The previous column was C8 column. Now I am using Chiral AMY1 column. The same HPLC device. The same lamp and wavelength.
I read before that changing flow rate affects peak area. Are there any other factors that affect the area significantly?
Ofcourse Yes.
Column chemistry plays major role in the analysis of samples.
The area of standard peak might be different, or may vary that majorly depend upon the type and quality of Stationary Phase used in column packing, also Brand to brand or Manufacturer as well.
Changing flow rate also results in variation in the standard peak response. Too low flow rate may broadening the peak which results peak shape problems directly affect area.
While changing column, you must know about the working limit pH range of particular column.
This is Normal, for different chromatography condition you may get different peak area.
Chromatography conditions should be same to compare area of standards and samples.
Even different days area comparison for same chromatography conditions is not acceptable. You have to inject standard and sample same day without break in system operation.
Tabrez Shaikh Thank you very much for your answer.
Yes, I understand this but is the column type from the factors that affect the area significantly if I keep the other conditions constant? or it might be a mistake made. I did not find a literature about this. Do you have any information about this?
I really appreciate your help.
Yes, Haya Aljazmaty you changed the type of HPLC column support used (a completely different one!) so recording very different area amounts would be 100% normal. This is a basic fundamental of HPLC (found in most books on the topic). You could see no sample peak at all or maybe even 100x as much. No one would ever expect similar results using a completely different HPLC column such as a Chiral amylose (from a conventional RP C8). Sample methods must be optimized for the mobile phase and column. The column support is one of the key variables in HPLC method development. You changed the support so the injected sample diffused onto a different material. *Please have someone experienced in HPLC assist you with this project. Professional training is required to use these complex analytical tools.
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