Fluorescence spectrometry INDEX

For detection of carbohydrates in principle, ultraviolet (UV), laser-induced fluorescence, refractive index, electrochemical, amperometric, and mass spec-trometric detection can be used. Mass spectrometry, with its various ionization methods, has traditionally been one of the key techniques for the structural determination of proteins and carbohydrates. Fast-atom bombardment (FAB) and electrospray ionization (ESI) are the two on-line ionization methods used for carbohydrate analysis. The ESI principle has truly revolutionized the modern mass spectrometry of biological molecules, due to its high sensitivity and ability to record large-molecule entities within a relatively smaU-mass scale. 

Passive optodes shown in Figure 17-34 are used for Raman or fluorescence spectrometry. 1 vo types of monofiber optode have been described [166], as shown in Figure 17-34a. They require the use of a pierced mirror and suitable optics to separate the source laser and fluorescence emission beams at the fiber-measuring instrument junction. With a single fiber the observation of fluorescence is divided into four zones in order to calculate the effective optical path (/,). For a fiber with radius rg and numerical aperture A and observation in a medium with refractive index n , this path was evaluated by Deaton [191] as... 

Table 28-1 lists the most common detectors for IIPLC and some of their most important properties. The most widely used detectors for IX arc based on absorption of ultraviolet or visible radiation (see Figure 28-8). Fluorescence, refractive-index, and electrochemical detectors are also widely used. Mass spectrometry (MS) detectors are currently quite popular. Such IX /MS systems can greatly aid in identifying the analytes exiting from the HPI.C column as discussed later in this section. 

The main purpose of the detector in a field-flow fractionation (FFF) system is to quantitatively determine particle number, volume, or mass concentrations in the FFF size-sorted fractions. Consequently, a number, volume, or mass dependent size distribution of the sample can be derived from detection systems applied to FFF [e.g., (UV-Vis) fluorescence, refractive index, inductively coupled plasma ionization mass spectrometry (ICPMS)]. Further, on-line light scattering detectors can provide additional size and molecular weight distributions of the sample. 

Although UV detection is most commonly used in the quahty control of drug substances, other detectors such as fluorescence, electrochemical, near infrared, refractive index, evaporative light scattering, or mass spectrometry may be used as appropriate. 

Other techniques previously described for general investigation of tautomeric equilibria (76AHC(S1)1> involve heats of combustion, relaxation times, polarography, refractive index, molar refractivity, optical rotation, X-ray diffraction, electron diffraction, neutron diffraction, Raman, fluorescence, phosphorescence and photoelectron spectroscopy, and mass spectrometry. The application of several of these techniques to tautomeric studies has been discussed in previous sections. Other results from the more important of these will be referred to later in this section. 

The components in a mixture separate in the column and exit from the column at different times (retention times). As they exit, the detector registers the event and causes the event to be recorded as a peak on the chromatogram. A wide range of detector types are available and include ultraviolet adsorption, refractive index, thermal conductivity, flame ionization, fluorescence, electrochemical, electron capture, thermal energy analyzer, nitrogen-phosphorus. Other less common detectors include infrared, mass spectrometry, nuclear magnetic resonance, atomic absorption, plasma emission. 

UV/Vis Absorption Fluorescence Amperometric Conductometric Refractive index (RI) Mass spectrometry (MS) Transport (FID)... 

Current IPC detectors are on-stream monitors. HPLC detectors range from (1) non selective or universal (bulk property detectors such as the refractive index (RI) detector), characterized by limited sensitivity, (2) selective (discriminating solute property detectors such as UV-Vis detectors) to (3) specific (specific solute property detectors such as fluorescence detectors). Traditional detection techniques are based on analyte architecture that gives rise to high absorbance, fluorescence, or electrochemical activity. Mass spectrometry (MS) and evaporative light scattering detectors (ELSDs), can be considered universal types in their own right... 

