Figure 2 shows some of the minor peaks in the spectrum taken after step 13. Small amounts of deletion peptides are seen in addition to small peaks representing residual Pbf (Arg-protecting group). These are due to released Pbf attacking, for example, the Serine residue. Small contributions from cationization with sodium and potassium are also observed.
Figure 3 shows the MH+ region of the completed and deprotected peptide. Neurotensin (MW 1,673) and ACTH 18-39 (MW 2,465) were used as internal calibrants. The average error of the mass measurement was +/- 0.03 Da (5 replicates). This measurement is more than would be required to differentiate between a carboxyl and an amide (one Da difference). All masses are monoisotopic since the resolution of the instrument was sufficient to resolve isotopes at these masses.
1 hour deprotection is not required to determine if the synthesis is proceeding correctly. Figure 4 shows the peptide data after step 10 with a 10 minute deprotection step. The Pbf protecting group on Arg is clearly still present but some fully deprotected product (Fmoc peptide) is visible. The t-Bu protecting group on Thr is completely absent. No t-Bu group was detected after > 5 minute treatment with TFA. The trityl group used to protect Gln behaves similarly.
The result in Figure 5 is from the deprotection of the synthesis of the peptide SEHFLANES-OH. In this case the deprotection step was for 5 minutes only, and the presence of trityl and up to 3 t-butyl groups can be detected.
The full time course experiment up to 1 hour is seen in Figure 6. After 1 hour the unprotected peptide is the principal peak in the spectrum.