Coupling of narrow-bore liquid chromatography to thin-layer chromatography : Part I. Interfacing

The coupling of liquid chromatography (l.c.) on narrow-bore columns to thin-layer chromatography (t.l.c.) is described. The effluent from a l.c. column can be deposited on a t.l.c. plate after a normal-phase or reversed-phase separation without serious loss of chromatographic information. Both silica and alkyl-modified silica plates can be used for storage. The interface is a fused silica capillary which connects the column outlet to the spray jet assembly of a Linomat applicator for t.l.c. The stored chromatogram can serve as starting point for a new separation, but also allows the use of detection principles which are normally not compatible with l.c. The chromatography of some polynuclear aromatic hydrocarbons is used to illustrate the possibilities of the combinations.     

Use of narrow-bore high-performance liquid chromatography for microanalysis of protein structure

We report here systematic approaches to microanalysis of protein structures using narrow-bone high-performance liquid chromatography of protein, peptide and derivatized [phenylthiocarbamyl (PTC-) and phenylthiohydantoinyl (PTH-)] amino acids. The utilization of columns with small diameters (2 mm or less) has improved resolution and sensitivity and enabled protein structure analysis at the low pmol level. Preparative isolation of proteins and peptides of pmol quantity is achieved and separation and identification of PTC- and PTH-amino acids can be routinized at the low pmol to subpmol level. The use of diode-array detection enables simultaneous multiple-wavelength monitoring and spectral retreat, which greatly enhances flexibility and usefulness of the present methods. In combination with protein microsequencing techniques, these narrow-bore high-performance liquid chromatographic procedures can facilitate structural analysis of minutely available proteins of biochemical and physiological significance.     

New background correction method for liquid chromatography with diode array detection, infrared spectroscopic detection and Raman spectroscopic detection

A new method to eliminate the background spectrum (EBS) during analyte elution in column liquid chromatography (LC) coupled to spectroscopic techniques is proposed. This method takes into account the shape and also intensity differences of the background eluent spectrum. This allows the EBS method to make a better estimation of the background eluent spectrum during analyte elution. This is an advantage for quantification as well as for identification of analytes. The EBS method uses a two-step procedure. First, the baseline spectra are modeled using a limited number of principal components (PCs). Subsequently, an asymmetric least squares (asLS) regression method is applied using these principal components to correct the measured spectra during elution for the background contribution. The asymmetric least squares regression needs one parameter, the asymmetry factor p. This asymmetry factor determines relative weight of positive and negative residuals. Simulations are performed to test the EBS method in well-defined situations. The effect of spectral noise on the performance and the sensitivity of the EBS method for the value of the asymmetry factorp is tested. Two applications of the EBS method are discussed. In the first application, the goal is to extract the analyte spectrum from an LC-Raman analysis. In this case, the EBS method facilitates easy identification of unknown analytes using spectral libraries. In a second application, the EBS method is used for baseline correction in LC-diode array detection (DAD) analysis of polymeric standards during a gradient elution separation. It is shown that the EBS method yields a good baseline correction, without the need to perform a blank chromatographic run.     

Miniaturization of solid-phase reactors for on-line post-column derivatization in narrow-bore liquid chromatography

Summary The use of solid-phase reactors for post-column derivatization in narrow-bore HPLC (1.0mm i.d. analytical columns) is evaluated. Two systems are described, viz. for the determination of N-methylcarbamate pesticides and for that of urea and ammonia. The solid-phase reactor is packed with a strong anion exchange resin and urease immobilized on silica, respectively, to effect the catalytic hydrolysis of the solutes eluting from the analytical column. In both systems, the hydrolysis product is reacted with o-phthalaldehyde followed by fluorescence monitoring. Analytical data are presented and band broadening from various parts of the reaction detector system is discussed. An on-line trace enrichment procedure via a micro precolumn is descried for the trace level determination of N-methylcaramates in surface water samples.     

Interfaced d.c. argon-plasma emission spectroscopic detection for high-pressure liquid chromatography of metal compounds

A d.c. argon-plasma emission spectroscopic system is described for directly interfaced metal-specific detection of eIuates in high-pressure liquid chromatography. Differing approaches are needed with regard to solvent systems used in normal-phase or reversephase chromatography. A simple nebulization system is adequate for reverse-phase applications of polar solvents. However, a novel design of an impact nebulizer-interface is required to accommodate the hydrocarbon and halocarbon solvents typically encountered in normal-phase and adsorption chromatography. The d.c. plasma detection system has been applied to the h.p.l.c. of a range of transition metal β-diketonate, β-ketoamine and diethyldithiocarbamate complexes, with good linear ranges for metalspecific detection and with detection limits in the sub ng s^-1 range of metal eluted in the complexes.     

