moving peak

[smkh]

what cause the problem of peak retintion time shifting (decreasing) within only 3 days on a new column , with acolumn oven temp about 35
m.ph:88% 0.05 buff and 12% acn , ph;3.50
and samples ppt using perchloric acid column:phenomenx gemini
how can i stop this moving?!

[zokitano]

smkh,

Give us more information about the composition of your buffer you're using in the mobile phase. Also is your column insulated from the lab environment? In other words are you sure that you had constant column temperature conditions over the days you've observed retention time shifts?

Regards

[danko]

Hi smkh,

A single question: Is the retention time restored to the original/expected, when you change the mobile phase and start a new analytical series in general?
If the answer to the above is “Noâ€

[bisnettrj2]

danko - Are you "dissing" Phenomenex columns? What reasons do you have to dislike their columns? I was hired into a lab that uses Phenomenex columns in my primary analysis, and I'm not particularly happy about it (not a problem of quality, but availability). If I can provide extra details from other users of their columns, maybe I can weasel my way into another manufacturers columns....

[grzesiek]

what about column contamination? Do you wash your column after each use/day? how do you know that all that was in the sample is visible in the chromatogram?

[smkh]

zokitano,
The Buffer is 0.05M NaH2Po4.
I use closed column oven to isolate it from the enviroment.

danko,
about column brand , in my opinion that phenomenex is agood brand and much better than ACE , but may water, agilent better than it
did I need aspecial type of column fo these conditions or just an ordinary C18 , what the suggestions of aspecial type from water or agilent.

bisnettrj2
I agree with you about avaliability problems of phenomenex.

grzesiek,
About column contamination , it is anew one and I use it for only 3 days .

Best regards to all.

[HW Mueller]

I am bewildered by all the people who have a severe problem, but don´t answer the questions of someone who wants to help them. For instance, Dancho asked the legitimate question of whether the problem exists with different mobile phase preps (on phenomenex: I have never seen stability problems).
The absolute first thing to do is to make sure that the peaks are really from my analyte and not some phantoms.
The next thing which I check when I have a decreasing retention time is flowrate (I no longer assume everybody else does that also, and that one need not mention it anymore).
Then would come the checking on mixing if it is done on-line. If premixed I would check whether equilibrium mixing was attained.
Then I would suspect a change in pH (here the buffering capacity is close to nil).
Only after that would I check into contamination of the column, chemical deterioration etc., if I did something where these could happen at all.

[zokitano]

As HW Mueller said, your buffer pH is not in the optimal pH buffer range for phoshate buffer (H3PO4/H2PO4-). Consider changing the pH of the mobile phase into the buffer pH range (e.g 1.2-3.2).
What is the composition (the matrix) of your standard/sample solutions.
How many microliters of them do you inject in your HPLC?

Regards

[danko]

Are you "dissing" Phenomenex columns? What reasons do you have to dislike their columns?

In this particular context, I would first and foremost emphasize their instability. And that’s what I believe Smkh is dealing with. In addition to that, I’ve experienced (first hand) their poor performance (Efficiency, Resolution and Reproducibility) in a couple of comparative studies that I conducted myself. The reference brands utilized in my tests were Waters and Ace (for the RP mode) and then TSK and Waters (for the SEC mode). I can promise you, they all easily outperformed their Phenomenex counterparts.

did I need aspecial type of column fo these conditions or just an ordinary C18 , what the suggestions of aspecial type from water or agilent.

Hi Smkh,
The conditions you describe are quite mild, so I don’t think you need to search for a specially designed column (referring to the stability). You should be aware of the fact though, that I (or anybody else) can not guarantee you an identical selectivity if you pick up a column of another brand. But the Phenomenex column’s reproducibility is so poor (according to my experience) that you are not guaranteed a perfect match either, even though you keep using them. On the other hand there is a reasonable chance of finding a column that both separate better and is much more stable if you just pick up one C18 made by Waters or Agilent or ……(just examples, so don’t feel limited to those two only). So just try – you may be pleasantly surprised. Remember to choose the same column dimensions!
By the way, your buffer is out of buffer range, so you don’t have a real buffer effect at pH 3.5
It’s not that big problem if you inject acidic samples, but if your samples were basic you should consider revising your “bufferâ€

[grzesiek]

"About column contamination , it is anew one and I use it for only 3 days . " - so?

[Fiz]

I saw people who get manage to completely clog new columns within a single week. So, 3 days for retention shifting sounds realistic to me in case of contamination. Do you use column guards?

For my feeling your buffer is not optimal. I would suggest to prepare new buffer and check the retention time.

[smkh]

zokitano,HW Mueller
Actually the PH of m.ph is:3.00 not 3.50 which is typed by error
usually it is adjusted after mixing it with ACN and then filtered , is this agood practice?
my samples are plasma ppt with perchloric acid and 100 microliter injected to HPLC

danko,
I have C18 column from Agilent and I will test it, is the PH of 3.00 is good or I need to go lower?!

grzesiek
[- so?]
I want utilize from all expert to tell me if I do a bad practice , is it a rquirement to do daily column washing (did you do it daily?) or it is needed for a special cases and what is the best washing solvent in this case?!

Fiz
I use gaurd columns from phenomenex, what did you mean by new buffer
new prepared one?! or change its type.

Thanks alot.[/quote]

[Uwe Neue]

It is always better to measure the pH before you mix with the organic solvent (reason: the pH scale changes when you add the organic solvent).

I do not subscribe to any column washing unless there is a need (need: late eluting contaminants, increasing backpressure, ...)

To get the best performance AND the best protection of the column, you should use guard columns made from the same material as in your analytical column (same brand, same manufacturer).

[danko]

Hi Smkh,

Since you adjust your buffer after addition of acetonitrile, the pH of the buffer (if it was adjusted prior to ACN addition) would probably have been ca. 2.9, so you don’t currently need to think about the buffer.
Yep… go for the test of the Agilent C18 column that you have. Make sure the dimensions and the particle size are the same – for the sake of comparison.
Btw, the fact that you're using a guard column indicates that the problem isn’t due to a contamination.
And I would advise you (taking the nature of your samples into account) to keep the guard column installed under the test of the Agilent column. If you achieve the required chromatography (separation etc.) with the Agilent column and choose to continue using it, then it’s advisable (kind of necessary) to acquire a matching guard column (i.e. the same brand, stationary phase etc. as the analytical column) as Uwe points out.

Good luck

[HW Mueller]

Your buffer is of low concentration and thus still not very strong. Are you injecting the supernatant directly with all that perchloric? How many times did you do this anyway (how many times did you see the shift in rt)?