I am trying to analyze short peptides containing hydrophobic aromatic amino acids. The samples are insoluble in water or any organic solvent but only soluble in a very acidic solution such as water with more than 30% acetic acid.
So, in this case, I made a 20% acetic acid in DI water, which has a very low pH (1.8). I used it to dissolve my sample, resulting in a 0.1 mg/ml concentration. The HPLC mobile phase, however, is just an isocratic flow of 20% acetonitrile in water. I use a C18-column that tolerates pH in the range of 2-8 and the injection volume is 10 uL.
- Does the C18 column get hydrolyzed when my sample solvent has a pH that is outside the lower limit of the column pH range?
- In the case of me using a completely different mobile phase and sample solvent, how does it affect the peak shape? I expect to first see an "acetic acid" peak at the front" and then a compound peak and so on.
- Also, what happens if my compound is soluble in the sample solvent but much less soluble or insoluble in the mobile phase?
Yes, the pH of the sample solvent affects HPLC results. Basically, It alters the ionization state of analytes, influencing their retention time and separation.
C18 columns may experience hydrolysis at pH 1.8, potentially affecting performance. Switching to a mobile phase incompatible with your sample solvent can lead to poor peak shape or no detection due to solubility issues. Adjusting the mobile phase composition may help improve solubility and chromatographic results.
If you're applying samples which are insoluble in your mobile phase yu are depositing gunk to the top of your column (or precolumn) if your smart and over time you will see the backpressure increase until at some point your system shuts down. I ruined many a column in grad school in just that fashion.
You may need to change to a more suitable column, use a more acidic buffer. There is no one size fit all column. After fhanhing rhe column need to flush and condition the column by chainfing it gradually over time.
Yes, the pH of the sample solvent does indeed matter in HPLC. It affects the retention time and peak shape
- Badges
- Science topic
- Similar topics
- Computer Science and Engineering
- Programming Languages
- Python