What are the most common among them? Also what are their operation conditions?
Hi Hussam,
most commonly used is a Acetonitrile/Water mixture with the addition of 0.1% TFA or 0.1% formic acid (e.g. we use solvent A: 5% AcN/95% water + 0.1 % HCOOH, solvent B: 95% AcN/5% water + 0.1 % HCOOH). You always have to consider that for DAD detector, you need a low absorbance in UV wavelength below 300 nm for the eluent system. Otherwise you will not see the analyte molecules eluting from the column, the solvent just "overabsorbs" that. Acetonitrile and water are good for that purpose and AcN is strong enough to remove even quiet hydrophobic compounds (always depends on compounds you want to analyze - very hydrophobic compounds would simply stick to the C18 material and there is less chance to remove it - C8, phenyl, CN... phases are available as well).
I attached a nice link where to find a lot explanations in the field of HPLC analysis. The second one is for different stationary phases.
http://www.sepscience.com/Techniques/LC/Articles
http://www.phenomenex.com/Products/HPLCDetail/luna
Acetonitrile-water (0-100%) with 0,1 % trifluoroacetic acid. Methanol-water, ethanol-water, propanol-water.
There are various water miscible solvents can be used for RP HPLC chromatography. Most common solvents are Acetonitrile, Methanol, Ethanol,IPA ,THF in varying combination with water .Use of buffers,acids, and ion pairs and percentage depends on the nature of analyte you are trying to separate with C18 column.
By mentioning "toluene", I assume you are using it as a solvent. This can be used for C18, for "non-aqueous reverse phase" It works by using a polar solvent in place of water, and an organic solvent, such as hexane, toluene, or another miscible solvent, as the strong solvent. This works because silica-based C18 is chemically bonded to the silica. This technique works well for carotenes and similar compounds. Suitable solvent pairs include alcohol/dichloromethane, alcohol/acetone, alcohol (except methanol)/hexanes, alcohol/toluene.
Do not use this technique on polymeric reverse phases since those columns swell with the solvents used.
See: Snyder, L.R.; Kirkland, J.J.; Glajch, J.L.; Practical HPLC Method Development, 2nd Ed, John Wiley & Sons, 1997, p.264
Also see:
Prep scale: http://www.isco.com/WebProductFiles/Applications/101/Poster_and_Paper_Reprints/Facile_Isolation_of_Carotenoid_Antioxidants.pdf
Fullerene purification with 2-propanol/toluene on C18:
http://www.isco.com/WebProductFiles/Applications/101/Application_Notes/AN92_Purification%20of%20C60%20Fullerene.pdf
General overview:
http://www.isco.com/WebProductFiles/Applications/101/Application_Notes/AN58_Non-aqueous_Reverse_Phase_with_RediSep_Rf_Gold_C18.pdf
I'm actually using methanol 80 percent as a solvent or mobile phase. what I need is I salute sample to inject and analyze through the HPLC.
Ok- I understand now!
Injecting toluene won't harm the column. It may harm your sample resolution.Toluene is a "strong" solvent for reverse phase, stronger than methanol. This means the sample could be carried, at least partially, through the column without interacting with the column surface. This reduces separation of your analyte from impurities. Depending on the amount of toluene, you may be able to get away with it though.
See the link below, page 4, for an example of loading in a strong solvent. Although this is flash C18, it holds for HPLC as well.
http://www.teledyneisco.com/en-us/liquidChromatography/Chromatography%20Documents/Application%20Notes/RediSep%20Rf%20C18%20Flash%20Column%20Loading%20Techniques%20App%20Note.pdf
Edited to update link 11 Aug 2017
and is rp hplc compatible with a RI detector with isocratic pumping?
and why is not compatible the RI detector with gradient pumping?
For gradient pumping in HPLC you constantly change the refractive index of the solvent (mixture), so there is no stable baseline.
@M. Viviana Gamboa A.
Isocratic is fine with RI detector, normal phase or reverse phase doesn't matter.
As mentioned above, the solvents used (again, normal phase or reverse phase) have differing refractive indices. This means that the RI changes during a gradient, so you won't get a stable baseline.
You will almost certainly want to thermostat your column and perhaps the solvents as well.
Please see the link below:
Article Avoiding Refractive Index Detector Problems
Having said that, maybe there are new detectors on the horizon that will work with a gradient:
Article Refractive Index-Based Detection of Gradient Elution Liquid ...
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