Fast HPLC with non-porous packing materials in the pharmaceutical industry

Summary This paper demonstrates the possibilities in transfering HPLC methods from porous to non-porous stationary phases. The procedures are transferred from 125 or 250 mm columns packed with porous stationary phases to 33 mm columns packed with non-porous particles. It is demonstrated that fast HPLC using non-porous columns reduces analysis times by a factor four to eight. Precision is comparable to HPLC with porous stationary phases. The costs for HPLC with porous and with nonporous packing materials are similar. The implementation of these fast HPLC columns is easy because standard equipment can be used.     

全多孔球形硅胶基质高效液相色谱填料研究进展

结合本实验室的研究工作,评述了以全多孔球形硅胶为基质的正相反相色谱填料、手性色谱填料、离子色谱填料以及核一壳型色谱填料的研究进展．     

Correlation between the chromatographic poperties of silica phenyl packing materials and the retention behaviour of methylated β-cyclodextrins

Summary The chromatographic properties of five columns packed with phenyl-bonded phases were characterized by an approach based on Tanaka's method for column to column comparisons in order to develop an LC analysis of partially methylated β-cyclodextrins. The retention behaviour of β-cyclodextrin, heptakis(2,6-di-O-methyl)-β-cyclodextrin and heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin was examined in order to evaluate the different interactions between phenyl stationary phase and this family of compound. Chromatograms show that the residual silanol activity of the packing materials plays a beneficial role in the partially methylated β-cyclodextrins separation process. It is concluded that knowledge of the interactions involved allows one to make a reasoned choice of the stationary phase in order to obtain the best possible analysis of three different commercial samples of partially methylated β-cyclodextrins.     

Resolving power in liquid chromatography: A trade‐off between efficiency and analysis time

This review describes chromatographic dispersion and different plate‐height models frequently used to assess the chromatographic performance of ultra‐high‐pressure liquid chromatography column technology. Furthermore, different performance indices, including the resolution, the separation impedance, and kinetic plots are discussed allowing to quantify and visualize the resolving power in liquid chromatography. The construction of kinetic plots is explained, and different visualization approaches are highlighted. Finally, key instrument and column‐technology developments to advance the kinetic performance limits are discussed and selected state‐of‐the‐art applications are highlighted.     

Theoretical and experimental comparison of mobile phase consumption between ultra-high-performance liquid chromatography and high performance liquid chromatography

Ultra performance liquid chromatography (UPLC) using small particles and very high pressure has demonstrated higher resolution and speed compared with conventional HPLC. An additional benefit of UPLC is the significantly reduced consumption of mobile phase. This report discusses how column length, particle size, inner column diameter, extra column void volume, and capacity factor contribute to the reduction of mobile phase consumption in UPLC compared with HPLC. In addition, theoretical and experimental comparison of mobile phase consumption was made between isocratic HPLC and UPLC as well as between gradient HPLC and UPLC. Both theoretical and experimental results indicate that UPLC typically saves at least 80% of mobile phase in isocratic and gradient conditions when compared with HPLC.     

Evaluation of the van Deemter equation in terms of open‐ended flow to chromatography

Plate theory and adsorption theory are the main tools available for understanding chromatographic experiments. Both theories predict a Gaussian distribution of solute molecules within the tubular system. However, Gaussian concentration distributions are observed predominantly at slow linear flow rates, while asymmetric concentration distributions are observed at the linear flow rates most used in chromatography. Allegedly, this asymmetry originates from an inhomogeneous distribution of grain sizes in the column and column overload. However, it is an experimental fact that the distribution of chemicals within an injected volume of solute changes as a function of time, while the response is measured simultaneously. Accordingly, the obtained signal cannot be compared to the theory before some type of time‐based deconvolution of the data has been performed. Adjustments to high‐performance liquid chromatography data were thus proposed through empirical equations that describe the relevant time values, peak height, peak area, and parameters of the van Deemter equation. It was proposed that the transfer of solute from the front to the rear part of the pulse during laminar open‐ended flow occurs at rate that depends on the linear flow rate, and to a lesser extent, on properties of the response function.     

