Transcript Purification of 5 Hydroxytryptophan using the Varian 940-LC

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
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
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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
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
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Packing Material
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.
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Analytical to Preparative
50 μL analytical injection of 5HTP standard
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Diode Array Chromatogram
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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
Instrument Scale up Chromatograms
50 μL analytical injection of legume extract
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4 mL preparative injection of legume
extract, full scale. Note the extended
range absorbance maximum of 8.5 abs
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
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