A simplex optimization of the experimental parameters in preparative liquid chromatography

Summary A study of the optimization of the experimental conditions for the purification or extraction of pure compounds by liquid chromatography is presented. Optimum values of the parameters of overloaded elution are derived for maximum production rate, using a Simplex algorithm and the procedure previously described for the simulation of the elution profiles of binary mixtures. The mobile phase flow velocity and the sample size have been optimized together in a first step, simulating the procedure followed in practice, when a column is available. In a second part, the influence of the column length and the average particle size of the packing material on the column performance as well as the trade-offs between the production rate and the yield are discussed.There are three major conclusions in this work. First, the optimum experimental conditions are often very different, depending whether one is primarily interested in the first or in the second eluted component of a mixture. Second, the column efficiency under analytical conditions is very important: it is traded-off for high flow rates, hence short cycle time and increased production rate. Third, the production rate depends strongly on the maximum pressure at which the equipment can be operated. Finally, the optimum production rate varies rather smoothly with the mobile phase velocity and the sample size, so a high yield (70% or more) can usually be obtained with a limited loss in production rate (30 to 60%).     

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.     

Variations of particle size and bed voidage distributions in expanded bed during transient operation processes

Changes in bed expansion are frequently encountered during an expanded bed adsorption, such as during the initial bed expansion, feed loading and washing processes. We have here studied the changes of local particle size distribution and bed voidage of an expanded bed in the initial bed expansion process as well as those during the changes in mobile phase viscosity, which imitated feed loading and column washing processes. Using a glass column modified with three side sampling ports and Streamline AC as the solid phase, experimental measurements on a series of operation moments during the transient processes were carried out by sampling the particles from within the column at different axial positions. In the initial bed expansion process, the gradual formation of an axial classification from a settled bed to a stable expanded bed was first displayed. By changing the mobile phase from water to 10% (w/w) glycerol solution or vice versa, the variations in both the particle size distribution and bed voidage corresponding to the increase or decrease of the bed height caused by the changes of the mobile phase viscosity were examined as well. The transient changes of the local particle size distribution and bed voidage first occurred in the bed bottom and then progressed from bottom to top along the axial direction. However, the changes of bed voidage at different axial positions were not unidirectional. That is, by changing the mobile phase to the high-viscosity glycerol solution, a constant increase of the bed voidage was observed in the bed bottom, while a distinct decrease of the bed voidage before its increase was involved at the middle and top positions. This is ascribed to the compression effect caused by the upward movement of the lower part particles.     

Resolution concept for separation of curium from californium by ion-exchange chromatography

Different factors affecting separation of the tervalent actinides curium and californium by ion-exchange chromatography are discussed in the light of the resolution factor. The fact that resolution is inversely proportional to resin particle diameter and directly proportional to column length has been confirmed experimentally. The effects influencing resolution due to changes in flow-rate of the mobile phase used are dependent on resin particle diameter. Changes in temperature affect a number of variables influencing resolution.     

Nano and rapid resolution liquid chromatography–electrospray ionization–time of flight mass spectrometry to identify and quantify phenolic compounds in olive oil

The applicability of nanoLC‐ESI‐TOF MS for the analysis of phenolic compounds in olive oil was studied and compared with a HPLC method. After the injection, the compounds were focused on a short capillary trapping column (100 μm id, effective length 20 mm, 5 μm particle size) and then nanoLC analysis was carried out in a fused silica capillary column (75 μm id, effective length 10 μm, 3 μm particle size) packed with C18 stationary phase. The mobile phase was a mixture of water + 0.5% acetic acid and ACN eluting at 300 nL/min in a gradient mode. Phenolic compounds from different families were identified and quantified. The quality parameters of the nanoLC method (linearity, LODs and LOQs, repeatability) were evaluated and compared with those obtained with HPLC. The new methodology presents better sensitivity (reaching LOD values below 1 ppb) with less consumption of mobile phases, but worse repeatability, especially inter‐day repeatability, resulting in more difficulties to get highly accurate quantification. The results described in this article open up the application fields of this technique to cover a larger variety of compounds and its advantages will make it especially useful for the analysis of samples containing low concentration of phenolic compounds, as for instance, in biological samples.     

