Hi Omprakash,

1. first of all, you should take a few days to equlibrate your instrument. Depending on your solvent system you used before, you can stepwise switch from Chloroform or Dichloromethane to Isopropanol (at least 10-20 volumes of your pump degasser/solvent lines of the instrument). Then stepwise switch to Acetonitrile and at the end to water. For the second solvent line, you can stop when using Acetonitrile. You must be sure that all hydrophobic solvents are gone, otherwise you create an emulsion in your column and this is bad for the column. However, I'm not sure if your HPLC system is suitable to run both normal and RP solvents - you should clarify this before starting the procedure. Maybe your manufacturer has special protocols for this, just ask the company.

2./3./4. You should always have a certain amount of water in your mobile phase, otherwise the silica of your column may collapse. For this reason, we use 5% Acetonitrile/95% H2O with 0.1 vol-% HCOOH as solvent A; and 9 5% Acetonitrile/5% H2O with 0.1 vol-% HCOOH as solvent B. For column washing, we go to 100% solvent B, which is still 5% H2O. Depending on your samples and the column (which column do you want to use?), you can do gradients starting from 0% B, then go to 60% B after 30 min, then 95 % B after 32 min and again 0% B after 35 min. This is just a starting point which can be optimized in terms of elution time and separation. You can also play around with the flow rate and gradient. Your samples should elute in the range from 8-25 min for the given gradient program. We routinely use a C18 column with 5 μM particle size and 3×250 mm for our analyses (Peptides, hydrophilic polymers (e.g. PEG and POx) and proteins).

In your case this may also work out, but there are analytical papers for Nucleotides and beta cyclodextrins around, I'm sure.

Good luck,

Marcus

Hi Omprakash,

I'm not sure what you exactly mean, but this question is very general... in fact, there is no "standard" method for purification/analysis of various compounds using reverse phase HPLC.

Usually, the stationary phases used are silica based C18 or C8 (hydrophobic) columns, depending on the hydrophobicity of the compounds. There are also phenyl/hexyl or fluorinated columns available. In RP HPLC, the mobile phase is usually water (+ 0.1 vol-% trifluoro acetic acid or formic acid) with a gradient of organic solvent, mostly acetonitrile. Also buffer/methanol gradients are used sometimes.

In any way there is no clear answer to your question, the method strongly depends on the type of molecules you try to analyze. I attached some links FYI.

Best regards,

Marcus

https://www.researchgate.net/post/How_can_I_choose_mobile_phase_for_reverse_phase_HPLC

https://www.researchgate.net/post/Can_anyone_tell_me_what_are_the_differences_between_Preparative_columns_and_analytical_columns

https://www.researchgate.net/post/When_is_it_important_to_add_drops_of_trifluoroacetic_acid_during_hplc_analysis

https://www.researchgate.net/post/Can_we_run_our_crude_plant_fraction_directly_on_recycle_preprative_HPLC_without_subfractionating_on_conventional_column_chromatography

Dear Marcus 

Many thanks for your supportive comments and highlights. 

My specific questions are related to use Reverse  phase columns in normal combiflash instrument.  Here they are 

1. How can we shift from normal phase  to reverse  phase? When we shift to RP should combiflash be equilibrated with specific solvent system. 

2. Regarding solvent  system: Can we start  with 100 % water and then  increase non polar solvents (ACN,  Methanol)

3. How much percentage of ACN/Methanol,  we can increase during purification process and column washing. 

4. What is the best condition for purification of Nucleotides and beta cyclodextrins? 

Please elaborate. 

Regards 

Hi Omprakash,

1. first of all, you should take a few days to equlibrate your instrument. Depending on your solvent system you used before, you can stepwise switch from Chloroform or Dichloromethane to Isopropanol (at least 10-20 volumes of your pump degasser/solvent lines of the instrument). Then stepwise switch to Acetonitrile and at the end to water. For the second solvent line, you can stop when using Acetonitrile. You must be sure that all hydrophobic solvents are gone, otherwise you create an emulsion in your column and this is bad for the column. However, I'm not sure if your HPLC system is suitable to run both normal and RP solvents - you should clarify this before starting the procedure. Maybe your manufacturer has special protocols for this, just ask the company.

