Amino acid analysis column life time

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

37 posts Page 2 of 3
Multidimensional wrote:
This applies to all HPLC methods: A proper "wash" should be stronger than the mobile phase and do a better job of fully dissolving everything injected.

"100% B (45/45/10 ACN/MeOH/H2O) and held for 3 min". So that means your column never "sees" more than 45% ACN / 45% MeOH (basically, 45% ACN since that is the strongest solvent). Therefore, you should use a wash solution which exceeds those values.

Perhaps 10% Water plus 90% ACN (or 5% Water/95% ACN); or a mixture of 80% ACN plus 15% MeOH plus 5% water. You should experiment to see what works best.


Thanks for the suggestions. The "wash" is stronger than the mobile phase to my understanding, because analytes are eluted with up to 52% B and then it's ramped to 100% B.

With this instrument I'm not able to wash with anything stronger than the 45% during the run because with 1 line occupied by the needle wash solution, 1 for the buffer, 1 for the ACN/MeOH/H2O mixture and 1 for water, there's no room for an ACN bottle. But I can definitely try manually switching the bottle after the end of a sequence and flushing with a higher ACN %.
Hi Crong,

You said,

Dissolution of the stationary phase could definitely explain what I'm seeing. Although I do wonder if that is the case, I should be expecting to see retention time shift forward with each sample right? For me the Rt only shifts forward when the instrument is shut down and restarted next time. During continuous runs in the same sequence the Rt is stable. That just makes me think I'm doing something wrong in the shut down process...

I'm looking into Agilent's columns since their derivatization process is pretty much the same, and Waters AccQ Tag seems very expensive. I recently watched a webcast from Agilent about their amino acid columns and it does advertise chemical modifications to the silica for high pH resistance. However in the same webcast they also recommend to recalibrate for retention times and response factors weekly, which to me says - expect peaks to shift forward with use. So how much better is it really?!

Thank you again for your insight.


It seems, to me, dependent upon the kinetics of the eluent's interaction with the stationary phase. I don't think that the Hypersil Gold phase would dissolve so quickly as to show reduced retention/lost peak resolution during a sample sequence--depending on the length of the sequence.

You make a good point. Perhaps the post-run wash with water to remove the buffer could be increased in length (likely longer than the 5.5 minutes indicated at the end of the gradient program for post-run re-equilibration) and the pH of the effluent checked (pH paper likely would be okay for this) to ensure that it is no longer basic [> 7] before flushing the column with a strongly-eluting solvent (such as ACN/MeOH or neat ACN) to store the column in between runs.

Agreed with your assessments of AccQ.Tag (expensive) and Agilent's AAA solution (I looked at their webcast, too)...the best you can do is to minimize exposure of the stationary phase to that eluent in-between runs. It would be a pain in the behind to change the analysis method mid-stream, that is for certain.

You have got a process that gives you the data you need--just want to see if you can increase column life as you can whilst being frugal as possible. Best wishes!
MattM
The column wash occurs after the analysis run, not during it. We wash our columns AFTER each analysis. Don't make the wash or re-equilibration part of the analysis method. The wash method and re-equilibration are separate steps. They should occur outside of the analysis method. Run the method until it hits whatever solvent max is called for, then HOLD it for a period of time, then end the analysis. Next, follow the analysis method with a column wash method. When that is complete, reload the initial analysis method and wait while it re-equilibrates at the "initial" conditions, then start the next analysis. These are the steps.
mattmullaney wrote:
Perhaps the post-run wash with water to remove the buffer could be increased in length (likely longer than the 5.5 minutes indicated at the end of the gradient program for post-run re-equilibration) and the pH of the effluent checked (pH paper likely would be okay for this) to ensure that it is no longer basic [> 7] before flushing the column with a strongly-eluting solvent (such as ACN/MeOH or neat ACN) to store the column in between runs.


Hi Matt, sorry for the confusion. That 5.5 min at the end of the gradient program is re-equilibration to mobile phase A which is the buffer. The 90% water is only used at the end of the whole sequence before shut down. What I did was after all the samples in the sequence I add one injection of H2O without derivatization and set the mobile phase to 90% H2O 10% B for 30 min at the same analysis flow rate, after which it is changed to 70% H2O 30% B, reduced flow rate, and the instrument shut down initiates. Shut down method turns off the oven and waits until oven temperature is below 35 C before turning off the pump (maybe another 5-10 min). All in all, the column is flushed with mobile phase that doesn't contain buffer for at least 35 min. I will definitely check the eluent pH next time to see if it's still basic, thanks for that suggestion! And flush with higher % ACN prior to storing the column. Hopefully that will have some improvement.

