S2B, Ethyl Acetate, 450 mL

The most common cause is aged S2B (ethyl acetate). Trace contaminants can develop over time and these seem to preferentially reduce lysine yields; more significant contamination will also reduce methionine and hydrophobic amino acid yields, and produce an overall reduction, sometimes quite significantly, in repetitive yield.

If the lot number indicates that the S2B bottle is close to a year old or more, it should not be installed. You can either replace the S2B or add high-purity DTT (dithiothreitol) to the S2B to correct this problem. The recipe for this calls for weighing out 4 mg of DTT, dissolving it in 1 ml of ethyl acetate (S2B), and then adding approximately 225 ul of this solution per full, 450-ml bottle of S2B.

In other cases, old R4 with oxidized DTT can also cause low lysine yields, and in this case such reduced yields can also be seen in the standards.

Tryptophan is one of the most difficult amino acids to sequence since it is readily oxidized. Oxidized Tryp (W) does not sequence. Often times you will see a derivative of W, kynuranine, which elutes between DPTU and W. It can account for 0-100% of the W signal. Replace the S2 bottle to improve recovery of Tryptophan. An alternative is to add DTT (an anti-oxidant) to S2 to increase the yield of W, or other forms of anti-oxidant can be added to R3. If you do add DTT to S2 (ethyl acetate) you may see a PITC-DTT adduct peak eluting close to Proline (P).