Detection limits molecular fluorescence

For an analyte of molecular weight 5000 and good chromatographic conditions, most photometric detectors can be expected to provide detection limits of 2—5 ng. Improvement into the mid-picogram or lower range normally requires the use of more sensitive detection means such as fluorescence or electrochemical detectors. 

The dye is excited by light suppHed through the optical fiber (see Fiber optics), and its fluorescence monitored, also via the optical fiber. Because molecular oxygen, O2, quenches the fluorescence of the dyes employed, the iatensity of the fluorescence is related to the concentration of O2 at the surface of the optical fiber. Any glucose present ia the test solution reduces the local O2 concentration because of the immobilized enzyme resulting ia an iacrease ia fluorescence iatensity. This biosensor has a detection limit for glucose of approximately 100 ]lM , response times are on the order of a miaute. 

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... 

Raman spectroscopy has enjoyed a dramatic improvement during the last few years the interference by fluorescence of impurities is virtually eliminated. Up-to-date near-infrared Raman spectrometers now meet most demands for a modern analytical instrument concerning applicability, analytical information and convenience. In spite of its potential abilities, Raman spectroscopy has until recently not been extensively used for real-life polymer/additive-related problem solving, but does hold promise. Resonance Raman spectroscopy exhibits very high selectivity. Further improvements in spectropho-tometric measurement detection limits are also closely related to advances in laser technology. Apart from Raman spectroscopy, areas in which the laser is proving indispensable include molecular and fluorescence spectroscopy. The major use of lasers in analytical atomic... 

For the cationic surfactants, the available HPLC detection methods involve direct UV (for cationics with chromophores, such as benzylalkyl-dimethyl ammonium salts) or for compounds that lack UV absorbance, indirect photometry in conjunction with a post-column addition of bromophenol blue or other anionic dye [49], refractive index [50,51], conductivity detection [47,52] and fluorescence combined with postcolumn addition of the ion-pair [53] were used. These modes of detection, limited to isocratic elution, are not totally satisfactory for the separation of quaternary compounds with a wide range of molecular weights. Thus, to overcome the limitation of other detection systems, the ELS detector has been introduced as a universal detector compatible with gradient elution [45]. 

Derivatization is never used in the analysis of the different phospholipid classes present, but is very widely used in the analysis of molecular species. The main reason for (mostly precolumn) derivatization in this case is to reduce significantly the detection limits. To this end, both UV-ab-sorbing and fluorescent derivatives are frequently used, as described in more detail in Secs. IV.B and V.3. 

Riboflavin (vitamin I 2) is an important chemical species that occurs in many complex biologically related samples. The substance is chiral and strongly fluorescent and the coupling of these molecular properties into the FDCD-HPLC detection system greatly enhances both the specificity and the limit of detection capabilities of the method. In the specific case of riboflavin an HPLC system using FDCD detection where excitation is done at 325nm with a HeCd-laser a detection limit of 170 pg of riboflavin [30] is attainable. This level is about 10 times better than what is possible with a laser excitation -transmission CD-HPLC system [29]. 

Fluorescence. The use of molecular fluorescence spectroscopy for the quantitation of enzyme reaction products has resulted in detection limits that are several orders of magnitude lower than those achieved by standard absorbance methods. At low analyte concentrations, fluorescence emission intensity is directly proportional to concentration, and its value depends on both the molar absorptivity of the analyte at the excitation wavelength, and the fluorescence quantum yield of the analyte, under the assay conditions. 

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