.2 mL/min (with 4:1 splitting) and for the C18 column system was 1.2 mL/min (with 3:1 splitting). The monolithic column system had a run time of 5 min and the conventional C18 column system had a run time of 10 min. The methods on the two systems were found to be equivalent in terms of accuracy, precision, sensitivity and chromatographic separation. Without sacrificing the chromatographic separation, sensitivity, accuracy and precision of the method, the reduced run time of the monolithic column method increased the sample throughput by a factor of two.     

Ultrahigh pressure liquid chromatography using elevated temperature

Fast liquid chromatographic (LC) methods are important for a variety of applications. Reducing the particle diameter (d(p)) is the most effective way to achieve fast separations while preserving high efficiency. Since the pressure drop along a packed column is inversely proportional to the square of the particle size, when columns packed with small particles (<2 microm) are used, ultrahigh pressures (>689 bar) must be applied to overcome the resistance to mobile phase flow. Elevating the column temperature can significantly reduce the mobile phase viscosity, allowing operation at higher flow rate for the same pressure. It also leads to a decrease in retention factor. The advantage of using elevated temperatures in LC is the ability to significantly shorten separation time with minimal loss in column efficiency. Therefore, combining elevated temperature with ultrahigh pressure facilitates fast and efficient separations. In this study, C6-modified 1.0 microm nonporous silica particles were used to demonstrate fast separations using a temperature of 80 degrees C and a pressure of 2413 bar. Selected separations were completed in 30 s with efficiencies as high as 220,000 plates m(-1).     

Capillary Electrochromatography as a Method Development Tool for the Liquid Chromatographic Separation of DUP 654 and Related Substances

Capillary Electrochromatography (CEC) offers a rapid, economical, and efficient means for resolving nonionic compounds in the reversed phase mode on octadecylsilane (ODS) columns. A CEC optimization on a Hypersil ODS capillary column was employed to identify a suitable mobile phase for the pressure-driven (reversed phase ODS) separation of the anti-inflammatory 2-phenylmethyl-1-naphthol (DUP 654), and its related substances. The proportions of mobile phase modifiers methanol, acetonitrile, and water as well as pH were employed as variables in a stacked mixture design. Comparable response surface profiles were obtained for the CEC separations at pH 4 and pH 8. However, subtle differences were evident in the quality of separations obtained in the liquid chromatographic (LC) mode when using a specially-prepared column packed with exactly the same stationary phase as used in the CEC experiments. A mapping of the response surface for separations on a commercially available Hypersil ODS LC column revealed obvious differences. The differences indicate that the transfer of ODS based separation methods between CEC and LC involves more than simply transferring the conditions from one mode to the other.     

HPLC‐ELSD analysis of spectinomycin dihydrochloride and its impurities

A simple, rapid and reliable reversed‐phase ion‐pair chromatography method by HPLC coupled to an evaporative light scattering detector (ELSD) has been developed to simultaneously determine chloride, spectinomycin and its related substances in a sample. The column was a TSKgel ODS‐100V. The mobile phase was ACN/aqueous solution of 15 mM ammonium acetate adjusted with TFA to pH 3.0 (2:98 v/v), in an isocratic mode. The drift tube temperature was set at 50°C and the nebulizing gas flow rate of air was 3.5 L/min for ELSD detection. Almost all of the reported degradation compounds of spectinomycin such as actinamine, actinospectinoic acid and biosynthesis intermediates such as dihydrospectinomycin diastereoisomers were baseline separated. MS was utilized for the identification of spectinomycin and its seven related substances. The method for the assay of spectinomycin was successfully validated with respect to accuracy, precision (RSD less than 2%), linearity (throughout the linear range 0.025–3 mg/mL, r=0.9993), sensitivity (LOD: 100 ng on column) and robustness. The experimental results demonstrated that the simultaneous determination of chloride, spectinomycin and related substances is feasible in a single run, which suggests applicability in routine assays.