Ultra high-performance liquid chromatography of porphyrins in clinical materials: column and mobile phase selection and optimisation

Ultra high-performance liquid chromatographic (UHPLC) systems on columns packed with materials ranging from 1.9 to 2.7 μm average particle size were assessed for the fast and sensitive analysis of porphyrins in clinical materials. The fastest separation was achieved on an Agilent Poroshell C(18) column (2.7 μm particle size, 50 × 4.6 mm i.d.), followed by a Thermo Hypersil Gold C(18) column (1.9 μm particle size, 50 × 2.1 mm i.d.) and the Thermo Hypersil BDS C(18) column (2.4 μm particle size, 100 × 2.1 mm i.d.). All columns required a mobile phase containing 1 m ammonium acetate buffer, pH 5.16, with a mixture of acetonitrile and methanol as the organic modifiers for optimum resolution of the type I and III isomers, particularly for uroporphyrin I and III isomers. All UHPLC columns were suitable and superior to conventional HPLC columns packed with 5 μm average particle size materials for clinical sample analysis.     

Microbore liquid chromatographic determination of cadralazine and cephalexin in plasma with large-volume injection

The application of microbore systems (15 cm X 1 mm I.D. columns filled with Nucleosil C18, 5 microns particle size) to the determination of cephalexin and cadralazine in plasma was investigated. Factors such as mobile phase flow-rate, detector flow-cell volume and injection volume were examined with regard to the needs of routine drug analysis. Mobile phase flow-rates of 50-60 microliters/min were used. A flow cell with an optical path length of 6 mm and an intermediary volume (2.4 microliters) was selected for UV detection in order to obtain sufficient sensitivity. Large volumes of non-eluting solvent containing the drug were injected on the column. The addition of an ion-pairing reagent to samples containing cephalexin and cefroxadin prior to the injection was found to improve the chromatographic performance. The blood sample size required for analysis with microbore columns was smaller than that with conventional columns. The analysis time was similar and the limit of quantitation was also similar, provided that large sample volumes were injected on the microbore column.     

Serum Injection of the HPLC Column for Carbamazepine Assay

Abstract

Serum was injected directly on an HPLC column packed with C₁, 6.5 μm particle size, 300 Å pore-packing material for carbamazepine determination. The use of a wide-pore column with low hydrophobicity eliminated excessive pressure buildup in the column from protein precipitation which is usually caused by the high concentration of organic solvents in the mobile phase. This simple approach can be utilized for the determination of other drugs and endogenous compounds in serum.     

Controlling selectivity of polymer‐based monolithic stationary phases

In this work, we aimed to prepare a monolithic capillary column that allowed an isocratic separation of ten dopamine precursors and metabolites in a single run. Segments of five zwitterion sulfobetaine polymer monoliths have been modified by zwitterion phoshorylcholine by using an ultraviolet‐initiated two‐step photografting. Columns with 0, 33, 50, 66, and 100% of modified length were prepared. Effect of length of the modified segment and mobile phase composition has been tested. All columns provided dual‐retention mechanism with reversed‐phase retention in highly aqueous mobile phase and hydrophilic interaction mechanism in highly organic mobile phase. The retention mechanism was controlled by the composition of the mobile phase and has been described by a three‐parameter model. We have used regression parameters to characterize the retention of analyzed compounds and to study individual pathways of dopamine metabolism. Comprehensive optimization of mobile phase composition allowed to find an optimal composition of the mobile phase and stationary phase surface chemistry arrangement to achieve desired separation. Optimized columns provided an isocratic separation of all tested compounds in less than nine min.     

Comparison of different types of stationary phases for the analysis of soy isoflavones by HPLC

Nowadays, there are new technologies in high-performance liquid chromatography columns available enabling faster and more efficient separations. In this work, we compared three different types of columns for the analysis of main soy isoflavones. The evaluated columns were a conventional reverse phase particle column, a fused-core particle column, and a monolithic column. The comparison was in terms of chromatographic parameters such as resolution, asymmetry, number of theoretical plates, variability of retention time, and peak width. The lower column pressure was provided by the monolithic column, although lower chromatographic performance was achieved. Conventional and fused-core particle columns presented similar pressure. Results also indicate that direct transfer between particle and monolithic columns is not possible requiring adjustment of conditions and a different method optimization strategy. The best chromatographic performance and separation speed were observed for the fused-core particle column. Also, the effect of sample solvent on the separation and peak shape was evaluated and indicated that monolithic column is the most affected especially when using higher concentrations of acetonitrile or ethanol. Sample solvent that showed the lowest effect on the chromatographic performance of the columns was methanol. Overall evaluation of methanol and acetonitrile as mobile phase for the separation of isoflavones indicated higher chromatographic performance of acetonitrile, although methanol may be an attractive alternative. Using acetonitrile as mobile phase resulted in faster, higher resolution, narrower, and more symmetric peaks than methanol with all columns. It also generated the lower column pressure and flatter pressure profile due to mobile phase changes, and therefore, it presents a higher potential to be explored for the development of faster separation methods.     

