Application of long-lived luminescence for detection in liquid chromatography

Summary Quenched and sensitized lanthanide luminescence as detection in liquid chromatography has been investigated. An important advantage in comparison with phosphorescence is that the long-lived luminescence as applied does not require deoxygenation of the samples. In order to obtain a high luminescence intensity Tb(III) complexes with acetylacetonate have been formed, after which indirect excitation of Tb(III) can be realized via the ligands. The potential of Tb(III) luminescence as a detection method in ion chromatography has been shown for chromate, which is an efficient quencher. Sensitizing of the Tb(III) luminescence has been applied for thiol-containing analytes. These compounds are derivatized with maleimidyl salicylic acid to complexes that sensitize the Tb(III) luminescence. From a comparison of the results obtained with normal fluorescence detection and time-resolved sensitized Tb(III) luminescence detection it has become clear that the last method has a higher sensitivity, but in particular a higher selectivity.     

Room temperature phosphorescence in the liquid state as a tool in analytical chemistry

A wide-ranging overview of room temperature phosphorescence in the liquid state (RTPL¹) is presented, with a focus on recent developments. RTPL techniques like micelle-stabilized (MS)-RTP, cyclodextrin-induced (CD)-RTP, and heavy atom-induced (HAI)-RTP are discussed. These techniques are mainly applied in the stand-alone format, but coupling with some separation techniques appears to be feasible. Applications of direct, sensitized and quenched phosphorescence are also discussed. As regards sensitized and quenched RTP, emphasis is on the coupling with liquid chromatography (LC) and capillary electrophoresis (CE), but stand-alone applications are also reported. Further, the application of RTPL in immunoassays and in RTP optosensing—the optical sensing of analytes based on RTP—is reviewed. Next to the application of RTPL in quantitative analysis, its use for the structural probing of protein conformations and for time-resolved microscopy of labelled biomolecules is discussed. Finally, an overview is presented of the various analytical techniques which are based on the closely related phenomenon of long-lived lanthanide luminescence. The paper closes with a short evaluation of the state-of-the-art in RTP and a discussion on future perspectives.     

Solid-surface room-temperature phosphorescence detection for high-performance liquid chromatography

In the present study, we have optimized the use of a two nebulizer system for solid-surface room-temperature phosphorescence (SSRTP) as a selective, permanent record detector for high-performance liquid chromatography (HPLC). The chromatographic parameters and the analytical figures of merit of five well known phosphorescent compounds were compared to those obtained by ultraviolet detection. Calibration curves with satisfactory linear dynamic ranges and limits of detection in the nanogram and subnanogram level showed the feasibility of the SSRTP detector for HPLC. In addition, overlapped compounds were individually identified demonstrating that the selectivity of the proposed detector can be a useful feature in case of incomplete chromatographic separations of complex mixtures.     

Some aspects of room-temperature phosphorescence in liquid solutions

Experimental requirements for room-temperature phosphorescence measurements in liquids (RTPL) are discussed. Phosphorescence quantum yields and triplet lifetimes of some brominated naphthalenes and halogenated biphenyls at 77 K in 2-methyltetrahydrofuran and at room temperature in hexane are reported and compared. Surprisingly the naphthalenes show only little loss in quantum yields in going from 77 K to room temperature. Sensitized phosphorescence is discussed as a means of expanding the analytical potential of RTPL. Results with a model system of benzophenone as a donor (analyte) and 1,4-dibromonaphthalene as an acceptor are presented.     

Comparison of different alcohols in a caged lumophore for measuring supersaturated dissolved oxygen

Intense phosphorescence is observed when an alcohol is introduced to an aqueous solution containing 1-bromonaphthalene and a cyclodextrin complex. The enhancement of the phosphorescence intensity of 1-bromonaphalene is related closely with the alcohol’s effectiveness in shielding the photo-excited lumophore from dissolved oxygen. The phosphorescence intensity decreases and the phosphorescence decay rate increases as the dissolved oxygen concentration increases. In this paper, we measure the phosphorescence intensity and decay rate of the complex using different alcohols and explain their respective advantages. By relating the concentration of oxygen to the intensity and the lifetime of the phosphorescence, the dissolved oxygen concentration can be measured. The sensitivities and measurement ranges with the complexes containing different alcohols are compared. Currently, this technique can measure dissolved oxygen concentrations from 0 to 40.2 mM (31,400% saturation). The range of the measurement is 40 times larger than previous studies.     