Kinetic performance optimisation for liquid chromatography: principles and practice

This HPLC tutorial focuses on the preparation and use of kinetic plots to characterise the performance in isocratic and gradient LC. This graphical approach allows the selection of columns (i.e. optimum particle size and column length) and LC conditions (operating pressure and temperature) to generate a specific number of plates or peak capacity in the shortest possible analysis time. Instrument aspects including the influence of extra-column effects (maximum allowable system volume) and thermal operating conditions (oven type) on performance are discussed. In addition, the performance characteristics of porous-shell particle-packed columns and monolithic stationary phases are presented and the potential of future column designs is discussed.     

Analysis of therapeutic proteins and peptides using multiangle light scattering coupled to ultra high performance liquid chromatography

Analysis of the physical properties of biotherapeutic proteins is crucial throughout all the stages of their lifecycle. Herein, we used size‐exclusion ultra high performance liquid chromatography coupled to multiangle light scattering and refractive index detection systems to determine the molar mass, mass‐average molar mass, molar‐mass dispersity and hydrodynamic radius of two monoclonal antibodies (rituximab and trastuzumab), a fusion protein (etanercept), and a synthetic copolymer (glatiramer acetate) employed as models. A customized instrument configuration was set to diminish band‐broadening effects and enhance sensitivity throughout detectors. The customized configuration showed a performance improvement with respect to the high‐performance liquid chromatography standard configuration, as observed by a 3 h column conditioning and a higher resolution analysis in 20 min. Analysis of the two monoclonal antibodies showed averaged values of 148.0 kDa for mass‐average molar mass and 5.4 nm for hydrodynamic radius, whereas for etanercept these values were 124.2 kDa and 6.9 nm, respectively. Molar‐mass dispersity was 1.000 on average for these proteins. Regarding glatiramer acetate, a molar mass range from 3 to 45 kDa and a molar‐mass dispersity of 1.304 were consistent with its intrinsic peptide diversity, and its mass‐average molar mass was 10.4 kDa. Overall, this method demonstrated an accurate determination of molar mass, overcoming the difficulties of size‐exclusion chromatography.     

An investigation of 3μm reverse-phase octadecylsilane packing materials for high-performance liquid chromatography

Summary The efficiency of high-performance liquid chromatography columns packed with 5 m and 3 m reverse-phase octadecylsilane packing materials has been evaluated using four test mixtures. Both stopped flow and valve injection have been used and typical efficiencies are given.     

A validated stability‐indicating ultra performance liquid chromatography method for the determination of potential process‐related impurities in eplerenone

A simple, sensitive, and accurate stability‐indicating analytical method has been developed and validated using ultra high performance liquid chromatography. The developed method is used to evaluate the related substances of eplerenone (EP). The degradation behavior of EP under stress conditions was determined, and the major degradants were identified by ultra high performance liquid chromatography with tandem mass spectrometry. The chromatographic conditions were optimized using an impurity‐spiked solution, and the samples, generated from forced degradation studies. The resolution of EP, its potential impurities, and its degradation products was performed on a Waters UPLC BEH C₁₈ column (50 × 2.1 mm, 1.7 μm) by linear gradient elution using a mobile phase consisting of 10 mmol/L ammonium acetate adjusted to pH 4.5, methanol and acetonitrile. A photo‐diode array detector set at 245 nm was used for detection. The flow rate was set at 0.3 mL/min. The procedure had good specificity, linearity (0.02–3.14 μg/mL), recovery (96.1–103.9%), limit of detection (0.01–0.02 μg/mL), limit of quantitation (0.03–0.05 μg/mL), and robustness. The correction factors of the process‐related substances were calculated.     