Siliceous mesocellular foam for high-performance liquid chromatography: Effect of morphology and pore structure

Spherical siliceous mesocellular foam (MCF) particles with an average particle size of 4.8 μm have been successfully prepared. These spherical particles were tailored in pore sizes and surface areas. They were functionalized with C₈ or C₁₈ groups, and applied towards reversed phase high-performance liquid chromatography (HPLC) column separations. Their high surface areas gave rise to very good retention characteristics, as illustrated in the separation of a series of alkylbenzene solutes with increasing chain length. The highly interconnected porous structure and ultralarge pore size of MCF allowed the columns to be used at high flow rates without much loss in column efficiency. The column efficiency and peak symmetry were further improved by eliminating the micropores of the stationary phase. The reversed phase column packed with C₁₈-modified spherical MCF particles provided for excellent separation of different deoxynucleosides, illustrating the broad applicability of these materials due to their controlled pore size.     

Ion-pair reversed-phase high-performance liquid chromatography analysis of oligonucleotides: retention prediction

An ion-pair reversed-phase HPLC method was evaluated for the separation of synthetic oligonucleotides. Mass transfer in the stationary phase was found to be a major factor contributing to peak broadening on porous C18 stationary phases. A small sorbent particle size (2.5 microm), elevated temperature and a relatively slow flow-rate were utilized to enhance mass transfer. A short 50 mm column allows for an efficient separation up to 30mer oligonucleotides. The separation strategy consists of a shallow linear gradient of organic modifier, optimal initial gradient strength, and the use of an ion-pairing buffer. The triethylammonium acetate ion-pairing mobile phases have been traditionally used for oligonucleotide separations with good result. However, the oligonucleotide retention is affected by its nucleotide composition. We developed a mathematical model for the prediction of oligonucleotide retention from sequence and length. We used the model successfully to select the optimal initial gradient strength for fast HPLC purification of synthetic oligonucleotides. We also utilized ion-pairing mobile phases comprised of triethylamine (TEA) buffered by hexafluoroisopropanol (HFIP). The TEA-HFIP aqueous buffers are useful for a highly efficient and less sequence-dependent separation of heterooligonucleotides.     

Comprehensive two‐dimensional monolithic liquid chromatography of polar compounds

Two‐dimensional liquid chromatography largely increases the number of separated compounds in a single run, theoretically up to the product of the peaks separated in each dimension on the columns with different selectivities. On‐line coupling of a reversed‐phase column with an aqueous normal‐phase (hydrophilic interaction liquid chromatography) column yields orthogonal systems with high peak capacities. Fast on‐line two‐dimensional liquid chromatography needs a capillary or micro‐bore column providing low‐volume effluent fractions transferred to a short efficient second‐dimension column for separation at a high mobile phase flow rate. We prepared polymethacrylate zwitterionic monolithic micro‐columns in fused silica capillaries with structurally different dimethacrylate cross‐linkers. The columns provide dual retention mechanism (hydrophilic interaction and reversed‐phase). Setting the mobile phase composition allows adjusting the separation selectivity for various polar substance classes. Coupling on‐line an organic polymer monolithic capillary column in the first dimension with a short silica‐based monolithic column in the second dimension provides two‐dimensional liquid chromatography systems with high peak capacities. The silica monolithic C₁₈ columns provide higher separation efficiency than the particle‐packed columns at the flow rates as high as 5 mL/min used in the second dimension. Decreasing the diameter of the silica monolithic columns allows using a higher flow rate at the maximum operation pressure and lower fraction volumes transferred from the first, hydrophilic interaction dimension, into the second, reversed‐phase mode, avoiding the mobile phase compatibility issues, improving the resolution, increasing the peak capacity, and the peak production rate.     

10°锥角台锥型液相色谱柱尺寸等比例放大和柱内谱带流型的可视化研究

摘要利用改进的可视化装置, 研究了10°锥角的台锥型液相色谱柱内的谱带流型与柱参数变化的关系. 将有机玻璃柱管加工成内圆台外方型的一体结构, 选择折射率一致的色谱固定相硅胶和流动相环己烷, 使整个色谱柱成为高度清晰的透明体, 能直接观察柱中彩色样品谱带的动态三维流型. 研究结果表明, 在实验条件范围内, 流动相流速对谱带流型无影响, 填料的形状和性质对塞子状流型有一定程度的影响. 比较了柱长为5cm和等比例放大后柱长为10cm锥型柱内的流型, 发现放大后的锥型柱内仍然保持塞子状流型, 总柱效等比例增加. 表明继续按比例放大成为工业规模色谱柱后仍能保持塞子状流型.     

