Thin layer HPLC detector

Neurochemicals in tissue samples (subpicomole levels) HPLC strong cation exchangers thin layer electrochemical detector Christensen and Le Roy Blank [417]... 

Thin-layer chromatography (TLC) is used both for characterization of alcohol sulfates and alcohol ether sulfates and for their analysis in mixtures. This technique, combined with the use of scanning densitometers, is a quantitative analytical method. TLC is preferred to HPLC in this case as anionic surfactants do not contain strong chromophores and the refractive index detector is of low sensitivity and not suitable for gradient elution. A recent development in HPLC detector technology, the evaporative light-scattering detector, will probably overcome these sensitivity problems. 

Bergstrom et al. [63] used HPLC for determination of penicillamine in body fluids. Proteins were precipitated from plasma and hemolyzed blood with trichloroacetic acid and metaphosphoric acid, respectively, and, after centrifugation, the supernatant solution was injected into the HPLC system via a 20-pL loop valve. Urine samples were directly injected after dilution with 0.4 M citric acid. Two columns (5 cm x 0.41 cm and 30 cm x 0.41 cm) packed with Zipax SCX (30 pm) were used as the guard and analytical columns, respectively. The mobile phase (2.5 mL/min) was deoxygenated 0.03 M citric acid-0.01 M Na2HP04 buffer, and use was made of an electrochemical detector equipped with a three-electrode thin-layer cell. The method was selective and sensitive for mercapto-compounds. Recoveries of penicillamine averaged 101% from plasma and 107% from urine, with coefficients of variation equal to 3.68 and 4.25%, respectively. The limits of detection for penicillamine were 0.5 pm and 3 pm in plasma and in urine, respectively. This method is selective and sensitive for sulfhydryl compounds. 

The same can be said for the sections concerning the instrumental techniques of GC, IR, NMR, and HPLC. The chromatographic techniques of GC and HPLC are presented as they relate to thin-layer and column chromatography. The spectroscopic techniques depend less on laboratory manipulation and so are presented in terms of similarities to the electronic instrumentation of GC and HPLC techniques (dual detectors, UV detection in HPLC, etc.). For all techniques, the emphasis is on correct sample preparation and correct instrument operation. 

Because polyphenolics show chemical complexities and similar structures, isolation and quantification of the individual polyphenolic compounds have been challenging. Many traditional techniques (paper chromatography, thin-layer chromatography, column chromatography) have been used. HPLC, with its merits of exacting resolution, ease of use, and short analysis time, has the further advantage that separation and quantification occur simultaneously. A reversed-phase HPLC apparatus equipped with a diode array detector makes possible the easy isolation and separation of many polyphenolics. For enhanced performance of HPLC separation, the polyphenolics should first be isolated into several fractions to effectively separate the individual polyphenolics (Jaworski and Lee, 1987 Oszmianski and Lee, 1990). 

Whether eluted from columns or from thin-layer plates, the quantitative determination of sugars was traditionally based on colorimetric reactions involving the use of chemical reagents, e.g., anthrone. These detection methods have been largely replaced in modem HPLC by the refractive index detector, although ultraviolet detectors are also employed. Recently we have also seen the introduction of other types of detector (e.g., the mass detector), as will be discussed later. 

HPLC-based electrochemical detection (HPLC-ECD) is very sensitive for those compounds that can be oxidized or reduced at low voltage potentials. Spectrophotometric-based HPLC techniques (UV absorption, fluorescence) measure a physical property of the molecule. Electrochemical detection, however, measures a compound by actually changing it chemically. The electrochemical detector (ECD) is becoming increasingly important for the determination of very small amounts of phenolics, for it provides enhanced sensitivity and selectivity. It has been applied in the detection of phenolic compounds in beer (28-30), wine (31), beverages (32), and olive oils (33). This procedure involves the separation of sample constituents by liquid chromatography prior to their oxidation at a glassy carbon electrode in a thin-layer electrochemical cell. 

Because of this concern, a variety of methods have been developed for the determination of NOC in foods. These include thin-layer chromatographic (TLC) (11,12), gas chromatographic (GC) (13-15), GC-mass spectrometric (GC-MS) (15-17), and high-performance liquid chromatographic (HPLC) (18-20) techniques. The purpose of this article is to review various HPLC methods developed for this purpose. Unfortunately, however, only limited advances have been made in this area, mainly because of the lack of sensitive and specific detectors. Most published methods for NOC reported to date are GC-based techniques. Therefore, this review will be a brief one and will emphasize the most recent HPLC developments. 

Note TLC, thin-layer chromatography HPLC, high-performance liquid chromatography GLC or GC, gas-liquid chromatography AA, atomic adsorption NPD, nitrogen phosphorus detector FPD, flame photometric detector GC/MS, gas chromatography/mass spectrometry. 

Notes LOD, limit of detection MeOH, methanol EtOH, ethanol ACN, acetonitrile EtAC, ethyl acetate SPE, solid phase extraction HLB (hydrophilic lipophilic balanced) TFA, trifluoroacetic acid GC, gas chromatography TMS, trimethylsilyl MS, mass spectrometry HPLC, high-performance liquid chromatography DAD, diode array detector NMR, nuclear magnetic resonance ESI, electrospray ionization APCI, atmospheric pressure chemical ionization CE, capillary electrophoresis ECD, electrochemical detector CD, conductivity detector TLC, thin layer chromatography PDA, photodiode array detector. 

Likewise, the luminescence properties of many analytes can be altered in the presenoe of surfactant aggregates (4,7.,8.). Consequently, addition of micelle-forming surfactants (present either in the LC mobile phase or added post-column) can improve the sensitivity of fluorimetric LC detectors (49,482). Micellar spray reagents have been utilized to enhance the fluorescence densitometric detection of dansylamino acids or polycyclic aromatic hydrocarbons (483). The effect was observed for TLC performed on cellulose or polyamide stationary phases with the micellar spray reagent being either CTAC, SB-12, or NaC (483). More recently, use of nonionic Triton X-100 has been found to improve the HPLC detection of morphine by fluorescence determination after post-column derivatization (486) as well as improve the N-chlorination procedure for the detection of amines, amides, and related compounds on thin-layer chromatograms (488). 

Analytical thin-layer chromatography on silica gel indicated some separation with n-hexane or with n-pentane as eluents, but not with cyclohexane. Analytical HPLC performed with hexanes (S-gm Econosphere silica. Alltech/Appiied Science) gave a satisfactory separation (retention times 6.64 and 6.93 min for 50 and C70, respectively, at a flo.w rate of 0.5 mL/mln detector wavelength, 256 nm), indicating the content of C70 to be approximately 15% for the Los Angeles samples. Two other minor peaks, possibly other unidentified C species, were observed (retention times 5.86 and 8.31 min), but they constituted less than 1.5% of the total mass. 

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