Contents A comparison of natural products

activity screening using SFE

versus HPLC

MICROSEP Technology Conference 31 July 2014

Nial Harding

nmharding@csir.co.za

2

Contents

1. Description of Activity Screening

2. Using HPLC to generate fractions

3. Why Supercritical Fluid Extraction?

4. Concluding remarks

3

Activity Screening

• Testing an extract, fraction or pure compound for activity

against a targeted entity normally by means of a bio-

assay.

• Points to consider:

– Cost of the assay – influences the number of samples

that can be tested.

– The amount of sample required for the assay –

determines the mass of plant material needed to

prepare the extract / fraction / pure compound.

– Selectivity of the assay.

4

Using HPLC to generate fractions

• Activity profiling – imported from Department of

Pharmaceutical Sciences, University of Basel, Prof M

Hamburger.

• Generic conditions:

– 10mg/ml sample is prepared in DMSO

– SunFire RP-18 Column (3.5µ, 3 mm x 150 mm)

– Injection volume 35 µl

– Column temperature ambient

– Collect 35 x 1 minute fractions into 96 well plates

5

Using HPLC to generate fractions

• Gradient table

Time (min) 0.1% FA in HOH

0.1% FA in ACN

Flow (ml/min)

Curve

0 90 10 0.5 1

30 0 90 0.5 6

35 0 90 0.5 6

6

Using HPLC to generate fractions

• Acknowledged challenge: Not possible to overlay collections due to

variability in fractions

• Possible solutions:

Time (min) 0.1% FA in HOH

0.1% FA in ACN

Flow (ml/min)

Curve

0 90 10 0.5 1

1 90 10 0.5 1

31 0 100 0.5 6

36 0 100 0.5 6

37 90 10 0.5 6

48 90 10 0.5 1

7

Using HPLC to generate fractions

• Flow rate perhaps a bit low? Equilibration time & wash.

• Column temperature?

• Injection volume.

Column id (mm)

2.1 3.0 3.9 4.6 7.8 10 19.0 30.0 50.0

Min Flow (ml/min)

0.2 0.4 0.7 1.0 2.6 4.5 16.4 41 113

Max Flow (ml/min)

0.3 0.6 1.0 1.4 4.1 6.8 24.5 61 170

Load (mg) 0.01 0.02 (0.35)

0.03 0.05 0.14 0.23 0.82 2.0 5.7

8

Using HPLC to generate fractions

9

Using HPLC to generate fractions

Some challenges:

• Class of compounds & targeted activity vs solubility.

• Solubility in DMSO.

• Chromatographic effect of the solvent the sample is

dissolved in.

• Required mass for activity screen / bio-assay vs load on

column.

• Flow rate vs collection vessels size.

• Evaporation of mobile phase

10

Why Supercritical Fluid Extraction?

• Cost of evaporation of high percentage aqueous solvent

fractions

– In terms of time

– Capital cost of equipment

– Running costs of equipment

• Less waste and more environmentally friendly

• Possibility of producing fractions from plant material

• Fractionate into classes?

©2013 Waters Corporation 11

CO2 tunable parameters and polarity for selectivity

Control of Tunable Extraction Parameters Critical to Optimizing and Reproducibility

12

Why Supercritical Fluid Extraction?

13

Concluding Remarks

• The MV-10 isn’t an HPLC

• SFE operating parameters:

– Extraction pressure affects selectivity. Increased pressure increases solvent power, but reduces selectivity.

– CO2 flow rate

– Temperature @ pressure constant - decreases CO2 solvent power but increase compound vapour pressure.

– Co-solvent.

– Particle size of sample matrix • internal mass transfer resistance

• Channelling

• Water content

– Duration of the extraction.

14

Concluding Remarks

The challenge:

– To develop a generic method on the MV-10 that will sequentially extract (classes ?) of compounds.

– The method should produce reproducible fractions from the same sample material

– To date - lots of learning but not there yet

Thank you

Name (email@csir.co.za)