Effect of particle size in preparative reversed-phase high-performance liquid chromatography on the isolation of epigallocatechin gallate from Korean green tea

To isolate epigallocatechin gallate (EGCG) of catechin compounds from Korean green tea (Bosung, Chonnam), a C18 reversed-phase preparative column (250x22 mm) packed with packings of three different sizes (15, 40-63, and 150 microm) was used. The sample extracted with water was partitioned with chloroform and ethyl acetate to remove the impurities including caffeine. The mobile phases in this experiment were composed of 0.1% acetic acid in water, acetonitrile, methanol and ethyl acetate. The injection volume was fixed at 400 microl and the flow rate was increased as the particle size becomes larger. The isolation of EGCG with particle size was compared at a preparative scale and the feasibility of separation of EGCG at larger particle sizes was confirmed. The optimum mobile phase composition for separating EGCG was experimentally obtained at the particle sizes of 15 and 40-63 microm in the isocratic mode, but EGCG was not purely separated at the particle size of 150 microm.     

High-speed gel-permeation chromatography

To establish optimum operating conditions for high-speed gel-permeation chromatography (GPC), the effects of column packing particle size, solvent flow rate, and column length on the separation efficiency have been investigated by using monodisperse polystyrene samples and polystyrene gel columns (TSK-GEL column, Type-H). Decreasing the particle size of the column packing reduces the time required to obtain a given resolution. Monodisperse polystyrene standards were measured under the optimum operating conditions established (gel particle size 5 μ, column length 2 ft, flow rate 2.5 ml/min). The molecular weight distribution of a polymer mixture was determined in less than 10 min with the same accuracy as by the conventional GPC. Such short analysis time enables one to use GPC for in-plant quality control.     

Simultaneous detection of cholyl adenylate and coenzyme A thioester utilizing liquid chromatography/electrospray ionization mass spectrometry.

It has been proposed that acyl adenylate is first formed during activation of the carboxy group into the acyl CoA thioester, an intermediate in the formation of amino acid conjugates. Acyl CoA synthetases may be responsible for this acyl adenylate formation. Recently, we hypothesized the preferential formation of cholic acid adenylate, a major bile acid, preceding production of the corresponding CoA thioester in incubations with rat liver microsomal fractions. To verify this biosynthetic mechanism, monitoring of the incubation mixture of acyl adenylate together with both substrate and acyl CoA thioester is needed. We have developed a detection method for the simultaneous detection of these cholic acid derivatives utilizing liquid chromatography/electrospray ionization mass spectrometry. The CoA thioester of cholic acid forms a chelation complex with the divalent cations remaining on the silica gel packed into the analytical column. Both the addition of a chelating agent, such as EDTA, to the mobile phase and an adjustment of the mobile phase pH to a weak alkaline effectively removed such chelate formation, producing a sharp CoA thioester peak. For a simultaneous mass spectrometric analysis of cholic acid, the corresponding adenylate and CoA thioester, the combined use of a 300 A particle diameter ODS column and 20 mM ammonium acetate buffer (pH 9.0)/2-propanol/acetonitrile as the mobile phase have been proved to be preferable. To avoid any degradation of the chemically unstable adenylate produced in the incubation, we employed a direct injection of the sample onto a preconcentration column. The obtained results indicated a high sensitivity of this method.     

Quantitative analysis of specific target DNA oligomers using a DNA-immobilized packed-column system

Although a DNA-immobilized packed-column (DNA-packed column), which relies on sequence-dependent interactions of target DNA or mRNA (in the mobile phase) with DNA probes (on the silica particle) in a continuous flow process, could be considered as an alternative platform for quantitative analysis of specific DNA to DNA chip methodology, the performance in practice has not been satisfactory. In this study, we set up a more efficient quantitative analysis system based on a DNA-packed column by employing a temperature-gradient strategy and DMSO-containing mobile phase. Using a temperature-gradient strategy based on T _m values of probe/target DNA hybridizations and DMSO (5%)-containing mobile phase, we succeeded in the quantitative analysis of a specific complementary target distinguishable from non-complementary DNA oligomers or other similar DNA samples. In addition, two different target DNA oligomers even with similar T _m values were separated and detected quantitatively by using a packed column carrying two different DNA probes.     

一种新型C18酯型反相高效液相色谱填料的制备

 发展了一种用于制备反相高效液相色谱填料的实用、经 济的方法。以γ-环氧丙基氧丙基三甲氧基硅烷为中间偶联试剂,将十八烷基脂肪酸 键合至硅胶上。元素分析、色谱测试验证了该工艺的可行性和填料的反相色谱行为。