One of my project goals is to analyze the profile of sugar alcohols (mannitol, sorbitol, etc.) that can act as osmolytes in green alage. During the growth phase of the algae, they are grown in 30 PSU liquid medium. When harvesting by filtering for HLPLC analysis, some NaCl is retained on the filter and subsequently in the final analyte solution.
Our basic processing steps are so:
1) Vacuum filter algae solution onto weighed filters, dry. Algal cells are not disrupted by Tissuelyser etc.
2)Take filters, extract in 70% ethanol. SpeedVac to recover pellet,
3) Re-suspend in HPLC-quality water. Move probe solution into HPLC vials for analysis.
Phenomenex REZEX ROA column, 5mM H2SO4 mobile phase
Would it be possible to wash out salt at any stage, either mechanically or chemically, while retaining a high amount of the sugar alcohols for the final analysis? Does there exist a salt-specific pre-column?
Thank you!
Not enough info to answer your question. How much salt? "Strong" injection solutions, relative to the mobile phase, may distort peak shape or change the chromatography. What is your HPLC mobile phase? Depending on the amount of salt in your sample relative to your mobile phase, it may or may not matter at all. *BTW: High concentrations of salt do not "hurt" an HPLC system (you must of course take proper care of your HPLC and always flush all buffers from the system every day to prevent corrosion or plugs).
Optionally, we us conventional dialysis to remove salts during sample prep.
I used Sephadex-15 columns to desalt sugar solutions. However, Sodium concentrations will not affect a regular C18 column but will kill a carbohydrate column like a Waters Sugarpak.
I suggest installing an of-line pre-column trap on the HPLC system selection valve, if you have enough ports, and wash loaded sample with solvent A for several minutes before switching the trap in-line with the your analytical column and performing separation. Your HPLC instrument vendor should be able to recommend required accessories, if you do not have them on hand. Note, this should be so-called pre-concentration setup, not just an in-line column filter.
Alternatively, there numerous SPE protocols and tools that allow purification of your re-suspended samples prior HPLC separation. Phenomenex company can be helpful in recommending their SPE product suitable for your application, and their application scientists are very helpful, should you contact them. Best wishes in your research!
We use C18 cartridge materials to remove salt from the matrix while quantifying amino acids and peptides.
You could run your sample(s) through a mixed-bed type ion exchange resin cartridge (a mixture of cation exchange and anion exchange resins), or two separate ion exchange resin cartridges (anion exchange cartridge then cation exchange cartridge, or vice versa). This will remove any ionic products present in the sample, while all neutral carbohydrates will pass through. Mind you that any charged carbohydrates (eg sugar acids) would also be removed from the sample!
- Badges
- Science method