Regeneration of FLARE C18 Mixed‐Mode Column after Exposure to TFA Diamond Analytics Technical Note: DA-11 Chuan‐Hsi Hung, 1 David S. Jensen, 2 Andrew E. Dadson, 2 Matthew R. Linford 1 1 Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT‐84602, USA 2 Diamond Analytics, 1260 S 1600 W, Orem, UT‐84058, USA Introduction The FLARE C18 Mixed-Mode (MM), core- shell HPLC column is stable at extremes of pH and at elevated temperature. 1-3 In addition, the column has mixed-mode properties, showing both weak anion exchange and hydrophobic retention mechanisms. 4 Because it contains an amine- based polymer, its stationary phase is protonated (charged) or deprotonated (neutral) over different ranges of pH. This amine-containing polymer is not expected to behave like a molecular amine because the charges on the polymer interact as the column is protonated. 4 Thus, it becomes increasingly difficult to protonate the column as it is more and more charged. Accordingly, one would expect a difference in the column at pH 2 and 7, while no such difference would be expected for a molecular amine. Changes in the protonation state of the FLARE column change its retention mechanism. Thus, mobile phase pH is a useful lever to control column selectivity. At lower pH (pH < 10), the FLARE column retains acidic analytes through both hydrophobic and ionic interactions. At higher pH (pH > 10), it retains basic analytes through hydrophobic interactions. Neutral analytes are retained over a wide pH range, although this retention is also affected by the protonation state of the column – whether it is more protonated (more hydrophilic) or more deprotonated (more hydrophobic). A common problem with amine-containing stationary phases is their retention of acidic additives like trifluoroacetic acid (TFA). Given the acid-base properties of TFA and the amines in the FLARE C18 MM stationary phase, one would expect that TFA could be removed from the column at elevated pH. This type of approach would not be an option for silica-based columns because of their lack of stability under these conditions. A separation was performed on the FLARE C18 MM column at neutral pH (phosphate buffer at pH 7). The same separation was then performed at pH 2 using a TFA- containing mobile phase. With this change in pH, the retention of the neutral analytes dropped. Nevertheless, the column could be regenerated at pH 11, and this regeneration was repeatable. Thus, TFA, a common additive, can be used with the FLARE C18 MM column. Experimental Analytes: Alkylbenzene mixture (-ethyl, -butyl, and hexylbenzene) HPLC system: Waters 1525 Binary HPLC pump with a UV detector Column: Diamond Analytics FLARE C18 MM column (4.6 x 33 mm, 4 µm) Injection volume: 5.0 µL