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A simple, fast, sensitive, and environmental friendly micro-method is described for the determination of desmosterol and cholesterol in milk samples using isocratic normal phase high performance liquid chromatography and diode array detection. After sample micro-saponification, the chromatographic separation is achieved in 10 min, further reduced to 6 min when only cholesterol is expected to be present. The method is accurate, presenting cholesterol and desmosterol recoveries >96% and inter-day RSD lower than 3%. Quantification limits were clearly below the requirements for any milk sample, with 40 and 20 μg/100 mL for cholesterol and desmosterol, respectively. Several raw and UHT commercial milk samples, infant powder formulas, and human breast milks were analyzed, with cholesterol and desmosterol (present only in the latter) contents within reported amounts. The proposed method proved to be simple and feasible for determination of a large number of samples, requiring less solvent consumption (9–13 mL of hexane for all saponification/extraction/chromatographic steps) and simpler equipment than most ones reported, and a further possibility of partial solvent recycling due to the isocratic chromatographic mode.

The purpose of this study is to improve the detection limit for trace herbicides, and to separate all samples within a short time with the combination of temperature and flow programming with micro‐reversed‐phase liquid chromatography (RPLC). The optimum condition for separation was ACN:0.05 M NaH2PO4 (46:54) (v/v), pH = 4.3, 40°C. The mass sensitivity is in the range between 0.086 × 10−13 g and 3.34 × 10−13 g in micro‐HPLC (HPLC). Micro‐HPLC exhibits higher mass sensitivity and better heat transfer than conventional HPLC. Thus, in the case of micro‐HPLC, the retention behavior of herbicides is easily determined according to the change of temperature. Especially, retention of triazines is decreased to a lesser extent with the increase of column temperature than that of other herbicides. This tendency has been accounted for the thermodynamic parameters ΔH° and ΔS°. The ΔS° for triazines is less negative than that for phenoxy acids, esters, and others. The chromatograms of herbicides were obtained ...

A micro high-performance liquid chromatography (HPLC) method has been developed for the determination of the anti-coccidial drug nicarbazin in chicken tissues, eggs, poultry feeds and litter. The 4,4′-dinitrocarbanilide (DNC) component of nicarbazin was extracted from foods, feeds and litter with acetonitrile. The extracts were purified by liquid-liquid partitioning, evaporated to dryness and taken up in methanol-acetonitrile-water (50:30:20, v/v). Micro HPLC of the 4,4′-dinitrocarbanilide (DNC) portion of nicarbazin is performed isocratically using a small bore column (1 mm I.D.) packed with reversed-phase and a UV detector set at 340 nm. The average recoveries of nicarbazin added to muscle, liver and egg were 92.8, 84.3 and 85.2%, respectively, 95.9% in poultry feed and 76.8–95.9% in different litters. The limit of detection was 25 pg, based on a detector signal-to-noise ratio of 3. These results were achieved with a simplified step extraction without the solid-phase extraction used by various researchers. This method offers a sensitive, selective, rapid, and less expensive alternative to conventional HPLC for such evaluations.

The generation of a micro-gradient for micro HPLC capillary columns is described. The system used is a modified and improved version of a split flow gradient elution device. Gradient tests have been performed and have shown good reproducibility under all conditions. The system has been successfully coupled with a particle beam LC-MS interface and the analysis of a mixture of twenty seven basic or neutral pesticides is reported.

