Chromatography Forum

Hello,

I have a problem with extraction of melittin - a 26 amino acid peptide from petroleum ointment.

My method:

1.~0.250 g of melittin ointment is dissolved in 2 ml of n-hexane
2. LLE is conducted - 2 ml of 25%ACN is added and solvents are stirred for 30 minutes
3. The 25%ACN layer is centrifuged
4. 100ul HPLC injection is made.

Problem: Recovery is only 80% (i need 95-105%)

I tried various concentrations of ACN and 25% ACN was, unfortunately, most effective.
I tried also Isopropanol, and methanol and they both were weaker than ACN.
I tried adding 2x 1ml of 25% ACN for 15 minutes and the recovery was the same

Any suggestions?

Cheers,

Dorian

Hi Dorian

How are you calculating recovery ?

Peter

Thanks for response,

I prepare spike :

1.~0.250 g of blank petroleum is dissolved in 2 ml of n-hexane
2. 2ml of peptide solution of expected* concentration in 25% ACN is added, and solvents are stirred for 30 minutes
3. The 25%ACN layer is centrifuged
4. 100ul HPLC injection is made.

Recovery is mean area of peak from ointment/mean area of peak from spike *100%

* The ointment is concentration is 0,5mg/1g so
in 250 mg of ointment there is 0,125 mg of melittin so
concentration of peptide solution was 62,5ug/ml

Dorian

You might try tweaking the pH of the extraction solvent. Look at the sequence and see whether you have a significant number of acidic or basic residues and adjust pH to ionize a majority of them. That might shift the equilibrium enough for your purposes.

Hi Dorian

* The ointment is concentration is 0,5mg/1g

How do you know that ?

What result do you get if you spike the vaseline (not the vaseline solution) with mellitin ?

Peter

Hi,

sorry for the delay in response. Thank you both tom jupille and Peter Apps for your help. After i applied your advices to my extraction, the recovery became ~100%.

* The ointment is concentration is 0,5mg/1g

How do you know that ?

I know that concentration is 0,5mg/1g, because i made this ointment

Dorian