2./3./4. You should always have a certain amount of water in your mobile phase, otherwise the silica of your column may collapse. For this reason, we use 5% Acetonitrile/95% H2O with 0.1 vol-% HCOOH as solvent A; and 9 5% Acetonitrile/5% H2O with 0.1 vol-% HCOOH as solvent B. For column washing, we go to 100% solvent B, which is still 5% H2O. Depending on your samples and the column (which column do you want to use?), you can do gradients starting from 0% B, then go to 60% B after 30 min, then 95 % B after 32 min and again 0% B after 35 min. This is just a starting point which can be optimized in terms of elution time and separation. You can also play around with the flow rate and gradient. Your samples should elute in the range from 8-25 min for the given gradient program. We routinely use a C18 column with 5 μM particle size and 3×250 mm for our analyses (Peptides, hydrophilic polymers (e.g. PEG and POx) and proteins).

In your case this may also work out, but there are analytical papers for Nucleotides and beta cyclodextrins around, I'm sure.

Good luck,

Marcus

 Many thanks for explaining  in detail.  

Regards 

I would disagree with Marcus on one point. Under reverse phase conditions you should always maintain a certain amount of organic solvent in your eluent otherwise the functional groups attached will essentially mat down. There are columns that will tolerate an eluent with little (1%) organic solvent with the Phenomenex Gemini C-18 being an example. It is advisable to run your acetonitrile as a 90% solution with deionized water because acetonitrile tends to polymerize. I have replaced many quaternary valves that became coated with this polymer. I have also found a bottle of it that sat for quite some time with a ring of clear material at the surface.

Switching back and forth between normal phase and reverse phase is not recommended. You should have one instrument dedicated to each.

Hi Michael,

I may used a undefined method description for changing the solvent system in my post before, important point from your side. However, we routinely add 5% Acetonitrile into our aqueous solvent (solvent A), just because of the reasons you mentioned.

Marcus

Michael Dong recently proposed a universal RP UPLC method. I think it was in LCGC magazine. Does one size fit all?

HPLC stands for 'High Performance (was Pressure) Liquid Chromatography'.  Thus, your target molecule(s) must be soluble in a mobile phase solvent and detectable.  

Assuming it has a UV spectrum (not all molecules do!) dissolve the standard (target molecule) in an organic solvent like Methanol, Acetonitrile, Isopropyl Alcohol. Assuming this is reverse phase chromatography (C18 column) inject 10 uL into the HPLC and note the retention time (RT).  The main peak should elute at the injection front.

Add water and note that the main peak (your target?) elutes later.  Optimize the RT so it elutes at 5-10 minutes for a 5 cm column (at 1.0 mL/minute flow) OR 10-15 minutes for a 25 cm column (at 0.5 mL/minute flow).

Sharpen the peak by adjusting the pH of the mobile phase to >pKa of the sidegroups of the target molecule.  These sidegroups are usually amino or carboxyl groups and may be protonated (thus pH control).  By manipulating the pH you assure yourself you only have 1 form (protonated versus anion).

Note; that a REGULAR C18 column can only tolerate a pH range of 2-7.  Going above 7 ensures that the column WILL be destroyed by cleaving the silanol groups on the stationary phase.

Dear Bruce, 

Actually, I am discussing about RP column chromatography in combiflash not HPLC method development. May be this technique is useful as reference purpose.

Is anybody used RP column chromatography in combiflash?

Thanks 

Hi Ompeakash,

Not that I have seen.  But there are application that are available.  See;

http://www.selectscience.net/products/combiflash-rf-200/?prodID=194973#

https://www.google.ca/#q=combiflash+method+development&*

 Hi

I do agree that reverse phase is very common however i would recommend to use the Silica Hydride columns. I'm using this type and has proven its usefulness for the separation of both polar and non-polar compounds. Read the below paper.