I'm trying to understand and challenge the root causes of the many HPLC failures we have in our Lab, where we have 8 instruments (1090 to 1100 Agilent and Gilson) and perform ~80 tests/mth (all validated methods).

I'd like to know what is an acceptable % of failures on HPLC analysis (our current is above 25%).

How can the product mix impact on each instrument performance? I mean, is "different mobile phases frequently alternated" a big issue?
Which parameter is most critical in re-distributing different analysis, among
"Organic Vs. Non Organic"
"Ph"
"Buffer Vs. Non Buffer"?

Thanks
Vincenzo

What kind of failures do you mean?
Are you talking about assays that are falling out their limits? Or about checkvalves, high pressures, ...?

To answer your question:
The product mix can't be the reason for your troubles, I work in a company with a couple of hundred products, and we usually perform 3 different active analisis a day, so we often change the mobile fase and column.

But to resolve your problems, I have to know your problem exactly.

I've been thinking about this, too. If you have records, could you give the % and type of failure you would like to consider? I think we'd all like to compare with our experiences.

Thanks.

Am I correct assuming you have 80 different methods for the 8 machines? Sounds like a busy place.

I was talking about ~80-90 analysis per month,
with ~20-25 different methods (columns, mob. phase..).
Most of the failures are about repeatibility (RSD and %difference in the std.s during the test),then because of high pressure, and column troubles
I know it is pretty general and several causes could be involved..
do you really think an. distribution is not an issue? Our Automation & Instrumentation Unit suggest this is a matter of
- lack of training in young analysts, std procedures not followed (like correct washing or good initial conditioning)
- too many different mob. phase one after the other. Are there some "must" to consider in this situation (e.g. 90% phosphate buffer solution.. followed by 95%ACN and methanol..)?

Do you agree Best working conditions are 24 hrs a day - 7 days a week?
Do you think weekend washing program could help?

Regards
Vincenzo

Our std procedure is to do after each time we have done our analysis to clean up our columns:
Flow: 1.0 ml/min.
first 15 min with 90 % water, 10% acn
then slightly going to 50/50 water acn
(ramp for 10 min), then 35 min equilibrate with this mobile phase, then flow to zero, and we can store our columns as they are.

For our tubings, every time we change to another solventbottle for a tubing, we first hang our solventtubing and filter in a beaker, filled with water, and do a wet prime (Alliance) for 3 min with a flow of 7.5 ml/min. When this is done, we can change to another solvent.

We use this practice for many years, and never had troubles (except for the ones who doesn't follow these procedure).

I don't have too many rules, but here they are:

1) One column per mobile phase.

That is, one column for water/MeOH or ACN, and one column for each buffer. I've read numerous reports about problems going from buffers back to reverse phase, etc. This avoids any potential chemistry problems, precipitation problems with buffers, or other difficult problems.

2) For 'routine' analyses, dedicated columns and any connecting tubing, filters, etc. that you use.

And of course, you can't move methods from system A to system B without testing to see if you get the same results. I have been very successful for many years putting in a known, trusted column assembly, and running the next batch of samples. I would guess you probably need about 10-12 columns for your 8 instruments. At the most, 1 column per analysis. I've found that it is far easier to just install a trusted column than it is to re-develop a method on a system that SHOULD work, but doesn't for any number of reasons.

High pressure problems may be a the result of switching from buffers to high organic mobile phases, which causes precipitation. One of our groups had to replace the tubing in the LC (at great expense!) because of precipitation in the lines. Washing and storage instructions for buffer systems MUST be followed.

One of our labs has separated instruments into buffer and non-buffer instruments. Again, it helps avoid any diastrous plugging problems, and makes life a lot easier, with fewer possible mistakes.

I use reverse phase, non-buffered phases almost exclusively. They are simple and forgiving. I install the column assembly (filters, tubing, etc.), and check for leaks. To 'condition' the system, I run two or three gradients. The first has no injection, but simply runs the system through one full cycle. The second and third gradient runs are mid-level standard runs to check for acceptable retention times, and any other problems. Usually, everything is GO at this point, and I run the batch.

When washing these columns, I either include a wash-out method as the last run, or just run high organic concentration--usually 100% MeOH--for 20-30 minutes (depends on column size) while I organize the data package or do other things around the lab. No fancy gradients, no particular time beyond the minimum. A wash-out method works very well at the end of the run to prepare a column for storage. It's easy to use, and usually just uses night time that would be wasted. No mistakes, not much thought required, always the same.

3) Don't leave standing water in the systems.

