Detectors, HPLC linear range

Table 30.8, provides a comprehensive comparison of various typical detector characteristics invariably used in HPLC, such as response, concentration expressed in g ml 1 and the linear range. However, the linear range usually refers to the range over which the response is essentially linear. It is mostly expressed as the factor by which the lowest factor (i.e., Cn) should be multiplied in order to obtain the highest concentration. 

In contrast with UV methods where the linear range is approximately 1-2 orders of magnitude at best, the HPLC method with UV detection typically has a linear range of 3-4 orders of magnitude due to the narrow path length of the detector flow cell. For example, a simple... 

Cassidy and Frei [23] designed a microflow cell for the Turner Assoc. Model III fluorimeter for use with HPLC. Nanogram quantities of fluorescent materials could be detected. The volume of the flow cell was only 7.5 jul. The detector was unaffected by the flow-rate or composition of the solvent. This gives this detector a decided advantage over refractive-index or UV detectors. The peak shapes were symmetrical and the linear range of response was 2-3 orders of magnitude. 

Another common reason for having separate assay and impurity methods is the need to use more concentrated samples with the impurity assay to increase sensitivity for minor impurities. Modern HPLC systems have been shown to adequately detect low-level impurities (i.e., 0.05%) in chromatograms where the parent peak is still on scale (that is, within the linear range of the detector). This level of detection is usually adequate for screening methods therefore, the assay for loss of parent compound and the measurement of the increase in impurities can typically be done using a single HPLC method. 

The detectors utilized for HPLC are designed to respond to the solute being eluted. HPLC detectors can be classified into two broad categories universal and selective. Selective detectors respond to some physicochemical property of the solute, while universal detectors respond to aU solutes independent of their physicochemical properties. The ideal detector would be highly universal and highly sensitive, have a wide linear range, and not be affected by change in temperature or mobile phase composition. Commercially available detectors possess some of these characteristics but not aU. 

Most commercial fixed-wavelength UV detectors take advantage of the intense line source of 254-nm radiation in the low-pressure mercury arc lamp. The high intensity of the radiation provides excellent detectability for the small-aperture microvolume flow cells required in HPLC. Concentration of most of the radiation in a narrow-wavelength band places less demand on optical filters and enhances the linear range of the detector. 

HPLC Detector Commercially Available Mass LOD (typical) Linear Range (decades)... 

The fundamental properties of fluorescence make this a particularly attractive basis for an HPLC detection system [27], for whereas photometers depend upon the measurement of fairly small differences between the intensity of a full and slightly attenuated beam the measurement of fluorescence starts in principle from zero intensity. At sufficiently low values of concentration (<0.05 absorbance) then the intensity of fluorescence is directly proportional to concentration with a linear range of three to four decades. Consequently, fluorescence detectors are more selective and sensitive than ultraviolet detectors in LC by a factor of 10 giving noise equivalent sensitivities of better 1 ngmP. ... 

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]. 

The ideal HPLC detector should have the same characteristics as those required for GC detectors, i.e. rapid and reproducible response to solutes, a wide range of linear response, high sensitivity and stability of operation. No truly universal HPLC detector has yet been developed but the two most widely applicable types are those based on the absorption of UV or visible radiation by the solute species and those which monitor refractive index differences between solutes dissolved in the mobile phase and the pure mobile phase. Other detectors which are more selective in their response rely on such solute properties as fluorescence, electrical conductivity, diffusion currents (amperometric) and radioactivity. The characteristics of the various types of detector are summarized in Table 4.14. 

The linearity of a method is its ability to obtain test resnlts that are directly proportional to the sample concentration over a given range. For HPLC methods, the relationship between sample concentration and detector response (peak area or height) is nsed to make this determination. 

HPLC methods can usually be transferred without many modifications, since most commercially available HPLC instruments behave similarly. This is certainly true when the columns applied have a similar selectivity. One adaptation, sometimes needed, concerns the gradient profiles, because of different instrumental or pump dead-volumes. However, larger differences exist between CE instruments, e.g., in hydrodynamic injection procedures, in minimum capillary lengths, in capillary distances to the detector, in cooling mechanisms, and in the injected sample volumes. This makes CE method transfers more difficult. Since robustness tests are performed to avoid transfer problems, these tests seem even more important for CE method validation, than for HPLC method validation. However, in the literature, a robustness test only rarely is included in the validation process of a CE method, and usually only linearity, precision, accuracy, specificity, range, and/or limits of detection and quantification are evaluated. Robustness tests are described in references 20 and 59-92. Given the instrumental transfer problems for CE methods, a robustness test guaranteeing to some extent a successful transfer should include besides the instrument on which the method was developed at least one alternative instrument. 

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