Question

How could solubility of an analyte impact its results in HPLC? How would the peak differ, for example? How would a more soluble molecule's peak differ from a less soluble one?

Also, what are the peak areas based on in HPLC? Why is one peak sharper and higher than another, for example? Does that relate to how well a molecule is retained by the stationary phase in the chromotography?

If you change the detection wavelength, how would the peak area of the same molecule change?

What does having a larger response factor mean in the internal standard method in HPLC? does it just relate just to the ratio of the two molecules?

Answer 1

How could solubility of an analyte impact its results in HPLC? How would the peak differ, for example?

Ans: If the analyte is less soluble, it will not be eluted completely from the column and will give less peak area (% content if quantified), and will give the wrong results.

How would a more soluble molecule's peak differ from a less soluble one?

Ans: The more soluble molecule’s peak will show higher peak area compared to the less soluble molecule.

What are the peak areas based on in HPLC?

Ans: The peak areas are related to the quantity of the particular molecules which are eluted and detected in the HPLC as an absorption output by UV/Vis detection or other any other suitable detector like RI, FLD, MS etc.

Why is one peak sharper and higher than another, for example? Does that relate to how well a molecule is retained by the stationary phase in the chromotography?

Ans: Yes, the sharper peak relates to the better adsorption/retention and elution as well by the suitable mobile phase. The poorly retained molecules give broad peaks.

If you change the detection wavelength, how would the peak area of the same molecule change?

Ans: Every UV/Vis light absorbing molecule has different absorption spectra and its maximum absorption wavelength. At particular wavelength some molecule may have higher absorption and the other may have lower absorption. This is directly related to the output peak areas and peak heights. Hence, changing the detection wavelength the absorption of each eluted molecule will change, and consequently the peak areas.

What does having a larger response factor mean in the internal standard method in HPLC? does it just relate just to the ratio of the two molecules?

Ans: The larger response factor mean the larger area of the particular peak. In internal standard method it is the area ratio of the analyte peak divided by internal standard peak. Hence, the larger response factor in internal standard means lower peak area of internal standard compared to the analyte peak. But this ratio is very useful for quantification as it is not much affected by the external factors like variation in injection volume, temperature ect., as the ratio always remains constant.