A Simple and Efficient Approach for Estimating Recovery of a Preparative Reversed Phase HPLC Purification

The widely applied reversed phase high-performance liquid chromatography (RP-HPLC) is an indispensable purification technique in drug discovery. During drug discovery, recovery was usually calculated based on the weight of the purified product after drying over the weight of the crude material multiplied by the assumed purity from HPLC/UV area percent of the product. Such a purity assumption can be off significantly when the crude material contains water, solvents, other UV-inactive impurities and inorganic salts. In this paper, we report a simple and efficient way to estimate recovery of preparative HPLC purification process. It is based on the ratio of the HPLC/UV peak area measured for the product in the crude solution and that in the final collected fraction with both accounted for their volumes. This approach eliminates not only the need for drying of the collected fraction to calculate recovery but also the inaccuracy associated with the true content in the crude sample using the traditional method. A systematic study was conducted to verify this method using caffeine mixed with various UV-active and -inactive impurities. The calculated recoveries using this approach were found to be consistent within 4% with the true recoveries based on dry weight estimation. The approach has been successfully applied for our in-house purifications. Furthermore, the approach was extended to library purifications, where in many cases heart-cutting the desired peaks is used to meet the purity requirements.

     

反相液相色谱对多肽的分离、纯化与制备

用反相高效液相色谱（RPLC）对两种化学合成多肽－－32肽和21肽进行了分离、纯化和制备。在用分析型RPLC色谱柱对多肽样品的制备过程中，对其进样量和洗脱梯度进行了选择。每次进样量为5mg，在最优化色谱条件下，用RPLC一步就可对21肽进行分离纯化，其纯度达到98.6%。而32肽由于样品组分更加复杂，RPLC一步纯化后其纯度仅有80％。通过对色谱分离条件的再次优化，对32肽进行二次分离纯化，纯度达杂，RPLC一步纯化后其纯度仅有80％。通过对色谱分离条件的再次优化，对32肽进行二次分离纯化，纯度达到96.4%。在其最优化条件下，通过多次样品收集和冷冻干燥，分别制备了高纯度的21肽和32肽各100mg。     

反相制备液相色谱分离白花败酱草异戊烯基黄酮

用半制备型反相高效液相色谱(RPLC)对白花败酱草氯仿萃取物进行了分离制备。色谱柱为YWGC18(200 mm×10.0 mm i.d.,10μm),流动相为乙腈∶水∶乙酸(55∶45∶1,V/V),检测波长280 nm,流速4.0mL/m in,进样750μL(样品液浓度30 g/L)。在此条件下一次分离得到5个单体化合物,经理化反应和光谱分析分别鉴定为bolusanthol B(1),(2S)-5,7,2,′6-t′etrahydroxy-6-lavandu lylated flavanone(2),(2S)-5,2,′6-′trihydroxy-2,″2-″d im ethylpyrano[5,″6″∶6,7]flavanone(3),orotin in(4)和orotin in-5-m ethyl ether(5)。其中2和3为新化合物,1、4和5为首次从败酱属植物中分离得到。     

A Mixed Retention Model of Reversed Phase HPLC

The retention behavior of solutes in reversed phase chromatographic system, especially containing hydrophobic ions in mobile phase, has been studied by many authors, but the silanol effect on alkyl-modified silica surface was neglected. Actually, because of stereo hindrance, numerous unreacted (residual) silanol groups, which can interact with some solutes, are left within the bonded phase after the silica surface has be modified. A mixed retention model, which considers adsorbed hydrophobic ions on the bonded phase can decrease the hydrophobicity and mask residual silanol groups, is proposed, based on hydrophobic distribution of neutral solutes, ion-pair distribution of ionic solutes and coulombic attraction between cationic solutes and the dissociated silanols.     

反相高效液相色谱法分析无机阴离子研究

本文研究了提高ＲＰ－ＨＰＬＣ测定无机阴离子的选择性的方法，设计了利用对离子试剂和背景试剂兼作ｐＨ调节剂，建立了几种分离分析系统和实际样品分析方法。     

Variation with Temperature of Phase Ratio in Reversed Phase HPLC for a Methanol/Water Mobile Phase

Chromatographia - The ratio between the volume of the stationary phase and the void volume of column in HPLC is known as the phase ratio (denoted by Φ). In almost all the thermodynamic studies...     

