I would like to perform HPLC/MS of a plant extract. Can anyone suggest me with the method of doing it?

Jack Silver Popular answer

I'll try to help, but I'll try not to do your work for you. Since this is analytical chemistry, I'm assuming you guys are looking for the presence of the compound in your extract. In no particular order:

Do a literature search on the compound in question. You can then use the method described in the paper(s) you find.

If the literature reference only describes purification of the target compound, it is relatively easy to convert that to an analytical system if the compound was purified on C18

Also look up the plant that you extracted in the literature and look for similar compounds

If you don't have the C18 column listed in any literature references, remember that although C18 columns aren't all the same, neither are they so different that you can't fine tune a method for the column you already have. In other words, don't tell your professor "we need "X" column" until you can prove that your column doesn't work.

Look up the eluting power of acetonitrile compared to C18 (hint- see the link below, Ref 1.). You may find that a method using acetonitrile may work with methanol, if you don't have acetonitrile.

Look up how and why solvent modifiers work (such as trifluoroacetic acid, formic acid, acetic acid). When could they be used to improve chromatography?

Also learn about "Limits of detection" and "limits of quantitation". You might not be able to say "that compound isn't in the extract". You may only be able to say "If it is there, the concentration is less than XXX". Alternatively, you might say "I see a peak indicating a concentration of less than XXX"

As for negative and positive ESI, that really depends on your compounds. Acids tend to show in ESI-, bases in ESI+, OH groups might go either way. Some compounds are visible as a sodium or potassium adduct, some lose a group (loss of water or ammonia, see Ref 2 for an example that isn't visible in ESI and loses water). Again, your literature search may provide this information. Also look up similar compounds.

Make sure the equipment is working! Make sure your column isn't "worn out", the pumps are running properly, and the detectors are detecting. Run standards and see that they retain properly- look up the column you are using if a QC test wan't provided, or call the vendor.

Do #1 first, and #9 second, before doing any real chromatography.

Ref 1: http://www.teledyneisco.com/en-us/liquidChromatography/Chromatography%20Documents/Application%20Notes/Acetonitrile-Methanol%20Substitution%20in%20C-18%20Reverse%20Phase%20App%20Note.pdf#search=solvent%20substitution

Ref 2: http://www.teledyneisco.com/en-us/liquidChromatography/Chromatography%20Documents/Application%20Notes/Mass-directed%20Purification%20of%20Steroids%20with%20APCI%20and%20CombiFlash%20PurIon%20App%20Note.pdf#search=APCI

Jack Silver

What sort of help do you need?

What sort of analytical LC? Normal phase or reverse phase?

I am going to assume that C18 is being used since that is the most common HPLC method. Compounds extracted into hexane are very non-polar and, depending on the solvent system used, may well "stick" to the column. One can try "non-aqueous reverse phase" using silica-based C18 columns to elute compounds. Don't use this technique on polymeric reverse phase since you might ruin the column!

The poster below describes the technique for preparative LC, but it works for analytical as well. You may need to use an APCI probe instead of ESI.

http://www.teledyneisco.com/en-us/liquidChromatography/Chromatography%20Documents/Posters/Facile%20Isolation%20of%20Carotenoid%20Antioxidants%20from%20Solanum%20Iycopersicum%20Poster.pdf#search=non-aqueous

Imam Hossen

By help I meant to say is the method development.

I want to do analytical LC with reverse phase C18 column. I am confused whether to use acetonitrile with water or methanol with water as mobile phase. I am also unsure about the column temperature to set, 25 degrees/30degrees/35degrees?

Also the ionization method. Which one I would set? I want to set ESI, but whether I set "ESI positive" or "ESI negative"?

My teacher suggested me to use methanol with water as mobile phase.

Jack Silver

Navid J. Ayon

Hello Imam,

You can select the MS mode based on the structure of the compounds you are expecting to see in your plant extract. If it’s totally unknown, then look at the UV chromatogram if you have already run an analytical sample and check the fractions corresponding to UV peaks in both MS positive and negative mode by infusion. We generally prefer and use water and acetonitrile in our lab because of low viscocity and better volatility, higher eluting strength, less appearance of ghost peaks. But LC grade acetonitrile is expensive than methanol, so you have to see the support you have. You can also check the following reference.

Thanks.

https://www.shimadzu.com/an/hplc/support/lib/lctalk/35/35lab.html

Jack Silver

Do a literature search on the compound in question. You can then use the method described in the paper(s) you find.

If the literature reference only describes purification of the target compound, it is relatively easy to convert that to an analytical system if the compound was purified on C18

Also look up the plant that you extracted in the literature and look for similar compounds

Look up the eluting power of acetonitrile compared to C18 (hint- see the link below, Ref 1.). You may find that a method using acetonitrile may work with methanol, if you don't have acetonitrile.

Look up how and why solvent modifiers work (such as trifluoroacetic acid, formic acid, acetic acid). When could they be used to improve chromatography?

Do #1 first, and #9 second, before doing any real chromatography.

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