手性流动相添加法拆分酮康唑外消旋体

采用C18反相色谱柱,利用在流动相中加入手性选择剂的方法实现酮康唑对映体的拆分。研究了手性选择剂的种类及浓度、流动相pH值、甲醇比例和柱温等因素对酮康唑手性分离的影响,结果表明磺丁基-β-环糊精可以使酮康唑对映体完全分离,最后选择的流动相组成为甲醇-0.02 mol/L磷酸二氢钠(体积比为60∶40,含0.02%三乙胺和1.0 mmol/L磺丁基-β-环糊精,用稀磷酸调节pH值到3.00)。酮康唑对映体在6 min内得到基线分离,分离度为2.05。方法简便,分离效果好,对酮康唑对映体的拆分具有应用价值。     

Evaluation of mobile phase additives in LC-systems using chemometrics

Chromatographia - A strategy for general optimization of the mobile phase in LC-systems is described in this study. The influence on column efficiencies and capacity factors of three uncharged...     

Separation of peptides with an aqueous mobile phase by temperature-responsive chromatographic column

Peptide separation technology is significant and is still an analytical challenge in proteomic studies. We report a simple preparation method for poly(N-isopropylacrylamide) grafted silica through the copolymerization of N-isopropylacrylamide with acetyl moieties immobilized on the silica surfaces. Differential scanning calorimetry results indicated that the prepared silica exhibited a sharp phase transition at 35.03°C. Silica grafted with poly(N-isopropylacrylamide) was evaluated as a temperature-responsive chromatography medium for the separation of peptides using 0.2 M NaCl solution as a mobile phase. Results indicated that at 10°C, the peptides were not resolved, but baseline separation with prolonged retention time at 50°C was attained. Particularly, a mixture of four peptides was efficiently separated within 8 min. The theoretical plate number of every peptide was more than 2500, and the resolutions were more than 3.40. The increased selectivity of the temperature-responsive column resulted from the temperature-modulated hydrophobic interaction with peptides. The retention times of these peptides were related to their hydrophobicities. This protocol provided a reliable set of chromatographic tool usable across all research and development applications that required isolation and analysis of peptides. It may represent a step forward in the complex analysis of hydrophobic and other proteins.     

激光诱导荧光检测微流控芯片毛细管电泳分离多肽的研究

微流控分析系统是分析科学及分析仪器重要的发展前沿,是90年代初发展起来的微分析系统的主要组成部分及目前较为活跃的领域.将微流控分析系统应用于电泳分离,与传统的电泳分离手段相比较而言,具有微型化、可集成化、速度快、进样量小等特点.它的检测方法有多种,其中激光诱导荧光(Laser induced fluorescence,LIF)法因其灵敏度高,成为微流控芯片分析检测目前最广为采用的方法[1].     

Liquid chromatographic separation of enantiomers using chiral additives in the mobile phase

Addition of an optically active compound to the mobile phase is an attractive method for resolving enantiomers in liquid chromatography. The technique is practical, easy to use and allows rapid screening for new chiral complexing agents as well as for optimal separation conditions.     

Liquid chromatographic determination of alcohols in food and beverages with indirect polarimetric detection using a beta-cyclodextrin mobile phase.

An indirect polarimetric detection method for the determination of alcohols has been proposed in liquid chromatography (LC). Optically active mobile-phase additives, such as beta-cyclodextrin (beta-CD), could be used to visualize optically inactive alcohols in reversed-phase LC. The visualization of alcohols is based on a perturbation of the partition of beta-CD caused by the alcohols. The detection limits of the present system at a signal-to-noise ratio of 3 were 0.031, 0.019, 0.018, 0.013, 0.011, 0.008 and 0.008% (v/v) for ethanol, 2-propanol, 1-propanol, 2-methyl-2-propanol, 2-butanol, 2-methyl-1-propanol and 1-butanol, respectively. The method was successfully applied to the determination of ethanol present in food and beverage samples.     

Ion-exchange chromatography using vanadyl and vandate salts as mobile phases with indirect detection

Summary Vanadyl sulfate, VOSO₄, was characterized as the mobile phase for the ion exchange separation of Li⁺, Na⁺, NH ₄ ⁺ , and K⁺ using indirect photometric detection at 254 nm. Detection limits ranged from 0.2 ppm for Li⁺ to 1 ppm for K⁺. Indirect electrochemical detection of these separated cations by reduction of VO (II) to V³⁺ was compared to spectrophotometric detection. The potential of the vanadate species, HVO ₄ ^2– , for the separation of F^–, Cl^–, and SO ₄ ^2– , with indirect photometric detection was also demonstrated.     

Separation of peptides by HPLC using a surface-confined ionic liquid stationary phase

A butylimidazolium bromide surface-confined ionic liquid stationary phase was synthesized in-house. The synthesized phase was investigated for the separation of five peptides (Gly-Tyr, Val-Tyr-Val, leucine enkephalin, methionine enkephalin, and angiotensin-II). The peptides were successfully separated in less than 5 min. The effect of trifluoroacetic acid (TFA) on the separation of peptides was evaluated with results confirming that TFA was not acting as ion-pairing agent in separation of peptides on this phase.     

