Mass spectrometry micro HPLC

Martin, S. E., Shabanowitz, J., Hunt, D. R, and Marto, J. A., Subfemtomole MS and MS/MS peptide sequence analysis using nano-HPLC micro-ESI Eourier transform ion cyclotron resonance mass spectrometry, Analytical Chemistry 72(18), 4266 274, 2000. 

Although this section provides a brief description of most commonly nsed detectors for HPLC, most of the focus is on a few detection modes. Optical absorbance detectors remain the most widely nsed for HPLC, and are discnssed in some detail. We also focns on flnorescence, condnctivity, and electrochemical detection, as these methods were not widely nsed for HPLC in the past, bnt are especially well suited to micro- and nano-flow instrnments becanse of their high sensitivity in small sample volumes. Mass spectrometry has also come into wide and rontine nse in the last decade, but as it is the subject of another chapter, it will not be fnrther discnssed here. Miniaturization has been particularly important for capillary and chip-based electrophoresis, which often employs sub-nanoliter detection volnmes [36,37]. 

Interfacing HPLC or HPCE (capillary electrophoresis) to mass spectrometry is technically more complex than. with a GC because these techniques use a solvent that is often aqueous (water is a poison to mass spectrometers). The use of microcolumns in HPLC is desirable for coupling to MS because micro-columns operate at very low flow rates. They are also compatible with different ionisation techniques for the analysis of high molecular-weight species. 

As a rule, a separation method should be used for both purification and concentration of the sample. The classic method for peptides and proteins is a reverse-phase liquid chromatography preparation of the sample, followed by a concentration step (often lyophiliza-tion) of the fraction of interest. During those steps performed on very small quantities of sample, loss on the sample can occur if care is not taken to avoid it. Lyophilization, for instance, can lead to the loss of the sample absorbed on the walls of the vial. The use of separation methods on-line with the mass spectrometer often are preferred. Micro- or nano-HPLC [32,33] and capillary electrophoresis [34], both coupled mainly to electrospray ionization/mass spectrometry (ESI-MS), are used more and more. 

Peak identification and interpretation of mass spectra Mobile phases for proanthocyanidins contain 2% (v/v) of acetic acid, and it suppresses electrospray ionization at negative mode. This can be overcome by adding ammonia acetate as an ionization enhancer. The ammonia acetate (lOmM in methanol, 0.1 mL/min flow rate) can be added into the flow via a three-way micro-splitter ( P-445, Upchurch Scientific, WA) just before the mass spectrometry. It can be delivered by a separate HPLC pump or by a syringe pump. 

Here we present some strategies to remove ionic and non-ionic ddter-gents during sample preparation at the low picomole level for micro HPLC chromatography and mass spectrometry. 

The characterization of the expressed constituent of a VCL is greatly facilitated by the availability of efficient analytical methods such as mass and NMR spectrometries, capillary electrophoresis, micro-HPLC, etc. These can be applied either to the isolated constituent or even directly to its complex with the target (for the use of mass spectrometry, see [10]). It is clear that the development of micro-methods and laboratory-on-a-chip [11] procedures will have a strong impact on the implementation of combinatorial chemistry approaches. 

Today, micro-LC is a valuable analytical tool for sample-constrained applications such as proteomics and bioanalysis. The microliter flow rates are ideally suited for direct, splitless coupling with electrospray ionization mass spectrometry (ESI-MS). Many benefits can be demonstrated from well-established HPLC theory, which allows for direct method transfer to micro-LC. 

Cappiello s research group applied micro-HPLC to the screening of water samples for pesticide contamination. With electron ionization mass spectrometric detection and the use of two microcolumns packed with a Cig silica-based stationary phase, they performed the preconcentration of water samples and successfully detected trace levels of pollutants in similar samples. They also showed that ion-interaction micro-HPLC on a Cig stationary phase with hexylamine as the ion-pair reagent and coupling to particle beam mass spectrometry could be successfully used for the analysis of herbicides ranging from acidic species such as... 

Martin, S.E., Shabanowitz, J Hunt, D.F, Marto, J.A. (2000) Subfemtomole MS and MS/MS Peptide Sequence Analysis Using Nano-HPLC Micro-ESI Fourier Transform Ion Cyclotron Resonance Mass Spectrometry. Anal. Chem. 72 4266-4274. 

Reviews of methods include all modem tools applied, including mass spectrometry, HPLC (in its various forms), capillary electrophoresis, biosensors, bioelectroanalysis, fluorescence, IR/Raman, and other optical spectroscopies, NMR radiometry, and methods related to bioimaging. In particular the series volumes provide reviews on perspective new instmmental approaches as they apply to bioanalysis, and on the use of micro-/nano-materials such as micro- and nanoparticles. Articles on p-total analytical systems (p-TAS) and on labs-on-a-chip also fall into this category. 

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