Switching mobile phases

[Yummes]

Hello, I am a lab analyst and although I have used an HPLC before, there's so much I still don't know. So my questions may seem simple but please bear with me, I need the information! I'd so rather ask questions here than wait on hold for Agilent customer service and sound like a moron.

I'm using an Agilent 1200, our lab has had it for about a month now. I need to switch mobile phases from Acetonitrile with 0.05% Formic Acid to a 10 mM Phosphate buffer. I also have to switch columns, the new column is a Zorbax C18 100 x 4.6 with 3.5 um particle size.

1- How long do I need to let the system run so that the colum is equilibriated and any bubbles are out of the system and the column?

2- Do I need to keep that "black twisty knob" on the outside of the pump tightened, or loosened, when I'm running the new mobile phase through the new column? (Forgive me but I cannot remember what that black knob is called).


If you guys can "walk" me through this, I'd really appreciate it!
Yummz

[zokitano]

Dear Yummes,

That "black twisty knob" is the purge valve. It should be opened (loosened) when you switch between mobile phase solvents prior start using/working the/on the changed column. Any bubbles present in the solvent lines would (in that way) be pumped off column in the waste.
When you're sure that there are no other bubbles present in solvent line then you could close (tighten) the purge valve and run the solvent through the column installed.
By this you'll keep the bubbles out of your column and wouldn't face problems with them (of course the degasser unit must be on all the time you're working with the instrument).

Regards

[tom jupille]

1- How long do I need to let the system run so that the colum is equilibriated and any bubbles are out of the system and the column?

zokitano answered the second part of the question. Once you have primed the system as he described, any residual air bubbles downstream from the pump (e.g. in the column) will gradually either dissolve or work their way out of the system. If you have the detector connected and operating you will see a large number of sharp spikes as the air bubbles pass through the detector cell. These spikes will gradually become less frequent and finally disappear. At that point, the air has been purged from the system.

For changes of solvent in a primed system, a good rule of thumb is that 10 times the system volume is usually safe.

[mbicking]

You did not tell us which pump you have: binary (two solvents) or quaternary (four solvents), so your exact procedure might be a little different.

These are some additional instructions that I give to new users of the Agilent 1100/1200 systems:

1. If you want to use a new mobile phase solution on Channel A, and you have a binary pump, take the mobile phase tubing (does it have a filter on the end?) out of the mobile phase bottle, and put it into a small flask that contains your new mobile phase (about 50 - 100 mL). This prevent contamination of the new mobile phase by the old solution.

2. Open the purge valve.

3. Set the pump flow to 5 mL/min and 100 %A (set all other channels to 0%) and turn on the pump. If you have the standard degasser, you must pump at least 25 mL through the degasser to replace the old solvent.

4. Turn off the pump. Transfer the mobile phase tubing to your mobile phase bottle. Set the pump to your desired conditions (flow and composition) and turn the pump on for about one minute.

5. Turn off the pump. Close the purge valve and now start the pump again. Your column will now start equilibrating with your new mobile phase.

If you need more help, just ask us.

[Yummes]

Thanks so much! I haven't made the switch yet but your replies help more than you can imagine. The books that came with the instrument are more conceptual and really don't have a "step-by-step" method listed.

To answer a previous question, my instrument has a quaternary pump. Does this change any of the answers that you guys have provided? I don't think so, but let me know if it does.

One more question: (a very longly-worded one, apologies!)

I have read, and been told by both the Agilent installation tech and a more experienced HPLC analyst, that it isn't good to let the phosphate buffer remain in the column after I'm done running samples. Apparently the salts can ruin the column, or at least shorten its life. With this in mind, I was planning on switching back to 100% Acetonitrile in between runs and at the end of the day. Is this appropriate? How long should I let the system run 100% ACN (how long, about, until I can be confident that the buffer is washed out of the column)?

This question ties into my initial questions from my first post, in that I don't exactly know how long, time-wise - OR how many mLs (with regard to flow rate) it takes for solvents to completely get through the system. I don't know my "system volume." I will look for this information, but at the moment I am unsure. I do recall the Agilent installation tech telling me that a good rule of thumb is 30 mLs; so if I set the pump for 5 mL/min, 30 mLs would run through the system in 6 minutes. What do you think? What should I do?

Thanks again and waiting patiently for replies--

Yummz

[tom jupille]

I was planning on switching back to 100% Acetonitrile in between runs and at the end of the day

Be sure to run an intermediate wash with buffer-free mobile phase (i.e., the usual water+ACN, but no buffer) before switching over to 100% ACN. Buffer salts have been known to precipitate in ACN .

A good rule of thumb is 10X the column volume, plus whatever it takes to flush the rest of the system. You can estimate the column volume from:
Vm ≈ 0.5 * L * dc² where
Vm = column volume (mL)
L = column length (cm)
dc = column internal diameter (cm)

I do recall the Agilent installation tech telling me that a good rule of thumb is 30 mLs; so if I set the pump for 5 mL/min, 30 mLs would run through the system in 6 minutes. What do you think?

That sounds about right (assuming your column can tolerate the flow rate).