Questions on protein analysis

[alicee!]

Note: these were originally 8 separate posts combined by the Admin.

a mobile phase containing salt/high salt.. how does it work in SEC?

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hi all..
i have protein recovery problems in analytical size exclusion chromatography? what are the chanegs i can make to the existing method/ mobile phase? its an ammonium bicarbonate buffer, pH 7. and why? thanks.

or how do i have a more sensitive method in such a case?

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hii.. can some1 explain to me the differences and reasons for use of the following in Size exclusion chromatogarphy?

Mobile phase:
1. Ammonium bicarbonate buffer, pH 7 at a flow rate of 0.5 ml/min, detection at 215 nm.
2. Phosphate buffer,Ph 7
3. Phosphate buffer with 110 mM sodium chloride, Ph 7
4. Phosphate buffer with 110 mM sodium chloride, Ph 7 with 10% ethanol

all 4 with Same column, at a flow rate of 0.5 ml/min, detection at 215 nm. for all.

and what is the best prefered diluent in all these cases?
please let me know why for all!
thanks!

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hii..
how to increase recovery of low amount of protein impurities in reverse phase hplc methods?

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hiya!

what is the kinda system suitabiliyt you guys have for quantitation methods using liquid chrom.. ?

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hi all..
pls help me with this..
how do u determine accuracy for a test for impurity quantitation by Liquid Chrom.? give me an idea of the experimental design and data interpretation pls.. confusions!

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hi all..
for a purity test using say, rp-hplc or sec.. generally LC.. is it recommended or needed to demonstrate linearity across the range?

i mean.. if my sample injection amount is 30 micrograms, call it 100%, do i need to demonstrate linearity for 120% amount to 2% or less.. i m claiming impurity detection up to 2%? if yes why?

secondly, if there is no linearity in the response what are the effects?

third, for eg..
if i get an underestimation / overestimation at the level of 5-1% impurity amounts.. and they fall in a linear range, while the 80-120% amounts fall in another linear range but all together do not.. does it affect anything?

in such a case how do you calculate accuracy for impurity detection?

pls let me know.. hyper-confusion and difference in opinion with others!
thanks!

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hii all..
what do u guys do to define yr method varation?

say i have a purity test, where i quantitate my impurities.. by LC.
i get an accuracy of 70% ie. compared to my 100% standard injection, my impurities which is say 5% (ie a low amount) is detected with 30% inaccuracy.. of course there is a variation above this. when i reanalyse the sample.. my precision varies to detecting 20% variation ie. the 5% is estimated between 20%RSD of the 5% imppurity (4.something to 5.something types). what is my method variation here?
would it be 20%, 30% or 50%? and WHY?
suppose i have a low accuacry of say 50% at my 2% impurity levels, which has a variation - imprecision of 10% how do i do it there? what are the kind of limits you guys set?

suppose i need to define the signal and noise for DOE experiments for process characterization how do i do it?

thanks!

[tom jupille]

Alicee!, you had about 8 questions posted in sequence, each of which requires an involved explanation. I exercised "adminstrator's privilege" and combined them into a single post.

If anyone wants to address one or more of the questions posed by Alicee!, please start a new thread on that topic.

I would suggest you look in the following places for information:

For information about protein SEC, the book Basic HPLC and CE of Biomolecules by Cunico, Gooding, and Wehr has answers to all your questions. Here is a link to it on Amazon:
http://tinyurl.com/6xd3eo

For guidelines on accuracy, precision, etc., you need to start with ICH Q2A: http://tinyurl.com/5snz6c
A complete listing of the ICH documentation is here:
http://www.ich.org/cache/compo/363-272-1.html

[Uwe Neue]

Re: your problem(s)

what is your SEC column?
what is your mobile phase?
Why do you think you have recovery problems?

Let us start with this...