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.     

The applicability of fluorescence line-narrowing spectroscopy in combination with thin-layer chromatography

Laser excitation at low temperatures of compounds on thin-layer chromatographic plates yields, also after elution, highly resolved spectra. The fluorescence line-narrowed spectra permit identification of spectroscopically alike compounds, as is demonstrated for pyrene and halogenated pyrenes. Detection limits are in the low nanogram region even with relatively simple instrumentation. It is shown that quantitative application is possible if an internal standard is used.     

Persistent spectral hole-burning effects in fluorescence line-narrowing spectroscopy. Analytical implications

Persistent spectral hole-burning (PSHB) effects on fluorescence line-narrowing (FLN) spectra of tetracene in glassy and polymer matrices have been studied with emphasis on analytical implications. Attention has been paid to the time-evolution of the FLN spectrum under prolonged laser irradiation as well as to the matrix, laser intensity, temperature and excitation wavelength dependence of hole-burning effects. It is shown that PSHB, that generally is an unwanted phenomenon in analytical FLN spectroscopy, can be reduced or completely removed by using a suitable matrix material, by reducing the laser intensity, by working at somewhat elevated temperatures and by employing excitation to the vibronic region of the analyte.     

Temperature effects on fluorescence line narrowing spectra of tetracene in amorphous solid solutions. Analytical implications

Temperature effects in the highly specific vibrationally resolved fluorescence line-narrowing (FLN) spectra of tetracene as a model compound have been investigated with emphasis on analytical implications.Contrary to what is generally observed in conventional solid state methods, in FLN when excitation in the 0-0 region is employed, the lowest temperatures in most cases do not produce the largest peak heights and intensities. For excitation in the vibronic S₁ region the conventional behaviour is observed. The analytically useful temperature interval in FLN spectroscopy generally is more limited than in techniques that use crystalline matrix materials.The spectral bands in FLN have a predominantly Lorentzian shape for excitation in the 0-0 region in our experimental set-up where, at the temperatures used, the excitation source does not contribute significantly to the lineshape. For excitation in the vibronic S₁ region a stronger Gaussian contribution to the lineshape is observed. No temperature dependent shift of the narrow lines occurs, contrary to what is generally found in the conventional solid state methods. In addition, the position of the lines in FLN is not solvent dependent.To explain these observations it is crucial to realize that in FLN, high resolution is achieved via selective excitation, so that the spectral emission features are determined by the excitation as well as the emission process.     

Preparation and spectral characteristics of anthracene/tetracene mixed crystals

A series of tetracene-doped anthracene crystals with different doping concentrations (the highest molar ratio 100 1) are grown from solution. Crystal structures and optical characteristics of the above mixed crystals are investigated at room temperature. By changing the doping concentrations, the fluorescence can be adjusted from blue-green to green and even to yellow-green. The emission spectra of anthracene/tetracene (An/Te) mixed crystals reveal the sensitized fluorescence of tetracene and the partial quenching of anthracene emission. The data of transient photoluminescence (PL) decays illustrate that in An/Te mixed crystals, the decay of anthracene becomes faster, while the PL lifetime of tetracene is longer than that of the tetracene single crystals. All above experimental results suggest that there is excitation energy transfer from anthracene to tetracene in the mixed crystals. Supported by the National Natural Science Foundation of China (Grant No. 5057 3039) and the National Key Basic Research and Development Program of China (Grant No. 2006CB806200)     

A VARIABLE TEMPERATURE, U.V. LUMINESCENCE SPECTROGRAPH FOR SMALL SAMPLES

Abstract— A variable temperature spectrometer suitable for recording phosphorescence, fluorescence and luminescence spectra is described. The instrument has been chiefly used for emission spectroscopy at temperatures between 80° and 370°K but may be used for excitation spectroscopy as well. The samples are contained in quartz tubes with inside diameters of 1.5 mm and are generally optically thick at the excitation wavelength. The sample volume can be as small as 10μl. Absolute quantum yields may be obtained with ease and the use of phase sensitive detection makes it possible to record in 30 sec a fluorescence spectrum with 30 Å resolution and a quantum yield of 10^--⁴ with a signal to noise ratio of 20. The instrument has also been useful for photochemical irradiations.     

