I'm trying to synthesize a highly polar compound which is uv inactive (In TLC charring at the bottom- 0.1RF/20%MeOH:DCM ,MP) . I have confirmed the compound by simple mass and H'NMR spectrums but I want to know the purity of the compound by HPLC. When I try to perform HPLC for which, no peak eluting at all ( MP A : 0.1 %FA, MP B : ACN). How can I know the purity of the compound by HPLC (despite of Q-NMR). Please suggest any method available.
It would help to know exactly what the sample name is. HPLC analysis with isocratic refractive index detection (RID) offers the simplest approach / solution to your question. Alternatively, MS detection modes are also commonly used for samples with no or very weak UV chromophores. MS may provide the most data once a suitable method has been developed. Optionally, ELSD or CAD detection, for stable, non-volatile samples may be possible. *The sample's own properties, solubility and stability will suggest which methods and modes are most suitable.
Highly polar compounds can be retained using RP mode. Several specialty columns are available to aid in retention of these types of compounds without the use of any ion-pairing agents (avoid the use of these, esp if MS detection is used). One example of a more retentive column is the "Waters Atlantis dC 18 column" (not a recommendation, just an example). A keyword search using your sample name and "HPLC" on the web will provide you with many example articles, papers, application notes to review. Additionally, you should contact a local experienced chromatographer (or lab) to assist you develop an HPLC method that follows good fundamentals.
18h ago
Gaddam Srinivas,
You can use HPLC with the detector attached refractive Index (RI). Alternate hyphenated technique can be use like LC-MS/MS.
To assess the purity of your UV-inactive polar compound via HPLC, consider using derivatization or alternative detectors like refractive index (RI), evaporative light scattering (ELSD), or charged aerosol (CAD) detectors. Additionally, optimize the mobile phase and explore different column chemistries such as HILIC. If HPLC is not effective, quantitative NMR (qNMR) can provide a reliable purity assessment.
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