Tailing issues - damaged columns?

[shaolinseperation]

all of a sudden my lab has had problems with tailing.

we have 2 machines operating.

both started tailing around similar times and have switched both columns.

with the new colums one is beginning to show signs of tailing again while the other is running like a dream.

the columns are BPX70s
50m x 32 mm

what could be degrading the colums? could leaks in the septa or something like that have allowed air in which degraded it?

septa are usually changed every 100 samples but was left in there for approx 350 samples on the new column. could this have done the damage?

the alternative is some new members of lab running samples which may have contamination with water. i have looked at their sample tubes which have sodium sulphate in them and it has started clumping which means some water was in there. perhaps too much for the sodium sulphate to mop up. im not sure.


could anything else could be damaging the columns?

how likely are the above options responsible for causing the damage.

when damage occurs to a column does it take a while to reveal itself or will the next 50 or so samples immediately reflect it?

i can give more details where required. just raising a few of my ideas on what could be causing the problem and interested to hear what experiences and thoughts you guys have.

cheers in advance.

[Bruce Hamilton]

BPX70 is fairly robust, and the co-incidence of both failing certainly would point to a common factor, especially if they are performing different analyses and purchased at different times.

I read into your comments that you have puchased new columns and they are starting to show signs of tailing?

I would investigate whether the instruments are fed by the same carrier gases and the line traps are in good condition, and you haven't had a bad cylinder of carrier gas. I'd monitor the tailing over time, trying to ascertain whether it correlates with samples and/or injections.

It's possible that somebody is putting cruddy samples down both columns, but if they were working fine before, then I'd initially focus on the possibility of exposue to air at elevated temperatures. It certainly would be a very good idea to fix the sample preparation to ensure only clean samples are injected.

Most likely sources would be carrier gas quality, or leaking injector/septa/column connections, or an operator turning off the carrier gas before the columns have cooled. You just have to identify what has changed.

Please keep having fun,

Bruce Hamilton

[Peter Apps]

What kind of compounds are tailing ? If it is polar compounds only the ikely cause is oxygen or water attacking the stationary phase. If it is all the peaks it is more likely to be contamination from dirty samples.

How often do you change or clean your inlet liners ?


Peter

[shaolinseperation]

cheers for replies

all the compounds in our standard are tailing which is about 30+ compounds from 8 carbons to 24 carbons. so could be contamination then hey?

i dont think its the carrier gas because that hasnt been replaced in a long time now.

if it was a problem with the carrier gas the new column wouldnt have degraded so rapidly.

unless the trap has gone. we have got a "big universal trap" (what its called in agilent catalogue) and write the date on it every time we change the helium and it is only half way through its life.

inlet liners were changed recently and changed again with the new column.

FID has been cleaned recently and is looking all good.


how long does it take for damge to a column to be seen in the results?

i set up a run of about 100 samples and can see pretty much all of the tailing develop over that run. the first samples are okay (though a tiny amount of tailing) but when i overlay the results for each next sample it gets progressively worse quite quickly.

the samples at the start of this run were from a person whose extraction skills concern me...

[Bruce Hamilton]

Did you run standards at the beginning, end, and throughout the run?. If the standard peak shapes are tailing during a run of 100 samples, it certainly point to junk from some or all of the samples affecting the column.

The BPX70 is typically used for FAMEs, are you using it for that?. If you are, then I suspect that sample preparation is inadequate, as the column is quite tolerant of normal FAME preparation methods.

A trace of water should not have such a significant effect, unless you are operating near the maximum temperature. Residues of acidic or basic reagents can have serious effects on the column, especially if temperatures are relatively high ( 150+C ).

I assume all the other properties, such as retention time were consistent. I would be discussing sample preparation, trying to minimise injection volume, and ensuring the columns are operated at the lowest possible temperature.

A basic rule is the use the column of lowest polarity that will provide separation, so if you can go to a lower polarity column that will also help greatly.

Bruce Hamilton

[shaolinseperation]

yeah its for FAMES

the method is one that was set up a few years ago by someone who had a more detailed knowledge of what was going on. since then there were two research assistants who followed those methods without questioning it.

(if it aint broke dont fix it mindsets)

im the new guy who likes to tinker more than those two (i have a y chromosome) and have been changing ramps and flowrates to maximise peak seperation but from what you are saying i might wanna have a look at generating an entirely new method from scratch.

the current method starts at 140 (the machines are constantly on standby at 140). then there is a 5 degree per minute ramp up to 220, hold for 5 minutes then ramp at 20 degrees per minute up to 260 which is held for 8 minutes (i am told this is to make sure the column is cleaned)

pretty high temps.

from your last post im wondering if this is doing the damage

also increasingly worried about someone methylating the fatty acids with KOH. if this got into the colum due to bad technique im guessing some real damage would occur yeah?

[Peter Apps]

If you are getting KOH on your column the deterioration will be distressingly rapid.

Try changing the inlet liner and septum without changing anything else - if you are anlysing FAMES I presume that they are derivatised fats or oils, and there could be a substantial quanity of non-volatile muck in each sample that rapidly builds up in the inlet. Reducing the starting temperature of the column programme might also help, but ultimately you will probably need to adrees sample preparation and clean-up.

Peter

[shaolinseperation]

thanks for all the help guys. much appreciated.

may all your chromatograms have symmetrical peaks, good seperation and run in the shortest time possible.

[shaolinseperation]

very confused and annoyed right now.

we isolated our good machine and have only run samples from our experienced lab technician who is doing the same stuff she has done for 10+ years. yet tailing is starting to develop again.

so no KOH this time and fresh septa and liner.

any ideas?

[Bruce Hamilton]

I assume you are still confident that the problem occurs from samples, and it's not some other instrument changes - such as the start up and close down procedures, or switching off of carrier gas when not in use.

It's really important to be able to isolate the problem to the instrument, or to the samples. We are working on the assumption that the instrument is totally consistent, and nothing has changed, including settings like syringe volume, septum purge, split ratio, etc..

The fact that a new column solved the problem for a while suggests you are putting rubbish onto the column.

If tailing is developing due to the samples being processed, and your technician hasn't changed anything in the procedure, I would suggest it's either something in the samples, or some change in the process timing. Possible causes are samples not left as long before sampling or dilution, faster derivatisation steps ( not allowing phases to separate ), more vigorous mixing = more emulsions that don't break, or one of the reagents degrading faster.

If you are extracting the oils, has the extraction process changed?. A more aggressive extraction will bring out more polar lipids and other material that may accumulate in your injector or column.

You have to carefully review what has changed, and there's no point in us listing 101 possible causes, as the method used to work, and only you can sort out what has changed.

Please keep having fun,

Bruce Hamilton

[shaolinseperation]

been making more calls and trying to sort this one out and it looks like its a gas contamination problem.

the universal trap may have gone and previous people may not have been ordering ultra high purity gas. (records show some pretty cheap bottles but not sure if thats due to a recent price decrease or wrong orders)

getting some new traps in and trying to find out from suppliers the exact impurity ppm of the gas we are using.

will update on how all that goes. fingers crossed it will fix it.

[shaolinseperation]

btw: found out that we have been treating the big universal trap incorrectly.

got advice that with gas that is 5 ppm total impurities it is good for about 36-60 cubic meters of helium (6-10 bottles @ size g)

with 10ppm impurity gas it is more like 3-5 cylinders before a change is required.