HPLC General Operationg Questions

[Jumpshooter]

I have been using HPLC (1090Agilent) for one week and am clearly a "Novice". I have been doing real-time PCR for the last 5 years, thus this field is entirely new to me. A question I have is: Why is it necessary to "equilibrate the instrument for 1 hour with pumping thru Mobile Phase"? That seems like an incredible waste of expensive Mobile Phase. When I did PCR work, we optimized and minimized the use of expensive reagents in our assays. So, it seemed counter-intuitive that one would have to just waste away a lot of expensive mobile phase for the ostensible purpose of "equilibrating" the instrument. Any ideas?

[chromatographer1]

There could be a lot of reasons why the writer of the method did this.

Whenever one writes a method for someone else to execute there is a tendency to be overly cautious.

It may be needed to truly clean off a previously used column or to saturate a column with an ion pairing reagent so that the results will be reproducible, especially when there is a 'novice' running the method for the first time.

The best answer to your question is to ask the person who wrote the method. Is it chemistry? temperature? or what?

No one in industry wants to waste time or money. Your question is valuable and I hope you get an answer that is worthy of it.

best wishes,

Rod

[Kostas Petritis]

When you speak about instrument equilibration do you speak about the HPLC only equilibration or the column as well? In the latter case, equilibration will depend on the nature of mobile phase and stationary phase that you are using and the type of chromatography that you are doing. I would say that reversed phase conditions with monofunctional bonded silica base short columns are equilibrated the fastest...

Anyways, I do not know if the HP1090 needs some special handling, personally I always like to start by purging each solvent for about 5 min (if the instrument has been recently been used) and then equilibrate the column accordingly. In a sequence I always start with a blank gradient...

If you are concerned about the solvent consumption, you might want to use 2.1 ID columns where the typical flow rates are 5 times lower than the 4.6 columns... (0.2 instead of 1 mL/min). Having said that, unless you use some very special mobile phase, I wouldn't consider the HPLC mobile phases very expensive...? After all, 1 hour at 0.2 mL/min is just 12 mL... I think that generally people discard much more than that (especially people that want to always have fresh made mobile phases)...

[Jumpshooter]

the stated flow rate is 2 ml per min. that is a LOT of MP going thru. But OK, I guess that's what I should do?

[Mark Tracy]

As I recall, the application here is normal-phase chromatography of retinol palmitate. The long equilibration is what is needed to get the column into a stable state starting from an "unknown" condition. If the column is stored in the LC, filled with mobile phase and at temperature, the next-day startup should be much shorter. You can do a quick study in the morning by injecting standards and see how long it takes to get consistent retention times.

If your pump were anything besides an HP1090, I would recommend keeping a maintenance flow of 0.05 mL/min overnight, and keep the column oven warm. Alas, the booster pump will wear out the diaphragm prematurely under those conditions, so you just have to turn it off at the end of the day.

These long equilibration times are characteristic of normal-phase chromatography. One of the reasons that reversed-phase chromatography became more popular is its has much faster equilibration.

[Dan]

The instrument electronics need a little bit of warm up time. This is usually 30 minutes (it can be a little more or a little less depending on the instrument model/make/age).

It is mostly the detector that needs the warm up. About 30 minutes for the lamp and electronics to get to a stable condition. You can keep the detector on with the lamp off when not in use for when there is no run. That way you just turn the lamp on and you can be injecting in 10-15 minutes.

Just remember that instruments usually only perform the self-diagnostics after first being turning on. You can usually run the self-diagnostics via a control command; a full re-start is necessary. The manufactuers usually recommend a once-a-month running of the self diagnostics.

Regards,
Dan

[Jumpshooter]

*Hi I'm a clear novice user: My standards (retinyl palmintate) looks like it has Tr of 3.4 min. With increasing concentration injections (increasing volume injections from one concentrated standard)--I have noticed that the Tr increases by about 0.1 min between respective calibrant concentrations (20 USP, 40 USP, 80 USP, 160 USP). What problem does this portend in the ultimate standardization of the run?

[Bruce Hamilton]

*Hi I'm a clear novice user: My standards (retinyl palmintate) looks like it has Tr of 3.4 min. With increasing concentration injections (increasing volume injections from one concentrated standard)--I have noticed that the Tr increases by about 0.1 min between respective calibrant concentrations (20 USP, 40 USP, 80 USP, 160 USP). What problem does this portend in the ultimate standardization of the run?

You can often expect a slight increase in Tr as the peak size increases, and 0.1 might just be that effect, depending on peak width, but it sounds a little too large for such an early retention...

Provided the behaviour is consistent at the beginning and end of sequence, it may be acceptable, but if Tr is drifting throughout the run, you need to fix it enough to meet whatever chromatographic quality criteria apply to the method. Likely causes are temperature and column not at equilibrium at start of run.

Please keep having fun,

Bruce Hamilton

[Jumpshooter]

hmmmm

[JA]

You can often expect a slight increase in Tr as the peak size increases

I don't like posting in contrast to respected members of the forum (it's a bad habbit of mine ), but I was only aware that retention time can decrease as the peak size (let me equate this directly to concentration) increases as an example of mass overload. Only if the calibration solutions were injected as a series of increasing volume can I grasp the idea of increasing RT due to the dispersion which occurs during injection

For the benefit of others, is this related to the "wet hexane" thread?

[Bruce Hamilton]

You can often expect a slight increase in Tr as the peak size increases

I don't like posting in contrast to respected members of the forum (it's a bad habbit of mine ), For the benefit of others, is this related to the "wet hexane" thread?

I'm not a respected member, I just comment from my experience. Please don't be inhibited in challenging my comments/opinions, we are all here to learn.

My observation could well be wrong, but the original poster will soon find out by running a sequence, and comparing the standards at the beginning and end, both with regard to peak shape and retention. Once we have some more data, we could all suggest possible solutions.

My recollection was that the original poster was analysing Vitamin A Palmitate in a fortified product. It's a very long time since I analysed Vitamin A by normal phase - the Waters silica column came in a nicely polished brown wooden box with brass? hinges and catches.

Please keep having fun,

Bruce Hamilton

[DR]

...I'm not a respected member...

Wrong! It just isn;t your day, is it?

[Jumpshooter]

this a.m. I will run my standards and then re-run them to see if there is drift. It may be due to the fact I only equilibrated the column for 30 mins. But today we will see. I prepare a stock standard of 180 USP Vit. A, then task the instrument to inject varying volumes (12.5, 25, 50, 100 uL). A calibration is built off of this approach. So today I will have data for you to inspect.