Detectors, HPLC sensitivity

Some luminol derivatives have been developed as CL labeling reagents. Analytes prelabeled with luminol derivatives are separated by HPLC, mixed with postcolumn reagents such as hydrogen peroxide and an alkaline solution of potassium hexacyanoferrate (III), and then detected by a CL detector. Highly sensitive determination is possible by optimizing the conditions to increase the CL reaction efficiency for each analyte. 

An HPLC method was described for the residue analysis of TBZ in meat (53). The recovery varies from 62 to 75%. The TBZ was extracted from the tissue using 3 mol HC1, eluted from the Extrelut 20 column with dichloromethane and then injected onto a C, 8 column. The TBZ was detected with fluorescence and UV detectors. The sensitivity is such that TBZ can be determined at a level of 5 yug/kg meat (53). Table 4 summarizes the HPLC conditions. 

Nassar, A. E., and Lee, D. Y. (2007b). Novel radio-HPLC detector for sensitive metabolite profiling and structural elucidation in support of drug metabolism studies. Drug Metab. Rev. 39(Suppl. 1) 73. 

Due to the high sensitivity it is favorably to couple a nanoHPLC to an ESI-source. As mass spectrometers are concentration dependent detectors, the sensitivity of an instrumental setup is mostly determined by the peptide concentration of the eluate but not by the peptide amount. Thus a nanocolumn with a flow rate of 300 nL/min provides an about thousand times higher sensitivity than a microbore column with a flow rate of 300 (xL/min. As an alternative to buying a nanoHPLC system it is also possible to use a relatively inexpensive flow splitter after the pump and before the injection valve and the column. Thereby the flow rate can be reduced to use a capillary column (flow rate 4 (xL/min) on an analytical HPLC system or a nanocolumn on a capillary HPLC system. Instead of a flow-splitter it is preferred to couple a nanoHPLC to an ESI-source. Thereby, the flow rate is split according to the column backpressure, i.e., mostly the column volumes if the same packing materials are used. However, these low-cost setups are less reliable than a nanoHPLC and the reproducibility is worse. 

A nonselective detector more sensitive than the RID and easier to use with a small contribution to band broadening is thus desirable in HPLC. The mass spectrometer would be a good solution if it were not so complex [10] and expensive. The electron-capture detector (BCD) [11] and flame-based detectors have been suggested [12]. Both are very sensitive and could be made with very small volumes. Unfortunately, the... 

Very often baseline problems are related to detector problems. Many detectors are available for HPLC systems. The most common are fixed and variable wavelength ultraviolet spectrophotometers, refractive index, and conductivity detectors. Electrochemical and fluorescence detectors are less frequently used, as they are more selective. Detector problems fall into two categories electrical and mechanical/optical. The instrument manufacturer should correct electrical problems. Mechanical or optical problems can usually be traced to the flow cell however, improvements in detector cell technology have made them more durable and easier to use. Detector-related problems include leaks, air bubbles, and cell contamination. These usually produce spikes or baseline noise on the chromatograms or decreased sensitivity. Some cells, especially those used in refractive index detectors, are sensitive to flow and pressure variations. Flow rates or backpressures that exceed the manufacturer s recommendation will break the cell window. Old or defective source lamps, as well as incorrect detector rise time, gain, or attenuation settings will reduce sensitivity and peak height. Faulty or reversed cable connections can also be the source of problems. 

Detection of tocopherols and tocotrienols after HPLC separation is based on their ability to absorb ultraviolet light and create fluorescence. Tocopherols and tocotrienols show typical UV spectra with maximum absorption at 290-300 nm (Table 1.6). If the samples contain sufficient amounts of analytes, e.g., vegetable oils and supplemented products, a UV detector is sensitive enough. When higher sensitivity and better selectivity is needed, a fluorescence detector is the commonly used detector. With a fluorescence detector, it is possible to analyze tocopherols... 

The detector can be considered as the "soul" of a HPLC system. Connected to the outlet end of the column, its role is to monitor the column effluent in real time. Detectors can be the most sophisticated and expensive component of the system. Classification of detectors is of two sorts, selective detectors which give different responses depending on the molecular structure of the sample under analysis, or universal detectors, for whom the response is similar for most compounds. Absorbance and fluorescence detectors are termed selective detectors, while the refractive index (RI) is a "universal detector". The Ultraviolet-Visible (UV-Vis) detector is more selective and sensitive, being able to detect amounts as low as lO g/mL, while the RI detector s sensitivity is in the range of lO g/mL. Therefore selective detectors can be used to minimise interference from unwanted components. As for fluorescence detectors, their sensitivity is in the range of lO i g/mL for... 

Continuous post-column derivatization has reached a considerably lower degree of development in gas chromatography than it has in HPLC owing to the difficulty in carrying out gas-phase reaction on the one hand and to the fact that the performance of GC detectors exceeds that of typical HPLC detectors in sensitivity, response, etc. 

Diode array detector HPLC multichannel ultraviolet-visible detector consisting of a linear array of photodiodes onto which the full spectrum falls, i.e. 200 400 nm ultraviolet, 400-800 nm visible regions. Thus a complete spectrum can be obtained in less than 0.1 s, also several wavelengths can be monitored simultaneously to achieve maximum sensitivity for each component. 

UV detector HPLC detector based on an ultraviolet-visible spectrophotometer using microsample flow cells (10 pi). Response to components depends on their absorption spectrum and absorptivity coefficient as defined by the Beer-Lambert law. Detector wavelength is set to give maximum sensitivity ideally at Amax however an optimum wavelength, Aopt at which all the components have a satisfactory absorbance, may be used. 

As mentioned in Section 4, detection limits in CE are generally poorer than HPLC or GC due to the narrow detection path length for UVA is detection and the nanoliter injection volumes. Various approaches to improve CE sensitivity such as the use of LIED detectors, low wavelength detection, and so forth have been covered in Section 4.4. Some comparisons of CE and HPLC sensitivity for pharmaceutical analysis are illustrated in Table 4.8. 

Once the chromatographic separation on the column has been conducted, the composition of the eluent at the column end must be determined using a detector. In all HPLC detectors, the eluent flows through a measuring cell where the change of a physical or chemical property with elution time is detected. The most important parameter of the detector is sensitivity, which is influenced by the noise and baseline drift, the absolute detection limit of the detector, the linearity, the detector volume (band broadening), and the effects of pressure, temperature and flow (pulsation, gas bubbles). 

It is universal for aU anions or cations in solution (i.e., a bulk property of mobile-phase detector), nondestructive, and a concentration detector compound sensitivities differ over an order of magnitude range, useable primarily with isocratic RP-HPLC without buffer salts (unless subsequent suppression column is used) and it is a premier detector for ion chromatography (IC). 

G.V. Melzi d Eril, G. Achilli and G.P. Cellerino, A new microprocessor controlled multi-mode multi-electrode electrochemical detector for HPLC Sensitivity and selectivity enhancements in neurochemical measurements. Inti. LabMate, 1992 (February), 17-18. 

Additional detectors available for HPLC analysis include fluorescence detectors, high-sensitivity diode-array detectors, refractive index detectors, and electrochemical detectors. 

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). 

There is a wide range of selective and sensitive detectors for HPLC. Ranking detectors for sensitivity is impractical as response is a function of molecular structure. The most common detectors will now be described. 

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. 

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