Fluorimetric detectors

Assessing the resources available for method development should also be done before beginning a project. The resources available include not only HPLCs, detectors, and columns, but also tools for sample preparation, data capture and analysis software, trained analysts, and especially samples representative of the ultimate analyte matrix. Also, it should be considered whether a fast, secondary method of analysis can be used to optimize sample preparation steps. Often, a simple colorimetric or fluorimetric assay, without separation, can be used for this purpose. A preliminary estimate of the required assay throughput will help to guide selection of methods. 

The peak symmetry, resolution, and detector response are directly dependent on the concentration of the sample. As the concentration of a sample increases, the retention time, separation, and peak symmetry generally decrease. These phenomena are due to isotherm nonlinearity. The detector response may also be nonlinear above or below certain concentrations. In some cases, small amounts of a dilute component are irreversibly adsorbed to the column, leading to reduced recovery. Above some concentration, the response of any detector will cease to be linear. The UV-VIS is one of the most linear detectors, generally exhibiting at least three decades of linearity, while RI, electrochemical, and fluorimetric detectors have a markedly narrower range of linearity. 

The modem HPLC system is a very powerful analytical tool that can provide very accurate and precise analytical results. The sample injection volume tends to be a minor source of variation, although fixed-loop detectors must be flushed with many times their volume in sample to attain high precision. Assuming adequate peak resolution, fluorimetric, electrochemical, and UV detectors make it possible to detect impurities to parts per billion and to quantitate impurities to parts per thousand or, in favorable cases, to parts per million. The major sources of error in quantitation are sample collection and preparation. Detector response and details of the choice of chromatographic method may also be sources of error. 

Fluorescence detectors, discussed in Chapter 1, are extremely sensitive picogram quantities of sample can sometimes be detected. However, most polymers (with the exception of certain proteins) are not fluorescent and thus these detectors are rarely used in GPC. Proteins, particularly those containing tryptophan, fluoresce intensely and are readily detected. Because both the IR and the fluorimetric detector are selective for certain functional groups, rather than being sensitive to analyte mass, there are many pitfalls in quantitation. These and other detectors have been reviewed.177178... 

Consider one small molecule, phenylalanine. It is an essential amino acid in our diet and is important in protein synthesis (a component of protein), as well as a precursor to tyrosine and neurotransmitters. Phenylalanine is one of several amino acids that are measured in a variety of clinical methods, which include immunoassay, fluorometry, high performance liquid chromatography (HPLC see Section 4.1.2) and most recently MS/MS (see Chapter 3). Historically, screening labs utilized immunoassays or fluorimetric analysis. Diagnostic metabolic labs used the amino acid analyzer, which was a form of HPLC. Most recently, the tandem mass spectrometer has been used extensively in screening labs to analyze amino acids or in diagnostic labs as a universal detector for GC and LC techniques. Why did MS/MS replace older technological systems The answer to this question lies in the power of mass spectrometer. 

The main advantage of fluorescence techniques is their sensitivity and measurements of nanogram (10—9 g) quantities are often possible. The reason for the increased sensitivity of fluorimetry over that of molecular absorption spectrophotometry lies in the fact that fluorescence measurements use a non-fluorescent blank solution, which gives a zero or minimal signal from the detector. Absorbance measurements, on the other hand, demand a blank solution which transmits most of the incident radiation and results in a large response from the detector. The sensitivity of fluorimetric measurements can be increased by using a detector that will accurately measure very small amounts of radiation. 

The equipment required to develop this type of sensor is very simple and resembles closely that used to implement ordinary liquid-solid separations in FI manifolds. The only difference lies in the replacement of the packed reactor located in the transport-reaction zone with a packed (usually photometric or fluorimetric) flow-cell accommodated in the detector. Whether the packing material is inert or active, it should meet the following requirements (a) its particle diameter should be large enough (< 80-100 fim) to avoid overpressure (b) it should be made of a material compatible with the nature of the integrated detection system e.g. almost transparent for absorbance measurements) and, (c) the retention/elution process should be fast enough to avoid kinetic problems. 

High-performance LC was also used for determination of TBZ after its extraction from marmalades and curds with ethyl acetate (13). The use of a buffered mobile phase improved the response of the UV detector, and column performance remained constant throughout 2 months of daily use with a detection limit of 100 ppb. Three detectors (UV, fluorimetric, and electrochemical) were used for the determination of OPP, BP, and TBZ in plant materials (45). The compounds were extracted with dichloromethane and separated on an RP-18 column with a methanolic formic acid buffer as eluent. It was not possible to determine TBZ using an electrochemical detector, although the extraction recovery varied between 80 and 95%. 

