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Method Development Strategy for a Polar Compound
Using Anatoxin-a
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printable Application Sheet
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Notes: Anatoxin-a (ANTX-A) is a cyanobacterial neurotoxin, implicated in many animal and human poisoning incidents. ANTX-A blocks
neurotransmission causing death by respiratory arrest. The presence of this toxin in freshwater has to be monitored in order to prevent
fatalities.
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Method Conditions
| Column |
Cogent Diamond Hydride™, 4µm, 100A |
| Catalog No. |
70000-15P-2 |
| Dimensions |
2.1 x 150 mm |
| Solvents |
| A: | 50% MeOH/ 50% DI H2O/ 0.1% formic acid |
| B: | Acetonitrile/ 0.1% formic acid |
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| Gradient |
| time (min.) |
%B |
time (min.) |
%B |
| 0 |
70 |
6 |
30 |
| 5 |
30 |
7 |
70 |
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| Post Time |
5 min |
| Injection Vol. |
1 microL |
| Flow rate |
0.4 mL/min. |
| Temperature |
25°C |
| Detection |
ESI - POS - Agilent 6210 MSD TOF MS |
| Peak |
Anatoxin-a, 166.1226 m/z (M + H)+ |
| t0 |
0.9 min |
Discussion
Figures A, B, and C illustrate the work flow in developing a method for analysis of polar compounds using Cogent™ TYPE -C columns. The steps
of method development are as follows:
- Injection at 50%A/50%B mobile phase composition (Figure A). In the case of Anatoxin-a, considerable retention is observed.
- Injection at 60%B (Figure B). As expected, the retention of Anatoxin-a is longer and the peak shape is broader.
- Based on the above results, a simple linear gradient is designed to achieve the desired retention of the compound and excellent peak shape
(Figure C).
If shorter retention time is desired it can be accomplished by changing the starting concentration of solvent B to 60%, designing a steeper
gradient, or using a shorter column (2.1 x50 mm).
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