High-speed liquid and thin-layer chromatography of polychlorinated biphenyls.

High-speed liquid chromatography in the system silica gel/dry n-hexane and ultraviolet spectrometry have been used to study the composition of various types of commercially available mixtures of chlorinated biphenyls. Special attention has been paid to the analysis of highly chlorinated products. In addition to data previously published, retention times are recorded for 11 individual polychlorinated biphenyls. The results of high-speed liquid chromatography are compared with those obtained in several normal and reversed-phase thin-layer chromatographic systems.     

Application of iodine-azide reaction for detection of amino acids in thin-layer chromatography

The iodine-azide reaction was employed to TLC detection of sulphur-containing derivatives of protein and some non-protein amino acids. The derivatization reaction with phenyl isothiocyanate (PITC) took place directly on the plate before the developing step. Subsequently, the plates were sprayed with a mixture of sodium azide and starch solution in NP-TLC and in the case of RP-TLC sodium azide solution with starch incorporated into mobile phase and then exposed to iodine vapour. The spots became visible as white spots on violet-grey background. The obtained detection limits of PTC-derivatives have been compared with other visualizing techniques commonly used in TLC practice (UV254 and iodine vapour). The iodine-azide system has been proved to be the most favourable and enabled to detect quantities per spot in the range of 1-60 pmol (HPTLC) and 3-100 pmol (TLC).     

Coupling solid-phase microextraction to liquid chromatography. A review

Solid-phase microextraction (SPME) is a technique for extraction of organic compounds from gaseous, aqueous, and solid matrices. SPME is rapid and simple, ideal for automation and for in situ measurements, and no harmful solvents are needed. The principle of SPME involves equilibration of the analytes between the sample matrix and an organic polymeric phase coated on a fused-silica fiber. SPME is traditionally combined with analysis by gas chromatography (GC) and this combination has proved sensitive, accurate, and precise for quantitative analysis of different classes of volatile compound. More recently SPME has been coupled with liquid chromatography to widen its range of application to non-volatile and thermally unstable compounds also. This article reviews the status of SPME coupled with liquid chromatography. It focuses on different applications of the technique, e.g. environmental samples, biological fluids, and food samples, to show that SPME-HPLC has great potential in the analysis of a wide range of compounds in different matrices.     

High-performance liquid chromatography with nuclear magnetic resonance detection applied to organosilicon polymers. Part 2. Comparison with other methods

LC-NMR utilizing (1)H and (29)Si NMR spectroscopy is ideally suited for the analysis of silicones. It is shown that reversed phase gradient LC-NMR surpasses standard gel permeation chromatography (GPC) and diffusion ordered spectroscopy (DOSY) in the analysis of model hydride terminated polydimethylsiloxane. (1)H and (29)Si NMR in the stopped-flow arrangement leads to full identification of the components. Concentration gradient introduces a dependence of the (29)Si shifts on solvent composition, this dependence can be substantially reduced by a proposed method of referencing. It is shown that the ADEQUATE version of powerful but insensitive 2D INADEQUATE experiment can be used for complete line assignment.     

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.     

A comparison of new techniques for the detection and determination of triazine herbicides separated by thin-layer chromatography. III

Summary Various factors affecting the fluorescence quenching properties ofs-triazines on fluorescing silicagel layers have been investigated. An analytical method has been developed which permits the quantitative analysis of these triazines, after Chromatographic separation, with an accuracy of ±5%. The instrumental detection limit for atrazine, based on a 21 signal to background ratio, ranges from 0.40 to 0.25g per spot depending on the type of silicagel used. The method compares favourably with three other techniques used for the determination of identical samples.

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Zusammenfassung Verschiedene Umstände, die die fluoreszenzlöschenden Eigenschaften vons-Triazinen gegenüber fluoreszierenden Kieselgelschichten beeinflussen, wurden untersucht. Eine Methode zur Bestimmung dieser Triazine nach deren chromatographischer Trennung mit ±5% Genauigkeit wurde ausgearbeitet. Die instrumentelle Erfassungsgrenze für Atrazin mit einem Signal-Untergrundverhältnis von 2l liegt zwischen 0,40 und 0,25g pro Fleck je nach der Art des verwendeten Kieselgels. Das Verfahren ist besser als drei andere, zur Analyse identischer Proben angewandte Methoden.

