The General Rule in HPLC
So this is in response to a video I did about the use of guard columns. The general rule in HPLC is that we always want to dissolve the sample in the mobile phase—that is the ideal situation. If the sample solvent is the same as the mobile phase, it will just disappear; it will blend in.
In this case, they are doing an extraction and are worried about the pH of the sample affecting the mobile phase.
The Question
Let me read the question:
“What about highly acidic or basic samples? How will that affect the C8 ligands on the column? I work with human serum and plasma that are treated with acid, precipitated, and the supernatant is injected, so it’s not the same as the mobile phase.”
Short Answer: You’re Probably Fine
Okay, so my easy answer is: you’re probably fine.
When we say the sample should be the same as the mobile phase, that’s the ideal situation. However, chromatographically, it’s more about the solvent strength—the amount of methanol or acetonitrile in your sample.
For example, if your sample were 100% acetonitrile and your mobile phase were 50/50 acetonitrile/water, that would cause peak shape issues—especially early in the chromatogram—because the mobile phase strength is very different from the sample solvent strength.
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Does pH Of The Sample Matter?
In your case, you have an aqueous sample—a plasma sample that you’re precipitating with acid or base. The pH of your sample, for the most part, has no effect on the chromatography because the sample volume is so small that it quickly takes on the pH of the mobile phase as soon as it mixes.
Even if you inject a sample with a pH of 12, it will immediately adjust to, for example, pH 2 if that is the pH of your mobile phase.
When Does pH Become a Problem?
Extreme cases where pH could damage the column include injecting pure nitric acid or pure hydrochloric acid. Believe it or not, I’ve had customers who do that! There aren’t many, but in those cases, you may see damage at the top of the column.
A guard column could help extend the life of the main column in those situations, but again, this is an extreme case.
Key Takeaways
If your sample is aqueous and has a pH between 1 and 14, I wouldn’t worry about it.
- You’re injecting 5 microliters, and your flow rate is 1–2 mL/min.
- The sample will quickly mix with the mobile phase and take on its pH.
- In normal cases, pH won’t damage your column.
Great question!
