Amino acid analysis column life time

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37 posts Page 3 of 3
Hi Crong,

Maybe the next things to think about are how you are cleaning the LC solvent reservoirs and how the pH is being measured. In the AccQTag method (the old one using NovaPak columns) a stock aqueous eluent was prepared (stored in a refrigerator, warmed to room temperature) and diluted 1:11 prior to use.

This could help with pH consistency between reparation runs.
MattM
mattmullaney wrote:
Hi Crong,

Maybe the next things to think about are how you are cleaning the LC solvent reservoirs and how the pH is being measured. In the AccQTag method (the old one using NovaPak columns) a stock aqueous eluent was prepared (stored in a refrigerator, warmed to room temperature) and diluted 1:11 prior to use.

This could help with pH consistency between reparation runs.

That's pretty similar to how I prepare it. I make a 10x concentrate, filter through 0.2 um and store at room temp (initially tried to store in fridge but that caused precipitation). I adjust pH after diluting 10x on the day of analysis. The inconsistency is because the method says to adjust pH with fuming HCl and it's impossible to get it to 7.8 exactly. But if there was any Rt shift due to pH, it would have been insignificant compared to the forward Rt shift that accompanies every run, prior to yesterday. For cleaning the solvent bottle, I always use the same one for this mobile phase and just clean by rinsing with water several times then rinse once with a small amount of mobile phase.

I was thinking of trying a mobile phase that's lower in pH (say pH 7.5) just to see what happens. Do you think that would be worthwhile? Also I'll try doing the final pH adjustment with 1:1 HCl:H2O so there's more control.
Hi Crong,

As to the reservoir cleaning, seems pretty good to me though maybe I'd use hot HPLC water (say bi-weekly) followed by HPLC-grade methanol just to keep any "bugs" from growing.

You wrote,

I was thinking of trying a mobile phase that's lower in pH (say pH 7.5) just to see what happens. Do you think that would be worthwhile? Also I'll try doing the final pH adjustment with 1:1 HCl:H2O so there's more control.


If it's a phosphate buffer, maybe it'd be okay to try out at 7.5, yes...as well as to use a more dilute HCl solution for adjustment.

Using a concentrate is reasonable--did Thermo indicate any stability for their concentrate?
MattM
Hello -

I was hoping the OP would have an update on this situation.

I have been tasked with determining the source of an identical problem. Around the same time, our Hypersil columns from Thermo started having the same problems. In 2017, our columns lasted over 400 injections. In 2018, we rarely got more than 200. Our average number was 192. We have a dedicated system using almost the exact same method the OP used.

We discussed extensively with Thermo and Agilent (many of our standards and reagents are purchased from Agilent). Most people we discussed this with had no Amino Acid Analysis experience, but one applications specialist remembered how many years ago, column lifetime was affected by OPA. How long it was open for and the amount used. We were unable to get any movement on this idea.

We have varied sources of reagents, eluents, wash, and how to store column. We use this dedicated instrument daily for amino acid analysis.

Any updates would be appreciated.

Best,
Tracy
The columns must have been costing you a fortune, even before this decline in column life-time (400 injections is a dreadfully short lifetime for a modern C18 column; they should survive thousands of runs). Wouldn't it be more cost-effective to use one of the many very good kits?
We've been doing OPA/FMOC amino acid analysis for about six months. Thousands of samples later, with a mobile phase at pH 8.2, we're still using the same column we started with. We have had to change the guard column three times. We can overlay the chromatogram from the first week with the current, and they're nearly identical. We've ended up using the full Agilent OPA/FMOC line, including all reagents. We're analyzing protein hydrolyzates primarily from biomass samples, so it's not that our samples are super clean. The column is their AdvanceBio AAA column. It's robustness has far exceeded our expectations. But we do carefully adhere to all the recommendations for longevity (fresh reagents, filtered solvents, etc.).

No, I don't work for Agilent, and YMMV
What is the pH of that buffer? Thermo claims a pH range of 2-9 for Hypersil Gold, but I would expect lifetime to start dropping above pH 8.

At this point, you will probably need to replace your existing column. Going forward, changing guard cartridges more frequently may help, but you'll have to balance that cost against the cost of frequent column changes.
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