Towards functional group-specific detection in high-performance liquid chromatography using mid-infrared quantum cascade lasers.

A distributed feedback quantum cascade laser was applied for the first time as a powerful light source for mid-infrared (MIR) detection in liquid chromatography. Fructose and glucose in red wine were separated with an isocratic HPLC system, which was connected to a custom-made flow cell. This flow cell was constructed of two diamond windows with adjustable spacing and two hollow wave-guides for guiding the incoming and outgoing light. The HPLC column based on an ion-exchange resin with calcium(II) counter ion was run at 80 degrees C with 0.04% formic acid as the mobile phase. Under these conditions the carbohydrates could not be completely separated from the organic acids also present in wine. However, the emission of the laser at 1067 cm(-1) matches the absorption maximum of fructose and glucose, whereas the organic acids do not absorb appreciably at this wavenumber. Thus group-specific detection could be achieved. Additionally, the optical path length could be increased from 25 to 125 microm, which is very promising in gaining enhanced sensitivity compared to Fourier transform IR detection.     

A silica-based monolithic column in capillary HPLC and CEC coupled with ESI-MS or electrospray-atmospheric-pressure laser ionization-MS

We describe the successful coupling of CEC and capillary HPLC with the recently developed atmospheric-pressure laser ionization (APLI) method. APLI is suitable for selectively and sensitively ionizing nonpolar aromatic compounds at ambient pressure for subsequent mass-selective detection. The polycyclic aromatic hydrocarbons used as analytes are first separated either by CEC on a silica-based monolithic column or by capillary HPLC. The eluent, along with a sheath flow, is volatilized by microelectrospray and then selectively ionized by excimer laser (KrF*) radiation via two-photon excitation. A QTOF-MS is used as mass-selective detector. This interface combination makes soft ionization of thermally labile nonpolar aromatic analytes possible.     

Low-level flow counting of liquid chromatography column eluates

Summary The principal parameters which determine the operation of a high-resolution, high-sensitivity radioactive flow monitor are described: a) Sample preparation to ensure adequate recovery of radiolabelled sample, metabolites and internal standard. b) The instrument background count rate, when no sample or radiolabel is present in the flow cell, is a function of shielding and a reduction in noise obtained with a coincidence time below one microsecond. c) The minimum detectable amount of label depends upon the machine background, HPLC eluent and scintillator flow, whether or not packed flow cells are used, flow cell geometry, and the scintillator used. d) Three types of flow cell have been designed to cover the majority of HPLC and isotope applications. e) The performance of solid and liquid scintillators. It is shown that an instrument has been designed taking account of these parameters. The resulting design satisfies present high sensitivity counting requirements and maintains the resolution of current HPLC procedures when detection is by simultaneous flow radioassay and by optical methods.     

A Crystalline Quartz,Laser-Excited Fluorescence,Photoacoustic, and Two-Photon Photoionization Detector for HPLC

Abstract

A crystalline quartz flow cell has been developed for simultaneous fluorescence, photoacoustic effect, and two-photon photoionization detection of aromatic compounds in HPLC eluents. Excimer laser-excited three mode detection of naphthalene, 7,8-benzoflavone, N-ethylcarbazole, and anthracene in 70/30 v/v acetonitrile/water is exhibited. The flow cell is suitable for use with deoxygenated solvent systems and is orientation independent.     

Light scattering detection in aqueous high-performance gel permeation chromatography (HPGPC): Relative molecular mass distribution of eye lens proteins

Summary Light scattering techniques have been greatly improved since laser light sources, enabling dynamic light scattering and measurements at low angles have been available. Relative molecular mass determinations can be performed at low solute concentration, extrapolations to zero-angle are no longer required and, because of the low scattering volume, a flow-through cell construction has become possible where dust particles only interfere as spikes [1]. For HPLC detection the low-angle laser light scattering (LALLS) signal corresponds to the product of relative molecular mass and concentration; combination with a concentration detector (for example, differential refractometer, UV absorbance) enables calculation of relative molecular mass [2]. The sensitivities of LALLS and the usual concentration detectors are comparable for (bio)polymers with relative molecular masses near 100,000. The minimum relative molecular mass to be determined in this way is near 10,000 and, therefore, the only mode of HPLC using the LALLS detector to date is gel permeation (size exclusion) chromatography.     

