3.3.2.1. JH-III standard

As commercial JH III (Sigma or other supplier) is usually not sufficiently pure and there is degradation over time, it is recommended that an aliquot of commercial JH III is first purified by thin layer chromatography (TLC). To do so:

1.“Wash” a glass backed TLC plate (5x20 cm or 20x20 cm, Merck silica gel 60 F254) in ethanol. This is done by placing it in a TLC glass tank with 1 cm of ethanol in the bottom; when the solvent reaches the top of the plate, remove and dry it; it may then be stored.

2. Prior to the actual separation, put a washed TLC a plate into a tank containing a 1 cm high volume of toluene: ethylacetate (95:5) solvent mix and run it in the tank until it reaches the top of the plate. This will clean and activate the plate.

3. Mark the top to indicate plate orientation.

4. Dry the plate.

5. Activate it by heat treatment at 60oC for 10 min.

6. On the bottom side, using a soft pencil, draw a line across at 1.5 cm.

7. At the sides, use the edge of a fine spatula to scratch a line at 0.5 cm from each edge to prevent edge migration distortion.

8. Streak an aliquot of the commercial JH III on the 1.5 cm bottom line.

9. Completely evaporate solvent with an air stream.

10. Run the plate in toluene: ethylacetate (95:5) solvent mix until the solvent front has risen approximately 5 cm into the plate.

11. Remove, air dry and return the plate to the solvent to complete the run to about 17 cm of the total plate length.

12. Air dry the plate.

13. Visualize the JH band under UV light (254 nm).

14.  Mark its position with a pencil.

15. Scratch the silica layer corresponding to the JH band from the plate using a spatula, collecting the scraps on a piece of aluminium foil.

16. Transfer scraps to a screw cap glass vial containing 5 ml of hexane/ethyl acetate 85:15.

17. Agitate on an orbital shaker overnight.

18. Filter solvent through a glass syringe fitted with a glass filter.

19. Dry the solvent by vacuum centrifugation.

20. Re-dissolve in toluene containing 0.5% propanediole (final conc.). This will be the purified JH solution for storage at -20oC.

For preparing the JH-III standard for preparation of standard curves:

1. Take a 5 µl aliquot of this purified JH.

2. Dilute 1:100 in methanol.

3. Quantify JH-III by UV spectroscopy at λ = 217 nm.

At this wavelength an absorption of 0.502 corresponds to a JH III concentration of 10 µg/ml.

4. Correcting for dilution (step 2), dilute an appropriate aliquot of the purified JH in toluene/propanediol to a final concentration of 50 pg/µl. This will be the stock solution for preparing standard curves.

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