25 Eluotropic strength
Acetonitrile has a higher elution strength than methanol for reversed-phase chromatography, therefore shorter analyte retention can be expected for equal proportions of organic to water (Figure 2). In this example, for the separation of these neutral analytes, approximately 1.7 x longer retention is obtained using methanol/water. In principle, it could be possible to increase the proportion of methanol in B to obtain a similar separation to that shown in Figure 2A (i.e. matching the eluotropic strength of the acetonitrile/water mix). Indeed, during the recent acetonitrile shortage in 2008/9, some laboratories attempted to replace acetonitrile with methanol in the mobile phase. For some applications this approach may be successful; although caution should be exercised as large changes in selectivity and elution order may occur.
Selectivity
One of the most useful aspects of the availability of both acetonitrile and methanol is that they have differing solvent properties. Methanol is a polar- protic solvent, whereas acetonitrile is a polar-aprotic solvent and possesses a stronger dipole moment. This means that the organic modifier used in the mobile phase can have a powerful effect on chromatographic selectivity. Varying the organic component of the mobile phase can therefore be a powerful method development tool. Figure 3 shows the same gradient separation run using methanol and acetonitrile as the organic modifier. In this example several co- elutions are observed when acetonitrile is used. When methanol is used different selectivity is produced, and all sample components are fully resolved. For some analytes, the change in relative retention is large and several complete reversals in elution order are observed (e.g. peak pairs 9/10 and 16/17). It is therefore highly recommended that both acetonitrile and methanol are assessed during method development to determine the most suitable solvent and to help optimise the separation.
As methanol and acetonitrile are fully miscible with one another, they can also be blended to fine-tune a separation. Figure
Figure 2: Comparison of the separation of neutral analytes using a mobile phase containing A) 75:25 v/v acetonitrile/water and B) 75:25 v/v methanol/water on a C18 column, 100 x 3.0 mm (flow rate: 0.43 mL/min, temperature: 30°C, injection volume: 0.5 µL, detection: UV 254 nm).
4 shows an example gradient separation of basic analytes where neither methanol or acetonitrile as the organic modifier provides a full separation. When methanol is used as the organic modifier, peaks 5 and 6 co-elute, whereas with acetonitrile, peaks 3 and 4 are not resolved. In contrast,
by blending the two solvents together with water as a ternary mixture, it is possible to obtain intermediate selectivity and separate all seven components.
Figure 3: Gradient separation of a 17-component test mix using A) acetonitrile and B) methanol as the organic modifier. Column: ACE 3 C18-AR 50 x 2.1 mm, flow rate: 0.6 mL/min, temperature: 40°C, gradient: 3-100% B in 6.5 mins, detection: 214 nm. Sample: 1. 3-Hydroxybenzoic acid, 2. Methyl phenyl sulfoxide, 3. Quinoxaline, 4. Salicylic acid, 5. Benzylcyanide, 6. 1,2-Dimethoxybenzene, 7. Ethylparaben, 8. 1,4-Dimethoxybenzene, 9. Bendroflumethiazide, 10. Piroxicam, 11. Benzylchloride, 12. Thioanisole, 13. Sulindac, 14. Chrysin, 15. Ibuprofen, 16. 1,2,3-Trichlorobenzene, 17. Meclofenamic acid.
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