Thanks again.
If you suspect your column is dissolving at high pH, you could test this by trying one of the many C18 columns available that will tolerate high pH.

Don't write off the AccQ tag method too quickly on grounds of cost. How much money have you spent buying columns, and how valuable is the time you're wasting on a method that is giving you trouble? AccQ tag is extremely reliable, and the derivatised samples are stable almost indefinitely; it gives good separation at column-friendly pH, and your columns really should last for 2000 injections with AccQ-tag. You don't have to buy the entire kit including running buffers and column. You can get the derivatising chemicals and buffers alone. The column is just a C18 column, but Waters guarantee the retention times and performance if you use their buffer and designated column. (I don't work for Waters!).

Just to clarify, 45/45/10 ACN/MeOH/water is a much, much stronger solvent than 45% ACN in water.

Someone asked for a 2nd opinion on guard columns. I like them. If you don't change them, then there is no point, because eventually the extremely hydrophobic dirt they are designed to extract will start to wash through onto the analytical column. In terms of using them as a filter; you need to balance the cost of filtering versus the improved reliability and column life-time. It shouldn't be hard to change the guard column. The financial side depends a lot on manufacturer. Phenomenex Luna guards used to be very cheap, I can't remember.
Hi Crong,

You said,

Hi Matt, sorry for the confusion. That 5.5 min at the end of the gradient program is re-equilibration to mobile phase A which is the buffer. The 90% water is only used at the end of the whole sequence before shut down. What I did was after all the samples in the sequence I add one injection of H2O without derivatization and set the mobile phase to 90% H2O 10% B for 30 min at the same analysis flow rate, after which it is changed to 70% H2O 30% B, reduced flow rate, and the instrument shut down initiates. Shut down method turns off the oven and waits until oven temperature is below 35 C before turning off the pump (maybe another 5-10 min). All in al0l, the column is flushed with mobile phase that doesn't contain buffer for at least 35 min. I will definitely check the eluent pH next time to see if it's still basic, thanks for that suggestion! And flush with higher % ACN prior to storing the column. Hopefully that will have some improvement.

Thanks again.


Actually--I wasn't confused on either count (seems to me to be some terminology inconsistency on my part, I'll try to be clearer). Just suggesting that to remove all of the basic eluent may take longer than that re-equilibration step in-between separation runs (and thanks, now I see you've been routinely using 30 minutes of 0.7 mL/min for the post-sequence run step of 90:10 water/ACN) within a sample sequence and at the same flow rate of 0.7 mL/min. I also understood from the first that the 90:10 water/ACN wash you perform occurs at the end of the ENTIRE sample sequence.

If you have pH paper handy, it still may make sense to check the effluent pH after the 30 minutes of pumping the 90:10 mix just as a check. Please let me know what you find with the pH check of the pH of the post-sequence run effluent--just seems that based on what you're observing, this could be why the stationary phase may be being adversely affected.

I agree with Imh too, to cause further confusion...I've had the easiest time with AccQ.Tag derivatization myself (life is full of these oxymorons, seemingly). I used to prepare the eluents for AccQ.Tag on my own using Steven Cohen's reference paper...these days that AccQ.Tag separation method has changed a bit (I think a BEH stationary phase is used these days rather than NovaPak). Oh...I don't work for Waters (or Agilent) either...just hard to change something that is working (Thermo's OPA/MPA and separation) for something new--time and money.

Is there a point of return on costs for injections on the Hypersil Gold columns/guards that could be satisfactory to your lab's needs?

Anyhow--I continue to wish you well, Crong!

Hi Rndirk: As to these guard columns, if they helped with something I was trying to develop, or were required to execute something I needed to run...I used them. If not, then not--guess it's safe to say that I liked them when they were specified for use or made a difference in what I was doing.

For example, the older AccQ.Tag method specified a guard for the NovaPak analytical column...there is no such specification for the modified AccQ.Tag separation with the BEH phase.
MattM
mattmullaney wrote:
If you have pH paper handy, it still may make sense to check the effluent pH after the 30 minutes of pumping the 90:10 mix just as a check. Please let me know what you find with the pH check of the pH of the post-sequence run effluent--just seems that based on what you're observing, this could be why the stationary phase may be being adversely affected.

Thank you Matt for your continued advice! I think I'm wrong about the pH being the issue.

I tested the effluent during a few points:
  • At the beginning of one sample where the gradient is at 98% buffer. pH paper showed pH 8.
  • Near the end of one sample where the gradient is 100% B. pH 6.
  • At the end of the sequence, 22 minutes into the 90:10 H2O:ACN rinse, pH 6. 35 min in, still pH 6.
  • Just DI H2O, pH 6.