高纯橙皮素的制备及HPLC纯度分析

建立了一种基于反相硅胶纯化的高纯橙皮素制备方法,充分优化了上样量、流速及洗脱液极性等因素,采用红外光谱法和核磁共振对纯化后产物进行定性分析,高效液相色谱法检测纯化前后橙皮素的纯度,最终产物的纯度达99.87%。该方法快速简便、制备量大、分离效率高,每天制备效率可达到克级,为制备橙皮素标准物质提供了技术支撑。     

反相高效液相色谱法分离重组促红细胞生成素

利用反相高效液相色谱法对重组促红细胞生成素进行了纯化。结果表明,利用Ｃ４反相柱和乙腈－三氟乙酸流动相在洗脱梯度和上样样品纯度等条件都较适当时,可以简单、快速、高效地从粗品中分离出重组促红细胞生成素,获得的产品纯度高,接近１００％;比活性好,约为１．９６×１０８ＩＵ／ｇ蛋白。     

高压液相色谱内表面反相填料的发展及其应用

内表面反相填料（ＩｎｔｅｒｎａｌｓｕｒｆａｃｅｒｅｖｅｒｓｅｄｐｈａｓｅＩＳＲＰ）是１９８５年首次研制出来的一种高压液相色谱填料，其微孔内表面接疏水基团，外表面接亲水基团，当含蛋白质或其它大分子的样品进入柱子后，蛋白等大分子不被系统保留，而小分子物质则可以进入微孔得到分离。这一填料的研制和发展使血清样品直接进样成为可能，在药物分析方面有独特的优点。本文综述了ＩＳＲＰ的特点、合成方法、工作原理及其应用。     

Electroanalytical Determination of Enrofloxacin in Reversed Phase HPLC

The electrochemical behavior of a glassy carbon electrode is investigated towards the oxidation of enrofloxacin in aqueous solutions of different pH. Two oxidative waves are observed at a potential range from 0.70 V to 1.20 V (vs. SCE), the intensity of each depending on the pH of the solution. These results are used to set the parameters for the amperometric detection of enrofloxacin in HPLC analysis. A wide linear range and high sensitivity are obtained for a calibration plot using the optimized experimental conditions. The limit of detection (S/N=3) obtained using those conditions is approximately 0.2 pmol. The applicability of the methodology is demonstrated in the analysis of enrofloxacin in fortified milk samples.     

Analysis of Plasma Angiotensins by Reversed Phase HPLC and Radioimmunoassay

Abstract

The analysis of biologically active angiotensin peptides in blood plasma by high performance liquid chromatography in a weakly non-polar reversed phase (C₂) chromatographic system combined with quantification of chromatographically isolated peptides by radioimmunoassay has been developed. This system is able to resolve each of seven closely-related peptides of the angiotensin group. The chromatographic system was applied to plasma samples which have been prepared for chromatographic analysis by C₁₈ cartridge extraction. Samples were reconstituted in HPLC solvent prior to injection into the HPLC system. Separated angiotensin were collected by fraction collector and the volatile components of the solvent system were blown off under an air stream. The content of several of the various angiotensin peptides in the fractions was then determined by radioimmunoassay using an appropriate antiserum. Antiserum to angiotensin II (octapeptide) was used to quantify the biologically active components angiotensin II, angiotensin III (hepta-peptide) and C-terminal hexapeptide. Recovery of angiotensin II in the C₁₈ cartridge extraction has been assessed at 85.0 ± 0.9% (mean ± SEM) using I¹²⁵-labelled angiotensin II, and 82.2 ± 4.45% using synthetic unlabelled angiotensin II. Recovery of standard preparations of angiotensin II in the HPLC system have been estimated at 67.5 ± 6.08%. The application of this technique to evaluating some components of the angiotensin in normal plasma is presented.     

A Direct Approach to Insulin Isotherm Analysis in Reversed Phase Chromatography

There is a need to demonstrate the application of a simple isotherm method and a process chromatography optimization methodology with its approximations and assumptions that is useful when quick application is required. This paper presents an explanation of a basic procedure for the optimization of the acetonitrile concentration under isocratic condition with insulin as the model compound, using the retention time method for single component isotherm measurement. The retention time method was used to measure the adsorption isotherm over the concentration range of 26–29% acetonitrile. The assumption of a single component isotherm for insulin is appropriate in this study since the shift of the peak front due to a tag-along effect of the des-amido insulin degradation product is minimal. This RTM method assumes a Langmuir isotherm model. This model is an accurate representation under these experimental conditions as revealed by the shape of the measured shock front time as a function of amount loaded. Analysis of the isotherm results shows the ‘a’ parameter decreases with increasing volume per cent acetonitrile and the column saturation capacity decreases with increasing volume per cent acetonitrile. The cycle time is represented by ‘a’ parameter and the loading factor by the column saturation capacity. Since the ‘a’ parameter increases with decreasing volume per cent acetonitrile, the cycle time increases and the production rate decreases. On the other hand, the column saturation capacity increases with decreasing volume per cent acetonitrile; therefore, the optimum loading factor increases, thus the production rate increases. This trade-off leads to an optimum acetonitrile concentration at low k′ until practical operational limits are achieved.     

