Thursday, March 30, 2017

HPLC related Interview Question

1. How the pH of mobile phase changes?

The pH of a mobile phase pH can change if u keep it for long hours due to the effect of CO2 from the atmosphere to affect pH.

Similarly, volatile reagents such as TFA may also selectively evaporate, thus changing the eluent pH.
A Buffer can help to reduce this effect to a certain extent.

2. Reagents used for modifying pH:

Mobile phase modifiers such as trifluoroacetic acid (TFA) or triethylamine (TEA) or Acetic Acid or Formic Acid or Ammonia or Triaryl methane can b used for this purpose.But the selection should help maintain pH  properly.
Therefore, we need buffers.

3. Effect of pH in HPLC:

The pH of your mobile phase can affect the
 Retention time and
Chromatographic peak shape issues like peak Tailing, Fronting, Shoulder etc.

4. Use of Buffers in HPLC:

A buffer is a solution that will resist a change in pH when small volumes of acid or alkali are added to it or when it is diluted with water.

Buffers are used in mobile phases to keep n maintain the pH constant after adjustment to the desired pH.

5. How to select a buffer:

For best results, use a buffer that has a pH at least 2 units away from the pKa for the analyte or eluant of interest in ur analysis.

6. Why is  Phosphoric Acid selected for pH adjustments of mobile phases:

Phosphoric acid is a weak acid compared to HCl, sulphuric acid etc .

But, the pka value for phosphoric acid is 2.12, 7.2 and 12.3. We can use for maximum  pH adjustments.Similarly, it has got a suitable UV cut-off value/Transparency.

Therefore, Phosphoric acid n its salts r used for pH adjustments or for the preparation of Buffers.

7. The effect of pH on stationary phase n chromatograms:

The column stationary phase is also affected by pH. At very low pH (<2) the bonded stationary phase will be stripped from the silica support. At high pH (>8) the silica itself will be damaged by dissolution.

If the pH of the mobile phase is too close to the analyte’s pKa, you might observe split peaks or shoulders.