Mobile phase containing acid

[amaryl]

hey all

In some papers the mobile phase has no buffer (water : MeOH ) but they contain certain percentage of acid like acetic acid.

how does this acid provide a good separation if the analyte is ionisable... a weak acid.

Doesn't it cause peak broadening?

Looking forward to complete explanation and if possible some good article.

Thanks in advance.

Regards,

Amaryl.

[amaryl]

http://www.ncbi.nlm.nih.gov/entrez/quer ... med_DocSum

This is the link to the abstract.

[tom jupille]

do an "any keywords" search here on the forum using the keywords "formic" and "acetic". All will be revealed.

[amaryl]

do an "any keywords" search here on the forum using the keywords "formic" and "acetic". All will be revealed.

Sir, so many posts...that the end result is confusion nothing else. I loose the track of my question.

Is the answer to long to type. Please guide me.

Thanking you,

Regards,

Amaryl.

[Uwe Neue]

in the paper that you are referring to, the analyte is neutral. The matrix is biological. Therefore the acid in the mobile phase is used to control the retention of the residual stuff in the matrix, not the analyte.

[amaryl]

in the paper that you are referring to, the analyte is neutral. The matrix is biological. .

Sir, how did you state off its neutral molecule?

Other papers have used buffers for the same analyte and even used the salt form of the analyte with sodiumphosphate.

check this

http://www.ncbi.nlm.nih.gov/entrez/quer ... med_docsum

Therefore the acid in the mobile phase is used to control the retention of the residual stuff in the matrix, not the analyte.

We use buffer to provide a constant pH and to prevent peak broadening (rugged and robust method).

how does this acid controls retention for the matrix residual peak when the pH can't be stable.

I may be wrong somewhere. I have not worked with biological samples.

Thanking you.

Regards,

Amaryl.

[Uwe Neue]

You gave a reference to dexamethasone. I assume that this is your analyte...
It is a steroid, structure on Wikipedia. Unless the structure is wrong, I can't see how this could be an ionizable analyte under normal circumstances...

The pH of an acid is not as stable as the pH of a buffer at equal concentration, but it is still better than doing nothing, right?

[Uwe Neue]

The second reference that you gave is to the phosphate ester of dexamethasone. So what is your analyte? And where are you trying to analyze it? In a biological matrix, or in a formulation?

[amaryl]

The second reference that you gave is to the phosphate ester of dexamethasone. So what is your analyte? And where are you trying to analyze it? In a biological matrix, or in a formulation?

Sir, analyte is dexamethasone. The matrix contains phospholipids. So what would be better choice if the analyte is neutral ( as stated by you )...mobile phase containing certain percentage of acetic acid or using a acidic buffer for lipids in matrix.

How does one work out to resolve the matrix peaks.

Regards,

Amaryl.

[Uwe Neue]

I still do not yet know what your matrix is, and what you do to clean up the matrix (if for example it is plasma).

You need some pH control in your monile phase to control the retention of ionizable or ionic interferences such as phospholipids.

The best way to deal with phospholipids though is to try to work with a suitable sample preparation technique to remove them. Mixed-mode techniques such as an Oasis mixed-mode cation exchanger might do best.

For a decision on the best approach, I would need to know more about your analytical tools. Are you using LC/MS or LC/UV?

[HW Mueller]

Acids at a pH of 2 and especially below are good buffers, actually very good buffers toward more acidity.

[amaryl]

I still do not yet know what your matrix is, and what you do to clean up the matrix (if for example it is plasma).

You need some pH control in your monile phase to control the retention of ionizable or ionic interferences such as phospholipids.

The best way to deal with phospholipids though is to try to work with a suitable sample preparation technique to remove them. Mixed-mode techniques such as an Oasis mixed-mode cation exchanger might do best.

For a decision on the best approach, I would need to know more about your analytical tools. Are you using LC/MS or LC/UV?

Thanks for the suggestions.

I was just trying to help out someone. Later i needed to clarify some points.

Its liposome of analyte. Lipids removal is worked out. Its HPLC-UV.


Is ethanol a good diluent for HPLC if mobile phase contains ACN ...both of same solvent strength.

what extractive methods are used in bioanalysis? Some good link to this.

Regards,

Amaryl.