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HPLC is a means of separating complex mixtures of non-volatile organic molecules in the solution phase. Organic compounds partition between a mobile phase comprised of a combination of aqueous and organic solvents and a stationary phase comprised of a non-polar (e.g. C18 for reverse phase) material. HPLC columns are available with a wide variety of stationary phases.

There are several separation techniques amenable to HPLC. In addition to Reverse Phase separations, there are Normal Phase Chromatography and Size Exclusion Chromatography. For safety reasons, CEST does not allow Normal Phase experiments because of the use of large volumes of strongly organic solvents.

Reverse Phase Chromatography uses a polar mobile phase (usually combinations of water, methanol, and/or acetonitrile) and a non-polar stationary phase column (C4, C8 or C18). Compounds separate in the column due to differences in their hydrophobicities.

Size Exclusion Chromatography separates compounds based on the differences in their hydrodynamic volumes in solution.

The HPLCs at CEST have 3 different detectors to measure these separated compounds:

  • Photodiode Array Detector: A photodiode array detector measures the absorbance of photons in the UV-Visible spectrum by the compounds in solution.

  • Fluorescence Detector: Compounds that fluoresce using specific excitation and emission wavelengths.

  • Mass Spectrometer Detector: CEST has a triple quadrupole mass analyzer equipped with electrospray ionization. In addition to MS measurements, three types of MS/MS experiments are possible: product ion scans, precursor ion scans, and constant neutral loss scans. Product ion scans identify all the fragment ions which originate from a selected parent ion. Precursor ion scans identify all parent ions which generate a specific fragment ion. Constant neutral loss scans identify parent ions which undergo the loss of a neutral fragment.

Ion Chromatography (IC) is a specialized form of liquid chromatography in which the separation of charged molecules in solution are facilitated by a buffered mobile phase moving the sample across a polar stationary phase which interacts with the sample to separate those charged molecules by the interaction with the stationary phase. The greater the interactions, the further from the origin the separation peak will be.