A critical guideline to follow in liquid chromatography is to always inject your filtered sample in either the initial mobile phase or a slightly weaker solution. *Samples must be fully dissolved in solution before loading onto the head of the column. Injecting a sample dissolved in an immiscible solvent such as Hexane (vs ACN/ water) may lead to possible precipitation and analysis errors. Such a method would generally fail any validation.
Questions you need to ask:
Is your sample only soluble in normal phase solvents such as Hexane?
Is your sample soluble in ACN/Water?
Hi Samer,
Would appreciate if you can elaborate on HPLC condition, particularly wavelength.
If you wish to analyse with RP HPLC only, then
Alternate 1 - Evaporation of hexane from extract and dissolve in ACN (if soluble) and make up with mobile phase.
Alternate 2- Add some 1,4 Dioxan (Spectroscopy grade- UV cutoff 215 nm) to dissolve the extract, and make up with ACN/Water system. If your working wavelength is >220 nm, it will not be an issue. 1,4 Dioxan, Tetra Hydrofuran (THF-UV cutoff 212 nm) & Acetone are solvents which are miscible with polar & apolar solvents. However, Acetone has UV cutoff of 330 nm, hence not at all useful.
While I agree with William's response in routine practice, I do know that you can get reasonable results using small injection volumes (5 µL or less) for non-compatible solvents on non-UPLC columns (e.g., 4.6 x 150 mm, 5 to 10 µm). We routinely analyzed a toluene based reaction by HPLC, and these results were very reproducible. These analyses should always start with a significant portion of organic solvent in the initial mobile phase (analysis of the toluene reaction above started at 50% MeOH since initial mixing tests showed taht all componenets had better solubility in MeOH vs ACN). However, you definitely run a risk of precipitation in the system.
- Badges
- Science topic
- Similar topics
- Phytochemistry
- Extracts