Detectors, HPLC dynamic range

The performance of all HPLC detectors can be characterised by certain parameters such as sensitivity, noise, drift, limit of detection, linear and dynamic range, and detection volume. Other factors are more specific to individual types of detectors, and are discussed in their respective sections. 

HPLC provides reliable quantitative precision and accuracy, along with a linear dynamic range (LDR) sufficient to allow for the determination of the API and related substances in the same run using a variety of detectors, and can be performed on fully automated instrumentation. HPLC provides excellent reproducibility and is applicable to a wide array of compound types by judicious choice of HPLC column chemistry. Major modes of HPLC include reversed phase and normal phase for the analysis of small (<2000 Da) organic molecules, ion chromatography for the analysis of ions, size exclusion chromatography for the separation of polymers, and chiral HPLC for the determination of enantiomeric purity. Numerous chemically different columns are available within each broad classification, to further aid method development. 

Each laboratory prepared separate calibrants according with their own laboratory procedure. These were used for calibrating the detector within its dynamic range. Calibrants were prepared, avoiding serial dilution. A minimum of five calibration points were required. The coordinator supplied the participants with pure pesticides samples with certified purity. The participants were requested to use these calibrants or to check their own calibrants. For GC and HPLC analysis at least one internal standard was used for the final determination. 

A further important component of each HPLC system is the detection device. Often, Ultraviolet (UV)-detectors are used for this purpose because many analytes absorb light in the range of 200-350nm. UV detectors provide reasonable sensitivity and dynamic range as well as a low noise level. 

Electrochemical detection in HPLC has become established for some, more specialized, applications such as catecholamine analysis though it can be exploited for a far wider range of compounds. The work in this paper has attempted to investigate some of the basic properties of an electrochemical detection system and some more difficult applications. The detector has a linear dynamic range and precision that are comparable with those of other detectors for HPLC. It is, however, more dependent on temperature than, for example, the UV absorption detector and must be operated in a temperature controlled environment to obtain the lowest detection limits. For many electroactive compounds with moderate oxidation potentials, the electrochemical detector can yield sub-nanogram detection limits. 

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 available detectors for HPLC involve either bulk properties of the mobile phase (such as refractive index, conductance, dielectric constant) or specific properties of the solute, e.g. ultraviolet, visible or infra-red absorbance, fluorescence, or electrochemical characteristics. The latter class are generally more selective and have a wider dynamic range. 

Zhang et al. [21] used the HPLC-flow injection chemiluminescence method to determine methylparaben, ethylparaben, propylparaben, and butylparaben, based on the same chemiluminescence enhancement reaction by parabens of the cerium(IV)-rho-damine 6G system in sulfuric acid. The method was applied to orange juice, soy sauce, vinegar, and cola soda (Table 10.1). The advantage of this technique is that it showed greater sensitivity and wider dynamic linear ranges than the electrochemical and fluo-rometric detectors [21]. 

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