Highly-resolved fluorescence spectrometry of pyrene on a thin-layer chromatographic plate

Site-selection spectroscopy, by using suitable laser line excitation, gives epectra of molecules on thin-layer chromatographic plates comparable in quality to Shpol'skii spectra. Pyrene is investigated as a model compound.     

Comparison of the column performance of narrow-bore and standard-bore columns for the chromatographic determination of alpha-, beta-, gamma-, and delta-tocopherol

A comparison of the performance of narrow-bore (2.1-mm i.d.) and standard-bore (4.6-mm i.d.) analytical silica columns having the same length is completed for the resolution of alpha-, beta-, gamma-, and delta-tocopherol. The studies are performed on high-performance liquid chromatographic equipment with minimum extracolumn contribution. Column permeabilities are 1.16 x 10(-9) and 2.48 x 10(-9) cm2 for narrow and standard bore, respectively. The narrow-bore column gives up to a 7 times increase in sensitivity compared with a standard-bore column at equivalent running times for the analytes. Approximately one-third solvent savings can be achieved with the narrow-bore column. Theoretical plates of the standard-bore column are higher than that of the narrow-bore column.     

Determination of brodimoprim and its hydroxy metabolite in human plasma, blood and urine by high-performance liquid chromatography.

A high-performance liquid chromatographic method was developed to measure the concentration of brodimoprim and its metabolite, hydroxybrodimoprim, in small volumes of blood, plasma and urine. The procedure involved a simple extraction step with chloroform, followed by chromatographic separation on a short reversed-phase column deactivated for the analysis of basic compounds. The column effluent was monitored by fluorescence (excitation wavelength 290 nm, emission wavelength 340 nm). The recoveries of both compounds were similar in all three biological fluids, and averaged 84 and 72%, respectively. The detection limit for both compounds reached 5 ng/ml. No endogenous compound interfered in the assay. The linearity of the method and its within- and between-day precision were analytically satisfactory.     

A multivariate chemometric approach to fluorescence spectroscopy

A multivariate approach to the solution of problems often encountered in the spectrofluorometry of natural samples, utilising information from whole spectra is presented. (a) Piecewise direct standardisation is implemented and employed to transfer emission spectra measured with two different xenon lamps of different ages as if the spectra were measured with the same lamp. (b) It has been shown using a multivariate analysis approach that it is possible to use the raw data points instead of the smoothed data based on an algorithm included in the instrument software by the manufacturer. (c) It is documented that Raman scattering does not hamper the performance of multivariate calibration; on the contrary, in an experiment with sugar samples the concentration prediction errors become about five times lower by including the whole emission spectrum in the analysis instead of using a univariate calibration based on an emission wavelength that only reflects the analyte of interest. (d) An algorithm for variable selection is implemented and employed in the selection of optimal excitation wavelengths. Among 13 emission spectra recorded for a sugar sample at different excitation wavelengths, four of these are chosen that describe 98.51% of the total variance in the original data. (e) Finally the combination of fluorescence spectroscopy and multivariate calibration with conventional chemical data according to the near-infrared black box model is presented. The refined sugar quality parameter, the ash content and the fluorescence emission spectra are correlated by a partial least-squares regression model. Five experiments employing different monochromator slit widths and sugar concentrations are performed, and the best correlation obtained by full cross-validation of the 15 sugar samples is R = 0.98.     

Ultra-thin-layer chromatography mass spectrometry and thin-layer chromatography mass spectrometry of single peptides of angiotensin-converting enzyme inhibitors

The separation of structurally related angiotensin-converting enzyme (ACE) inhibitors lisinopril, cilazapril, ramipril and quinapril and their corresponding active diacid forms (prilates) by conventional TLC silica gel 60 plates was contrasted with that afforded by monolithic ultra-thin-layer chromatographic (UTLC) plates. For the use of UTLC plates technical modifications of the commercially available equipments for the sample application, development and detection were made. Plates were developed in modified horizontal developing chamber using ethyl acetate-acetone-acetic acid-water (4:1:0.25:0.5, v/v). Detection of the separated compounds was performed densitometrically in absorption/reflectance mode at 220 nm and after exposure to iodine also by image analysis. The obtained results showed that monolithic layer is more efficient for the separation of structurally similar polar compounds, such as prilates than conventional silica layers. Identification of the compounds was confirmed by ESI-MS after their on-line extraction from the UTLC and TLC plates by means of Camag TLC-MS interface.     