     

A comparison of new techniques for the detection and determination of triazine herbicides separated by thin-layer chromatography

Summary The use of the liquid scintillation counting method for the determination of¹⁴C labelled ametryne on thin-layer chromatograms has been thoroughly investigated. Aging, quenching effect of carrier and adsorbents as well as temperature stability of the compound in the adsorbed state have been studied. The method has a reproducibility of 1 to 2% and a detection limit of 0.005g per spot at a 3 1 signal to background ratio. Fluorescence quenching was used for the location of spots on the chromatogram. Recovery studies were carried out by this method. After 30 minutes development on a chromatoplate coated with silicagel, about 90% of the ametryne was recovered independent of solvent system used. About 75% of the ametryne was recovered from a two-dimensional chromatogram. Recoveries of ametryne from water and soil samples ranged between 78 and 88%.

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Zusammenfassung Die Flüssigkeits-Szintillationszählung zur Bestimmung von¹⁴C-markiertem Ametryn in Dünnschichtchromatogrammen wurde eingehend geprüft. Alterung, Löschungseffekt von Trägersubstanz und Adsorptionsmitteln, Temperaturbeständigkeit der Verbindung in adsorbiertem Zustand wurden untersucht. Die Reproduzierbarkeit beträgt 1 bis 2%, die Nachweisgrenze 0,005g pro Tüpfel, das Signal-Hintergrund-Verhältnis 31. Zur Lokalisierung der Flecken wurde die Fluoreszenzlöschung herangezogen. Die Ausbeute nach 30 Minuten Entwicklung auf Silikagelplatten war ungefähr 90% Ametryn, unabhängig vom Laufmittel. Bei zweidimensionaler Arbeitsweise wurden 75% wiedergefunden. Die Ausbeute aus Wasser- und Bodenproben lag zwischen 78 und 88%.

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Presented at the Pittsburgh Conference on Analytical Chemistry, Cleveland, U. S. A., March 1969.     

Microwave high performance liquid chromatography with UV-visible detection. Application to vitamins determination

The present work describes the first attempt to use microwave reversed phase high performance liquid chromatography (MW-HPLC) to carry out the separation of organic compounds. Biotin and riboflavin were selected for the characterization of the new separation technique. Additional vitamins (nicotinamide, pyridoxine and thiamine) were used as reference compounds. In order to perform the separation, a chromatographic column was placed inside a domestic microwave oven in a hanging position. The column particular location was an extremely critical point, since it precluded the actual power absorbed by the sample. In order to avoid magnetron damage, a heat well (i.e., water vessels) was used. Vitamins were detected using a UV-VIS detector. Results obtained showed that the application of microwave radiation, even at low power levels, gave rise to a significant modification in the characteristics of the chromatograms. It was found that retention times for biotin and riboflavin shortened as the power increased. Furthermore, the peak shape also changed, with the modification being more significant for the former vitamin than for the latter one. Furthermore, sensitivity also increased as the column was exposed to the action of microwave. Comparatively speaking, MW-HPLC was more efficient in terms of compound separation than when performed at room temperature or thermostatted at 45 °C HPLC. This was likely due to the combined action of a moderate and quick heating of the mobile phase with an increase in the analytes diffusivity caused by the radiation.     

Determination of biologically active low-molecular-mass thiols in human blood. III. Highly sensitive narrow-bore isocratic reversed-phase high-performance liquid chromatography with fluorescence detection

The fast isocratic narrow-bore reversed-phase high-performance liquid chromatographic method employing fluorescence detection is described for the precise reproducible simultaneous measurement of total homocysteine, cysteine and glutathione in human blood. Sample preparation involves conversion of disulfides to free thiols with triphenylphosphine, precipitation of proteins with sulfosalicylic acid, and conjugation of thiols with monobromobimane. Optimized sample preparation conditions as well as chromatographic conditions allowed to obtain reliable quantitative results within the concentration range corresponding to the levels of these thiols in human blood in norm and pathology. The detection limit was approximately 70 amol for all labeled aminothiols. The proposed method for these compounds analysis includes simple sample preparation, high selectivity, good linearity (r2>0.998), high reproducibility (within-run precision for derivatized aminothiol peaks area RSD<1.8% for three times consequently injected sample); high reliability and the small sample volume (1 microl) required for analysis make it suitable for clinical studies.