Separation and indirect detection of alkyl sulfonates and sulfates

Iron(II) 1,10-phenanthroline, Fe(phen)3(2+), salts are used as mobile phase additives for the liquid chromatographic separation of alkyl sulfonates and sulfates on the reversed-phase PRP-1. As alkyl chain length increases retention increases. For a given chain length an alkyl sulfate is more retained than the corresponding alkyl sulfonate. Major elution variables that affect retention are mobile phase solvent and counteranion concentration. Indirect photometric detection is used to detect alkyl sulfonates and sulfates at 510 nm where Fe(phen)3(2+) salts absorb. Conditions for isocratic and gradient elution of multicomponent mixtures are described. Detection limits depending on analyte approached 0.1 nmol for isocratic elution and 3 nmol for gradient elution.     

Separation and determination of carnosine-related peptides using capillary electrophoresis with laser-induced fluorescence detection

A capillary electrophoresis (CE) method with laser-induced fluorescence (LIF) detection was developed for the separation and detection of carnosine-related peptides (carnosine, anserine, and homocarnosine). A sensitive and fluorogenic regent, 3-(4-carboxybenzoyl) quinoline-2-carboxaldehyde (CBQCA) was selected as a precapillary labeling reagent for imidazole dipeptides to form isoindole derivatives. The optimized molar ratio between CBQCA and peptide was found to be 75:1, and 50 mmol/L borate buffer (pH 9.2) was used for the derivatization in order to achieve good efficiency. Three imidazole dipeptides were baseline-separated within 20 min by using 112 mmol/L sodium borate (pH 10.4-10.8) as running buffer. Concentration detection limits (signal-to-noise ratios) for carnosine, anserine, and homocarnosine were 4.73, 4.37, and 3.94 nmol/L, respectively. This method has been applied to the analysis of human cerebrospinal fluid (CSF) and meat dry powder of pig and sheep. Recoveries were in the range of 82.9-104.8% for homocarnosine in CSF. For carnosine and anserine, the recoveries are 98.3% and 80.2% in meat dry powder of pig and 111.2% and 112.8% in meat dry powder of sheep, respectively.     

Effect of amine mobile phase additives on chiral subcritical fluid chromatography using polysaccharide stationary phases

Increased retention and selectivity in the subcritical fluid chromatography (SFC) of various amine compounds on polysaccharide chiral stationary phases (CSP) was observed upon incorporation of cyclic amines into the modifier. The retention increases are most pronounced with 2-propanol and are almost absent when methanol is used as modifier. This suggests that the effect may arise from a restriction to the modifier access to the binding site required to effect elution. The effect of the amine additives in SFC does not remain after their removal from the mobile phase. Findings were applied to the development of a 5 min separation of amphetamine and methamphetamine enantiomers.     

Separation and determination of perfluorinated carboxylic acids using capillary zone electrophoresis with indirect photometric detection

Perfluorinated carboxylic acids (PFCAs) belong to anthropogenic fluoroorganic compounds that have been detected in the natural environment and living organisms including humans. A capillary zone electrophoretic method with indirect UV detection using 2,4-dinitrobenzoic acid (2,4-DNBA) as a chromophore probe has been developed for analysis of PFCAs (C6-C12) in water. Optimal analyte resolution and detection sensitivity was obtained with 50 mM Tris solution of pH 9.0 and 50% methanol as a background electrolyte (BGE). The baseline separation of C6-C12 PFCAs was obtained within 20 min with detection limits in the range from 0.6 to 2.4 ppm.     

高效液相色谱手性流动相添加剂法分离3种黄酮类化合物对映体

采用反相高效液相色谱法(RP-HPLC),以磺丁基醚-β-环糊精(SBE-β-CD)作为手性流动相添加剂,建立了二氢黄豆苷原(dihydrodaidzein)、雌马酚(equol)和山姜素(alpinetin)3种黄酮类化合物的手性拆分方法。考察了环糊精的种类和浓度、有机相的种类和比例、缓冲盐的种类和浓度以及pH对3种化合物手性拆分效果的影响。结果表明:采用Kromasil 100-5C₁₈(250 mm×4.6 mm, 5 μm)色谱柱,流动相为乙腈-10 mmol/L SBE-β-CD水溶液(含20 mmol/L KH₂PO₄, pH值到4.0)(体积比为20:80)的条件下,二氢黄豆苷原、雌马酚和山姜素的对映体都达到了基线分离,分离度分别为1.8, 1.9和1.4。该方法简便,分离效果好,对黄酮类化合物的拆分具有应用价值。     

Separation of rare earth elements by anion-exchange chromatography using ethylenediaminetetraacetic acid as mobile phase

Rare earth elements (REEs) form anionic complexes which can be separated isocratically by anion-exchange chromatography using ethylenediaminetetraacetic acid (EDTA) as the mobile phase. For easy detection and identification, inductively coupled plasma (ICP) MS was used as detection method. From pH 3.5 to 7.5, retention increases from La to Sm and then decreases again up to Lu. Above pH 8.5, the retention of the light lanthanides increased drastically. It seems that the stoichiometry and the charge of the REE-EDTA complexes change with the elution pH. This strange elution behaviour can be easily tuned for particular applications by selecting the elution pH. For example, at pH values between 5.5 and 7.5 most isobaric and polyatomic interferences which occur in ICP-MS detection of the lanthanides are eliminated. A mechanism for the stepwise formation of the REE-EDTA complexes as a function of pH is proposed.     

