Fluorescence column

Detection in CC may be visually for coloured compounds. Different methods can be used to monitor colourless compounds (collecting fractions addition of an inorganic phosphor to the column adsorbent). The detector choice is quite limited, with UV, RI and molecular fluorescence (F) emission being the most popular. A fluorescent column adsorbent is extremely... 

Figure Cl.5.14. Fluorescence images of tliree different single molecules observed under the imaging conditions of figure Cl.5.13. The observed dipole emission patterns (left column) are indicative of the 3D orientation of each molecule. The right-hand column shows the calculated fit to each observed intensity pattern. Molecules 1, 2 and 3 are found to have polar angles of (0,( ))=(4.5°,-24.6°), (-5.3°,51.6°) and (85.4°,-3.9°), respectively. Reprinted with pennission from Bartko and Dickson [148]. Copyright 1999 American Chemical Society.

Purification of anthracene. Dissolve 0-3 g. of crude anthracene (usually yellowish in colour) in 160-200 ml. of hexane, and pass the solution through a column of activated alumina (1 5-2 X 8-10 cm.). Develop the chromatogram with 100 ml. of hexane. Examine the column in the hght of an ultra-violet lamp. A narrow, deep blue fluorescent zone (due to carbazole, m.p. 238°) will be seen near the top of the column. Immediately below this there is a yellow, non-fluorescent zone, due to naphthacene (m.p. 337°). The anthracene forms a broad, blue-violet fluorescent zone in the lower part of the column. Continue the development with hexane until fluorescent material commences to pass into the filtrate. Reject the first runnings which contain soluble impurities and yield a paraffin-hke substance upon evaporation. Now elute the column with hexane-benzene (1 1) until the yellow zone reaches the bottom region of the column. Upon concentration of the filtrate, pure anthracene, m.p. 215-216°, which is fluorescent in dayhght, is obtained. The experiment may be repeated several times in order to obtain a moderate quantity of material. 

Despite their importance, gas chromatography and liquid chromatography cannot be used to separate and analyze all types of samples. Gas chromatography, particularly when using capillary columns, provides for rapid separations with excellent resolution. Its application, however, is limited to volatile analytes or those analytes that can be made volatile by a suitable derivatization. Liquid chromatography can be used to separate a wider array of solutes however, the most commonly used detectors (UV, fluorescence, and electrochemical) do not respond as universally as the flame ionization detector commonly used in gas chromatography. 

Detectors Most of the detectors used in HPLC also find use in capillary electrophoresis. Among the more common detectors are those based on the absorption of UV/Vis radiation, fluorescence, conductivity, amperometry, and mass spectrometry. Whenever possible, detection is done on-column before the solutes elute from the capillary tube and additional band broadening occurs. 

The analysis of cigarette smoke for 16 different polyaromatic hydrocarbons is described in this experiment. Separations are carried out using a polymeric bonded silica column with a mobile phase of 50% v/v water, 40% v/v acetonitrile, and 10% v/v tetrahydrofuran. A notable feature of this experiment is the evaluation of two means of detection. The ability to improve sensitivity by selecting the optimum excitation and emission wavelengths when using a fluorescence detector is demonstrated. A comparison of fluorescence detection with absorbance detection shows that better detection limits are obtained when using fluorescence. 

Pressure. Standard atmospheric pressure is defined to be the force exerted by a column of mercury 760-mm high at 0°C. This corresponds to 0.101325 MPa (14.695 psi). Reference or fixed points for pressure caUbration exist and are analogous to the temperature standards cited (23). These points are based on phase changes or resistance jumps in selected materials. For the highest pressures, the most rehable technique is the correlation of the wavelength shift, /SX with pressure of the mby, R, fluorescence line and is determined by simultaneous specific volume measurements on cubic metals... 

The most widely appHed colorimetric assay for amino acids rehes upon ninhydrin-mediated color formation (129). Fluorescamine [38183-12-9] and (9-phthalaldehyde [643-79-8] are popular as fluorescence reagents. The latter reagent, ia conjunction with 2-mercaptoethanol, is most often used ia post-column detection of amino acids separated by conventional automated amino acid analysis. More recently, determiaation by capillary 2one electrophoresis has been developed and it is possible to determine attomole quantities of amino acids (130). 

Post-column in-line photochemical derivatization permits fluorescence detection of the common aflatoxins Bl, B2, Gl, and G2 (60). Chromatographic evidence indicates that photolysis causes the hydration of the nonfluorescent Bl and Gl components to B2a and G2a components, respectively. Analysis of naturally contaminated com samples show no interfering peaks and permits the deterrnination of 1 and 0.25 ppb for Bl and B2, respectively. 

For more specific analysis, chromatographic methods have been developed. Using reverse-phase columns and uv detection, hplc methods have been appHed to the analysis of nicotinic acid and nicotinamide in biological fluids such as blood and urine and in foods such as coffee and meat. Derivatization techniques have also been employed to improve sensitivity (55). For example, the reaction of nicotinic amide with DCCI (AT-dicyclohexyl-0-methoxycoumarin-4-yl)methyl isourea to yield the fluorescent coumarin ester has been reported (56). After separation on a reversed-phase column, detection limits of 10 pmol for nicotinic acid have been reported (57). 

More specific methods involve chromatographic separation of the retinoids and carotenoids followed by an appropriate detection method. This subject has been reviewed (57). Typically, hplc techniques are used and are coupled with detection by uv. For the retinoids, fluorescent detection is possible and picogram quantities of retinol in plasma have been measured (58—62). These techniques are particularly powerful for the separation of isomers. Owing to the thermal lability of these compounds, gc methods have also been used but to a lesser extent. Recently, the utiUty of cool-on-column injection methods for these materials has been demonstrated (63). 

There have been compared the methods of mycotoxin control in food products with aflatoxin as an example, using both HPLC method with fluorescent detecting on the apparatus Thermo FL 3000 with a column BDS Hypersil C 2.1x150, as well as a chromatodensitometry method on the apparatus CAM AG TLS Scanner 3. 

Confirmation of the formation of the radicals during combustion reactions has been made by inuoducing a sample of dre flames into a mass spectrometer. The sample is withdrawn from a turbulent flame which is formed into a thin column, by admitting a sample of the flame to the spectrometer drrough a piidrole orifice, usually of diameter a few tenths of a millimetre. An alternative procedure which has been successful in identifying the presence of radicals, such as CHO, has been the use of laser-induced fluorescence. 

Diaminoanthraquinone [131-14-6] M 238.3, m 310-320°. Crystd from pyridine. Column-chromatographed on AI2O3 / toluene to remove a fluorescent impurity, then recrystd from EtOH. 

Indeed, great emphasis was placed on the presentation of compounds in crystalline form for many years, early chromatographic procedures for the separation of natural substances were criticized because the products were not crystalline. None the less, the invention by Tswett (3) of chromatographic separation by continuous adsorption/desorption on open columns as applied to plant extracts was taken up by a number of natural product researchers in the 1930s, notably by Karrer (4) and by Swab and lockers (5). An early example (6) of hyphenation was the use of fluorescence spectroscopy to identify benzo[a]pyrene separated from shale oil by adsorption chromatography on alumina. 

EC, electrochemical detection Flu, fluorescence detection MS, mass specu-omeu-ic detection pre-Flu, fluorescence detection after pre-column derivatization post-Flu, fluorescence detection after post-column derivatization UV, UV absorbance detection. 

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