Analysis of small droplets with a new detector for liquid chromatography based on laser-induced breakdown spectroscopy

The miniaturization of analytical techniques is a general trend in speciation analytics. We have developed a new analytical technique combining high pressure liquid chromatography (HPLC) with laser-induced breakdown spectroscopy (LIBS). This enables a molecule-specific separation followed by an element-specific analysis of smallest amounts of complex samples. The liquid flow coming from a HPLC pump is transformed into a continuous stream of small droplets (diameter 50–100 μm, volume 65–500 pl) using a piezoelectric pulsed nozzle. After the detection of single droplets with a droplet detector, a Q-switched Nd:YAG Laser is triggered to emit a synchronized laser pulse that irradiates a single droplet. The droplets are evaporated and transformed to the plasma state. The spectrum emitted from the plasma is collected by a spherical mirror and directed through the entrance slit of a Paschen–Runge spectrometer equipped with channel photomultipliers. The spectrometer detects 31 elements simultaneously covering a spectral range from 120 to 589 nm. Purging the measurement chamber with argon enables the detection of vacuum–UV lines. Since the sample is transferred to the plasma state without dilution, very low flow rates in the sub-μl/min range can be realised.     

A Simultaneous,Laser-Excited Fluorescence,Photoacoustic, and Two-Photon Photoionization Detector for Liquid Chromatography

Abstract

A laser-excited windowless flow cell has been developed for simultaneous fluorescence, photoacoustic, and two-photon photoionization detection of aromatic compounds in HPLC eluents. Sensitive three-mode detection of acridine, naphthalene, 7,8-benzoflavone, N-ethylcarbazole, and anthracene in 70/30 V/V acetonitrile/water is demonstrated with conservative detection limits in the nanogram range and below.     

Universal on-line detector for high-performance liquid chromatography via magneto-optical rotation

Detecting changes in magneto-optical rotation is useful as a universal on-line detector for high-performance liquid chromatography. Such apparatus is similar to a polarimeter except for the external magnetic field on a flow cell. Two modulation modes suitable for the magneto-optical rotation detector are discussed. Use of a semiconductor laser provides better sensitivity than a He-Ne laser. The detection limit is 0.006% (w/w) for polyethylene glycol 20000 in a 20-mul injection.     

Separation of diastereoisomers of podophyllum lignans by micellar electrokinetic chromatography

A technical solution and development of a method for on-line HPLC monitoring of bioreactor processes in a membrane reactor system are presented. Experiences in system design for the continuous coupling of a bioreactor system with capillary by-pass circuits using membrane flow cells and a dual HPLC system are reported. A continuously working integrated sample purification step by ultrafiltration with the membrane cell coupling is established. Using electrical switching valves and separated pumping and eluent systems, the dual HPLC system allows diode array detection as well as measurement of the refractive index. The application of the on-line HPLC monitoring system is demonstrated by measuring the anaerobic H-acid degradation kinetics. H-acid, 1-amino-8-hydroxynaphthalene-3,6-disulfonic acid, is one of the most important coupling components for a variety of direct, mordant, reactive dyes which remains in the process water and the textile dyeing effluents in high concentration.     

3-氨基-9-乙基咔唑衍生化寡糖混合物的高效液相色谱分离及激光解吸电离飞行时间质谱分析

建立了以3-氨基-9-乙基咔唑(AEC)为衍生化试剂对寡糖的标记方法。寡糖的还原端与AEC的伯氨基反应生成烯胺,再被NaBH3CN还原为二级胺,使得寡糖被AEC标记。衍生物通过反相高效液相色谱分离纯化,采用的色谱柱为Waters Symmetry C18柱(3.9 mm×150 mm,5 μm),乙腈和乙酸铵水溶液(pH 4.5)为流动相,梯度洗脱,在254 nm波长处检测,并以基质辅助激光解吸电离飞行时间质谱进行分析。在此衍生化条件和色谱条件下,葡寡糖衍生物分离良好,并且AEC衍生可显著提高葡寡糖的质谱检测灵敏度。该方法适用于寡糖的分离纯化和结构分析,并与生物质谱具有良好的兼容性,表明该方法在微量寡糖链分析方面有广阔的应用前景。     

