Dennis Hoobin Varian Australia Pty Ltd 679 Springvale Rd Mulgrave VIC Australia

1. Using Linear Scale-up Principles and the Varian 940-LC with Scale-Up Module for the Isolation of 5-Hydroxytryptophan from Griffonia Simplicifolia Dennis Hoobin Varian Australia Pty Ltd 679 Springvale Rd Mulgrave VIC Australia

2. 940-LC Instrument

3. Introduction • Griffonia simplicifolia is a tall bushy vine found in Ghana, Nigeria, Liberia, Ivory Coast, Cameroon and Congo • The vine produces pods containing 3 large legumes • The legumes are a rich source of 5-hydroxytryptophan (5HTP) • 5-hydroxytryptophan produced by the human body from the essential amino acid L-hydroxytryptophan (LT) • 5-HTP has been clinically used to increase serotonin production to combat many mental illnesses like depression and schizophrenia among others

4. Instrumentation • Varian 940-LC Semi-Prep HPLC (part no 02102201-00) • Binary gradient pumps • 128 position autosampler with 5mL syringe • Scale Up Module to allow seamless scalability from Analytical to Semi-preparative applications • Optimised Dual path flowcell • Diode Array detector • Accurate Fraction collection • Columns - Varian XRs C18 • Analytical column -150 mm, 4.6 mm ID, 5u • Preparative column - 150 mm, 21.2 mm ID, 5u

5. Instrument Features • Scale Up Module • Fully Automated unit allowing switching between analytical and preparative columns • software-controlled module switches from analytical to preparative operation to ensure that chromatograms are correlated • Flow cell • optimized dual path flowcell that spans analytical to prep flow rates (1–25 mL/min) • the dual path flowcell design provides the ability to work over an extended absorbance range (mAu to tens of Au)

6. Instrument Conditions Scaling Up • Scaleable System Elements Absolute System Elements • Flow Rate Packing Material • Tubing Area Column Length • Sample Injection Volume Sample concentration • Solvent Consumption Gradient Slope Run Time Back Pressure • Flow Rate Packing Material

7. Instrument ConditionGradient conditions • Mobile phase 85% A (H2O) 15% B (MeOH) • Flow rates: • 1 mL/min (analytical) • 21.2 mL/min (preparative) • Injection volume 50 μL analytical–4 mL preparative • Dual Array PDA detector: monitoring 2 channels at 230 and 280 nm.

8. Analytical to Preparative 50 μL analytical injection of 5HTP standard

9. Diode Array Chromatogram Diode array display. Quadrant 1: Chromatogram. Quadrant 2: 3D diode array data. Quadrant 3: Isogram. Quadrant 4: UV spectrum derived from chromatogram at cursor position

10. Instrument Scale up Chromatograms 50 μL analytical injection of legume extract 4 mL preparative injection of legume extract, full scale. Note the extended range absorbance maximum of 8.5 abs

11. Purity Check • Fraction between 3 and 3.5 min in the preparative chromatogram was collected resulting in a fraction volume of 10.1 mL • Reinjection of the collected fraction onto the Analytical column results in a single peak chromatogram. Overlayed with the PDA peak purity trace, the flat top of the purity trace indicates a pure compound

12. Discussion • A good estimate of the amount of the % of HTP in dry Griffonia seeds can be made • The entire 10 mL fraction contains 17.2 mg • 1gm of seeds contains 215mg of 5HTP • Approx. 20% dry weight • G. Simplicifolia seeds are a very rich source of 5HTP

13. Conclusion • Varian 940-LC is a powerful automated system to allow easy development of HPLC preparative separations • Developing an analytical method – seamless scale up to an optimised preparative columns • Excellent correlations between analytical and preparative chromatograms • Confidence in the purity of fractionated material