Detection modes chip-based

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]. 

Online intact protein separation was the same as for the Q-TOF LC-MS (above) for consistent protein retention times across platforms. For LC-MS/MS the eluent flow was split to a flow rate of 350 nL/min via the TriVersa NanoMate (Advion BioSci-ences, Ithaca, NY) chip-based nanospray source and analyzed with a LTQ-Oibitrap XL (Thermo Fisher, San Jose, CA) mass spectrometer. The instrument was operated in a top-three data dependent mode, with both MS spectra and collision-induced dissociation (CID) MS/MS spectra acquired at 60,000 resolving power in the Oibi-trap. CID collision energy was operated at 15 %. Each MS spectrum was composed of three microscans, and each MS/MS spectrum was the average of 10 microscans. To facilitate the analysis of intact proteins, the instrument was operated with the HCD gas off and the delay before image current detection shortened to 5 ms. 

By far most of the work discussed in this review has been based on LIF detection, usually with an 488 nm Ar-ion laser as the excitation source. Only very few other examples exist in the literature where other detection principles were investigated. One of these exceptions is an integrated detection cell for chip CE that has been described by Liang et al. [78]. In combination with the U-shaped separation channel, two additional well aligned channels to take up the excitation and collection fibers where micromachined in a glass plate. The U-cell provides a longitudinal path of 120 -140 pm in length parallel to the flow direction and can be used both for absorption and fluorescence measurements. The absorption detection limit was 0.003 AU ( 6 pM of a fluorescein dye) in the fluorescence mode a detection limit of 3 nM fluorescein (20 000 molecules) was achieved. 

A liquid prism was created on a PDMS chip for detection based on absorption and refractive index shift. The liquid prism was formed by filling a hollow triangular-shaped chamber with a liquid sample. Excitation and emission were arranged at the minimum deviation configuration. At a low concentration of fluorescein (< 100 pM), excitation light from an optical fiber was absorbed by the molecule, but there was no shift in the excitation maximum. In this absorption-only mode, the LOD of fluorescein was 6 pM. At higher concentration (i.e., 100-1000 pM), there is an additional shift in the excitation maximum This leads to a much sharper decrease in the measured intensity, which is more than can be accounted for simply by the absorption effect [714]. 

A quartz crystal sensor chip was bonded with a microfluidic glass chip for acoustic wave detection (see Figure 7.46). The sensor was operated in the thickness-shear mode (TSM). This has allowed rat heart muscle cell contraction to be studied based on the measurement of the resonant frequency changes [133]. 

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