Low-Energy Electronic Excitations of N-Substituted Heteroacene Molecules: Matrix Isolation Spectroscopy in Concert with Quantum-Chemical Calculations

N-Heteropolycycles are attractive as materials in organic electronic devices. However, a detailed understanding of the low-energy electronic excitation characteristics of these species is still lacking. In this work, the matrix isolation technique is applied to obtain high-resolution absorbance spectra for a series of tetracene and core-substituted N-analogues. The experimental electronic excitation spectra obtained for matrix-isolated molecules are then analysed with the help of quantum-chemical calculations. Additional lower energy excitation bands in the spectrum of the core-substituted N-derivatives of tetracene could be explained in terms of intensity borrowing from dipole-forbidden transitions due to Herzberg–Teller vibronic coupling. In the case of tetracene, evidence for the additional formation of London dimers (J aggregates) is found at higher tetracene concentrations in the matrix.     

A fluorescence detector for high-pressure liquid chromatography

We describe a fluorescence spectrophotometer adapted with a micro quartz flow cell to record the output of modern liquid chromatographs. The optical system is double beam in that the light source variations are cancelled out by a second photomultiplier, thus enhancing the sensitivity of the technique. The emission spectra may be scanned by stopping the flow in the chromatographic column and scanning the fluorescence detector. Many specific applications have been studied: polycyclic aromatic hydrocarbons, several vitamins, porphyrins, methyl anthranilate, etc. These are studied in natural samples and it is shown that the specificity of the fluorescence detector frequently obviates the need for sample preparation. The sensivitity available with the fluorescence detector for fluorescing compounds is often much greater than is available with variable-wavelength ultraviolet spectrophotometers. We report picogram-level detectability in real samples for many of the compounds that we have studied.     

Capillary electrophoresis of urinary porphyrins with absorbance and fluorescence detection

Urinary porphyrins are separated in a 72 cm x 50 microns I.D. fused-silica capillary by micellar electrokinetic capillary chromatography with 100 mM sodium dodecyl sulfate and 20 mM 3-(cyclohexylamino)-1-propanesulfonic acid at pH 11. Detection is accomplished by absorbance at 400 nm or fluorescence with excitation at 400 nm and emission at wavelengths above 550 nm. Substantial trace enrichment is found for porphyrins in urine samples or for porphyrin standards prepared without surfactant in the injection buffer. Limits of detection are in the 100 pmol/ml concentration range with an optimized fluorescence system. The method is shown suitable for the determination of porphyrins in clinical urine specimens. Comparisons are made between electrophoretic and chromatographic methods for the separation and detection of urinary porphyrins.     

Size-exclusion chromatography with on-line ultraviolet, proton nuclear magnetic resonance and mass spectrometric detection and on-line collection for off-line Fourier transform infrared spectroscopy.

The coupling of HPLC with UV detection and on-line NMR spectroscopy and mass spectrometry combined with a dedicated interface for the collection of the chromatographic eluent for subsequent Fourier transform (FT) IR has been investigated using a number of polymer additives as model compounds. Size-exclusion chromatography was performed using deuterated chloroform as eluent with the separation monitored on-line by UV detection at 254 nm and on-flow 1H-NMR and MS. The effluent from the NMR probe was directed to a dedicated HPLC interface where it was deposited on a germanium plate for subsequent FT-IR. NMR and MS spectra were successfully obtained for 2,6-di-tert.-butyl-4-methylphenol, octadecyl-3-(3,5-di-tert.-butyl-4-hydroxyphenyl) propionate (Irganox 1076) and diisooctyl phthalate on-line and FT-IR spectra for all three compounds were obtained off-line. Practical problems encountered with this multiple hyphenation are described.     

