Using Linear Scale-up Principles and the Varian 940-LC with Scale-Up Module for the Isolation of 5-Hydroxytryptophan from

Griffonia Simplicifolia

Dennis HoobinVarian Australia Pty Ltd

679 Springvale Rd Mulgrave VIC Australia

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940-LC Instrument

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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

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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

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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)

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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

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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.

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Analytical to Preparative

50 μL analytical injection of 5HTP standard

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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

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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

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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

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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

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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