Chromatographic detector, dynamic range

The dynamic range of protein expression represents a main obstacle since abundant proteins are seldom of interest and others such as transcription factors are only present in a few copies. There is no detector that is able to visualize all proteins at the same time so that prefractionation and the investigation of subproteomes is required. In fact, pre-MS sample preparation techniques exploiting electrophoretic, chromatographic, or chemical properties of the analyte are often the bottleneck of proteomics. 

With a modern variable-wavelength or PDA detector, it is safe to use a maximum absorbance of up to 1.0-1.5 absorbance units (AU). A typical UV detector today should have a baseline noise in the order of 10 micro absorbance units (10 AU). Therefore, modern UV detectors should have a dynamic range of 4-5 orders of magnitude. The sample concentration and injection volume can be adjusted to make it possible to quantify the major component and impurities at the 0.05% level in the same chromatographic run without changing detection wavelength, sample concentration, or injection volume. 

It is generally required that all methods allow for the monitoring of API and impurities/degradation products in the same chromatographic run, that run times per sample should not be too long, and that for precise and robust quantitative analyses, the separation of the peaks of interest should have target resolutions of >2.0. To allow for easy transfer, the detector response for the nominal concentration of the API (100%, w/w) should be about 75% of the qualified linear dynamic range of the detector. Methods should be temperature controlled (e.g., at 35°C,... 

For a detector to be of use in quantitative analysis, the signal output should be linear with concentration for a concentration-sensitive detector and with mass for a mass-sensitive detector. Some detectors have an additional time constant purposely introduced to remove the high-frequency noise. This should always taken into consideration, since a slow detector response can significantly broaden and attenuate chromatographic peaks relative to those actually sensed. Moreover, a versatile detector should have a wide linear dynamic range so that major and trace components can be determined in a single analysis, over a wide concenua-tion range. 

Bulk property detectors generally have neither the sensitivity nor the linear dynamic range of solute property detectors and, as a consequence, are less frequently used in modem LC analyses. Furthermore, none can be used with gradient elution, flow programming or temperature programming and so they place considerable restrictions on the choice of chromatographic system. They do, however, have certain unique areas of application, some of which have already been mentioned, but their use probably represents less than 5% of all LC analyses. 

Finally the sensitivity of the conductivity function to sodium chloride is 5 x 10 g/ml with a linear dynamic range of 3 x lo. This detector, is obviously very versatile and, perhaps a little surprisingly, is relatively inexpensive. The detector together with a column, sample valve, pump and recorder, which constitutes a basic liquid chromatograph, costs about 10,000. 

One of the most important decisions that is left to the analyst when operating a liquid chromatograph is the choice of detector sensitivity. In some instruments the output from the sensor is monitored continuously over its entire dynamic range and so sensitivity is not an optional experimental parameter. Nevertheless, in this case, the sample size determines the concentration range over which the eluted solutes are monitored and thus an optimum sample size must be chosen. The detector should never be operated at its maximum sensitivity unless such conditions are enjoined by limited sample size or column geometry. Provided that there is adequate sample available, and the sample concentration when eluted is within the linear dynamic range of the detector, the maximum sample size that the column can tolerate should be used. This ensures that the detector noise is always minimal... 

Figure 15-7. Detection limits and dynamic range of some chromatographic detectors.

The secondary electron multiplier (SEM) detector is the key to the role of mass spectrometry as an extremely sensitive analytical technique with wide dynamic range and compatibility with on-line coupling to fast chromatographic separations. The SEM was a natural development from the invention of the photomultiplier (Zworkin 1936, 1939), in which photoelectrons produced by photons falling on a conversion dynode with a photo-sensitive surface are amplified in an avalanche fashion by accelerating the original (first strike) photoelectrons on to a... 

Sulfur compounds play a major role in determining the flavor and odor characteristics of many food substances. Often sulfur compounds are present in trace levels in foods making their isolation and quantification very difficult for chromatographers. This study compares three gas chromatographic detectors the flame photometric detector, sulfur chemiluminescence detector and the atomic emission detector, for the analysis of volatile sulfur compounds in foods. The atomic emission detector showed the most linearity in its response to sulfur the upper limit of the linear dynamic range for the atomic emission detector was 6 to 8 times greater than the other two detectors. The atomic emission detector had the greatest sensitivity to the sulfur compounds with minimum detectable levels as low as 1 pg. 

An evaluation of gas chromatographic detectors typically involves a study of dynamic range, mimimum detectable level and selectivity. Dynamic range is one of the major response characteristics of detectors. It is the range of sample... 

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