I want to use HPLC to detect of particular material, but I don't have a standard.
So, what is the solution? please
Dear Ghada,
First, You have to scan the product in UV-Visible spectrophotometer to check maximum wavelength of the particular material then by using suitable chromatographic parameter like stationary phase and mobile phase material can be separated. Run this to HPLC with PDA detector to scan spectra. Your question is not having standard then you have to first collect the sample through preparative chromatography and then have to do characterization of particular material. Once material is characterized then you can use as standard.
First check UV absorbance of molecule by structure or available data then choose that uv in your method ,
start analysis
Do you have LC-MS? A combination of UV and MS detection will suggest which peak is probably the desired compound. Use a LC that provides a spectrum for both UV-vis and mass spectrometer. I assume that you can get an HPLC method from the literature that you can use to elute the compound of interest.
It is emphasized that this analytical technique according to the type of analyte, its available standards and ensurement of a specific instructure method are utilized and posed a series of differnt results. Hence, Before doing experiment, take a look to the following links for more information.
Regards
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6264234/
https://tigerprints.clemson.edu/cgi/viewcontent.cgi?article=3090&context=all_theses
https://encyclopedia.pub/entry/history/show/27266
https://rjptonline.org/HTMLPaper.aspx?Journal=Research%20Journal%20of%20Pharmacy%20and%20Technology;PID=2015-8-12-11
https://www.idosi.org/aejaes/jaes15(11)15/12.pdf
HPLC method development requires knowledge of your sample's known pKa value, chromophores, known impurities and solubility. Have you tried researching a known HPLC method for your compound of interest? A lot of the instrument manufactures have a column selection and method guidelines you can reference but it requires knowing the physical properties pKa, solubility, and chromophores.
A quick preparation of a 5-10 mg/mL in HPLC grade solvent like MeOH, followed by full UV scan blanked for your solvent will allow you to determine the correct UV/Vis wavelength setting for your detector in lieu of knowing its chromophore. But your compound could be one with poor UV/Vis absorption then it will require a different detector like refractive index, fluoresces, or electro chemical. The pKa value will help you with column and mobile phase selection. Then you can perform a series of solubilities test for for the correct mobile phase selection base off of its physical solubility properties.
I would recommend reading this reference, to better understand HPLC method development basics https://www.agilent.com/cs/library/eseminars/public/Method%20Development%20by%20the%20Numbers.pdf
You ask how "to detect a particular material" by HPLC, but do not specify any sample type or name. Start at the beginning. Identify (specify) the sample first, then research its chemical and physical properties to determine which analytical technique(s) and specific methods are appropriate for analysis.
If you want to use HPLC, you need a standard, but you can use other methods, such as spectrophotometry, to identify the compound. First of all, you need to specify which compound you would like to identify and based on the characteristics of that compound; it might be possible to recommend an identification method.
- Badges
- Science topic