Separation of Cyclic Dipeptides (Diketopiperazines) from Their Corresponding Linear Dipeptides by RP-HPLC and Method Validation
Table 1
Retention factor () for DKPs, dipeptides, and amino acids and separation factor () and resolution () of two adjacent peaks at chosen separation conditions.
Separation conditions
Substance
Retention factor
Separation factor
Resolution
Cyclo(Gly-Gly)
1.03
1.63
5.47
Gly-Gly
0.63
Cyclo(L-Ala-D-Ala)
2.34
1.18
3.26
Cyclo(L-Ala-L-Ala) and cyclo(D-Ala-D-Ala)
2.75
10% MeOH, 20°C, 0.7 mL min−1
L-Ala-L-Ala and D-Ala-D-Ala
0.67
1.25
1.83
L-Ala-D-Ala
0.84
Cyclo(L-Ala-D-Ala)
2.34
2.79
13.77
L-Ala-D-Ala
0.84
Cyclo(L-Arg-L-Arg)
1.08
1.36
1.55
L-Arg-L-Arg
0.79
10% MeOH, 15°C, 0.3 mL min−1
Cyclo(L-Asp-L-Asp)
0.61
1.15
1.04
L-Asp-L-Asp
0.53
Cyclo(L-Asp-L-Phe)
3.33
1.14
2.31
L-Phe
2.93
20% MeOH, 20°C, 0.7 mL min−1
L-Asp-L-Phe
2.10
1.40
5.45
L-Phe
2.93
L-Asp-L-Phe
2.10
1.59
5.88
L-Phe-L-Asp
1.32
Cyclo(L-Pro-L-Tyr)
3.75
2.26
9.44
L-Tyr-L-Pro
1.66
L-Tyr-L-Pro
1.66
1.76
4.23
L-Tyr
0.95
L-Pro-L-Tyr
0.93
1.02
0.22
30% MeOH, 30°C, 0.7 mL min−1
L-Tyr
0.95
Cyclo(Gly-L-Phe)
4.36
2.31
16.12
L-Phe-Gly
1.89
L-Phe-Gly
1.89
1.01
0.17
L-Phe
1.87
Gly-L-Phe
1.84
1.02
0.27
L-Phe
1.87
Hold-up time = 2.167 min (manufacturer information).
