General Shoulder Troubleshooting

[ChemistB]

A number of my reverse phase methods have started to show shouldering (post peak) where none existed before. What are some possible causes?

[gbalock]

Chemist,
Any number of things can cause a shoulder on the peak. Column voids being the most likely. Another is unidentified chromatographis garbage stuck to the active sites of the column. Clean your current column or replace it if the possibuility of a void exists.

[unmgvar]

ChemistB

if the shouldering appears to be on all the peaks in all the runs with all your different columns since a "given day" then it is most likely an hardware problem. void volume somewhere.
i would start by looking at your column connections. probably one of them as worn out and is creating a void.
run a short aplication with one of your columns and see if you get any improvement.

if it is not then it could be several causes and we would need to have more details.

[ym3142]

A number of my reverse phase methods have started to show shouldering (post peak) where none existed before.

if you are using different columns and difference LC's, all of them shoudered. it means there is ghost in your lab;
if you are using many columns in one LC all ofthem shouldered. It means you LC have a void.
if you are using one column for many methods, it may mean your column has some issue such as contaminated, void, frit dirty if not the above ;
if you are using one column for one method for many tests it may mean you have sample prep issue such as strong diluent if not the above

some times we do enjoy the guessing game because people like you who do not have time or interesting to tell us what actually happened in your methods in detail.
thanks for your sharing.

[PJ8]

We very much need more details. You say a number of methods, what column/columns? Same instrument or different. Give us as much info as you can so we have a chance of narrowing down to a route cause.

Cheers, Pat.

[ChemistB]

Okay, I need to do a little detective work to see if shouldering has been on the same instrument or any other common factors. I am the reviewer of the chromatography and the runs have been by a number of individuals. I can give this information and will get more.

1. we dedicate columns to methods
2. the most recent episode was isocratic acetonitrile:water (no buffer) analysis of glycine in our product which contains a lot of proteins on a C-18 column.

Thanks for your help. I initially made the question general so I can get a wider view of possible answers in case the most probably answer is not the answer.

[ym3142]

ChemistB,
are you saying I (Chemist B) am smarter than you guys by

I initially made the question general so I can get a wider view of possible answers in case the most probably answer is not the answer.

?

And are you satisfactory with these milliiiiions possibilities?

[PJ8]

Too wide, we need something to go on!

If you are using old Phenomenex Luna C18's there is a known ghost peak/hump issue (to do with frits wearing I believe) which develops in columns as they age. Peaks can look like anything from a shoulder to a huge lump and perhaps worst a small impurity peak on the tail of your main peak (we had QI's here in the past because of that.)

However, Phenomenex say they have fixed the problem now so I guess new Luna columns should be safe (time will tell.)

The general stance I have seen in this forum is that unless you are specific with your problem people wont waste their time guessing without plenty of information to go on. There is a wealth of experience here, but they don't have time to list every peak shape issue they've ever seen in case it's relevant to you. Have a browse through discussions past and you will see plenty of examples I'm sure, the search facility is pretty good.

Regards, Pat.

[ChemistB]

[/quote]are you saying I (Chemist B) am smarter than you guys by Quote:
I initially made the question general so I can get a wider view of possible answers in case the most probably answer is not the answer.
?

No, that was not my intention at all. If my general question offended anyone, I am sorry. I am saying, in fact, there's a lot I don't know and if I can learn general possibilities that will aid me more in the future rather than get a specific solution that might not be applicable next week, I'd rather learn the "million possibilities."

ChemistB

[ym3142]

Well, ChemsitB,
I am sorry too. I felt my questions may have offended you. Hope not though.

but any way, this is a good learning platform for any one but we are not expecting others to copy a text book into this forum so as that we can all everything about LC. That's my understanding. That 's the reason I prefer seeing a specific problem to a broad general question. The latter should be your alone home work such as read a text book. Thanks for your understanding.

[Jamie]

when I get a shoulder in my peaks I reverse the flow in my column and it usually gets rid of the shoulder. That means, as others have indicated there is something wrong at the top of the column packing . Either there is a void or it is contaminated.

Jamie

[danko]

ChemistB wrote

the most recent episode was isocratic acetonitrile:water (no buffer) analysis of glycine in our product which contains a lot of proteins

I’ve been working with proteins for a long time and one thing I can say for sure is: Protein in water/acetionitril mixture is not the best idea one can conceive. In fact the most proteins will precipitate in this environment. It is most often necessary to adjust pH away from the protein’s pI, which usually is in the range of the neutral pH. Another possibility is to add a limited amount of some salt (salting in). The latter is a little bit longer story so I won’t address it here.

My idea of what is happening in your case is: Although you are only interested in the glycine, you - in your own words – are loading some protein too. The protein precipitates on the column and stays there until something “helps it outâ€