26 February / March 2020


Figure 5: Loading study for amitriptyline at low and high pH. (A) Comparison of peak shape (normalised retention time) and (B) comparison of peak width as analyte load on column is increased. Note that mobile phase composition was set to obtain a retention factor (k) of approximately 5 at both pH values. Column: ACE Excel 3 SuperC18, 150 x 4.6 mm; Mobile phase (low pH): 1% TFA in MeCN/H2 (high pH): 0.1% NH3


O 44:56 v/v; Mobile phase in MeCN/H2 Detection: UV, 254 nm. O 77:23 v/v; Flow Rate: 1.0 mL/min; Injection Volume: 5 µL; Temperature: 40°C;


Conclusion


Natural Product SFC & HPLC Purification Columns


When working with ionisable analytes, the mobile phase pH can dramatically affect analyte retention behaviour. The mobile phase pH is therefore an important parameter to consider during method development. This article has shown how pH can be utilised during method development to alter selectivity and help optimise the separation. Appropriate pH selection also provides additional gains in retention, peak shape and sensitivity.


HPLC Separation Specialists


References


[1] ACE Knowledge Note AKN0005: The Fundamental Resolution Equation and the Impact of k, N and α (accessed at https://uk.vwr.com/cms/ace_ knowledge_notes)


ES Industries provides products for isolation and purification in HPLC & SFC for natural products. Purification


products include state of the art columns manufactured specifi- cally for the SFC isolation of cannabinoids from Cannabis sativa.


ES Industries • 701 S. Route 73 • West Berlin, NJ 08091 856-753-8400 • 800-356-6140 • Fax 856-753-8484 esindustries@msn.com • www.esind.com


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