Native fluorescence detector

A third type of detector is the intrinsic or native fluorescence detector that utilizes native fluorescence properties of amino acids. The sensitivity of this detector is between UV/PDA and LIF detection. The advantage of this technique over pre-labeling is that there is no pre-labeling step required therefore, the sample preparation is relatively simple, and the sensitivity is improved over UV/LIF. However, the intrinsic fluorescence detection relies on the presence of Tryptophan (Try), Tyrosine (Tyr), Phenylalanine (Phe), and this detector has just become commercially available. 

Since a relatively small number of analytes of interest have native fluorescent properties, derivatization reactions are frequently employed to enable this detection technique to be extended to a broader range of compounds. This is an excellent means of increasing the detectability for a whole range of molecules, but it is important to realize that there are certain limitations. First, it is difficult to obtain quantitative yields at low analyte concentrations. This implies that in some cases, the obtainable detection limit are not limited by the detector sensitivity, but instead by low yields in the derivatization reaction. Furthermore, to shift the equilibrium toward the product side at low analyte concentrations, as much as 104 times excess of fluorescent label may be necessary. Tow concentrations of impurities in the label can be present at levels greater than the analytes of interest and as a result, numerous interfering peaks in the chromatograms may be observed. These problems are discussed in detail in Ref. 181. 

Fluorescence is not widely used as a general detection technique for polypeptides because only tyrosine and tryptophan residues possess native fluorescence. However, fluorescence can be used to detect the presence of these residues in peptides and to obtain information on their location in proteins. Fluorescence detectors are occasionally used in combination with postcolumn reaction systems to increase detection sensitivity for polypeptides. Fluorescamine, o-phthalaldehyde, and napthalenedialdehyde all react with primary amine groups to produce highly fluorescent derivatives.33,34 These reagents can be delivered by a secondary HPLC pump and mixed with the column effluent using a low-volume tee. The derivatization reaction is carried out in a packed bed or open-tube reactor. 

The use of HPLC to analyze biogenic amines and their acid metabolites is well documented. HPLC assays for classical biogenic amines such as norepinephrine (NE), epinephrine (E), dopamine (DA), and 5-hydroxytryptamine (5-HT, serotonin) and their acid metabolites are based on several physicochemical properties that include a catechol moiety (aryl 1,2-dihydroxy), basicity, easily oxidized nature, and/or native fluorescence characteristics (Anderson, 1985). Based on these characteristics, various types of detector systems can be employed to assay low concentrations of these analytes in various matrices such as plasma, urine, cerebrospinal fluid (CSE), tissue, and dialysate. 

Fluorescence detection, alone or with the aid of derivatizing reagents to enhance detector responses and improve the chromatographic resolution, has also been used for the determination of biogenic amines. Lakshmana and Trichur (1997) used native fluorescence to analyze NE, DA, and 5HT in rat brain utilizing an isocratic separation on an ODS CIS column. The detection limits reported were 100-250 pg on column. 

For coumarins in orange fruits (115), the HPLC used a Zorbax Rx C8 (250-mm X 4.6-mm ID, 5 fim) column maintained at 25°C, and analysis was performed by binary-gradient elution using 0.1% HOAc in acetonitrile (eluent A) and 0.1% HOAc in HzO (eluent B). In the author s lab, standard coumarins could be separated by isocratic elution on Zorbax Rx C8 column with acetonitrile-0.1% HOAc in water (35 65) at 1.0 ml/min, as presented in previous work (1). The eluate from the column was passed to a UV detector (UV 330 nm) and then into a fluorescence detector (excitation at 340 nm, emission at 425 nm). As for the specificity, some of the coumarins do not have native fluorescence. Nine coumarins are separated under UV 330 nm, and three coumarins could not be detected with fluorescence detection. Detailed conditions for coumarin analysis in foods and absorption spectra of coumarins obtained by online diode array detector with HPLC were presented by Lee and Widmer (1). Since coumarins exhibit strong absorption in the ultraviolet region, absorption at approximately 313 nm has been used to estimate the dilution of cold-pressed lemon oil with distilled oil (12). Analysis of umbelliferone (7-hydroxy-coumarin) and scopoletin (6-methoxy-7-hydroxycoumarin) in citrus fruits was performed using... 

The inability to detect precludes the ability to develop a separation. The selected technique is defined by the required limit of detection. If low-pg/mL levels are needed, it is fruitless to use a UV/visible absorbance detector. Laser-induced fluorescence (LIF) is usually appropriate, provided derivatization reagents are available if the solute does not have significant native fluorescence [2], Limits of detection of 10 10 M are easily achieved using LIF, provided the solute absorbs at a laser emission wavelength and has a reasonable fluorescence quantum yield. 

LSD is an example of a drug where dosage is extremely low and a detection limit of less than lOOng must be obtainable. This excludes UV as a detection method, but fortunately LSD has a native fluorescence and therefore this detection level can be obtained very readily with a fluorescence detector. Again it is important that LSD can be resolved from other ergot alkaloids and this can be achieved on unmodifled silica by ion-exchange chromatography. 

This detector is of great utility in biochemical analysis. It can be used for compounds that have native fluorescence, e.g., indoles, catecholamines, porphyrins, or else derivatization can be used to produce or enhance fluorescence. This latter aspect will be discussed later in this chapter. 

The fluorescence detector is available as either a filter fluorimeter or as a continuous wavelength fluorimeter. The filter fluorimeters are less expensive, but in most cases low wavelength excitation is not possible with these instruments, and this makes, e.g., the determination of indoles and catecholamines by their native fluorescence impossible. The selectivity of the fluorescence detector is much better than that of the ultraviolet detector, and for favorable compounds the sensitivity may also be better. 