Another possible cause of pressure problems.....and this might address your 'weekend washing' question.
A wash-out method does not address water in the reservoir to pump delivery lines. If your systems spend a lot of time not running, it is imperative that you remove all water from every inch of tubing in the system. If not, 'slime' will grow in the lines, and then wash off and plug the columns. In an extreme case, it took two days pumping MeOH to get 99% of the stuff out of a system. After that episode, all my systems have had 'PLEASE! Remove ALL water from system if it is going to be idle for more than 48 hours!' signs on them. For the last several years I have been adding 1% MeOH to my water, and this has helped a lot, extending the time to perhaps a week. But the signs remain as a reminder. Your water reservoirs should be crystal clear, always. With the new vacuum de-gassers, this may be even more important. I have one, but it hasn't given my any heartache. It's new, so there's time.

You may want to re-evaluate the injection sizes. Injecting very small quantities (5 uL) is borderline for many systems, no matter what the specs say. If you want maximum reliablility, don't push the systems any harder than you must. I don't go below 10 uL, and use 20 if I can. Make sure that injector systems are well purged, if it appears to be a problem. I have one system where that is a problem, and two systems that insist on long sparging times after cold starts. I have also used an injector program to 'pump' the syringe plunger a few times, just as GC autosamplers do, when I seemed to have problems.

Are the concentrations of analytes you run causing the problems? If so, are the limits appropriate? Are the limits justified? Maybe the systems aren't up to the task expected of them.

I don't think running 'normal' HPLC systems 24/7, if they are not running real samples, is necessary. You will wear them out faster, that's for sure! We have very recent information that this might be a good idea for some LC/MS methods and analytes, but it's probably not a general rule. I have noticed that every time I cold start my HP 1050, even if I change nothing, I get a slightly different retention time. This is not a big problem, and I don't know if it is random (within the typical batch Tr variability) or if there is a pattern. It's just a little different.

You may be able to help your problems dramatically by having your people specialize in certain analyses or instruments. You probably have no one that gets it right every time--we certainly don't. Let them concentrate on a method until they do really well with it, then rotate or move them to a more difficult method or instrument. Eventually they will be able to do everything with great confidence, which is good for everybody. One of our groups with a similar problem has used this technique for years with great success. They always have an 'expert' for everything, and are highly productive under a difficult, highly variable workload.

Other management questions....
As an old guy, I'm entitled to this one, because I've had to do it myself...these youngsters can be sharp, just like us!!

Has your 'Automation & Instrumentation Unit' shown that they can do it better?

Do your 'young analysts' have time to follow the procedures? Anon 1:29, above, has a 1 hour pre-storage regimen. That's not that unusual, and it will always take 30 minutes to wash, remove, column, prime, purge, etc., for most 'normal' systems.

Do they know WHY this stuff is important, or is it all still the mystery it was to us all when we started? I'm still learning every day.

And loving it!!!

Here Anon 1:29 again,

When I have to explain HPLC to some new people, and I begin my explanation about why to clean up, and why begin with 90% water, I do a simple experiment:

I dissolve some KH2PO4 in a bit of water, shake until its clear, and then add some methanol, until it precipitates.

When the "students" see this, they usually understand the importance of a good clean up of the column.

1:29, I was wondering about the 90% water for so long. Obviously your lab runs lots of buffer. This sounds like an excellent regimen. If a lab wanted to use only one method for all purposes, this would a good choice. A pre-batch gradient would remove anything that might build up during the water wash and short, 50% pre-storage gradient.

Your simple salt demonstration would get my attention! I've read a similar suggestion that if there is any doubt about organic/buffer compatability, mix the components in the appropriate concentrations, as you suggest, and see if there is any precipitation.

Thanx ScottF.

If I'm not sure if my sample dissolves in mobile phase, I do the same thing, and sometimes you see some precipitation. Another case: when you run gradients, I always check the buffer/organic ratio in a beaker, so I'm quiet sure that I'm never surpriced with high pressures.

Thank you all for your quick and detailed suggestions, I've discussed some of these with Lab Managers, some implementation on going.
Just to further understand:
if you realise, by these precipitation experiments, that two mobile phases would cause salt precipitation, do you suggest to avoid to schedule them one after the other or, through an adequate washing cycle, there won't be any problem? This because it is impossible to us (by now) to dedicate each instrument only to buffer or to very organic phases.

regards
Vincenzo

Yes, that is exactly what we are suggesting. I'm not an expert in buffers, but I believe minimizing the number of times you change a system from buffers to organic will help with your problems, as well as save time.

If you can see a problem developing in a simple test tube or beaker test, imagine what's happening inside your tiny LC system tubing!

Dear Colegues,
what do you think about possible consequences of contackt of mobile phase (solution 9 g/L NaCl in water) with stainless steel parts of modern chromatograph.Time of contact is very long one and temperature is 30 0C?

Thank you in advance.

Michael Levin
Ukraine