Fluorescence detection at 284/310 nm (extinction/ emission wavelengths) leads to a detection limit of 1.3 mmol/L (0.14 mg/mL for / -cresol). Identification of phenol and /7-cresol may be confirmed by liquid chroma- tography/mass spectrometry. Because HPLC methods require only simple extraction, e.g., by ethyl acetate, and do not require further steps such as derivatization, they j are simple and rapid compared with gas chromatography or gas chromatography/mass spectrometry. Such methods I are useful for monitoring serum phenols in dialyzed patients as an index of hemodialysis adequacy. How- ever, the separation of the three isomers of cresol can only be performed by adding 3-cyclodextrin to the c liquid phase. q... 

There exist several different detectors suitable for detecting the analytes after the chromatographic separation. Some commercial detectors used in LC are ultraviolet (UV) detectors, fluorescence detektors, electrochemical detectors, refractive index (RI) detectors and mass spectrometry (MS) detectors. The choice of detector depends on the sample and the purpose of the analysis. 

Liquid chromatography (LC) has already been described and is an excellent separation technique for compounds that are nonvolatile, thermally unstable and relatively polar in nature. The usual detectors for LC are based on refractive index, conductivity, amperometry, light scattering, UV and fluorescence, all of which have been discussed in Section 3.2. However, sometimes it is desirable to have a more powerful detector attached to an LC instrument and, as such, the following combinations are possible LC-infrared spectrometry, LC-atomic spectrometry, LC-inductively coupled plasma-mass spectrometry, LC-mass spectrometry, LC-UV-mass spectrometry, LC-nuclear magnetic resonance and even LC-nuclear magnetic resonance-mass spectrometry. 

Spectroscopic techniques are popular as a means of detection on chips. Examples include the determination of flavins and DNA by fluorescence. Spectrophotometric techniques are often used for biological samples . Mass spectrometry has also been used. Benetton et al. coupled electrospray ionisation MS to a chip while Sillon et al. developed a low cost mass spectrometer which incorporated the ionisation chamber, filter and detector on the chip. A fibre optic coupler has been developed as a detector. The dual optical fibre configuration (one transmitting, one receiving, (Eigure 10.5)) in the chip forms the microchannel as well as the detector itself and measures refractive index changes but can also be used to measure absorbance . 

Potentiometric and refractive index detection are not affected by volume but are relatively insensitive in the nanolitre to picolitre range compared to amperometric detection (micro surface area) and fluorimetric detection (micro amount of material). At 1 pL, limits of detection are similar for potentiometry, amperometry and fluorescence. On-chip LC is very compatible with mass spectrometry due to the low volumes and flow rates required. Battery-operated ion trap MS has been reported but miniaturisation of MS offers no sensitivity or selectivity advantages. Electrospray ionisation (ESI) has been successfully integrated into a chip format allowing for many ESI nozzles on one chip. Arrays make pattern recognition possible. 

Ultraviolet absorption Fluorescence Electrochemical detection Refractive index Radioactivity Mass spectrometry... 

Fiber-optic applications in the chemical industry were first tried over 15 years ago with colorimetry. However, concepts related to remote spectrometry and refractometry with optical fibers are of much more recent date. In the former area, it was necessary to overcome problems due to coupling of teleconununication fibers with spectrometric analyzers for absorption [165], fluorescence [166], and Raman scattering [167]. The last area was studied at the same time as the intrinsic FOS, with the measurement of the variation in amplitude of an optical signal as a function of the refracdve index of the optical dber cladding which consists of the liquid to be measured [168]. A characteristic example is the vehicle battery acid concentration indicator [169]. 

The most commonly used analytical technique for sugars is HPLC with a refractive index detector (RID). Although the HPLC-RID method is simple, the RID lacks sensitivity and selectivity. Therefore, UV and fluorescence detection is frequently used, coupled with pre- or postcolumn derivatization, for analysis with higher sensitivity. Liquid chromatography-mass spectrometry (LC-MS) using electrospray ionization also requires pre- or postcolumn derivatization. LC-MS using atmospheric pressure chemical ionization does... 

