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Topic: Mistery tailing in rp-hplc  (Read 12315 times)

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Offline coquim

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Mistery tailing in rp-hplc
« on: March 18, 2008, 10:36:26 AM »
 Hi people, here i am, my head is burning....i can´t solve this tailings that i´ve attached...sorry but...i´m all out of ideas...i really don´t know how....i´ve changed solvents, and it´s %, Ph, i´ve turned column in the inverse flow in order to find out some inner change in it but i´ve got the same results...if somebody could give a clue ´may be i could solve this mistery...
Thanks...!!! ??? ??? ??? ??? ??? ???

Offline Arkcon

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Re: Mistery tailing in rp-hplc
« Reply #1 on: March 18, 2008, 11:13:28 AM »
Presumably this is a run of the analysis you mentioned previously:  Folic acid and pangamic acid, yes?  It would help if you included the text in your question, so people doing Google searches find this forum.

I'd also help if you were as specific as possible about your separation:  column type, manufacturer, eluent composition, and what you've tried.

I'd also like to direct you to http://www.chromforum.com/ , they're a great group of professionals, who specialize in these sort of questions.  Don't get me wrong -- I love talking about separation science, and could do this all day. They're just better at it than I am, that all. :)

Now, for my specific help:
Folic acid tails badly.  It just does.  If you look at some of the promotional literature for the more advanced, and expensive columns, from such companies like FMC, Waters, etc., you see pretty high tailing, like 1.7.  And that's with ion pair reagents, which, as I recall, you prefer not to use, right?

Now, some simple HPLC tips, that you may already know.  You want to analyze folic acid, presumably by reverse phase.  An acid is by definition, charged at high pH.  If your eluent pH is close to the pKa of folic acid, you are analyzing two things -- folate, and folic acid, so you will get bad tailing.  To analyze just one on reverse phase, you will have to lower the pH, so the folate is protonated, and behaves more like a non-polar molecule and less like an acid.

Now as for pangamic acid, as I recall from your other post, it's structure is more than a little different than folic acid, so improving one separation may harm the other separation. Heh.  I'd need to look up the pKa to see for sure.  However, I think an ion pair reagent may be just what your separation needs, if you're not using it already.

And another tip.  I don't much care for Word documents as attachments.  They can carry viruses, and I don't care to scan so carefully.  The plain graphics, as attachments, are easier to handle.
« Last Edit: March 18, 2008, 11:26:38 AM by Arkcon »
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Offline JGK

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Re: Mistery tailing in rp-hplc
« Reply #2 on: March 18, 2008, 11:28:19 AM »
If you are certain that you have a tailing issue, it may be caused by "channeling" in your column. I would check an alternate column of the same type, if you get silmilar chromatography the it rules out that cause.

Are you sure that what you are seeing is "Tailing"? 

From the look of it, it  seems you have two sets of double peaks that are not completely resolved rather than peak tailing issue. Do you get the same chromatography from standard materials?

Experience is something you don't get until just after you need it.

Offline coquim

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Re: Mistery tailing in rp-hplc
« Reply #3 on: March 18, 2008, 03:28:02 PM »
yes, i was thinking that they are a couple of peaks, and that´s because the ph it´s no acid enough...and it´s true that i continue working with Folic and Pangamic, and also 7 drugs at the same time in order to obtain clean peaks...
i´ve started use IPC regeants (tetrabutylammonium hydrogen sulphate) and by the way, i have a doubt about, how can this regeant could alter the column selectivity...
JGK thanks, i dont know what channeling means, i think have a light idea but i´m not sure...
Arkcon, thanks for your time, honestly, you´ve told me so much....i´m working about your info right now...
 i must say THANKS, but i feel it´s no enough...as i always say, selfless help has no price and it´s the more important thing that someone could do...i´ll appreciate so much, for all data, thanks again.! when i get resolve the problem i let you know....

Offline Arkcon

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Re: Mistery tailing in rp-hplc
« Reply #4 on: March 18, 2008, 03:55:43 PM »

i´ve started use IPC regeants (tetrabutylammonium hydrogen sulphate) and by the way, i have a doubt about, how can this regeant could alter the column selectivity...

You are in for a surprise then, these used to be called PIC reagents from Waters, back in the pre-history of chromatography, the late 1980's, when dinosaurs roamed the Earth.  People would just put a little bit of this magic reagent in their eluent and viola, peak shoulders would disappear.  We've since learned how they work, and plan our separations better, and our column media has advanced, to remove the non-specific binding of nitrogenous compounds to the silica media underneath the bonded reverse phase media.

Quote
JGK thanks, i dont know what channeling means, i think have a light idea but i´m not sure...

