Detectors, HPLC selectivity

In order to achieve detection limits below the ng mL-1 range only amperometric, chemiluminescence, radiometric, or conventional fluorescence (CF) can be applied (Table 4.41). Fluorescence detectors are generally about 100 times more sensitive and more selective than UV detectors. The selectivity of fluorescence detection is due to the fact that only aromatic and conjugated molecules can be analysed, and by applying specific excitation and emission wavelengths the selectivity can even be increased. Pre- or postcolumn derivatisation in HPLC is a technique that is most commonly performed prior to UV absorption or fluorescence detection... 

These are by far the most popular detectors in hplc. The principle is that the mobile phase from the column is passed through a small flow cell held in the radiation beam of a uv/visible photometer or spectrophotometer. These detectors are selective in the sense that they will detect only those solutes that absorb uv (or visible) radiation. Such solutes include alkenes, aromatics and compounds having multiple bonds between C and O, N or S. The mobile phase we use, on the other hand, should absorb little or no radiation. 

In-line Detector It broadly helps to sense the separated solutes, after they exit through the column. Invariably the detector is an electrical signal whose variation is displayed on a potentiometer recorder or a computing integrator or a video-screen. Modem HPLC units are provided with detectors having selective-devices thereby categorically restricting the response to all the solutes present in a mixture. 

Second detector confirmation is another compound confirmation technique. Two detectors with selectivity to different functional groups are connected in series to one column or in parallel to two columns. For example, two detectors, a UV/VIS detector and a fluorometer, connected in series to the HPLC column are used in the EPA Method 8310 for analysis of PAH compounds. If the second detector is connected to a second column of a dissimilar polarity, then the confirmation becomes even more reliable. An example of such a configuration is organophosphorus pesticides analysis the samples may be initially analyzed with an NPD, and then confirmed on a different column with an ECD or a FPD. 

Supercritical fluid chromatography is compatible with both HPLC and GC detectors. As a result, optical detectors, flame detectors and spectroscopic detectors can be used. The FID is the most common detector used. However, the mobile phase composition, column type and flow rate must be taken into account when the detector is selected. Some care must also be taken such that the detector components are capable of withstanding the high pressures of SFC. 

If necessary, a classical detector can be attached to the column outlet, preceeding the fraction collector. UV detectors are the easiest to install and use. A variable-wavelength detector allows selection of the monitoring wavelength. Such detectors should be equipped with the appropriate cell. Analytical cells, used on HPLC systems, have path lengths of 6-10 mm and are not suitable for use with preparative open columns, as the detector output will quickly become saturated. A more generally useful cell for open column detectors would be a cell with a path length of less than 0.5 mm. 

High-performance liquid chromatography (HPLC). HPLC is a chromatographic method that separates compounds from other compounds through differences in molecular size or affinity to a stationary phase. In the HPLC system, a sample solution passes though a column with a mobile phase at high pressure and separated compounds are monitored by a detector. HPLC has advantages in selectivity and sensitivity, and is thus widely used for determination of small molecules. 

In addition to the spectrometric detectors, HPLC has also been coupled with other detection systems, such as mass spectrometry (MS). Specificity can be highly improved using tandem mass spectrometry (HPLC-MS/MS) (Holler et al. 2006 Yomota and Ohnishi 2007). High sensitivity and selectivity can also be achieved by HPLC coupled with electrochemical detection (Kucera et al. 2007). 

Consequently, analysis of PAHs is mainly accomplished by GC-MS in simple or tandem mode (GC-MS/MS) and HPLC coupled with fluorescence detector (HPLC-FLD) since most of the PAHs are strong fluorophores. Multiple reaction monitoring (MRM) in a triple quadrupole GC-MS/MS is inherently more selective and sensitive than either scan or selected ion monitoring (SIM) as many matrix interferences are minimized or even removed. For this reason various groups have used tandem MS (Matozzo et al. 2010 Xia et al. 2012), however sufficient detection was accomplished also with GC-MS-SIM (Webster et al. 2006). HPLC-UV has also been used with less sensitivity as expressed by LOD values, although in one work LOD values with UV detection are reported quite low (Cravo et al. 2012). PAHs as in almost all UV or FLD methods were identified on the basis of retention time and quantification on an external standard method. 

Efavirenz [6] is a non-nucleoside inhibitor of FIIV-l reverse transcriptase. The metabolism of efavirenz in rats was studied using directly coupled HPLC-NMR-MS. This hyphenated technique utilizes an eluent splitter to also incorporate a mass spectrometer into the HPLC-NMR system in order to obtain additional information on molecular weight and to act as a detector for selection of peaks on which to perform... 

Description of Method. Fluoxetine, whose structure is shown in Figure 12.31a, is another name for the antidepressant drug Prozac. The determination of fluoxetine and its metabolite norfluoxetine. Figure 12.31 b, in serum is an important part of monitoring its therapeutic use. The analysis is complicated by the complex matrix of serum samples. A solid-phase extraction followed by an HPLC analysis using a fluorescence detector provides the necessary selectivity and detection limits. 

Hplc techniques are used to routinely separate and quantify less volatile compounds. The hplc columns used to affect this separation are selected based on the constituents of interest. They are typically reverse phase or anion exchange in nature. The constituents routinely assayed in this type of analysis are those high in molecular weight or low in volatility. Specific compounds of interest include wood sugars, vanillin, and tannin complexes. The most common types of hplc detectors employed in the analysis of distilled spirits are the refractive index detector and the ultraviolet detector. Additionally, the recent introduction of the photodiode array detector is making a significant impact in the analysis of distilled spirits. 

For selective estimation of phenols pollution of environment such chromatographic methods as gas chromatography with flame-ionization detector (ISO method 8165) and high performance liquid chromatography with UV-detector (EPA method 625) is recommended. For determination of phenol, cresols, chlorophenols in environmental samples application of HPLC with amperometric detector is perspective. Phenols and chlorophenols can be easy oxidized and determined with high sensitivity on carbon-glass electrode. 

The most common detectors in HPLC are ultraviolet, fluorescence, electrochemical detector and diffractometer. However, despite all improvements of these techniques it seems necessary to have a more selectivity and sensitivity detector for the purposes of the medical analysis. It should be therefore improvements to couple analytical techniques like infrared IR, MS, nuclear magnetic resonance (NMR), inductively coupled plasma-MS (ICP-MS) or biospecific detectors to the LC-system and many efforts have been made in this field. 

Quantitative accuracy and precision (see Section 2.5 below) often depend upon the selectivity of the detector because of the presence of background and/or co-eluted materials. The most widely used detector for HPLC, the UV detector, does not have such selectivity as it normally gives rise to relatively broad signals, and if more than one component is present, these overlap and deconvolution is difficult. The related technique of fluorescence has more selectivity, since both absorption and emission wavelengths are utilized, but is only applicable to a limited number of analytes, even when derivatization procedures are used. 

To appreciate the ways in which mass spectral data may be processed to utilize fully the selectivity and sensitivity of the mass spectrometer as a detector for HPLC. 

The application areas for LC-MS, as will be illustrated later, are diverse, encompassing both qualitative and quantitative determinations of both high-and low-molecular-weight materials, including synthetic polymers, biopolymers, environmental pollutants, pharmaceutical compounds (drugs and their metabolites) and natural products. In essence, it is used for any compounds which are found in complex matrices for which HPLC is the separation method of choice and where the mass spectrometer provides the necessary selectivity and sensitivity to provide quantitative information and/or it provides structural information that cannot be obtained by using other detectors. 

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