Optimisation and characterisation of silica-based reversed-phase liquid chromatographic systems for the analysis of basic pharmaceuticals

Reversed-phase liquid chromatography using silica-based columns is successfully applied in many separations. However, also some drawbacks exist, i.e. the analysis of basic compounds is often hampered by ionic interaction of the basic analytes with residual silanols present on the silica surface, which results in asymmetrical peaks and irreproducible retention. In this review, options to optimise the LC analysis of basic pharmaceutical compounds are discussed, i.e. eluent optimisation (pH, silanol blockers) and stationary phase optimisation (development of new columns with minimised ionic interactions). The applicability of empirical based, thermodynamically based and test methods based on a retention model to characterise silica-based reversed phase stationary phases, as well as the influence of the eluent composition on the LC analysis of basic substances is described. Finally, the applicability of chemometrical techniques in column classification is shown.     

色谱指纹图谱组合相似度的算法

色谱指纹图谱相似度是评价中药质量稳定性的有效手段之一,其中向量夹角余弦相似度应用最为广泛。但是当色谱峰面积分布范围宽时,夹角余弦对数据的差异响应不灵敏。以土茯苓色谱指纹图谱数据为实例证实了当两样本数据间比例相差较大时,夹角余弦相似度对共有峰和非共有峰响应的灵敏程度相差很大。组合相似度在共有峰的夹角余弦相似度基础上纳入了非共有峰的影响,分别计算非共有峰和共有峰对相似度的贡献,并以最大峰比例同态性为指标,确定非共有峰的合适权重。基于样本峰面积数据建立的组合相似度模型能灵敏地反映待测样本与参照样本内在成分的峰比例变化,适合衡量多个药材样本间的相似程度,已成为评价药材质量相似度的新方法。     

Estimation of chromatographic parameters on two silica-based columns connected in series under nonaqueous reversed phase liquid chromatographic conditions

Empirical equations were produced to relate important chromatographic parameters on two silica-based columns serially linked, in isocratic nonaqueous RP HPLC, to retention times and peak widths of the separated compounds on the individual columns. These equations were derived because the experimental data seemed to deviate from the values expected, applying basic chromatographic theoretical equations. The chromatographic parameters studied were retention time, peak width, resolution, number of theoretical plates, capacity factor, and separation factor. In addition, empirical linear relationships were produced for the estimation of the above mentioned parameters of the serial systems, in direct and reverse order, relating them to those obtained on each column, separately. The experimentally obtained values were in good agreement with those estimated by the derived equations.     

Comparison of silica-based and polymer-based cation exchangers for the ion chromatographic separation of transition metals

Silica gel- and polymer-based exchangers differ not only in the substrate but usually also in the structure of their sulphonic acid exchange group. The performance and chromatographic behaviour of modern macroporous poly(styrene-divinylbenzene) polymers of 5-μm particle size after surface sulphonation were examined. Further, two commercially available silica gel cation exchangers were investigated as references for what is now possible and how the chromatographic performance is influenced by the substrate and the structure of the exchange site. The influence of the capacity of surface-sulphonated exchangers for acid and complexing eluents, which are necessary when transition metals are to be separated, was studied. The interaction of polarizable metal cations with the π-system of the polymer depends on the resin capacity, and a comparable dependence for H⁺ and complexing eluents was found. The distance of the functional group from the core influences the adsorption effects dramatically. Lowering the non-specific interaction increases the chromatographic efficiency rapidly. The performance of silica gel-based exchangers is nearly one order of magnitude better than that of surface-sulphonated exchangers. The selectivity of the exchangers investigated is strongly dependent on the structure of the exchange site and on the resin capacity.     