So the column is not exposed to high pH during storage but resolution continues to decline. I'm baffled. :shock:
Hi Crong,

My thanks to you for taking a look into the matter--now we know the trouble doesn't seem to be an issue with the pH of the storage solution of the Hypersil Gold column in between runs.

For fun, I went back to the Thermo App note-the Macherey-Nagel, Nucleodur®
C18 Gravity column was specified:

http://ftp.mn-net.com/english/Instructi ... 165_EN.pdf

I went here to compare/contrast the Nucleodur Gravity with the Hypsersil Gold:

http://www.hplccolumns.org/database/compare.php

As an aside, it may be that these two phases' end-capping or ligand binding are different such that the Gravity can stand up better overall to this aggressive separation method. That said, the suggestion M-N gives for storage in between sequence runs was shown as anywhere from 60:40 to 80:20 ACN/water. This would be the next thing to try, I think, with the Hypersil Gold.

If it is not a dissolution of the silica backbone, all that remains is the possible modification or loss of bonded phase causing the observed retention/resolution loss... it still seems clear to me that something is happening to the stationary phase in between sequence runs. In any case--if a change in the final storage solvent for holding the column in between separation runs doesn't help, I'll be baffled and stumped at that point, too.
MattM
crong wrote:
mattmullaney wrote:
If you have pH paper handy, it still may make sense to check the effluent pH after the 30 minutes of pumping the 90:10 mix just as a check. Please let me know what you find with the pH check of the pH of the post-sequence run effluent--just seems that based on what you're observing, this could be why the stationary phase may be being adversely affected.

Thank you Matt for your continued advice! I think I'm wrong about the pH being the issue.

I tested the effluent during a few points:
  • At the beginning of one sample where the gradient is at 98% buffer. pH paper showed pH 8.
  • Near the end of one sample where the gradient is 100% B. pH 6.
  • At the end of the sequence, 22 minutes into the 90:10 H2O:ACN rinse, pH 6. 35 min in, still pH 6.
  • Just DI H2O, pH 6.

So the column is not exposed to high pH during storage but resolution continues to decline. I'm baffled. :shock:


Be careful drawing conclusions from pH measurements of organic solvents.
MattM
Hi Again, Crong,

I found this article...just suggests that it may be necessary to go to extremes to flush out buffers and store UHPLC stationary phases:

http://digital.findanalytichem.com/nxtb ... /2/OnePage

Page 3, overnight (8 - 10 hours) with HPLC- grade water, slow flow (0.3 microliter/min). Guess it works for those folks
MattM
mattmullaney wrote:
Hi Again, Crong,

I found this article...just suggests that it may be necessary to go to extremes to flush out buffers and store UHPLC stationary phases:

http://digital.findanalytichem.com/nxtb ... /2/OnePage

Page 3, overnight (8 - 10 hours) with HPLC- grade water, slow flow (0.3 microliter/min). Guess it works for those folks

Hi Matt,
Thank you for that reference. I think it's probably not the same issue because I don't have increasing pressure as they did in the article!
Hi Crong,

Agreed and understood, just thought the article was interesting. Unlike those fellows, you didn't observe unchanging high pressure under flow after the step you take to clear out the salt from the column after an AAA sample set run.

Any progress with other ideas for working with column longevity and the OPA/MPA method?
MattM
off-topic, but that's an insane method. I know it's not a new paper any more, but it's hard to imagine someone writing "The shorter column length and smaller internal diameter helped to achieve high speed separations..." about a 75 minute method on a 50mm column. Strewth, if they'd been using a 250mm column they'd still be pre-equilibrating it today!
mattmullaney wrote:
Hi Crong,

Agreed and understood, just thought the article was interesting. Unlike those fellows, you didn't observe unchanging high pressure under flow after the step you take to clear out the salt from the column after an AAA sample set run.

Any progress with other ideas for working with column longevity and the OPA/MPA method?

Hi Matt, not much progress but I did observe one additional strange behavior...

The retention times continued to shift forward with each time I use the column, but today I saw Rt shift to later! Resolution still bad in general but the first pair of merged peaks unmerged :shock:

I'm back to thinking about pH. I usually adjust the pH to 7.8x and today it was a little lower at 7.76! Edit - nevermind! I checked back further and have had pH 7.7x before.

Image
https://imgur.com/QQiwZ6X
Blue trace is latest (today).

PI for the project decided to keep using the same column/method. While waiting for a new column we're still using this old one and just quantifying merged peaks as a single peak group. I've been washing the column with 90% H2O for 45 min, then 25 min at 40% H2O before shut down.
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