Videofluorometric Analysis of Aging Human Collagen

Abstract

The development of fluorescent pigments in aging human collagen has been observed, but neither the source of these compounds nor their nature has been described. Recently two distinct fluorophores were isolated from aging insoluble human collagen rich tissue following a sequence of proteolytic digestions and chromatographic separations. Using the videofluormeter, which monitors the fluorescence intensity of a sample as a function of several excitation and emission wavelengths, the fluorescence of the collagen rich tissue at various stages of the separation process was analyzed to determine the number of fluorescent components in each of the samples and estimate their fluorescence spectra. The analysis indicated that the isolated fluorophores were indeed single-component samples and that the insoluble collagen-rich fraction contains two major fluorophores whose spectra are consistent with the spectra of the isolated compounds.     

Laser-excited fluorescence line-narrowing: an analytical study

The limits of detection obtained by fluorescence line-narrowing spectroscopy for 6 polynuclear aromatic hydrocarbons are compared with those obtained by low-temperature and room-temperature fluorescence, and the merits of the technique are discussed.     

Spectroscopic investigation of the solvation of organic molecules in superfluid helium droplets

The spectroscopy of molecules doped into superfluid helium nanodroplets provides valuable information on the process of solvation in superfluid helium. In continuation of an earlier report on emission spectra of various phthalocyanines showing a splitting of all molecular transitions in the range of about 5-12 cm(-1), the emission spectra of tetracene, pentacene, and perylene in superfluid helium droplets are presented. The new spectra and the results obtained for the phthalocyanines are explained by an empirical model which accounts for the existence of different metastable configurations of a nonsuperfluid solvation layer around the guest molecule.     

Photophysical properties of novel lanthanide (Tb3+, Dy3+, Eu3+) complexes with long chain para-carboxyphenol ester p-L-benzoate (L=dodecanoyloxy, myristoyloxy, palmitoyloxy and stearoyloxy)

In this paper, a series of 12 binary luminescent lanthanide coordination compounds with long chain p-carboxyphenol ester were assembled. Both elemental analysis and infrared spectroscopy allowed to determine the complexes formula: LnL3, where Ln=Tb, Dy, Eu; L=p-dodecanoyloxybenzoate (12-OBA), p-myristoyloxybenzoate (14-OBA), p-palmitoyloxybenzoate (16-OBA) and p-stearoyloxybenzoate (18-OBA), respectively. The photophysical properties of these complexes were studied in detail with various of spectroscopies such as ultraviolet-visible absorption spectra, low temperature phosphorescence spectra and fluorescent spectra. The ultraviolet-visible absorption spectra showed that some bands shift with the different chain length of p-carboxyphenol ester. From the low temperature phosphorescent emission, the triplet state energies for these four ligands were determined to be around 24,242 cm-1 (12-OBA), 24,612 cm-1 (14-OBA), 24,084 cm-1 (16-OBA) and 24,125 cm-1 (18-OBA), respectively, suggesting they are suitable for the sensitization of the above lanthanide ions, especially for Tb3+ and Dy3+. The fluorescence excitation and emission spectra for these lanthanide complexes of the four ligands take agreement with the above predict from energy match.     

RESOLVED FLUORESCENCE EMISSION SPECTRA OF IRON-FREE CYTOCHROME c

Abstract The fluorescence emission of iron-free cytochrome c (⁰Cyt c ) in a glassy matrix was investigated under conditions of very low temperature (4.2 K.) and narrow bandwidth laser excitation. Excitation into the vibronic band, Q _x (1,0) resulted in highly resolved emission spectra of linewidth 10-20 cm⁻¹. Using the model of selective excitation developed by Abram el al. (1975) and McColgan et al. (1978), the emission spectra of vibronic excitation afforded a method to investigate excited state vibrational structure. Furthermore, emission profiles have shown that in ⁰Cyt c , the site distribution (inhomogeneous broadening) has a width in the order of 200cm⁻¹.