Buffers and ACN concentration, collumn conditioning

[dragosb]

Hi,

I have a question concenrning the mobile phase I use and the storrage of the collumn.

I use a C18 Waters collumn to separate aminoacids from deproteinized plasma (30% sulphosalicilic acid SSA) but I use it once a week. I used to store the collumn in my mobile phase (Na2HPO4, NaH2PO4*H2O 10mM, pH 6. but I understood that it would be better to store it in 60-90% ACN/water solution. Is this true? Can I just switch directly to this ACN concentration at the end of the day or should I better pass by a lower ACN concentration first?
In my protocle I use an ACN concentration of 10% and at the end of each injection I rise it to 25% to rinse the collumn. Is this good or bad for the collumn?
I use a Waters Nova-Pak C18 Column, 60Å, 4 µm, 4.6 mm X 150 mm collumn.

Does anyone use a conditioning protocole for this type of collumn for aminoacid separation?

Thanks a lot for all the answers that you can give me

[tom jupille]

Several comments here:

1. Columns are cheap; your time is valuable.

2. Columns *are* a consumable item.

3. In general, follow the column manufacturer's storage recommendation to get the best column lifetime (in other words, ask Waters what *they* recommend).

4. In the absence of specific recommendations from the column vendor, the best long-term storage solvent for C18 columns tends to be 100% organic (usually ACN; sometimes MeOH).

5. Phosphate buffer is great for growing microbes.

6. I would not switch directly from an aqueous buffer to a high-organic mixture; it's a good idea to flush with buffer-free mobile phase first (minimizes the possibility of buffer crashing).

7. The maximum lifetime and the maximum useful lifetime are not necessarily the same. Your column will last longer if you store it in high organic, but you will waste some of that lifetime re-equilibrating it with buffer. If you are getting acceptable column lifetime now, keep doing what you are doing.

[Gerhard Kratz]

In addition to Tom's comments:
Just use your mobile phase with a low flow rate to flush the column overnight. That will cost you some solvent, but will save time to get started next morning.
When using inorganic buffer salts please flush first with high amount of water before flushing with ACN or MEOH.

[Mr.Brown]

We analyze a lot of dirty samples
So some gunk gets stuck on the column
So I just have a method for cleaning and bringing the column to storage conditions
We run our batches usually over night
So at the end of the batch I just inject the lowest amount of some solvent
And run my cleaning method
Very simple
I switch to 10% org solvent and 90% water and run it for several minutes to get rid of the buffer
Then I have a slow gradient to 100% org solvent e.g. 30 min from 10 to 100
Then I keep it at 100% for a few minutes and then go to 80% org. Solvent and 20% water
Which is my storage condition

In the morning everything is finished and I can store the column
No time wasted

[dragosb]

Thanks for the answers.

I will try to use Mr. Brown's washing technique.

I still have a question for Mr. Brown:
What is the maximum ACN conc. that you usually use in your experiments.
Can a 25% ACN do any harm (precipitation)?

Thanks

[Mr.Brown]

This depands on the buffer concentration you are using.

I usually go for a 10 mmmol/L ammonium acetate buffer (if possible and usuable)
this i have used my self with up to 40% ACN but even up to 75% ACN should not be a problem.

There is even a paper about this problem
http://www.chromatographyonline.com/lcg ... rticle.pdf

Table I shows that with 70% ACN you still can use a buffer up to 20 mmol/L
And this ist for the least soluble potassium phosphate buffer.

hope this helps

[thiazole]

Mr Brown, thanks for posting the link. You saved me some serious headaches today as I'm using a phosphate buffer for the first time and didn't consider that it would crash out if I didn't keep the ACN concentration from getting too high.

[paulw]

One thing to keep in mind with phosphate buffers and ACN. You can have a buffer/ACN mixture that is perfectly clear and free of precipitate once it is well mixed. That same combination when pump mixed may cause you issues, due to the fast mixing as well as the slight decrease in temperature when ACN mixes with aqueous (it might cause you some issues on a particularly cold day if your company goes cheap on the heating bill too).