Planar chromatographic separation of enantiomers and diastereomers with cyclodextrin mobile phase additives

A variety of racemic compounds were resolved using reversed-phase thin-layer chromatography (TLC) with mobile phases containing highly concentration solutions of beta-cyclodextrin (beta-CD). These include the drugs labetalol and mephenytoin, metallocenes, crown ethers, methyl-p-toluenesulfinate, nornicotine derivaties and several dansyl and beta-naphthylamide substituted amino acids. It was possible to resolve some racemates that could not be separated on beta-CD bonded phase liquid chromatography (LC) columns with this technique. Likewise there were some compounds that could be resolved with the LC approach that failed to separate with the present TLC method. In cases of racemates that could be resolved by either approach, it was found that the retention order was exactly opposite for the two methods. Enantiomeric resolution is highly dependent on mobile phase composition. In particular, the type and amount of organic modifier as well as the concentration of beta-CD affect the observed resolution. Possible reasons for the chromatographic behavior are discussed. Several diastereoisomeric compounds were separated as well, including steroid epimers and pharmaceutical compounds.     

Fluoroquinolone antibiotic determination in bovine,ovine and caprine milk using solid-phase extraction and high-performance liquid chromatography-fluorescence detection with ionic liquids as mobile phase additives

This paper describes the use of 1-ethyl-3-methylimidazolium tetrafluoroborate (EMIm-BF₄) as mobile phase additive for the analysis by high-performance liquid chromatography with fluorescence detection of a group of seven basic fluoroquinolone antibiotics (i.e. fleroxacin, ciprofloxacin, lomefloxacin, danofloxacin, enrofloxacin, sarafloxacin and difloxacin) in different milk samples. EMIm-BF₄ was found superior to 1-butyl-3-methylimidazolium tetrafluoroborate for the separation of the analytes from chromatographic interferences of the sample matrix. The optimized method was applied to the analysis of ovine, caprine and bovine milk, in the last case in either skimmed, semi-skimmed and full-cream milk after suitable acidic deproteination followed by a solid-phase extraction procedure. Recovery values between 73% and 113% were obtained for the three types of bovine milk samples, as well as for ovine and caprine milk (RSDs below 16% in all cases), which clearly demonstrates the applicability of the method to the three types of milk irrespective of the fat content of the samples. Limits of detection were in the range of 0.5–8.1 μg/L (approximately 0.5–25.9 μg/kg), well below the maximum residue limits established for these compounds by the current European legislation. A screening study of 24 different milk samples was also developed. In none of the samples, residues of the selected antibiotics were found.     

Chiral separation using cyclodextrins as mobile phase additives in open-tubular liquid chromatography with a pseudophase coating

The chiral separation of various analytes (dichlorprop, mecoprop, ibuprofen, and ketoprofen) was demonstrated with different cyclodextrins as mobile phase additives in open-tubular liquid chromatography using a stationary pseudophase semipermanent coating. The stable coating was prepared by a successive multiple ionic layer approach using poly(diallyldimethylammonium chloride), polystyrene sulfonate, and didodecyldimethyl ammonium bromide. Increasing concentrations (0–0.2 mM) of various native and derivatized cyclodextrins in 25 mM sodium tetraborate (pH 9.2) were investigated. Chiral separation was achieved for the four test analytes using 0.05–0.1 mM β-cyclodextrin (resolution between 1.11 and 1.34), γ-cyclodextrin (resolution between 0.78 and 1.27), carboxymethyl-β-cyclodextrin (resolution between 1.64 and 2.59), and 2-hydroxypropyl-β-cyclodextrin (resolution between 0.71 and 1.76) with the highest resolutions obtained with 0.1 mM carboxymethyl-β-cyclodextrin. %RSD values were <10%. This is the first demonstration of chiral open-tubular liquid chromatography using achiral chromatographic coatings and cyclodextrins as mobile phase additives.     

Effect of mobile phase amine additives on enantioselectivity for phenylalanine analogs

Curve fitting seems to be one of the best methods for the evaluation of chromatographic signals. As it is known, in this case mathematical function is fitted to digitized measured points. The most important task is to find the best mathematical function, which corresponds perfectly to the peak shape, and then to determine the parameters of the equation using a computerized least-squares method of approximation. In this work, a new mathematical function was sought for with the purpose of describing different chromatographic signals and it was fitted to the digitized measured points. The fitted curve is suitable for a quick evaluation of chromatographic information, noise filtering and correction of baseline drift. The fitting of gas chromatographic and high-performance liquid chromatographic signals were completed. The mathematical function, the generated chromatographic curves, the application of the function for describing real signals and the fitting process will be demonstrated in this study.