Simultaneous correlation chromatography,a new technique applied to calibration in high-performance liquid chromatography

Simultaneous correlation chromatography (SCC) is introduced as a technique capable of analyzing several different samples simultaneously on the same chromatographic column. The theoretical basis of the technique is outlined and a computer simulation demonstrates the feasibility of the method. The advantages and potential of the technique are discussed. The technique is applied experimentally in a calibration procedure for high-performance liquid chromatography. During the calibration, unknown sample and calibration standards are processed under the same conditions, resulting in very accurate calibration. Other applications are outlined.     

Preparative argentation reversed-phase high performance liquid chromatography

In this study, a preparative chromatography method named preparative argentation reversed-phase high performance liquid chromatography (Ag-RP-HPLC) was developed by adding silver ion to the mobile phase of preparative HPLC. Firstly, an analytical Ag-RP-HPLC method was developed, and the effects of silver content and acid content in the mobile phase on the column efficiency were studied. Based on the method of linear amplification, a preparative Ag-RP-HPLC technique with optimized separation conditions was developed. The new technique was applied successfully to the separation of the unsaturated aliphatic acid amide isomers contained in Asarum forbesii Maxim. Compared with the commonly used technique of argentation normal phase chromatography, this method with little solvent consumption is simple, fast, efficient, and flexible for the isolation and purification of the unsaturated compounds.     

Estimating measurement uncertainty in high-performance liquid chromatography methods

In every measurement procedure, it is important to know the components of measurement uncertainty affecting the quality of measured result and reliability of quantified result. The procedure for recognizing measurement uncertainty is not universal but depends on the method and sample type. It has to be made according to good laboratory practice. This paper aims at showing the comparison of measurement uncertainty component estimations for three methods using the high-performance liquid chromatography techniques: determination of the type and content of aromatic hydrocarbons in diesel fuels and petroleum distillates by normal phase high-performance liquid chromatography, determination of nitrates in water samples by ion chromatography, and determination of molecular weights of polystyrene by size exclusion chromatography technique. Both similarity and differences were found during the measurement uncertainty component estimation, and conclusions about influences of certain components on the result uncertainty were made.     

Sensitized phosphorescence as detection method for the enantioseparation of bupropion by capillary electrophoresis

A new CE detection method was developed for the chiral drug bupropion (a second-generation antidepressant), based on phosphorescence both in the direct and in the sensitized mode using pulsed laser excitation at 266 nm. Electrokinetic chromatography using 5 mM sulfated-α-CD as chiral selector in 25 mM phosphate buffer at pH 3 allowed the separation of bupropion enantiomers with a high chiral resolution (Rs>3). In the sensitized phosphorescence detection mode, excitation energy is transferred from the analyte to an acceptor (1-bromo-4-napthhalenesulfonic acid or biacetyl) followed by time-resolved phosphorescence detection under deoxygenated buffer conditions. Using 2 × 10(-4) M biacetyl as the acceptor an LOD of 2 × 10(-7) M was obtained for each enantiomer, about 40 times better than in the direct mode. Under these separation conditions, no significantly different phosphorescence lifetimes (measured on-line) were obtained for the two bupropion enantiomers. The suitability of the method was demonstrated with the quantification of bupropion in a pharmaceutical formulation and its determination in a spiked urine sample.     

High-performance precipitation liquid chromatography

High-performance precipitation liquid chromatography is based upon solubility differences and thus especially appropriate for polymers which have a narrow solubility window. The technique offers new possibilities for the analysis of copolymers according to their chemical composition distribution and can be used in combination with size-exclusion separation for chromatographic cross-fractionation.     

Complementary information from isotachophoresis and high-performance liquid chromatography in peptide analysis

Reversed-phase high-performance liquid chromatography is a valuable analytical technique to support the synthesis, isolation and purification of peptides, as is illustrated by some critical separations. In addition to this technique, capillary isotachophoresis can give useful information on the purity determination of peptides and on the presence of ionic compounds of a non-peptidic nature. With regard to the latter aspect, isotachophoresis proved to be a suitable technique as a check on the effective removal of salts after preparative high-performance liquid chromatography.     