As the column media dissolves, and gaps form, the bands of analytes may begin to double.  Sharp double peaks of analytes that used to be single are pretty obvious, but for the first few injections, bad tailing is all you see.  It's always funny, you start seeing a tail, then a shoulder peak, and you start wondering, "Is this a new decomposition product?"  Or maybe, you have a peak that fronts badly instead of tailing (simply because that's the method you've been given),  and suddenly it's symmetrical, and you think, "Damn, I am good, I fixed it."  But in either case, you're wrong, you're just watching a column die.  :D


Quote
Arkcon, thanks for your time, honestly, you´ve told me so much....i´m working about your info right now...
 i must say THANKS, but i feel it´s no enough...as i always say, selfless help has no price and it´s the more important thing that someone could do...i´ll appreciate so much, for all data, thanks again.! when i get resolve the problem i let you know....

Told ya, I love talking about column chemistry.
Hey, I'm not judging.  I just like to shoot straight.  I'm a man of science.

Offline coquim

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Re: Mistery tailing in rp-hplc
« Reply #5 on: March 19, 2008, 08:07:41 AM »
NOOO!! please don´t tell me this...seriously...i can´t believe that it´s happening to me...the column has only 4 months of use, i always use it with low pressure (under 200kgf), i wash it everytime i using 80%water and 20%Meoh in order to clean and protect it...the mobile phase i used to are the common solvents we know very well as Meoh and Acn and buffers as acetate and phophate, so, as i said at the begining, i can´t believe the column is not working anymore...is it would possible in only 4 months? and why this happen? is there any way to avoid the column die in 4 months? how is the useful lifetime of a column?
Thanks again!

Offline JGK

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Re: Mistery tailing in rp-hplc
« Reply #6 on: March 19, 2008, 08:28:40 AM »
NOOO!! please don´t tell me this...seriously...i can´t believe that it´s happening to me...the column has only 4 months of use, i always use it with low pressure (under 200kgf), i wash it everytime i using 80%water and 20%Meoh in order to clean and protect it...the mobile phase i used to are the common solvents we know very well as Meoh and Acn and buffers as acetate and phophate, so, as i said at the begining, i can´t believe the column is not working anymore...is it would possible in only 4 months? and why this happen? is there any way to avoid the column die in 4 months? how is the useful lifetime of a column?
Thanks again!

Can you test the column with a compund (or compounds) you are very familiar with and "know" will produce  single peak(s)? If you see problems in the chromatography of these analytes it will confirm if your column is the problem.

Column life can vary dramatically depending on the type of samples passed through them and the aggressivness of mobile phases.  Some last only a few hundred injections and some for several thousand.

A short column life sometime falls under the old adage "sh1t happens"  ::)
Experience is something you don't get until just after you need it.

Offline Arkcon

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Re: Mistery tailing in rp-hplc
« Reply #7 on: March 19, 2008, 08:55:47 AM »
Column media isn't only affected by high pressure, but by changes in pressure -- the media relaxes and expands when pressure is low or off, and compresses whenever any pressure is on it, each injection carries a pressure spike, etc, and the particles grind against each other, and then, gaps, and channels.  They taught me very early on, as an intern at Waters, that it's changes in pressure that does the worst damage to columns, not constant high pressure.  You can add a lot of life time to your columns, and your systems, if you set them to low flow, 0.1 ml/min, when idle.  That used to be the norm wherever I worked, but they're phasing that out more and more these days -- 0.1 mL/min*60min*24hrs*7days = lots of money spent on solvent waste.

Old style columns, with irregular particle sizes, were more prone to channeling.  The more modern spherical particles tend to form gaps at the head end, plus we've advanced our column packing techniques.  If there's a gap at the head of your column, reversing the column may solve double peaks, for a few more runs, until the gap reforms.  Heh.  At least you get your answer -- you determine conclusively that your column is going bad, and you might get the last of your analyses done.

What brand and model column are you using?  And like JGK: says, yes a heavily used column can sometimes last as little as a few months.  If you're switching from buffer/ACN to water/MeOH daily, methanol's higher viscosity is sending a pressure flux each time.  :o
« Last Edit: March 19, 2008, 09:24:47 AM by Arkcon »
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Offline coquim

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Re: Mistery tailing in rp-hplc
« Reply #8 on: March 19, 2008, 09:42:59 AM »
oh oh...ok...i see...
i´m using a shimadzu column (shim-pack vp-ods 250mmx 4,6) with a pre-column and i suppose that using this little component the column life would improve a lot, is´n it?
if I turn the column, how many inyections and what pressure may i use to try to fix the media column? if it would be possible to do, of course...
you say idle, but i dont understand so much, do you mean idle instead pump off? i mean, when the system are not being use in order to avoid the pump off for add lifetime to the equipment and column?
i wash the column at the end, aprox 1 hr with 80H20 20 Meoh no matter the solvent i´ve been using before, as i said, to clean it...i start work at 8 and finish at 17, may be is not necesary to clean it if i going to use it early next day...
may be i should say that i use 1ml/min flow almost always...