New Fluorous Reversed Phase Silica Gels for HPLC Separations of Perfluorinated Compounds

Two new perfluoroalkyl-modified stationary phases were prepared and compared with two commercially available perfluorinated stationary phases (Fluophase RP and Fluophase Pentafluorophenyl) and a C₁₈-RP column with respect to retention times of an array of perfluoroalkyl-tagged and untagged molecules. A few highly lipophilic compounds were also included in the study. They showed high retention times on C₁₈-RP columns, but not on perfluorinated support materials. Perfluoroalkyl-tagged compounds revealed a weak interaction with the pentafluorophenyl-modified support. The interaction between perfluoroalkyl-tagged compounds and perfluoroalkyl-modified stationary phases was strong, and dependent on the chain length of the perfluoro tags. Surprisingly, there was only a small difference between the retention times of perfluorinated compounds on C₁₈-RP and C₆F₁₃-modified support. Fluorous-fluorous interactions became prevalent only with C₈F₁₇-tagged compounds on C₈F₁₇ functionalized silica gel. Compounds with two perfluoro tags showed a drastic increase in retention time, which might be due to a cooperative effect. These results demonstrate the uniqueness of fluorous-fluorous interactions based on linear perfluoroalkyl chains and open up possibilities for the design of new perfluoro tags for purifications and noncovalent attachments of catalysts or biomolecules on perfluorinated solid supports.     

Analysis of Allopurinol and Oxipurinol in Plasma by Reversed Phase HPLC

Abstract

A reversed phase HPLC assay is described for the quantitation of allopurinol and oxipurinol in human plasma. The strategies for the development of this method are discussed. The analysis is performed on a C-18 column using an acidic aqueous mobile phase. Sample preparation consists of protein precipitation with a mixture of trichloracetic acid and perchloric acid. The detection limit of the assay for both substances is in the region of 30ng/ml. This method has been applied to an investigation of the relative bioavailability of two commercial preparations of 300mg allopurinol tablets in eight healthy volunteers.     

Determination of Polyphenylarenes and Polynuclear Aromatic Hydrocarbons by Reversed Phase HPLC

Abstract

Standard mixtures of polycyclic compounds are analyzed by reversed phase microcapillary liquid chromatography. Acetonitrile/water and acetonitrile/THF/water are employed as stationary phases, and capacity ratios are reported. Polyphenylarenes can be distinguished from polynuclear aromatic hydrocarbons by a large shift in capacity ratios when changing the mobile phase. The shift in capacity ratios is most significant for 1,3,5-triphenylbenzene, which is demonstrated by a gradient elution. The influence of THF and water on retention behaviour of solutes is described.     

不抑制电导检测的反相高效液相色谱法分析烷基磺酸盐

本工作采用反相高效液相色谱法分析表面活性剂驱油研究中所用C_(10)～C_(16)烷基磷酸盐。用含有低浓度磷酸二氢钠的移动相和电导检测器,不需抑制柱,直接测检洗脱的烷基磺酸盐,方法简单、快速、灵敏。     

Immobilized Polysaccharide-Based Stationary Phases for Enantioseparation in Normal Versus Reversed Phase HPLC

A set of 31 structurally different chiral pharmaceutical compounds was used as model analytes for investigation of the enantioselective potential of two immobilized polysaccharide-based chiral stationary phases under normal and reversed phase separation conditions. These chiral stationary phases differed in the polymeric backbone, amylose or cellulose, but possessed the same derivatization functionality. The results showed that the tris(3,5-dimethylphenylcarbamate) of amylose and cellulose have very broad, and often complementary, enantiorecognition abilities. In general, normal phase separation mode seemed to be more advantageous for separation of the majority of studied pharmaceuticals no matter if amylose- or cellulose-based columns were used. However, in certain cases the reversed phase separation system yielded better results. The combination of these two immobilized chiral stationary phases offers a powerful tool for enantioseparation of different types of pharmaceuticals in the normal and/or reversed phase mode.

     

Investigation of 2-Methoxyethanol as a Modifier in Acetonitrile for the Reversed Phase HPLC of Proteins

Abstract

The results of using 2-methoxyethanol as a mobile phase modifier combined with acetonitrile for the separation of a standard mixture of proteins is described. Selectivity comparisons are made between this system and similar mobile phase systems containing acetonitrile, methanol, n-propanol and iso-propanol.