Medical applications of organic-inorganic hybrid materials within the field of silica-based bioceramics

Research on bioceramics has evolved from the use of inert materials for mere substitution of living tissues towards the development of third-generation bioceramics aimed at inducing bone tissue regeneration. Within this context hybrid bioceramics have remarkable features resulting from the synergistic combination of both inorganic and organic components that make them suitable for a wide range of medical applications. Certain bioceramics, such as ordered mesoporous silicas, can exhibit different kind of interaction with organic molecules to develop different functions. The weak interaction of these host matrixes with drug molecules confined in the mesoporous channels allows these hybrid systems to be used as controlled delivery devices. Moreover, mesoporous silicas can be used to fabricate three (3D)-dimensional scaffolds for bone tissue engineering. In this last case, different osteoinductive agents (peptides, hormones and growth factors) can be strongly grafted to the bioceramic matrix to act as attracting signals for bone cells to promote bone regeneration process. Finally, recent research examples of organic-inorganic hybrid bioceramics, such as stimuli-responsive drug delivery systems and nanosystems for targeting of cancer cells and gene transfection, are also tackled in this tutorial review (64 references).     

Estimation of silanol groups in silica-based packings for liquid chromatography from gas chromatographic retention of cyclohexane and benzene

Summary Gas chromatographic retention of cyclohexane and benzene on microcolumns packed with silica-based materials for liquid chromatography has been investigated to elucidate the relationship with the surface properties of the packing materials. Differences in capacity factors (k) for these two hydrocarbons were related to the surface area of the packing materials. For untreated silica gels, the k value increased with increasing total surface area of the column employed and measurement of k for chemically-bonded stationary phases allowed estimation of accessible residual silanol groups.     

New approach on similarity analysis of chromatographic fingerprint of herbal medicine

A new approach to the construction and similarity analysis of chromatographic fingerprint for herbal medicine is presented in this paper. Samples of chuanxiong, a herbal medicine for headache, from three producing areas of China were used to evaluate the utility of this study. The samples were analyzed with high-performance liquid chromatography (HPLC) and the peak areas of the chromatograms were used to construct the fingerprints of the herbal medicines. A vector of differences was defined between the two fingerprints. The scalar mean of the difference vector was taken as a statistic and both the t-test and Bayesian hypothesis testing were implemented to provide a one-to-one comparison of the fingerprints. Compared with principal component analysis (PCA), correlation coefficient and vector cosine, the new method offers a better differentiation of the similarity or difference between the fingerprints from same sample of chuanxiong. When the new method was used in the similarity analysis of the fingerprints of chuanxiong from different production areas, a clear-cut signature was obtained that reveals the significant difference between them.     

Overloading control of gas chromatographic systems

Increasing the interlaboratory reproducibility of gas chromatographic retention indices requires avoiding measurements distorted by overloading effects. Several criteria of evaluating the limits of the mass overloading of gas chromatographic systems are compared and reconsidered. The criteria mostly appropriate for practical purposes are based on (i) the dependences of factors of peak broadening (ratio of peak height and its width) vs. amount of analyte injected into the chromatographic column and (ii) the dependence of parameters characterizing the peak distortion (asymmetry factor) vs. the amount of analyte. Both these criteria provide mutually comparable evaluations of the overloading limits for analytes of different polarity. At the same time, the dependence of retention indices vs. amounts of analyte injected in the chromatographic column cannot be recommended for overloading control, because the parameters of the corresponding linear regressions indicate temperature dependence. The interpretation of certain gas chromatographic anomalies requires the correct evaluation of overloading limits. For example, the unusual temperature dependence of retention indices of polar analytes on non‐polar stationary phases and the dependence of retention indices on ratio of amounts of target analytes and reference compounds.     

Non-ideal behaviour of silica-based stationary phases in trifluoroacetic acid-acetonitrile-based reversed-phase high-performance liquid chromatographic separations of insulins and proinsulins.

Several C18 stationary phases were found to behave non-ideally when insulins and proinsulins were eluted with shallow acetonitrile gradients in 0.1% trifluoroacetic acid, resulting in poor peak shapes or no elution at all. With triethylammonium phosphate or ammonium sulphate as buffer components, the insulins and proinsulins were eluted with excellent peak shapes, presumably owing to better masking of residual silanol groups on the stationary phases. Similar use of trifluoroacetic acid-acetonitrile gradients on the less hydrophobic C4 or C3 stationary phases resulted in excellent peak shapes. The difficult separation of rat proinsulin I and II, which are important for the study of rat insulin biosynthesis, was only achieved with two different stationary-mobile phase combinations.     

