I am trying to identify some metabolites from a recently sequenced strain of Streptomyces. The metabolites were extracted from the secreted products of the Streptomyces using methanol, chloroform and acetic acid. I wondered if there were HPLC conditions/gradient that separate a broad range of metabolites ? Or do i have to use several different gradients ? I am aware that methanol /chloroform will precipitate proteins.
Was the methanol, chloroform, and acetic acid mixed together somehow? I ask because this affects the advice that you'll be given. Was the Streptomyces grown on an agar plate or in suspension in culture media? If in suspended culture, the methanol and acetic acid probably left the chloroform and went into the culture media (assuming they were mixed somehow). Please give more details of your extraction procedure.
To start, I'd run a gradient of water/acetonitrile containing 0.1% TFA (5 0r 10% to 100% acetonitrile). If a silica-based column is used,I'd follow-up with a wash with dichloromethane (after the acetonitrile) to see any possible non-polar compounds, and then wash with acetonitrile again. The DCM wash is essentially non-aqueous reverse phase.
There are no universal chromatographic HPLC conditions. View the range of HPLC chromatographic conditions the same as the electromagnetic spectrum (x-rays to microwaves).
Thus, your main fallacy is jumping to HPLC 1st. I would have tried TLC 1st.
Dear Gerry,
One of my co-worker has worked on somewhat same system. He also wished to run an HPLC for metabolites from Streptomyces. As Prof. Bruce mentioned, you should run a TLC in various mobile phases i.e. EA:Hexane:Glacial AA (10:90:1) etc. and check the number of spots. The number of spots will help you to identify number of peaks in HPLC. Also, the wavelength for different spots could be judged from TLC plate which in turn will help in HPLC too. Likewise, you will have to select the mobile phase for your HPLC in a suitable manner such that the peaks will be separated at significant intervals. Addition of 0.1% Acetic Acid/Formic Acid to the mobile phase often promotes separation. I worked with some scientists from Agilent Technologies and they are usually using 10% ACN and if not then 10% Water in ACN along with 0.1% GAA/FA.
All the best
Thanks Jack.
It was a sequential extraction , extract with methanol , then extract with chloroform , then extract with acetic acid. The extraction was from a solid substrate (of mixed composition) which was used to absorb metabolites. I wanted to make sure I extracted all compounds , polar , non-polar , acidic , basic etc , so I tried a few more conditions. The media was washed from the organisms and they were rinsed in basic medium containing only a few buffer compounds. This was gently shaken for a period of time and then the liquid was seperated. I used this technique in an old paper to look at staphylococcus secretions. There are something like 20 antibiotic genes and maybe 15 antibiotic clusters from our sequencing of the organism so I wanted to fractionate the samples further so that I could analyse by mass spectrometry. I do not plan to identify everything, just some major metabolites.
Gerry- That helps a lot. Although, as mentioned earlier, there is no universal HPLC conditions, knowing the extraction conditions can suggest HPLC conditions.
Since you are looking for antibiotic compounds, Sachin's idea about using TLC is a good one. TLC can be run in different solvents and bioautograms can be run to find the solvent system that resolves the antibiotics from other compounds. When doing assay guided fractionation, TLC just says spots are resolved, but there is no indication which spot(s) are the active compound and if the active compound(s) are resolved from impurities. With bioautograms, you get a visual indication that the compound is resolved.
The methanol extract tends to get a good mix of compounds- polar, and non polar, acidic, and basic. I would run water/acetonitrile HPLC with an acid modifier. The acetonitrile is less polar than the methanol and should help to wash off the non-polar compounds.
The DCM wash is likely to contain mostly non-polar compounds. If you can run normal phase HPLC, hexane/ethyl acetate, this may resolve your compounds. If you are limited to reverse phase, use the solvent system above followed by a DCM wash in case anything sticks to the column (wash with acetonitrile, and this is only for silica based reverse phase. Don't use this wash on polymeric reverse phase).
The acetic acid wash is likely to contain polar, basic compounds. reverse phase water/methanol with an acid modifier would be my first choice here.
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