Trace analysis of pollutants in water using the photothermal interferometry as HPLC detector

A new procedure including high performance liquid chromatography in combination with photothermal interference spectroscopy as detection device (HPLC/PIS) has been proposed, optimized and its figures of merit for pesticide residue analysis are shown. The flowing sample under study is set in one arm of a Mach-Zehnder interferometer, and its refractive index is modulated by a periodically chopped continuous wave argon ion laser. As chopper, an acousto optical modulator has been introduced to switch the excitation laser beam between different lines (457 nm, 488 nm, 514 nm) simultaneously. Thus a multi component analysis can be realized either by using an HPLC-system in front of the PIS device or by a multi line Ar(+)-laser, directly. The limit of detection of the HPLC/PIS system reached 71 microg/l of the pesticide di-nitro-ortho-cresol (DNOC).     

Micro high performance liquid chromatography of aliphatic amines by means of resonance raman detection

A micro high performance liquid chromatography coupled with a resonance Raman detection system is described. For highly sensitive Raman detection, aliphatic amines were derivatized with dabsyl chloride (4-dimethylaminoazobenzene-4′-sulfonyl chloride). The derivatives were separated on an ODS micro column (0.5 mm i.d. × 145 mm PTFE tube). Chromatograms were obtained by measuring the intensity of Raman scattering at 1136 cm^?1 with the 488.0 nm line of an Arion laser. The lower detection limit was 1.5 ng and the RSD of relative peak height (n = 9) was 5.9% at 11 ng of methylamine derivative. Moreover, by stopping the flow of the micro HPLC system at the retention time of the individual derivatives, it was possible to measure their resonance Raman spectra.     

Characterization of organotin species using microbore and capillary liquid chromatographic techniques with an epifluorescence microscope as a novel imaging detector

The novel application of a UV epifluorescence microscope as an imaging detector for microbore and capillary high-performance liquid chromatography (HPLC) is reported. The microscope is focused on an in-line quartz flow cell incorporated down stream of a microbore HPLC column or directly on an optically clear portion of fused-silica capillary columns for analyte detection. The effect of different fluorescent ligand to analyte ratios on detection limits is also reported, as well as the effect of different image volume sizes produced by changes in microscope objective lens magnification power. Determination of relative sensitivities an detection limits for methyl- and butyltin compounds, complexed with fluorescent dyes, reveals that the organotins show decreasing sensitivity as the number of alkyl substituents on the tin atom increases, with minimum detectable amounts of 6-160 pg of analyte-ligand complex.     

Long pathlength, three-dimensional absorbance microchip

A long pathlength, three-dimensional U-type flow cell was microfabricated and evaluated for improved absorbance detection on a glass microdevice. A small diameter hole (75 μm) was laser etched in a thin glass substrate whose thickness (100 μm) defined much of the pathlength of the cell. This substrate was thermally bonded and sandwiched between two different glass substrates. The top substrate contained a typical injection cross and separation microchannel. Projecting out of the plane of the separation device was a 126 μm pathlength flow cell as defined by the laser etched hole and the attached microchannels. The flow cell was connected to a microchannel on the bottom substrate that led to a waste reservoir. The planar, flat windows on the top and bottom of this device made light introduction and collection a simple matter using a light emitting diode (LED) and microscope objective. The experimentally obtained detection limit for rhodamine B was determined to be 0.95 μM, which is nearly identical to the theoretical limit calculated by Beer's Law. A separation of three fluorescent dyes was performed, and direct comparisons were made between the transmittance changes through the narrow pathlength separation microchannel and the adjacent long pathlength, three-dimensional U-type flow cell.     