Reversible and irreversible denaturation of proteins in chromatographic systems

Protein denaturation in typical chromatographic mobile phases is examined from kinetic and equilibrium viewpoints, preliminary to consideration of the binding of proteins to stationary phases. Protein binding to a stationary phase may result in concomitant stabilization or destabilization. Experiments to clarify mechanisms and/or to lead to stabilization of proteins against denaturation are discussed. Among the work to be discussed are the ion exchange chromatography of α-chymotrypsinogen and hen egg lysozyme with mobile phases containing urea; reverse phase HPLC of RNase A and serum albumin with conventional mobile phases; and hydrophobic interaction chromatography of enolase, carbonic anhydrase, lysozyme, RNase A, and bovine pancreatic trypsin inhibitor, mediated by the surfactant CHAPS.     

Characterization and modeling of nonlinear hydrophobic interaction chromatographic systems

A general rate model was employed in concert with a preferential interaction quadratic adsorption isotherm for the characterization of HIC resins and the prediction of solute behavior in these separation systems. The results indicate that both pore and surface diffusion play an important role in protein transport in HIC resins. The simulated and experimental solute profiles were compared for two model proteins, lysozyme and lectin, for both displacement and gradient modes of chromatography. Our results indicate that a modeling approach using the generate rate model and preferential interaction isotherm can accurately predict the shock layer response in both gradient and displacement chromatography in HIC systems. While pore and surface diffusion played a major role and were limiting steps for proteins, surface diffusion was seen to play less of a role for the displacer. The results demonstrate that this modeling approach can be employed to describe the behavior of these non-linear HIC systems, which may have implications for the development of more efficient preparative HIC separations.     

A high-throughput three-capillary noise-cancelling viscometer for chromatographic systems

This paper describes an analytical three-capillary viscometer detector that eliminates the traditional viscometer ??hold-up?? volume (commonly found in four-capillary designs) while maintaining cancellation of short-term pump noise and long-term baseline drift of temperature and solvent flow rate that are inherent in chromatography systems. This improvement allows a staggered sample injection approach in chromatography, yielding a significant increase in sample throughput by cutting down the chromatographic run time. It also provides a more robust design as it does not require capillary rebalancing, complex purging, flushing or changing the hold-up volume to accommodate long-term chromatographic use.

General theory of similarity measures for library search systems

A general theory of similarity measures for library search systems is presented. It shows that once the spectral features to be used in the comparison of two spectra have been selected and their relative importance has been fixed, the characteristics of the similarity measure are fully controlled by two independent parameters. These two parameters control whether a forward or a reverse search and whether an identity, a similarity, or a classification search is conducted.     

Gradient elution in normal-phase high-performance liquid chromatographic systems

Gradient elution is widely used for separation of complex samples in reversed-phase HPLC systems, but is less frequently applied in normal-phase HPLC, where it has a notoriously bad reputation for poor reproducibility and unpredictable retention. This behaviour is caused by preferential adsorption of polar solvents used in mixed mobile phases, which may cause significant deviations of the actual gradient profile from the pre-set program. Another important source of irreproducible retention behaviour is gradual deactivation of the adsorbent by adsorption of even traces of water during normal-phase gradient elution. To avoid this phenomenon, carefully dried solvents should be used. Finally, column temperature should be carefully controlled during normal-phase gradient elution if reproducible results are to be obtained. Working with dry solvents at a controlled constant temperature and using a sophisticated gradient-elution chromatograph, reproducibility of the retention data in normal-phase gradient elution better than 2% may be achieved even over several months of column use. The retention data in gradient elution can be calculated accurately if appropriate corrections are adopted for the gradient dwell volume and for the preferential adsorption of the polar solvents using experimental adsorption isotherms. The average error of prediction for the corrected calculated gradient retention data was lower than 2% for a silica gel column and lower than 3% for a bonded nitrile column, which may be suitable for the optimization of separation. Further, a simple approach is suggested for rapid estimation of changes in the retention induced by a change in the gradient profile in normal-phase HPLC. For such a rough estimation, it is not necessary to know the parameters of the dependence of the solute retention factors on the composition of the mobile phase.     