The purpose of the study was to develop a micro HPLC system with physically changeable selectivity. This was done using two series-coupled beds of chemically differing stationary phases in a packed fused silica column. The selectivity change, in fine repeatable steps, was possible by the stepwise moving of a tube oven, covering only 50% of this “tandem column”. Each step of selectivity started with a position change of the oven, thus heating up a fresh position of the more polar and the non-polar column bed. As the chromatogram is thus repeated under these new selectivity conditions, multiple chromatograms result. For this reason we call the technique “Multi Micro HPLC”. This very simple mode of operation provides an analytical tool for developing separation conditions and which allows optimizing for a given special sample and checking for one of the most critical problems in HPLC: unidentified peak overlapping. The selectivity changes are nonlinearly correlated with the oven position with regards to temperature field distribution and are greater than is possible by simple change of column temperature. Micropacked columns are ideal for quick temperature changes. Elevated temperatures in HPLC help to shorten analysis time. The concepts discussed above provide an explanation of how a normal, formerly chemically homogeneous, column can become very temperature sensitive after detoriation at the inlet. In fact the column has become an unintentional “tandem column” and peak positions tend to move erratically as its selectivity changes drastically by inhomogeneous temperature changes along the separation path.

A new sampling procedure for individual raindrops with volumes of 4 nl - 4 μl is presented. It is demonstrated that analyte concentrations in small volumes can be analysed using micro-HPLC and capillary zone electrophoresis (CZE). Results for the analysis of cations such as Na+, NH4 +, K+, Mg2+, Ca2+, and anions such as Cl−, SO4 2-, NO3 −, NO2 − and organic acids are given and discussed.

This article reviews the works published since 2001 (included) on the micro/nano-HPLC analysis of bioactive and biomarker peptides. The main achievements related to the improvement of the detection sensitivity, quantitation repeatability and reproducibility, and separation selectivity are highlighted. A wide attention is paid to the application of micro/nano-HPLC to the analysis of bioactive peptides in biological matrices. The uses of micro/nano-HPLC in peptidomics to discover new endogenous bioactive peptides and to develop quantitation procedures to compare the levels of peptides of interest in two different biological samples are also considered. Finally, the application of micro/nano-HPLC to the analysis of biomarker peptides for various diseases is also included in this review.

A micro high-performance liquid chromatographic (micro-HPLC) procedure for the assay of digitoxin tablets and deslanoside injections has been developed. Micro-HPLC is performed on an ODS micro column, with acetonitrile-methanol-water (10:20:17) for digitoxin tablets and acetonitrile-water (21:70) for deslanoside injections. The effluent is monitored by UV absorption at 220 nm. Quantitation of cardiac glycosides in tablets and injections is carried out by the internal standard method. The composite assay results for digitoxin tablets and deslanoside injections provide average values of 101.2 and 99.9% with standard deviations of 1.2 and 0.93%, respectively. This micro-HPLC method is sensitive, quantitative, and reproducible. It is suitable for use in examining the content uniformity of pharmaceutical preparations.

Routine use of micro HPLC will need the development of several important constituents; the reproducible preparation of high-performance columns, small-volume pumps and gradient systems, and improvement of an injection system. Subjects to be studied are the development of high-performance monolithic silica columns for a variety of separation modes, multidimensional micro LC systems, and optimization of an interface between LC and MS instruments. Large peak capacities realized by highly efficient micro HPLC systems or multidimensional HPLC will greatly contribute to metabolomics studies when coupled with MS instruments.

A highly sensitive determination method for troglitazone stereoisomers was developed by high-performance liquid chromatography with electrochemical detection (HPLC-ECD). The oxidation behavior of troglitazone was investigated for the application of ECD by measuring the cyclic voltammogram. The separation was performed on a semi-micro chiral column (Chiralcel OJ-RH) using a mobile phase consisting of methanol-acetic acid (1000:1, v/v) containing 50mM LiClO4 at a flow rate of 20 microl/min. The peak areas of the stereoisomers separated from 0.1 to 50 ng/ml of troglitazone had good linearity with correlation coefficients of >0.999, and had similar response. The limit of detection was 1.3 fmol (signal-to-noise ratio of 3). This method was applied to the determination of troglitazone stereoisomers in rat plasma. The levels of troglitazone stereoisomers in rat plasma could be monitored until 24h after the oral administration.

