how to separate isomers(positional) which have same polarities
they are eluting at the same retention time,
we have tried with chiral chromatography
all the ratios possible with chiral solvents using chiral pak columns
Chiral method development by HPLC and/or SFC is an area that I know a bit about (and only applicable to true enantiomers). OK, first you need to start by running achiral chemical purity on the sample to determine what else besides the racemate is in the sample. This needs to be done on an achiral column, not chiral, using proper HPLC method development guidelines showing good K primes for all compounds present. This step must not be skipped. No point running chiral methods unless the sample is clean. If the chemical purity is high enough (>95%), then you can move on to identifying the best chiral phase to use. Solubility is key as the sample must be soluble in the mobile phase used. The most efficient way to do this is by using an automated chiral HPLC screening system loaded up with multiple chiral columns. In general, chiral HPLC has the broadest selection range for chiral samples (better than SFC or any other technique). This allows you test a wide range of different mobile phases (polarity and solubility) to find the column and method which works. Things need to be done in this order. ID column FIRST, then mobile phase. Trying to "force" a method to work on any specific column is a huge waste of time. Chiral methods just do not work the way regular achiral separations do (though in all cases, finding the best column first is still the best technique to use).
If you do not own the ten or twenty "best" chiral columns to try, then you may want to think about sending your sample out one of the column manufacturers (e.g. Daicel (CTI), Regis) and have them screen your sample against their columns to find a suitable support. That will save you from buying them all and testing them.
"Normal phase" encompasses many different stationary phases. These include silica, alumina, diol, cyano, and some of the chiral columns. I'm reading that you used both some form of normal phase, and also chiral columns.
Which normal phase(s) have your tried, and which solvents? This gives us an idea of what you have tried, and also the polarity of the compounds. What was the retention time? Chromatographic conditions? Gradient? Can you post a chromatogram?
"Chiralpak" is a trademark for a whole series of column phases (all with different selectivity) so we have no idea what methods or columns you tried, or even what form your sample is in.
Are you isomers actual enantiomers? If so, what is the chemical purity of the material as only chemically pure enantiomers are suitable for chiral HPLC analysis (see below for why). If they are not true enantiomers, then pursue routine HPLC method development, ion pairing or perhaps derivitization (used as last option).
These are very basic, but important questions which need to be answered before you proceed. Chiral columns are absolutely terrible (unsuitable) at resolving achiral compounds apart and would never be used for such applications. You need to perform proper HPLC method development to retain, then resolve your sample apart following acceptable chromatography fundamentals.
enantiomers.. and retention time around 30mins
we have used all modifiers like DEA,MSA,TEA Still we can separate tried all possible ways.
n i can't share chromatogrms as they are restricted for me too @William Letter
Chiral method development by HPLC and/or SFC is an area that I know a bit about (and only applicable to true enantiomers). OK, first you need to start by running achiral chemical purity on the sample to determine what else besides the racemate is in the sample. This needs to be done on an achiral column, not chiral, using proper HPLC method development guidelines showing good K primes for all compounds present. This step must not be skipped. No point running chiral methods unless the sample is clean. If the chemical purity is high enough (>95%), then you can move on to identifying the best chiral phase to use. Solubility is key as the sample must be soluble in the mobile phase used. The most efficient way to do this is by using an automated chiral HPLC screening system loaded up with multiple chiral columns. In general, chiral HPLC has the broadest selection range for chiral samples (better than SFC or any other technique). This allows you test a wide range of different mobile phases (polarity and solubility) to find the column and method which works. Things need to be done in this order. ID column FIRST, then mobile phase. Trying to "force" a method to work on any specific column is a huge waste of time. Chiral methods just do not work the way regular achiral separations do (though in all cases, finding the best column first is still the best technique to use).
If you do not own the ten or twenty "best" chiral columns to try, then you may want to think about sending your sample out one of the column manufacturers (e.g. Daicel (CTI), Regis) and have them screen your sample against their columns to find a suitable support. That will save you from buying them all and testing them.
PS: If you use your own chiral HPLC columns, do Not use any modifers with the new chiral columns UNLESS you can afford to buy dedicated versions of EACH chiral column to use with them (*that is we we do). Each time you use an ion paring agent or strong base/acid with those columns you change the surface chemistry of it, forever. It may no longer work to resolve past compounds and may not provide reproducible results again (we learned this from running thousands of racemates). Label any column used with such agents so they will only be used with those modifiers. If your compound permits it, run initially under neutral conditions only. If you have extra columns, then next you can try acidic and basic conditions too (we always run acidic, neutral and slightly basic when running automated chiral screening MD for NP or RP columns). Those additional permutations are tried as part of comprehensive chiral method development.
"Chiral HPLC and SFC Column Screening Strategies for Method Development"; https://hplctips.blogspot.com/2011/06/chiral-hplc-and-sfc-column-screening.html
This will be 'very' hard to do! While C18 columns do have some activity in separating cis-trans positional isomers, which have the same groups (more peak splitting than anything else). This separation is due to the large ODS fronds projecting into the mobile phase and interacting with the molecule of interest. Columns normally used for 'normal phase' like silica, amino, diol have a much smaller zone of interaction. Thus, you will be relying more on slight differences in solubility between the mobile phases. This is the basis behind gradient work. Good luck!
Please review the thread. Anil later stated he is trying to separate TWO ENANTIOMERS, so no C18 column or Buchi based cis/trans citral methods are relevant or will work. A chiral method of racemic resolution is required.
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