A sample-drying technique for room-temperature phosphorescence

The sample-drying procedure described improves the precision, decreases the time needed and reduces the expense of the solid-surface room-temperature phosphorescence technique. Samples are dried in a glovebg apparatus interfaced to the sample compartment of the spectrophotophosphorimeter. The samples are treated with sodium citrate to prevent moisture quenching the phosphorescence emission The relative standard deviations of measurements are reduced to less than 2% for large sets of samples. The total time required for 37 samples is only 5.5 h compared to 20 h by a conventional technique.     

Mechanistic studies on the photo-oxidation of commercial poly(butadiene)

Products from both the thermal and photo-oxidation of poly-butadiene have been studied using various analytical techniques such as infra-red and ultra-violet spectroscopy, gas-liquid chromatography and phosphorescence emission and phosphorescence excitation spectroscopy. Analyses have allowed a plausible mechanism to be formulated for the photo-oxidation of this polymer, consistent with all the observed physical and chemical changes. Both photo- and thermal oxidative polymer degradation initially result in the formation of α, β-unsaturated carbonyls which, under photo-degrading conditions, are subsequently photolysed to yield the observed final products.     

Porphyrin analysis by reversed-phase high-performance liquid chromatography: biomedical applications

Reversed-phase high-performance liquid chromatography has considerable advantages in porphyrin analysis since many of the extraction and esterification steps required for normal-phase high-performance liquid chromatography can be eliminated. This technique is of increasing importance for the diagnosis of porphyrias and in other clinical and experimental studies of porphyrins.     

Resolution of uranium isotopes with kinetic phosphorescence analysis

This study was conducted to test the ability of the Chemchek? Kinetic Phosphorescence Analyzer Model KPA-11 with an auto-sampler to resolve the difference in phosphorescent decay rates of several different uranium isotopes, and therefore identify the uranium isotope ratios present in a sample. Kinetic phosphorescence analysis (KPA) is a technique that provides rapid, accurate, and precise determination of uranium concentration in aqueous solutions. Utilizing a pulsed-laser source to excite an aqueous solution of uranium, this technique measures the phosphorescent emission intensity over time to determine the phosphorescence decay profile. The phosphorescence intensity at the onset of decay is proportional to the uranium concentration in the sample. Calibration with uranium standards results in the accurate determination of actual concentration of the sample. Different isotopes of uranium, however, have unique properties which should result in different phosphorescence decay rates seen via KPA. Results show that a KPA is capable of resolving uranium isotopes.     

Thermal aqueous liquid chromatography—the TALC technique

Thermal Aqueous Liquid Chromatography (TALC) using pure water as a mobile phase should be a very attractive technique promoting the use of liquid chromatography in routine analysis, either in the laboratory or in future on-line process control. This simple form of chromatography, the selectivity of which depends on the fixed phase only, would allow the use of flame ionization as an almost universal, low-cost detector in liquid chromatography. The potential of the TALC technique is illustrated by selected examples.     

Correlation between phosphorescence lifetimes and ratios of steady state phosphorescence to fluorescence intensity for biacetyl in various solvents

The phosphorescence of biacetyl in fluid solutions is quenched not only by oxygen but also by impurities resulting from the distillation procedure and from the freeze-pump-thaw degassing technique. A linear correlation between the measured phosphorescence lifetimes and the ratios of the phosphorescence to the fluorescence intensities is established; the slope affords reliable calculations of the biacetyl triplet lifetimes from steady state measurements and additionally the determination of the rate constant k_p of radiative triplet deactivation. This constant is dependent on the solvent, whereas the fluorescence constant k_F is not.     

药物发光分析的研究与展望

本文概述了国内目前药物发光分析的现状着重对荧光，化学发光和磷光光谱法在药物分析中的应用作了较详细的介绍，此外还介绍了作者近几年来用发光技术与薄层分离联用进行药物含量测定，中药微量元素测定，血药浓度监测和药代动力学研究的研究工作，提出了药物发光分析研究了目前主要是要把高分离度与高灵敏度检测相结合及连续操作。     

Use of ultra-performance liquid chromatography in pharmaceutical development

Ultra-performance liquid chromatography (UPLC) has been investigated as an alternative to HPLC for the analysis of pharmaceutical development compounds. We present data on three compounds showing that significant reductions in separation time can be achieved without compromising the separation quality. Results from precision and comparative studies indicate that UPLC is a suitable technique for routine pharmaceutical analysis.