Separation of mixed halocarbons of environmental interest on a new type of silica-based porous-layer open tubular capillary gas chromatographic column

A new type of capillary porous-layer open tubular (PLOT) column consisting of a hydrophobic silica layer on a fused-silica capillary has been tested for the separation of a mixture of environmentally sensitive halocarbons present in tropospheric air. The column shows high retention for a wide range of halocarbons, with elution orders following both boiling point order and hydrogen bonding capability. The resolution of the halocarbons is good and only one pair of halocarbons [CHFClCF₃ (HCFC 124) and CH₃CF₂Cl (HCFC 142b)] cannot be resolved on this column type at all column temperature profiles. Unlike alumina PLOT columns, the silica PLOT column does not dehydrohalogenate labile halocarbons. Excellent reproducibility of retention times and peak areas for halocarbons on the column are reported.     

Application of self-organizing maps for the classification of chromatographic systems and prediction of values of chromatographic quantities

The separation of a complex mixture of inorganic and organic anions by ion chromatography–capillary electrophoresis using a cationic polymer added to the background electrolyte and indirect UV detection has been studied. The addition of unmodified polymer to an electrolyte suitable for indirect detection resulted in the appearance of a system peak due to the counter-anion on the polymer and while the position of the analytes relative to this system peak could be changed, this was found to be an unacceptable approach for mixtures of large numbers of analytes. Although conversion of the polymer to replace the counter-ion with the indirect UV detection probe ion simplified the system, this approach restricted the flexibility of the system because the probe and polymer concentration were necessarily linked. This limitation could be overcome by selecting the appropriate type of probe ion, with probes having a low ion-exchange selectivity coefficient providing greater retention of analytes than probes with a high ion-exchange selectivity coefficient. Three electrolyte systems with different probes (benzoate, chromate and phthalate) were modelled using a previously derived migration equation and this was used to optimise the electrolyte composition to enable the separation of a mixture of 24 inorganic and organic anions within 7 min. The electrolyte composition was then optimised for the analysis of anions in Bayer liquor with the final separation selectivity being substantially improved for selected key analytes.     

Relating chromatographic retention and electrophoretic mobility to the ion distribution within electrosprayed droplets

Ions that are observed in a mass spectrum obtained with electrospray mass spectrometry can be assumed to originate preferentially from ions that have a high distribution to the surface of the charged droplets. In this study, a relation between chromatographic retention and electrophoretic mobility to the ion distribution (derived from measured signal intensities in mass spectra and electrospray current) within electrosprayed droplets for a series of tetraalkylammonium ions, ranging from tetramethyl to tetrapentyl, is presented. Chromatographic retention in a reversed-phase system was taken as a measure of the analyte’s surface activity, which was found to have a large influence on the ion distribution within electrosprayed droplets. In addition, different transport mechanisms such as electrophoretic migration and diffusion can influence the surface partitioning coefficient. The viscosity of the solvent system is affected by the methanol content and will influence both diffusion and ion mobility. However, as diffusion and ion mobility are proportional to each other, we have, in this study, chosen to focus on the ion mobility parameter. It was found that the influence of ion mobility relative to surface activity on the droplet surface partitioning of analyte ions decreases with increasing methanol content. This effect is most probably coupled to the decrease in droplet size caused by the decreased surface tension at increasing methanol content. The same observation was made upon increasing the ionic strength of the solvent system, which is also known to give rise to a decreased initial droplet size. The observed effect of ionic strength on the droplet surface partitioning of analyte ions could also be explained by the fact that at higher ionic strength, a larger number of ions are initially closer to the droplet surface and, thus, the contribution of ionic transport from the bulk liquid to the liquid/air surface interface (jet and droplet surface), attributable to migration or diffusion will decrease.