What is mobile phase in HPLC?

What is mobile phase in HPLC?

High Performance Liquid Chromatography (HPLC) is a form of column chromatography that pumps a sample mixture or analyte in a solvent (known as the mobile phase) at high pressure through a column with chromatographic packing material (stationary phase).

Why we use TFA in HPLC?

Because of its high resolving power, RP-HPLC is the preferred separation mode for peptides. Traditionally, TFA is used in the mobile phases for RP-HPLC peptide separations. First, typical concentrations of TFA (0.1% v/v) have high surface tension and prevent efficient spray formation (nebulization).

Why is pH important in HPLC?

When samples contain ionisable compounds, mobile phase pH can be one of the most important variables in the control of retention in a reversed‑phase HPLC (RP-HPLC) separation. Since most compounds analysed by RP-HPLC contain one or more acidic or basic functional groups, most mobile phases require pH control.

What is the pH of TFA?

Trifluoroacetic acid (TFA) also generates a mobile phase pH of ≈2 at 0.1% v/v (Table 2), and for many years was the additive of choice for LC-MS at low pH. TFA also acts as an ion-pairing reagent, and is widely used for protein and peptide separations.

Is CF3COO an acid or base?

The conjugate base of CF3COOH is CF3COO-, but the conjugate of the other acid is CH3OH (methanol, and NOT an anion).

What is the role of pH in HPLC mobile phase?

In LC separations, the mobile phase pH determines the ionisation state of ionisable analytes. The mobile phase pH can therefore be varied and used as a powerful tool to control analyte retention, peak shape and selectivity.

Why do you add formic acid to mobile phase?

First of all formic acid makes your mobile phase compatibile to MS. It could improve resolution in case of proteins or peptides because it acts as ion pair agent however not very strong. The disadvantage of formic acid comes from its higher UV cut-off comparing to phosphoric acid.