Purification Methods
If its colour has become strongly violet, then it will need to be reduced again. It is best to check the UV absorption in N HCl where it has a peak at ~265nm which drops sharply to zero having no absorption at ~340nm. The presence of absorption at 340nm indicates oxidation to quinonoid or 7,8-dihydropterin. If the absorption is weak, then dissolve it in the minimum volume of anhydrous trifluoroacetic acid (fume hood), add charcoal, filter, then add two drops of N H2SO4 followed by dry Et2O at 0o, allow the white tetrahydro salt to settle, collect, and wash it with dry Et2O, by centrifugation. Dry the residue in vacuo over P2O5 and KOH. Store it in aliquots in the dark at <0o. It has UV max at 265nm ( 16,980) at pH -1.0 (dication); 219nm ( 23,440) and 266nm ( 12,880) at pH 3.5 (monocation); 220nm ( 18,620), [260nm ( 4,270)sh] and 299nm ( 9,330) at pH 8.0 (neutral species); and 218nm ( 10,000), [240nm ( 5,500)sh] and 287nm ( 5,500) at pH 13 (anion). [Blakley Biochem J 72 707 1959, Asahi Yakugaku Zasshi (J Pharm Soc Jpn) 7 9 1557 1959, Pfleiderer in Pterins and Folate (Benkovic and Blakley Eds) J Wiley Vol 2 p 97 1985.]