Offline JGK

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Re: Mistery tailing in rp-hplc
« Reply #9 on: March 19, 2008, 09:57:10 AM »
if I turn the column, how many inyections and what pressure may i use to try to fix the media column? if it would be possible to do, of course

If your problem is a void or gap in the column you will not be able to "fix" it by reversing the column and playing about with mobile phases and pressure. As Arkcon said it may be a temporary fix but no more than that.

If your feeling very brave and have some of the column packing in loose form, you could try repacking the gap at the column head. However, this may not fully restore your column and is risky, especially with modern columns which, when opened can start to slowly expel the stationary pase from the column body (due to the packing techniques used) .

However, as I said previously make sure the column is the problem before you start trying for a cure.  Test it with known single peak methods and check that chromatography.
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Offline Arkcon

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Re: Mistery tailing in rp-hplc
« Reply #10 on: March 19, 2008, 11:07:24 AM »
I don't know a lot about the Shimadzu Shim-Pack VP-ODS model columns, their advertising blurb seems to reference typical column chemistry.  I know, for a fact that a Waters Brand μ-Bondapak is a 10 μm irregular particle column, which is more sensitive to channeling with abuse, as compared to a Water Nova Pak column, which has 5 μm spherical particles, so is easier to pack throughly in the column housing.  Waters has since the 1980's updated it's column chemistry with it's Symmetry line, with higher quality silica, and it's Xterra line, with embedded methyl groups.  If you can find the information from Shimadzu, about particle size and shape, you can make a value judgment on your column, 1970's-style or 1980's-style technology, and see what sort of lifespan and separation quality you should expect.

Like JGK: said, if there's a gap at the top of your column, reversing it only leaves you with a gapless column for a short while, as the ordinary HPLC pressure drives the media back to the bottom reforming the gap at the new top of the column.  High pressure and direction swapping can also compress channels, maybe, but you're just trying to prove what's inside the "black box" of the column.  You don't get to revive it from the dead.

I mean idle as whenever the column is not analyzing anything, overnight for example, or while you process data.  Keeping the piston heads moving lubricates the pump seals for longer lifetime, and avoids the pressure flux from zero to even low pressure on the column, which is really when particles begin to compress, and rub against each other.

Now, a pre-column is useful to protect a column from strongly bound compounds, that fowl the media.  These can be diverse and hard to understand.  They're not a panacea however, you should know the chemistry of your possible contaminants, to see whether it's worth sacrificing resolution with the pre-column in place.  Is your matrix, for example, biological fluids, or homogenized plant material, or fermentation vat contents?  then a pre-column may be warranted.  For general use, only a filter is needed between pump and injector to catch pump particles, if you also remember to filter samples.  But some people like the pre-column option as a catch all procedure.

Don't forget to consider -- the gap or channel may be in the pre-column, and it might be the entire source of the mini-double injection that is the cause of peak splitting.

Assuming you've excluded the possibility that you're getting these shoulders from sample chemistry on the column.  Again, like JGK: said, unless you check, you really can't assume there's a gap or channel.
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Offline JGK

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Re: Mistery tailing in rp-hplc
« Reply #11 on: March 19, 2008, 12:01:00 PM »
Arkcon,

It looks like our friend doesn't entirely believe us  ???

http://www.sepsci.com/chromforum/viewtopic.php?t=8093
Experience is something you don't get until just after you need it.

Offline Arkcon

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Re: Mistery tailing in rp-hplc
« Reply #12 on: March 19, 2008, 12:22:36 PM »
Well, I did send him there in the first place. ;)  I hardly ever go there anymore, nobody asks questions that are simple enough for me to solve.  :'(  Or they ask "gimmie gimmie" questions.

*[EDIT]*

Not exactly good advice over there, they don't even have the mobile phase composition, and they assume the pH is out of range.  I may have to go over there and take some of those guys to school ...
Hey, I'm not judging.  I just like to shoot straight.  I'm a man of science.

Offline coquim

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Re: Mistery tailing in rp-hplc
« Reply #13 on: March 19, 2008, 12:25:10 PM »
ja ja ja ja!!! No my friends, please dont think in that way, you don´t have idea how much i appreciate your help, seriously...JGK, it is true that i´ve posted my question to another forum that arkon linked me yesterday, that´s all, don´t be jelous man...anyway i got some important thing to say: the problem was......THE PRECOLUMN...YES!!!! YES!!!
We say a lot and i didn´t think before, and Arkon said about the gap in the precol. so i changed for the old precol. and.....EVERYTHING WORKS OK!!!....the rare thing is that the precol was a new one....so, i think it´s a manufacturer mistake....THANKS AGAIN MY FRIENDS!!!!!....What can i say?...i am indebted with you guys...if you came to argentina sometime i´ll cook an asado for you!!

Offline JGK

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Re: Mistery tailing in rp-hplc
« Reply #14 on: March 19, 2008, 01:17:51 PM »
I wasn't jealous, actually I contribute on both sites
Experience is something you don't get until just after you need it.

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