Mary: Hi Nick, I’ve been monitoring several reactions over the past few weeks with HPLC.
Suddenly, and with the same method,
my spectra look very different! Retention times are much longer and there is much more noise in the baseline.
The same sample runs on another machine with the same method produces the “correct” spectrum.
Can this be an indication that the column needs cleaning or replacing?
more noise in the baseline丨基线噪音变大
Nick: Just as a point of terminology, it’s a “chromatogram“,not a “spectrum“. With that nit-pick aside,
the problem could be either with the column or with the pumping system.
Since you have another instrument,
the easiest diagnostic is to switch columns and see if the problem follows the column or stays with the instrument.
With that nit-pick aside丨意思是“先把吹毛求疵地提意见放到一边”
Mary: Oh, okay! I see.
Nick: Another hint is to look at the dead time marker,
sometimes called the “solvent front”.
If the dead time and the retention times of all your analyte peaks changed “by the same percentage”,
that strongly suggests a flow problem.
Mary: A flow problem?Like what?
Nick: Like leaks, bad seals, bad check valves, air bubbles in the pump head – stuff like that.（泄漏，磨损的密封圈，故障的单向阀）
Mary: Is there anything else that could be the cause?
Nick: You can also check the compatibility of the reaction media and the column.
Make sure you adjust the sample pH to neutral or acidic before injecting it into the RP column.
Also, make sure other ingredients are not irreversibly sticking to the column.
Good luck, and let us know what you find out, so we can all learn from it!
Mary: Hey Nick, thanks for your help yesterday! Today, the problem is a little different.
I ran the same sample on 2 instruments this morning, including the one that was giving me problems yesterday,
which is “my instrument”.
My instrument produced a chromatogram that was identical to what I had seen in previous weeks.
The other instrument was similar,
but retention times were about a half a minute shorter and it picked up a minor peak that my instrument did not.
Nick: Did you check the compatibility issue?
Mary: The compound itself is compatible with the column and mobile phase and should not stick to the column.
There are unidentified impurities in the reaction mixture,
but I don’t think they should cause a problem either. Any suggestions?
Nick: If “your instrument” is back to normal, that suggests
that yesterday’s problem was caused by a flow problem. A trapped bubble would be my guess.
Mary: Then, what do you think caused the difference between the two instruments?
Nick: The instrument-to-instrument difference you’re seeing could be due to a couple of things.
The most likely reason is a slight difference in the selectivity of the two columns.
In many cases, retention and peak spacing can be affected by the very subtle differences of column chemistry
(including those brought about by the aging of the column).
A second possibility, if your separation involves the use of a gradient,
is a difference in the gradient delay volume between the two instruments.
If your separation is isocratic(meaning, the mobile phase composition remains constant during the run),
then you can ignore this.
the very subtle differences丨细微的差别
gradient delay volume丨梯度延迟体积
Mary: I forgot to mention, that I did try swapping the columns between the two instruments.
The retention times were constant to the column.
That is, I saw the same retention times with the columns as before,
but the different instruments.
So, your assessment of aflow problem seems most likely for the original problem.
The only question I have left is,
what could cause that type of problem?
Should any maintenance be done to prevent it from happening again?
swapping the columns丨交换色谱柱
Nick: Since the problem went away, the most likely culprit is a bubble.
the General remedy is to make sure you’re degassing effectively.
Also, check the “sinker” frit on the inlet line to make sure it’s not partially plugged,
and check the inlet lines themselves to make sure they’re not kinked – a flow restriction on the inlet side
can cause enough of a pressure drop to persuade residual dissolved air to come out as bubbles.
Mary: Ohh! I did not know that!
Nick: When all is said and done, “bubbles happen”.
If you’re getting longer-than-usual retention times and if all the peaks are off by the same percentage,
just re-prime the pump and run again.
When all is said and done丨总而言之，言而总之
Mary: Okay. That sounds simple enough.
Nick: Since you’ve pinned down your selectivity difference to the column,
you can now look at what might be causing “that”.
Are the columns from the same lot?
Are they different ages? Different histories?
All of those can contribute to selectivity differences.
If HPLC were easy, we’d all be unemployed!
Mary: I did check the inlet lines for kinks. I can see a minor kink,
but it doesn’t seem to be enough to reduce flow and drop the pressure.
Am I wrong? I mean, can a slight bend cause a flow restriction?
Nick: Yes, sometimes it can if the tubing is bent badly enough.
Mary: Hi, Nick. Selectivity difference between the two columns I tried doesn’t surprise me too much.
The”other” instrument I used belongs to someone else,
so it has a much different history.
I believe both are from the same lot,
but mine has been used more heavily.
Nick: It sounds like your system is basically ok.
Like I said earlier, “bubbles happen”!
If the problem doesn’t recur, don’t worry about it.
If it happens again, you may want to think about having your pumping system serviced.
Mary: I see. Thank you so much for your advice, Nick! I appreciate your help.
Nick: Of course, Mary! I’m glad we could resolve your issues together. Let me know if you have any more questions!
Mary: I sure will.Thanks again, bye!