Carbamates and substituted ureas are a numerous group of pesticides widely used to control weeds, pests, and diseases in fruit trees, vegetables, and cereals. Carbamate residues in foods are commonly extracted with water-miscible solvents and determined by using a liquid chromatograph equipped with a sensitive detector, frequently a UV detector. In addition, to obtain adequate detection selectivity, the postcolumn fluorimetric labeling technique is used for methyl carbamates. Substituted ureas are normally extracted from foods with organic solvents, and they can be determined directly by HPLC-UV or after postcolumn derivatization by fluorescence determination of their derivatives. 

One of the major disadvantages of HPLC, despite all of the progress in the past years, is a serious lack of detectors, particularly universal detectors, which can match the sensitivity of GC detectors. The best HPLC detectors currently available are (spectro) photometric and (spectro) fluorimetric detectors. It seems logical, therefore, to solve the immediate detection problems at least partially by UV- and fluorescence-derivatization techniques. Recent activities by several research groups show an increased interest in such an approach. Derivatization techniques are not restricted to these two detection modes. Reaction products which yield good MS signals (a favourable fractionation pattern) are... 

The oxidation detector for the fluorimetric analysis of carbohydrates in effluents from liquid chromatography columns provides a sensitive method of analysis in blood and urine [110]. The principle involves the reduction of cerium(IV) to cerium(III) by oxidizable compounds such as organic acids and many carbohydrates. The fluorescence... 

Mukhtar et al. [229] have described a laser fluorimetric method for the determination of uranium (and thorium) in soils. To determine uranium (and thorium) in soils, fluorescent X-rays were measured by the use of a germanium planar detector and chromometric techniques [227]. No sample preparation was required in this method. 

In the human biological system, rocuronium bromide is eliminated unchanged by the biliary and urinary routes [15], and has two metabolites as mentioned in Section 4. As detailed in Table 6.2, determination of rocuronium bromide in biological samples was mostly carried out using LC-MS detection [16-23], although some workers made use of electrochemical detector [24—26] and only one report used a fluorimetric detector [27]. 

The main advantage of using a fluorimetric detector is its inherently high sensitivity for pharmaceutical analysis, but very often application of this method to nonfluorescent drug substances requires derivatization with a strong fluorophore. The derivatization process can be performed either precolumn or postcolumn to increase the detection sensitivity [14]. 

Chevalier et al. analyzed dipyridamole directly in the blood samples by a reversed phase high performance chromatographic method with fluori-metric detection [68], The HPLC system uses a 15 cm x 4.8 mm column packed with Lichrosorb RP8 (10 pm) and a mobile phase consisting of 40 60 v/v acetonitrile-water. Detection was made on the basis of fluorimetric detection at 410 nm (excitation at 305 nm). Depending on the model of the detector used, the detection limit of the method was between 2 x 10 13 and 2 x 10 15 mole injected. The precision was 5% for a concentration of 180 ng/mL of dipyridamole, and the method was used for a biodisposition study of dipyridamole in dogs. 

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). 

Figure 4-33. Block diagram of capillary electro- carried out close to the cathode in a region phoresis equipment. Capillary electrophoresis where the capillary is transparent, allowing equipment consists of a thermostatted capillary photometric or fluorimetric analysis of the whose ends are placed in the electrode buffer eluate. The detector system is linked either to a chambers these contain the electrodes attached recorder/integrator or to a PC. to a high-voltage power supply. Detection is...

At excitation wavelengths and concentration ranges where the simple absorbance fluorescence is linear with concentration, the fluorimetric detector is susceptible to the usual interferences that hinder fluorescence measurements, mainly background fluorescence and quenching. 

Luminescence affords a very sensitive means of detection in flowing systems such as HPLC, electrophoresis, flow injection, and flow cytometry. HPLC fluorescence detectors are similar in operation to conventional fluorimeters. Most fluorescence detectors use filters for crude monochromation. Filters pass light in a wider band than do monochromators. This favors spectral sensitivity because more light excites the sample and is collected by the detector. Grating monochromators, on the other hand, favor selectivity. The fluorimetric detector is susceptible to the usual interferences that hinder fluorescence measurements, namely, background fluorescence and quenching. 

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