Phenolic compounds HPLC-fluorescence

The variety of detection modes available for HPLC analysis that provide additional information about the eluent as it exits the column greatly facilitates unknown characterization. The majority of analytical methods for phenolic compounds includes HPLC with spectrophotometric-based detection techniques (UV absorption, fluorescence, photo diode array—PDA) as well as HPLC with electrochemical detection. 

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. 

Fluorescence (FL) detection is another selective and sensitive technique. Vitamin E, a-, /3-, S-, and y-to-copherols have been analyzed by RP-HPLC using FL as well as UV detection. In addition, a newer technique, electrochemical detection, has been reported in the analysis of phenolic compounds of olive oil [7]. 

The detection and identification of phenolic compounds, including phenolic acids, have also been simph-fied using mass spectrometry (MS) techniques on-hne, coupled to the HPLC equipment. The electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) interfaces dominate the analysis of phenohcs in herbs, fmits, vegetables, peels, seeds, and other plants. In some cases, HPLC, with different sensitivity detectors (UV, electrochemical, fluorescence), and HPLC-MS are simultaneously used for the identification and determination of phenolic acids in natural plants and related food products.In some papers, other spectroscopic instmmental techniques (IR, H NMR, and C NMR) have also been apphed for the identification of isolated phenolic compounds. 

HPLC with TSP-MS and ISP-MS detection methods was used to identify phenolic glycoside components of olive leaves, such as oleuropein (40), directly from crude extracts . Phenolic compounds in extracts from freeze-dried olives were cleaned up by SPE and subsequently analyzed by HPLC with both fluorescence and ESI-MS detection. Oleuropein (40) was the major phenolic component in the fruit. ... 

Microspectrofluorometry was employed for mapping the location of phenolic substances in maize kernels. Autofluorescence due to phenolic acids was detected mainly in the embryo, aleurone and pericarp of maize kernel cross sections. Boric acid (H3BO3) reagent enhanced the fluorescence due to flavonoids in the aleurone layer. The amides of phenolic acids required derivatization with Ehrlich s reagent (168) to reveal fluorescence in the embryo and aleurone. The localization of phenolic amines was conflrmed by HPLC analysis. Phenolic compounds are important in the resistance of maize kernels to pests. Resistant maize types showed higher intensities of phenolic fluorescence but no unusual distributions of these compounds. ... 

A logical step would be HPLC separation of the extracts and measurement of the native fluorescence after chromatography. Enhanced sensitivity in the detection of the individual phenols may be achieved by chromatography of dansyl derivatives (Cassidy et al., 1974) in which case ng quantities of phenolic compounds were estimated in physiological fluids. 

LC in conjunction with fluorescence detection has been used to improve the sensitivity and selectivity for the determination of phenolic compounds in water. All the different techniques for the determination of phenolic compounds in water that use HPLC and fluorescence detection are summarized in Table 16.3. 

Fluorescence Detection with HPLC Separation for Determination of Phenolic Compounds in Different Water Matrices... 

Air is drawn through a midget impinger or a bubbler containing 0.1 ANaOH solution. Phenol and ciesols are trapped as phenolates. The pH of the solution is adjusted <4 by H2S04. The compounds are determined by reverse-phase HPLC with UV detection at 274 nm. An electrochemical or fluorescence detector may also be used. The solution may be analyzed by colorimetric or GC-FID technique. 

The answer lies in the nature of the chemical compounds that are of interest. For example, there is little difference in HPLC detector sensitivity between UV absorption and fluorescence emission for phenol, but a great difference exists for naphthalene. Naphthalene is a fused aromatic and allows for extensive delocalization of electrons and is quite sensitive when measured by RP-HPLC-FL. For both analytes of environmental concern being dissolved in water or dissolved in the RP-HPLC mobile-phase, a vivid illustration of this difference in HPLC detector sensitivity is shown in Fig. 4.56. A RP-HPLC-UV chromatogram is shown at the top and a RP-HPLC-FL chromatogram is shown at the bottom. The chromatograms are stacked and both the ordinate and abscissa axes are aligned. Effluent from a reversed-phase column was fed to the Model 2478 UV absorption detector and the effluent from this detector was made the influent to the Model 474 fluorescence detector as shown in... 

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