Is ii possible to run HPLC analytical crude extract of Hexane and DCM using C18 column? How to prepare extract sample and what will be the choice of solvent for mobile phase?
Hello,Yes, you can, sometimes, C18 column is very helpful in fractionation of non polar compounds but you can just elute your column with polar solvents such as methanol, acetonitril, water or gradient of them. So your extract may insoluble in these solvents. After testing your extract souloblity, I suggest you to prepare C18 TLC and observe your extract pattern on it (after developing with selected solvents)
Dear , applying C18 separation columns you can not inject samples in hexane or DCM because for separation in principle aqueous mixtures of water, methanol, acetonitrile or isopropanol are used. So, you could choose to change to normal phase separation or redissolve your samples in MeOh or Acetonitrile and separate them on a c18 column.
As per my knowledge, C18 columns are only used in Revers phase HPLC in which mobile phase is polar and stationary phase (column) is non-polar, better u go for silica column for better illusion. If u use, DCM or Hexane there is a chance of dissolution of stationary phase in that so better don't use C18 column for those separations....
As per my knowledge, C18 columns are only used in Revers phase HPLC in which mobile phase is polar and stationary phase (column) is non-polar, better u go for silica column for better illusion. If u use, DCM or Hexane there is a chance of dissolution of stationary phase in that so better don't use C18 column for those separations....
Yes it is possible to run C18 columns with very hydrophobic solvents. This is called NARP (non-aqueous reverse phase chromatography). There have been several examples on the analysis of non-polar species using ethanol, tert-butyl methyl ether, chloroform or hexane as mobile phases. An example is the analysis of carotenoids. You should simply not use solvents that are not miscibles (e.g. methanol and hexane). Water is not used in this mode and must be washed from the HPLC system before analysis.
An alternative for very hydrophobic compounds is the use of normal phase as mentioned above.
As mentioned above, one can run non-aqueous reverse phase. I like to use methanol and DCM as a mobile phase, although there are other solvent systems that would work. Non-aqueous reverse phase doesn't harm silica bonded C18 (remember the C18 is chemically bonded), but the solvents will harm polymer based columns (cause swelling and high back pressures). Remember that the more polar solvent takes the place of water because it is the weak solvent. Although written for flash chromatography, the link below goes to an application note I've written describing non-aqueous reverse phase and suitable solvent systems. I've also includes a poster demonstrating the purification of lycopenes with this method on a large-scale flash system.
http://www.isco.com/WebProductFiles/Applications/101/Application_Notes/AN58_Non-aqueous_Reverse_Phase_with_RediSep_Rf_Gold_C18.pdf
http://www.isco.com/WebProductFiles/Applications/101/Poster_and_Paper_Reprints/Facile_Isolation_of_Carotenoid_Antioxidants.pdf
I just want to say that hexane as typical solvent is a mixture of hexane isomers, besides it uses to be higly dirty (metal impurities, water and other solvents) even as reactant or analytical grade. Ensure you are using HPLC-grade hexane(s) when working at any step.
Yes you can.
You should prepare your samples in those solvent which are soluble in methanol or acetonitrile. as your mobile phase will constitute these solvent for c18 columns. to check miscibility add your sample in mobile phase and see for miscibility. if miscible then you can start your work on c18 column.
as your components are non polar they will have more retention time in RP- HPLC (more than 15min). so you better start with mobile phase metahnol: water 95:05 water.
Thank you everybody. Actually my extracts are Hexane and DCM. if not soluble in MethOH or ACN then how can I proceed?
inject them in the minimum DCM that you can. Either minimize the injection volume, or dilute it with the weak solvent (methanol, of you are using that in your gradient) until it is barely soluble.
I like DMSO and DMF as reversed-phase sample solvents. Their very high polarity (highly negative log P values) and excellent solvation properties enable them to dissolve many lipophilic compounds and "wet" the C18 surface effectively such that there is much more efficient sample component transfer from the injection solvent to the stationary phase. So, if you have an organic extract of hexane or DCM evaporate the solvent and redissolve in either DMSO or DMF.
- Badges
- Science method