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. 2 940-LC Instrument

3. 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. 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. 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. 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. 7 Instrument Condition Gradient 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. 8 Analytical to Preparative 50 μL analytical injection of 5HTP standard

9. 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. 10 Instrument Scale up Chromatograms 4 mL preparative injection of legume extract, full scale. Note the extended range absorbance maximum of 8.5 abs 50 μL analytical injection of legume extract

11. 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. 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. 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