Liquid chromatography of macromolecules at the critical adsorption point. II. Role of column packing: Bare silica gel

Liquid chromatography of macromolecules at the critical adsorption point (LC CAP) presents a potentially very powerful method for molecular characterization of complex polymers. However, LC CAP applicability is limited due to various experimental problems. The pore sizes and surface chemistry of the column packings belong to the most important weak points of the method. The LC CAP behavior of poly(methyl methacrylate)s was investigated using bare silica gels of 6, 12, and 100 nm pore sizes and with various amounts of surface silanols. Tetrahydrofuran as the adsorption suppressing liquid and toluene as the adsorption promoting liquid were mixed to form the “nearly critical” eluents. Both pore size and surface chemistry of silica were found to strongly influence the retentive characteristics of the system in the critical adsorption area. Macromolecules that were large enough to be excluded from the packing pores hardly followed the LC CAP rules: their retention volumes changed irregularly with the polymer molar mass and their recovery dropped sharply. The narrow pore silica gel-packed column governed the elution patterns of the whole column set composed of silica gels with different pore sizes. This makes the conventional LC CAP characterization of common polymers with broader molar mass distribution impractical and even not feasible. A hybrid column system was proposed containing narrow pore nonadsorptive column added in series to the meso- and macroporous LC CAP silica gels. This narrow pore column would allow separation of gas, impurities, and system peaks from the polymer peaks. The possible successive changes of the surface of silica gel, e.g., due to formation of silanols by hydrolysis or due to irreversible adsorption of some admixtures from the sample or eluent may make the LC CAP irrepeatable. Pronounced peak broadening was observed in the critical adsorption area and this effect increased strongly with the polymer molar mass. © 1998 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 36: 1363–1371, 1998     

An investigation of microbore reverse-phase columns containing 3 μm packing materials

A procedure for packing 15 cm × 1 mm id reverse-phase microbore columns with 3 μm silicas obtained from different manufacturers is described. The speed of analysis and detection limits are compared to those obtained with a 50 cm × 1 mm id column packed with 10 μm ODS. The effect of detector time constant on the system, and flow rates on column efficiency are also examined.     

Synthesis of monodisperse silica microspheres and modification with diazoresin for mixed‐mode ultra high performance liquid chromatography separations

Monodisperse silica particles with average diameters of 1.9–2.9 μm were synthesized by a modified Stöber method, in which tetraethyl orthosilicate was continuously supplied to the reaction mixture containing KCl electrolyte, water, ethanol, and ammonia. The obtained silica particles were modified by self‐assembly with positively charged photosensitive diazoresin on the surface. After treatment with ultraviolet light, the ionic bonding between silica and diazoresin was converted into covalent bonding through a unique photochemistry reaction of diazoresin. Depending on the chemical structure of diazoresin and mobile phase composition, the diazoresin‐modified silica stationary phase showed different separation mechanisms, including reversed phase and hydrophilic interactions. Therefore, a variety of baseline separation of benzene analogues and organic acids was achieved by using the diazoresin‐modified silica particles as packing materials in ultra high performance liquid chromatography. According to the π–π interactional difference between carbon rings of fullerenes and benzene rings of diazoresin, C₆₀ and C₇₀ were also well separated by ultra‐high performance liquid chromatography. Because it has a small size, the ∼2.5 μm monodisperse diazoresin‐modified silica stationary phase shows ultra‐high efficiency compared with the commercial C₁₈‐silica high‐performance liquid chromatography stationary phase with average diameters of ∼5 μm.     

Ultra high performance supercritical fluid chromatography coupled with tandem mass spectrometry for screening of doping agents. I: Investigation of mobile phase and MS conditions

The conditions for the analysis of selected doping substances by UHPSFC–MS/MS were optimized to ensure suitable peak shapes and maximized MS responses. A representative mixture of 31 acidic and basic doping agents was analyzed, in both ESI+ and ESI− modes. The best compromise for all compounds in terms of MS sensitivity and chromatographic performance was obtained when adding 2% water and 10 mM ammonium formate in the CO₂/MeOH mobile phase. Beside mobile phase, the nature of the make-up solvent added for interfacing UHPSFC with MS was also evaluated. Ethanol was found to be the best candidate as it was able to compensate for the negative effect of 2% water addition in ESI− mode and provided a suitable MS response for all doping agents.     

