1. Exanitide and its enantiomers
separation by RP-HPLC
WH, Kao; JR, Ai; YN, Lin; CW, Tsai; RC, Ruaan &
WY, Chen*
Bio-Recognition Engineering Lab., CME, NCU
2011/08/29

2. Condition of Exanitide Purification

3. Chromatogram
Impurity: peptide enantiomers:
racemization of Serine

4. Short Chain Peptide Isomer
• Identical molecular formula
– Sequence isomers (positional isomer)
• SFDENLGK
• SFEDNLGK
– Diastereomers
• IGLisoDCASSEFFK
• IGLDCASSEFFK
– Enantiomers
• LALARELEELN
• LALAD-RELEELN

5. Peptide Isomer Separation by RP-HPLC
• Peptide isomers
– Same hydrophobicity
– Difference in 3D-structure
• Structure flexibility
• Solution conformation energy
– Lower energy  short retention time
– Higher energy  long retention time
Lower energy
Higher energy
tR
Int.

6. Solution conformation energy
• Conformation Energy =
“Non-bonded Energy” + “Bonded energy”
• Non-bonded Energy= Eelectrostatic + EV.D.W.
• Bonded energy= bond energy + angle energy +
dihedral energy.

7. Our previous study:
RPLC retention of sequence isomers
• GELE (GELELKLKLEG)
• GELK (GELKLELKLEG)
• Solution conformation energy: GELK > GELE
23 24 25 26 27
-0.8
-0.4
0.0
0.4
0.8
1.2
1.6
2.0
GELE
GELK
lnk'
ACN (%) Tsai et. al., 2010 JPCB

8. Recently study:
Peptide enantiomers separation
0 2 4 6 8 10 30 40
-100
0
100
200
300
400
IL-K7F89
IL-K7F89 (DL2)
IL-K7F89 (DW4)
IL-K7F89 (DK7)
IL-K7F89 (DW11)
AU
Retention time (min)
Econf (Kcal/mol)
tR
e = 66
Sample in
30% ACN
ILK7F89 278.9 (2) 4.79 (2)
ILK7F89-D-L2 271.6 (5) 2.74 (5)
ILK7F89-D-W4 276.3 (3) 4.10 (3)
ILK7F89-D-K7 274.9 (4) 2.78 (4)
ILK7F89-D-W11 279.8 (1) 5.14 (1)

9. Prediction for Short Peptide Enantiomer
 Solution energy calculation
– LALARELEELN (135.747 kcal/mol)
– LALA-D-RELEELN (138.991 kcal/mol)

10. RP-HPLC
• Column: ACE C18-column
• Flow rate: 1.0 mL/min
• Detection: UV 215 nm
• Sample: 0.5 mg/mL
• Mobile phase:
– A: H2O + 0.1% TFA
– B: ACN + 0.1% TFA
– C: THF + 0.1% TFA

11. Effect of Elution Strength
0 2 4 6 8 10 12 14
-100
0
100
200
300
400
42 % ACN
40 % ACN
39 % ACN
38 % ACN
mV
Elution time (min)
Long RT & Broad
API

12. Co-elution of all peptide enantiomers
0 2 4 6 8 10
-200
-100
0
100
200
300
400
500
600
D-Ser11
D-Ser32
D-Ser39
API
mV
Elution time (min)
42 % ACN

13. Co-elution of D-Ser 39 & API
0 2 4 6 8 10
-200
-100
0
100
200
300
400
D-Ser11
D-Ser32
D-Ser39
API
mV
Elution time (min)
40 % ACN

14. Good Correlation between Energy & RT
Solution conformation
energy (kcal/mol)
Retention Time
(min)
D-Ser32 416.666 6.72
API 415.928 5.25
D-Ser39 414.096 5.16
D-Ser11 412.512 4.47
Energy
RT
Co-eleution
Model at 36 % ACN

15. Solubility Parameter of Solvent
Solvent
δ, MPa1/2
δt δd δp δh
Water 47.80 15.60 16.00 42.30
Methanol 29.60 15.10 12.30 22.30
Acetonitrile 24.40 15.30 18.00 6.10
Tetrahydrofuran (THF) 19.40 16.80 5.70 8.00
ACE column 23.96 15.80 15.73 8.79
Daiso column

16. 0 1 2 3 4 5 6 7 8 9 10
-200
-100
0
100
200
300
400
500
600
700
800
mV
Elution Time (min)
API_iso_0.85_MeOH_DAISO
API_iso_0.83_MeOH_DAISO
API_iso_0.80_MeOH_DAISO
API_iso_0.75_MeOH_DAISO
API_iso_0.72_MeOH_DAISO
API_iso_0.71_MeOH_DAISO
API_iso_0.70_MeOH_DAISO
Change the mobile phase to MeOH
• Low Separation Resolution

17. Change the resin to Daiso
• Low Separation Resolution
0 1 2 3 4 5 6 7 8 9 10
-50
0
50
100
150
200
250
300
350
mV
Elution Time (min)
D-Ser39_iso_0.40_ACN_DAISO
D-Ser39_iso_0.42_ACN_DAISO
D-Ser39_iso_0.44_ACN_DAISO

18. Sample solvent effect
200 220 240 260 280 300
-20
-15
-10
-5
0
5
10
15
20
H2
O (D-Ser39)
H2
O (API)
40% ACN (D-Ser39)
40% ACN (API)
(mdeg)
Wavelength (nm)
a-helix
Lower helicity
Similar structure

19. Effect of Sample Solvent Strength
Solution conformation energy (kcal/mol)
55% ACN 36% ACN 0% ACN
D-Ser39 413.612 414.096 412.914
API 416.898 415.928 415.748
D (D-Ser39-API) -3.286 -1.832 -2.834
Non-retention in column

20. Sample Solvent Effect
API
D-Ser39
5 6 7 8 9 10
0
50
100
mV
Elution time (min)
0
50
100
150
200
mV
0% ACN
40% ACN
Broad
40 % ACN

21. Solubility Parameter of Solvent
Solvent
δ, MPa1/2
δt δd δp δh
Water 47.80 15.60 16.00 42.30
Methanol 29.60 15.10 12.30 22.30
Acetonitrile 24.40 15.30 18.00 6.10
Tetrahydrofuran (THF) 19.40 16.80 5.70 8.00
ACE column 23.96 15.80 15.73 8.79

22. Change the mobile phase
0 1 2 3 4 5 6 7 8
-100
0
100
200
300
400
500
600
mV
Elution time (min)
D-Ser39
API
37% THF

23. 0 1 2 3 4 5 6 7 8 9 10
-50
0
50
100
150
200
250
300
350
400
450
mV
Elution Time (min)
Mixture_0.75_API_iso_0.40_ACN
Mixture_0.50_API_iso_0.40_ACN
Mixture_0.25_API_iso_0.40_ACN
Mixture of API and D-Ser39
40% ACN isocratic elution

24. 0 1 2 3 4 5 6 7
-50
0
50
100
150
200
250
300
350
400
450
mV
Elution Time (min)
Mixture_0.75_API_iso_0.37_THF
Mixture_0.50_API_iso_0.37_THF
Mixture_0.25_API_iso_0.37_THF
Mixture of API and D-Ser39
37% THF isocratic elution

25. Summary
• MD simulation for the solution conformation energy
calculation facilitated the prediction of the retention
behaviors of peptide enantiomer in RP-HPLC.
• Sample solvent could be utilized to tune the retention
behavior.