In terms of sensitivity of detection, electrochemical detectors are generally suggested to be approximately four times more sensitive than systems using native fluorescence for the detection of noradrenaline, DOPA and dopamine (Scratchley et al., 1979). However, for certain compounds native fluorescence provides equal or greater sensitivity than electrochemical detection. For example, while electrochemical detection is more sensitive than fluorescence for the measurement of homovanillic acid, it is equally sensitive for 5-hy-droxyindoleacetic acid and is less sensitive for the measurement of tryptophan (Anderson and Young, 1981). 

In conclusion, the specific detector chosen for the quantitation of the catecholamines is very dependent on the specific apphcation for which the system is to be used. For systems requiring high sensitivity either electrochemical or fluorescence derivatisation systems are probably optimal. If sensitivity is not a problem, then clearly the choice of detector is wider and the selected system could utiUse electrochemical, native fluorescence or even UV detection. 

Photometric detectors are the most popular in CE instruments including diode array detectors. Laser-induced fluorescence (LIE) detection and electric conductivity detectors are also popular. LIE is particularly sensitive and powerful for detecting low concentration analytes. However, most analytes are not natively fluorescent and some derivatizations are necessary. Conductivity detector is useful for the detection of non-ultraviolet (non-UV) absorbing analytes such as inorganic ions or fatty acids. Both LIE detection and conductivity detectors are commercially available and easy to interface with conventional CE instruments. Electrochemical detectors are also useful for selective high-sensitivity detection. Several techniques have been developed to circumvent the problem of strong effects of electrophoretic field on electrochemical detection, but despite this, commercial electrochemical detectors are not used extensively. 

Of the CE detectors available, fluorescence is by far the most sensitive. Mass detection limits have been reported as low as a single molecule. CLOD values routinely approach <10 moll. Unfortunately, most analytes are not natively fluorescent, making sample derivatization necessary. Labeling protocols for many functional groups, including amines, carboxylic acids, and thiols, have been developed. 

LIF detectors are more sensitive and selective than the UV-visible absorption ones, but only a few different laser sources (488 nm Ar ion, 442 nm He-Cd, and 324 nm He-Ca) are available. Direct detection of native fluorescence compounds separated by CE has been demonstrated for some fluorescent dyes. Fluorescence detection of other inorganic and organic contaminants is also achieved by indirect methods - either direct fluorescence by the formation of complexes or derivatives, or by incorporating a fluorophor into the BGE. 

The electrochemical detector has been used for the analysis of roquefortine and the zearalenones and refractive index detection has bean used for trichothe-cenes such as T-2 toxin with no native fluorescence or useful UV absorption. For molecules such as T-2 toxin, with a free hydroxyl group, it is possible to derivatize with a reagent such as an aromatic acid, which confers UV absorption on the derivative. 

A third and currently preferred option to detect K vitamers in LC is fluorescence detection. Vitamin Kj(20) does not show native fluorescence, and consequently a number of procedures have been developed to modify vitamin Ki(20) into a fluorescent molecule. A first procedure consists of the use of a coulometric detector as a postcolumn reactor to reduce vitamin Ki(20) to the fluorescent hydroquinone form. Reduction of vitamin Ki(20) can also be obtained by a chemical reaction, either by a reagent that is added postcolumn or one that is incorporated in the eluent itself. In both cases elevated temperature is necessary for the reaction (thermally induced wet-chemical reaction). Alternatively, and currently preferred, the... 

Fluorescence detectors. These are very sensitive and selective and can be used both with molecules which have native fluorescence and with molecules which have been derivatised to contain a fluorophore. This second class is most common. 

The small inner diameter of the separation capillary used in CE implies a short optical pathway, and the consequent poor concentration sensitivity when conventional UV detectiOTi is used. To overcome this drawback several techniques have been developed some of them consist in application of general approaches that are not specifically addressed to CE analysis of alkaloids. One is the use of LIF detector for analysis of alkaloids with native fluorescence [68, 69] or after their off-line derivatization [64, 88, 112]. Sample pretreatment, a second major approach, is popularly employed in combination with sample extraction and can be conveniently applied in analysis of alkaloids because they can be easily retained in cationic-exchange sorbents in solid-phase extraction (SPE) mode [113, 114]. It may be interesting to focus on more specific aspects to detect very low levels of analytes using limited amoimts of samples to this regard chemiluminescence reactions and the use of online preconcentration methods will be considered. 

Other important detector types are fluorescence detector (FL), light scattering detector (LS), and the refractive index detector (RI). The FL detection offers the higher sensitivity and the lower detection limits. Nevertheless, these properties are frequently counteracted by the limited number of compounds with native FL therefore, additional derivatization reactions of analytes are usually needed, and the cost of acquisition and maintenance. In addition, when FL is used in HPLC systems, the linear dynamic range is often small for many analytes (even when the dynamic range is relatively large) and care should be taken... 

The quantitation of riboflavin together with thiamine and niacin using HPTLC silica gel plates and methanol/water (70 30, v/v) as mobile phase was described by Diaz et al. (30). For riboflavin the native fluorescence was used and a preplate derivatization was applied for the other two vitamins (addition of a fluorescent tracer to label nicotinic acid conversion of thiamine into thio-chrome). The developed plates were scanned by a commercially available bifurcated flber-optic-based instrument that transferred the excitation and emission energies between the plate and the fluorescence spectrometer. Calibration curves for the determination of riboflavin 48 to 320 ng, thiamine 300 to 750 ng, and niacin 10 to 100 ng were established. The advantages of this method are that no elimination of excess oxidation reagent is necessary and that the simultaneous determination of vitamins with only one detector is possible. 

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