Liquid chromatography at the critical condition: Thermodynamic significance and influence of pore size

Liquid chromatography at the critical condition (LCCC) is a high performance liquid chromatography (HPLC) technique that lies between size exclusion chromatography and adsorption-based interaction chromatography, where the elution of polymers becomes independent of polymer molecular weight. At LCCC, the balance between the entropic exclusion and the enthalpic adsorption interactions between polymers and stationary phases results in the simultaneous HPLC elution of polymers regardless of molecular weight. Using C18-bonded silica chromatographic columns with 5 μm particle size and different average pore size (diameter = 300 Å, 120 Å, 100 Å, and 50 Å), we report (1) the thermodynamic significance of LCCC conditions and (2) the influence of column pore size on the determination of critical conditions for linear polymer chains. Specifically, we used mixtures of monodisperse polystyrene samples ranging in molecular weight from 162 to 371,100 g/mol and controlled the temperature of the HPLC columns at a fixed composition of a mobile phase consisting of 57(v/v)% methylene chloride and 43(v/v)% acetonitrile. It was found that, at the fixed mobile phase composition, the temperature of LCCC (T_LCCC) is higher for C18-bonded chromatographic columns with larger average pore size. © 2009 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 47: 2533–2540, 2009     

Preparation of HPLC chiral packing materials using cellulose tris(4-methylbenzoate) for the separation of chrysanthemate isomers

We investigated the separation of chrysanthemate isomers ( 1 ), particularly the (1R)-trans form, by high-performance liquid chromatography (HPLC) using polysaccharide derivatives, such as the phenylcarbamates and benzoates of cellulose and amylose, as the chiral stationary phases (CSPs). The chiral packing materials (CPMs) having a high chiral recognition for the chrysanthemic acid ethyl ester ( 1a ) were prepared by coating cellulose tris(4-methylbenzoate) ( 2a ) dissolved in solvents containing methyl benzoate or acetophenone as an additive on silica gel. The separation factor for 1a significantly depended on the preparation conditions of CPM 2a , such as the coating amount of 2a and the type and amount of additives. The chiral recognition ability created by imprinting the additives was lost when the CPM was heated at a high temperature, and was recovered by contacting it with the additive in a packed column. The structural change in 2a during these treatments was not clearly detected by spectroscopic methods. © 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 5087–5097, 2006     

Ultra high performance supercritical fluid chromatography coupled with tandem mass spectrometry for screening of doping agents. II: Analysis of biological samples

The potential and applicability of UHPSFC–MS/MS for anti-doping screening in urine samples were tested for the first time. For this purpose, a group of 110 doping agents with diverse physicochemical properties was analyzed using two separation techniques, namely UHPLC–MS/MS and UHPSFC–MS/MS in both ESI+ and ESI− modes. The two approaches were compared in terms of selectivity, sensitivity, linearity and matrix effects. As expected, very diverse retentions and selectivities were obtained in UHPLC and UHPSFC, proving a good complementarity of these analytical strategies. In both conditions, acceptable peak shapes and MS detection capabilities were obtained within 7 min analysis time, enabling the application of these two methods for screening purposes. Method sensitivity was found comparable for 46% of tested compounds, while higher sensitivity was observed for 21% of tested compounds in UHPLC–MS/MS and for 32% in UHPSFC–MS/MS. The latter demonstrated a lower susceptibility to matrix effects, which were mostly observed as signal suppression. In the case of UHPLC–MS/MS, more serious matrix effects were observed, leading typically to signal enhancement and the matrix effect was also concentration dependent, i.e., more significant matrix effects occurred at the lowest concentrations.