I want to purify compounds from extract of Emblica officinalis . I want to know about the columns used and what flow rate is required for eluting out the natural compounds ? Also, I want to know about the wavelength at which prep HPLC is done for plant extracts ?
I do not feel there is enough data to answer the question properly.
What sort of compounds? Do you know what you are purifying, or is it unknown compound(s)? Are you working with a pre-purified extract?
For Prep HPLC, C18 is commonly used, but the solvents used, and their concentration, depend on the compound polarity. For the phenolic compounds typically found in this plant (http://globalresearchonline.net/journalcontents/v24-2/25.pdf ), methanol/water/ 0.1% formic acid or TFA will work. (for an example with flash chromatography for similar phenolic compounds, see: http://www.teledyneisco.com/Documents/Liquid%20Chromatography/Posters/Purification%20of%20Phenolic%20Flavonoids%20with%20FC%20Poster.pdf#search=phenolic). As for the UV wavelength used, that is determined by the compounds (I used 214 and 278 nm in the referenced poster, close to the 226 and 280 nm mentioned above). For unknown mixtures, 214, 254 and 280 nm are commonly used.
If the compound is unknown, I like to do a solvent and column screen on the extract. The use of different solvents in chained gradients ("wide polarity range chromatography" ) maximized the possibility of the compound eluting and narrrows the possibilities of which solvents to use based on compound polarity. See: http://www.teledyneisco.com/Documents/Liquid%20Chromatography/Posters/New%20Techniques%20in%20Extract%20Column%20Screening%20Poster.pdf#search=screening and http://www.teledyneisco.com/Documents/Liquid%20Chromatography/Posters/New%20Techniques%20in%20Extract%20Column%20Screening%20II%20Poster.pdf#search=screening
Links updated 14 Jan 17
flow rate:1.0 ml/min
Column temperature: 37oc
Column Pressure: max 400 bar
signal detection Mode: UV/VIS
Detector wavelengths: 280 & 226 nm.
I do not feel there is enough data to answer the question properly.
What sort of compounds? Do you know what you are purifying, or is it unknown compound(s)? Are you working with a pre-purified extract?
For Prep HPLC, C18 is commonly used, but the solvents used, and their concentration, depend on the compound polarity. For the phenolic compounds typically found in this plant (http://globalresearchonline.net/journalcontents/v24-2/25.pdf ), methanol/water/ 0.1% formic acid or TFA will work. (for an example with flash chromatography for similar phenolic compounds, see: http://www.teledyneisco.com/Documents/Liquid%20Chromatography/Posters/Purification%20of%20Phenolic%20Flavonoids%20with%20FC%20Poster.pdf#search=phenolic). As for the UV wavelength used, that is determined by the compounds (I used 214 and 278 nm in the referenced poster, close to the 226 and 280 nm mentioned above). For unknown mixtures, 214, 254 and 280 nm are commonly used.
If the compound is unknown, I like to do a solvent and column screen on the extract. The use of different solvents in chained gradients ("wide polarity range chromatography" ) maximized the possibility of the compound eluting and narrrows the possibilities of which solvents to use based on compound polarity. See: http://www.teledyneisco.com/Documents/Liquid%20Chromatography/Posters/New%20Techniques%20in%20Extract%20Column%20Screening%20Poster.pdf#search=screening and http://www.teledyneisco.com/Documents/Liquid%20Chromatography/Posters/New%20Techniques%20in%20Extract%20Column%20Screening%20II%20Poster.pdf#search=screening
Links updated 14 Jan 17
Dear
Dr Silver is right, the experimental design for purification is not that simple it depends on may factors especially the type of compounds wanted and it's properties. However fractionation of extract with different solvents with gradual polarity using CC before HPLC can give better understanding of the nature of compounds.
Good luck
Just for the initial assessments of the Component behaviours, I will like to spend some time on the TLC of the Extracts, see the solvent system efficiency to separate the components , the go for the Analytical Hplc Column separation campaign & only then go for the Prep Hplc Column . this is so that we dont damage the Expensive prep Column. Off course , I will never use the unpurified extract directly on the Hplc Machines. Whatever technique we use for extract purification , we must go through this exercise first to have a slightly purified extract for the Columns. We may get into, esp. when we are the beginners, new problems of wasting time & energy & still getting no where. Read the Jacks proposals as well . you will find these exercises more productive in all respects.
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