Anthocyanins of red orange juices were analyzed by micro-HPLC coupled on-line with an MS detector equipped with an ESI source. The use of microcolumn HPLC greatly enhanced detection performance, allowing direct identification of the anthocyanins present in the orange juices. The use of a soft ionization technique allows detection of the molecular ions of the aglycons. Eight components were identified, five of them for the first time in red orange juice. Three additional anthocyanins were detected, of which only the aglycon was identified.

Monolithic silica columns having continuous gel skeletons and through-pores were prepared by a sol-gel method in fused-silica capillaries, and evaluated in reversed-phase liquid chromatography. The monolithic silica columns showed performance beyond the limit of conventional particle-packed columns in HPLC.

A dry-packing method has been developed which enables the preparation of packed capillary columns for micro HPLC from 250 μm i.d. tubing and 5 μm packing materials. Pressurized gases, such as hydrogen and argon, were used to transport the packing media, and ethanol or methanol were used as discharge agents. By changing the gas pressure, the packing density could be easily adjusted. It was found that, within experimental limits, the higher the packing density, the greater the column efficiency. Comparison between dry- and slurrypacked columns showed that the former had greatly improved stability; the efficiency of dry-packed columns was about the same as, or even better than, that obtained by slurry-packing.

In view of the importance of cyclodextrins in various pharmaceutical areas, methods for their determination in plasma are necessary for pharmacokinetic studies. Cyclodextrins show almost no UV absorption. This paper describes indirect detection of cyclodextrins in micro-HPLC

Recherche des parametres optimisants, qui sont nombreux: type de pompe, melange initial, nature des solvants, debits, geometrie de la cellule de melange, etc...

Detecteurs optiques, a barette de diodes, laser, electrochimiques, du type CG, a induction de plasma

The advantages of short microbore columns packed with a sorbent having a particle diameter of 3µm, such as the speed of analysis, the high solute concentration in the effluent and the detectability of small quantities are experimentally verified. The broadening in the valve is eliminated by the peak compression technique reached by adding a ion-pairing agent into the sample. The examples of catecholamines show the dependence of the height equivalent to a theoretical plate on the velocity of the mobile phase. The optimum is h=3 for a column 30mm long, i.d. 0.7mm, particle diameter 3µm. The minimal detectable quantity with a two-electrode amperometric detector with a gold electrode is from 30 to 40fmol for catecholamines.

Utilisation des techniques de spectrometrie IR a transformee de Fourier, de la chromatographie en couche mince avec de l'alumine et de la chromatographie HPLC en phase inverse afin d'identifier des composes de melanges complexes

High-performance micro packed fused-silica and open-tubular glass capillary columns were prepared and applied to separations of complex mixtures. Solvent-gradient elution proved quite useful for the separation of solutes with wide polarity. Instruments and some applications are described.

The capcity factors determined by reversed-phase micro high performance liquid chromatography (micro-HPLC) can be used as one of the descriptors in Quantitative-Structure-Activity-Relationships (QSAR) studies. The present method gives a more simple and convenient way to study relationships between physicochemical properties and biological activities of some drugs.

A miniaturized continuous gradient elution system with variable gradient profile was designed. A retention time reproducibility below 1% was achieved. The system was applied to the separation of phenolic compounds with a wide range of polarities.

Phenol-formaldehyde resins of the Novolak and Resol type were chromatographed on a reverse-phase micro-column packed with octadecylsilane on silica gel. A good separation of most components was achieved by the use of gradient elution with methanol-water; the contents of methanol in the mobile phase increased continually from 10 to 90%. The gradient was generated in a simple tubular mixer with an internal volume of 1.5 ml. Samples were applied to the column in a non-eluting solvent, using a six-port injection value with a 10 μl sample loop. The method, which shows a good reproducibility, is suitable for the characterization of phenol-formaldehyde resins. Phenol-Formaldehyd-Harze vom Novolak- und Resol-Typ wurden durch Chromatographie an einer mit Octadecylsilan auf Kieselgel bepackten Umkehrphasen-Mikro-Saule getrennt. Eine gute Auftrennung der meisten Komponenten wurde durch die Verwenndung der Gradientelution mit Methanol und Wasser erzielt. Der Gradient wurde in einer Rohr-Mischkammer (Volumen 1,5 ml) erzeugt; der Anteil von Methanol in der mobilen Phase wurde im Verlauf der Analyse von 10 auf 90% kontinuierlich gesteigert. Die Probenaufgabe erfolgte mit Hilfe eines Dosierventils und einer Probenschleife (Volumen 10 μl). Das Verfahren ergab reproduzierbare Ergebnisse; es erwies sich als gut geeignet zur Charakterisierung von Phenol-Formaldehyd-Harzen.