Koons RD, Peters CA, and Rebbert PS (1991) Comparison of refractive index, energy dispersive X-ray fluorescence and inductively coupled plasma atomic emission spectrometry for forensic characterization of sheet glass fragments. Journal of Analytical Atomic Spectrometry 6 451 56. 

Any of the methods of detection used in liquid chromatography can be used in IC, though some are more useful than others. If the eluent does not affect the detector the need for a suppressor disappears. Common means of detection in IC are ultraviolet (UV) absorption, including indirect absorption electrochemical, especially amperometric and pulsed amperometric and postcolumn derivatization. Detectors atomic absorption spectrometry, chemiluminescence, fluorescence, atomic spectroscopic, refractive index, electrochemical (besides conductivity) including amperometric, coulometric, potentiometric, polaro-graphic, pulsed amperometric, inductively coupled plasma emission spectrometry, ion-selective electrode, inductively coupled plasma mass spectrometry, bulk acoustic wave sensor, and evaporative light-scattering detection. 

In the introductory Section 2.1.3, it was discussed that an important aspect of optimization can be to improve a method for its applicability in trace analysis. The nature of the mode of detection is very relevant in this case whether the applied detector is concentration proportional like the very common UV detector or mass proportional hke nebulizer-based detectors, for example, evaporative light scattering detector (ELSD) or charged aerosol detector (CAD). This textbook contains dedicated chapters on nebulizer-based or aerosol detectors (Chapter 10 on trends in detection), as well as for the coupling of LC with mass spectrometry (Chapter 1). Here, the focus is on concentration proportional detectors UV detectors (VWD, DAD), fluorescence detectors (FLD), electrochemical detectors (ECD), and refractive index (RI) detectors. 

Detection methods applied in ion chromatography (IC) can be divided into electrochemical and spectrometric methods. Electrochemical detection methods include conductometric, amperometric, and potentiometric methods, while spectroscopic methods include molecular techniques (UVA is, chemiluminescence, fluorescence, and refractive index methods), and spectroscopic techniques such as atomic absorption spectrometry (AAS), atomic emission spectrometry (AES), inductively coupled plasma-optical emission spectrometry (ICP-OES), inductively coupled plasma-mass spectrometry (ICP-MS), and mass spectrometry (MS). ... 

To overcome the problem of detection in CE, many workers have used inductively coupled plasma-mass spectrometry (ICP-MS) as the method of detection. Electrochemical detection in CE includes conductivity, amperometry, and potentiometry detection. The detection limit of amperometric detectors has been reported to be up to 10 M. A special design of the conductivity ceU has been described by many workers. The pulsed-amperometric and cyclic voltametry waveforms, as well as multi step wave forms, have been used as detection systems for various pollutants. Potentiometric detection in CE was first introduced in 1991 and was further developed by various workers. 8-Hydroxyquinoline-5-sulfonic acid and lumogallion exhibit fluorescent properties and, hence, have been used for metal ion detection in CE by fluorescence detectors. Overall, fluorescence detectors have not yet received wide acceptance in CE for metal ions analysis, although their gains in sensitivity and selectivity over photometric detectors are significant. Moreover, these detectors are also commercially available. Some other devices, such as chemiluminescence, atomic emission spectrometry (AES), refractive index, radioactivity, and X-ray diffraction, have also been used as detectors in CE for metal ions analysis, but their use is stiU limited. 

SEC suffers from poor resolution and low sensitivity [5], while GC is limited by the high molecular weight and polar nature of many antioxidants and light stabilisers, which are designed to be reactive and so decompose when exposed to heat [9]. HPLC the most widely used instrumental method also has limitations [10-12]. HPLC lacks a simple sensitive universal detector that is compatible with all liquid mobile phases. UV or fluorescence detectors, which are commonly used, require that additives have a chromophoric moiety, while the universal refractive index detector only functions under isocratic conditions. As a result, Vargo and Olson have coupled HPLC with mass spectrometry (MS) for this type of application by using a moving belt interface [13]. 

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