微流控芯片系统流式细胞术及单细胞荧光检测

微流控芯片系统用于细胞分析是近年来该技术发展的一个热点,因而受到越来越多的关注．其主要原因在于微全分析系统具备高度微型化、集成化和设计灵活等特点,通过巧妙设计和精密加工能够实现细胞的培养、凋亡及检测等功能．流式细胞术(Flow cytometer,FCM)是一种在功能水平上对单细胞或其它生物粒子进行定量分析和分选的检测手段,     

Review. Use of laser detectors in capillary liquid chromatography

The main relationship of high-performance liquid chromatography (HPLC) are considered. It is shown that the optimum conditions of ultrasensitive trace analysis should be achieved by using packed capillary columns manufactured from flexible quartz capillaries with dc approximately less than 0.2 mm. The main features of these columns (v opt = 0.6 v opt of that for conventional HPLC columns with double the hydraulic permeability) make it possible to obtain two or three times higher plate numbers for the same analysis time and column pressure characteristic of conventional HPLC, as a result of using a submicrometre sorbent. The main features of laser detection in capillary liquid chromatography (laser-induced fluorescence and cross-beam thermal lens absorption detectors) are considered. The requirements that should be met by a modern capillary liquid chromatograph based on using flexible quartz capillary columns with a submicrometre sorbent and laser detectors are formulated. Examples of using these systems for femtomole and attomole analyses of biological samples (amino acids and prostaglandins) are given.     

A method for hydrogen content control of UO2 fuels

A new method for hydrogen content control over UO₂ based on laser microsampling in a noble gas flow as well as independent ways of hydrogen detection are proposed. Solid electrolytic cell and a metal-insulator-semiconductor sensor are used for hydrogen registration. The limit of detection is about 10^–11 g H₂... The results received by the two analytical procedures do not show considerable systematic discrepancy.     

Union of capillary high-performance liquid chromatography and microcoil nuclear magnetic resonance spectroscopy applied to the separation and identification of terpenoids

This paper describes the first coupling of a commercial capillary HPLC system with a diode array spectrophotometric detector and a custom-built nuclear magnetic resonance (NMR) flow microprobe. The eluent from a 3-microm diameter C18 HPLC column is linked to a 500 MHz 1H-NMR microcoil probe with an observe volume of 1.1 microl. The separation and structurally-rich detection of a mixture of terpenoids under both isocratic and gradient solvent elution conditions is presented. The lowest limits of detection yet reported for capillary HPLC on-line measurement (i.e., 37 ng for alpha-pinene) are achieved with this system. The complementary nature of diode array and NMR detection allows stopped-flow data collection from analytes which would otherwise go unnoticed in continuous-flow NMR. Moreover, stopped-flow NMR data is presented for the detection of a trace (sub-nmol) impurity in the sample mixture. Since NMR signals degrade and shift during solvent gradients, flow injection analysis studies are conducted with injected solvent plugs differing in mobile phase composition. The NMR signal degradation accompanying these injections is largely due to the variance in chemical shift with the solvent composition rather than to changes in magnetic susceptibility of the solvent. Characterization of such effects enables the development of improved NMR probes for the coupling of capillary HPLC and NMR.     

On-column infrared monitoring for reversed phase liquid chromatography with deuterated solvents

Infrared monitoring was accomplished in reversed phase HPLC on using deuterated solvents. The end of a micro HPLC column was flattend and used as a flow cell for on-column IR detection. Detection of alkylbenzenes and oil-soluble vitamins which were separeted on ODS packing with CD₃CN/D₂O and CD₃OD, respectively, was performed at 2940 cm^?1.     

新型高效液相色谱激光诱导荧光检测器的研制

采用共聚焦结构,研制了一种高效液相色谱激光诱导荧光检测器(HPLC-LIFD)。采用473 nm的半导体泵浦固体激光器为光源,设计了一种专用于共聚焦的HPLC-LIFD检测池。该池的物理体积为7 μL,有效体积约为0.1 nL,入口通道与出口通道的夹角为120°,无直角区和变径区,无滞留和积累气泡的死角,对谱带展宽影响小。以核黄素标准品为测试物评价了该体系并利用该系统测定了实际样品中核黄素的含量。