The combination technique of micro high performance liquid chromatography and inductively coupled plasma atomic emission spectrometry is demonstrated in the separation of organometallic compounds. Problems relating to the interfacing between micro HPLC and ICP would be elininated uti1izing the simple interfacing technique.

The chromatographic potential of very short fused-silica column (30 mm length) packed with 3-μm particles has been investigated. The results indicate that this kind of micro-column has high applicability for a rapid identification of several samples. This technique also meets the recent demands in HPLC, as low solvent consumption and faster analysis time.

Publisher Summary This chapter discusses the miniaturized voltammetric detectors. Micro high-performance liquid chromatography (micro HPLC) using packed columns of I.D. less than 1 mm and packed or open tubular capillary chromatography are attractive in that the consumption of the mobile phase and sample is much less than that in conventional HPLC. These extremely slow flow rates require small volume detectors and these have been designed for spectrophotometric detection by UV and fluorescence methods. The usefulness of electrochemical detectors has already been recognized. Numerous voltammetric detectors have been described for HPLC. These can be broadly divided into coulometric detectors that use a large surface area working electrode, and amperometric detectors that use a small surface area working electrode.

Publisher Summary This chapter discusses the combination of micro-HPLC and IR spectroscopy. High-performance liquid chromatography (HPLC) has become an extremely powerful technique for separating mixtures into their constituents , with the recent developments in the packing materials and pumps. HPLC permits separations of a wide variety of mixtures containing non-volatile or temperature-sensitive compounds which cannot be separated readily by gas chromatography (GC). However, no universal equivalent in HPLC to thermal conductivity or flame ionization detection in GC is available, because various mobile phases are used and the physical properties of both the mobile phase and the sample components are often quite similar. Although there are two detection methods based on UV absorption and refractive index, which are the most frequently used, they provide little structural information on the separated species. Infrared spectrometry ( I R ) is undoubtedly one of the most useful tools providing qualitative information.

The importance of dispersion in High Performance Liquid Chromatography (HPLC) is emphasised. The state of the art of Micro HPLC techniques such as high speed HPLC and narrow bore HPLC are reviewed.

Publisher Summary This chapter discusses the instrumentation and applications of micro-liquid chromatography/mass spectrometry. Micro high performance liquid chromatography (micro HPLC) is finally enjoying increased consideration and applications. Several manufacturers are now marketing either modified conventional HPLC equipment, or entirely new hardware designed to deliver micro LC flow rates ranging usually from 10-100 micro liters per minute. There are a variety of reported benefits or reasons for utilizing micro LC rather than conventional HPLC, but usually the most justifiable reasons for "bothering" with micro LC include HPLC solvent savings and on-line high performance liquid chromatography/mass spectrometry (LC/MS). This chapter reviews on micro LC/MS with emphasis placed upon practical applications which demonstrate the sensitivity gains available when total introduction of micro LC effluent is provided to the mass spectrometer.

The separation of polycyclic aromatic hydrocarbons (PAH) on various reversed-phase columns (phenyl, ethyl, octyl and octadecyl) have been compared. The correlations between the logarithm of capacity factor (log k′) and several descriptors, which show the molecular size, shape and physical property of a solute, were determined. The results indicated the high possibility to predict the retention of PAH in reversed-phase high performance liquid chromatography.