Diode array spectrometer HPLC detector

During the last decade, research efforts in the field of LC-MS have changed considerably. Technological problems in interfacing appear to be solved, and a number of interfaces have been found suitable for the analysis of flavonoids. These include TSP, continuous-flow fast-atom bombardment (CF-FAB), ESI, and APCI. LC-MS is frequently used to determine the occurrence of previously identified compounds or to target the isolation of new compounds (Table 2.11). LC MS is rarely used for complete structural characterization, but it provides the molecular mass of the different constituents in a sample. Then, further structural characterization can be performed by LC-MS-MS and MS-MS analysis. In recent years, the combination of HPLC coupled simultaneously to a diode-array (UV-Vis) detector and to a mass spectrometer equipped with an ESI or APCI source has been the method of choice for the determination of flavonoid masses. Applications of LC-MS (and LC-MS-MS) in flavonoid... 

Martens-Lobenhoffer et al. [119] used chiral HPLC-atmospheric pressure photoionization tandem mass-spectrometric method for the enantio-selective quantification of omeprazole and its main metabolites in human serum. The method features solid-phase separation, normal phase chiral HPLC separation, and atmospheric pressure photoionization tandem mass spectrometry. The internal standards serve stable isotope labeled omeprazole and 5-hydroxy omeprazole. The HPLC part consists of Agilent 1100 system comprising a binary pump, an autosampler, a thermo-stated column component, and a diode array UV-VIS detector. The enantioselective chromatographic separation took place on a ReproSil Chiral-CA 5 ym 25 cm x 2 mm column, protected by a security guard system, equipped with a 4 mm x 2-mm silica filter insert. The analytes were detected by a Thermo Scientific TSQ Discovery Max triple quadrupole mass spectrometer, equipped with an APPI ion source with a... 

A more definitive identification may be obtained by combining retention characteristics with more specific information from an appropriate detector. Arguably, the most information-rich HPLC detectors for the general identification problem are the diode-array UV detector, which allows a complete UV spectrum of an analyte to be obtained as it elutes from a column, and the mass spectrometer. The UV spectrum often allows the class of componnd to be determined but the... 

HPLC - Beckman 125 binary gradient pumps, 168 diode-array detector, 507 autosampler MS - Ion-trap mass spectrometer Finnigan LCQ equipped by APCI (atmospheric pressure chemical ionization), data analyzed in negative mode, spectra confirming found compounds were obtained from tandem mass spectromectry (MS/MS). 

Liquid chromatography/mass spectrometry analyses were performed with an ion trap mass spectrometer (LCQ, Thermo Fisher Scientific Inc., MA) equipped with an HPLC system (Agilent, CA Model 1100) connected with a diode-array detector (DAD, G1315A). The sample solution (1-5 p,L) was applied on an Inertsil ODS-3 column (2.1 x 150 mm, 3 p,m, GL... 

UV/VIS absorbance detector (single wavelength or diode array) placed in-line between HPLC column and mass spectrometer for additional characterization of eluting chlorophylls (optional)... 

These three examples illustrate technology developments over time (dual-channel detector, diode array detector, mass spectrometer). Note that while the overall methodology is very similar (methanolic extracts, methanol-based, acidified solvents used for HPLC, detection of eluted compounds), the exact conditions for successful separation need to be defined for each system. 

Spectroscopic detectors measure partial or complete energy absorption, energy emission, or mass spectra in real-time as analytes are separated on a chromatography column. Spectroscopic data provide the strongest evidence to support the identifications of analytes. However, depending on the spectroscopic technique, other method attributes such as sensitivity and peak area measurement accuracy may be reduced compared to some nonselective and selective detectors. The mass spectrometer and Fourier transform infrared spectrometer are examples of spectroscopic detectors used online with GC and HPLC. The diode array detector, which can measure the UV-VIS spectra of eluting analytes is a... 

Reversed-phase high-performance liquid chromatography (RP-HPLC) is the usual method of choice for the separation of anthocyanins combined with an ultraviolet-visible (UV-Vis) or diode-array detector (DAD)(Hebrero et al., 1988 Hong et ah, 1990). With reversed-phase columns the elution pattern of anthocyanins is mainly dependent on the partition coefficients between the mobile phase and the Cjg stationary phase, and on the polarity of the analytes. The mobile phase consists normally of an aqueous solvent (water/carboxylic acid) and an organic solvent (methanol or acetonitrile/carboxylic acid). Typically the amount of carboxylic acid has been up to 10%, but with the addition of a mass spectrometer as a detector, the amount of acid has been decreased to as low as 1 % with a shift from trifluoroacetic acid to formic acid to prevent quenching of the ionization process that may occur with trifluoroacetic acid. The acidic media allows for the complete displacement of the equilibrium to the fiavylium cation, resulting in better resolution and a characteristic absorbance between 515 and 540 nm. HPLC separation methods, combined with electrochemical or DAD, are effective tools for anthocyanin analysis. The weakness of these detection methods is a lack of structural information and some nonspecificity leading to misattribution of peaks, particularly with electrochemical... 

It was during the 1970 s and early 80 s that advances in technology transformed natural product discovery programmes. The advent of HPLC, and later coupling to UV diode-array detectors and then mass spectrometers, improved the efficiency of dereplication procedures. The ability to rapidly separate complex mixtures reduced the time from lead identification to natural product structure. The discovery of doramectin is a fine example of the use of such technology. 

A decade ago, a system such as the one described would require many PCs to control all the component parts. The HPLC, mass spectrometer, fraction collector and even the diode array would each require a separate PC and. separate software. Modem systems are controlled by more integrated software and the MS-prep system is now controlled by one computer and one software platform. The demands on this computer should not be underestimated. The computer controls the HPLC system including the diode array detector and gradient conditions, the mass spectrometer, the make-up pump and the fraction collector. The same computer is also required to acquire and store data from each of these component instruments. 

Rapid quantification of products and substrates in a fermentation process is essential for process development and optimization. Most fermentation laboratories have access to HPLC equipment with possibilities to couple them to quite inexpensive diode-array-detectors, and this equipment could be used for quantitative monitoring of the process. Because HPLC can allow multi-component analyses, i.e., several analytes in the same sample can be determined virtually simultaneously, and since it is often necessary to monitor more than one substance at a time, this technique is an important tool for bioprocess monitoring. HPLC coupled to expensive MS does not represent standard equipment at fermentation laboratories. Even if mass spectrometers are available, DAD is often sufficient for quantification because product concentrations are relatively high, so the MS could be used for other issues. In paper II the goal was to develop and validate a method for analytical quantification of both the product and the substrate to enable the proper characterization of the kinetics of the process i.e., the determination of the values of substrate conversion and product formation. 

---