|Abstract Title:||The impact of pump design and setting on trace level detection in HPLC with trifluoroacetic acid modified mobile phases|
|Session Choice:||Fundamentals in Separation Science & Sample Prep|
|Presenter Name:||Dr Mauro De Pra|
|Co-authors:||Dr Christoph Strobl|
Dr Matthias Krajewski
Dr Remco Swart
|Company/Organisation:||Thermo Fisher Scientific|
Abstract Information :
Trifluoroacetic acid (TFA) is the most common ion-pairing agent used in reversed-phase (RP) UHPLC for peptide and protein separations. It lowers the pH and modifies the interaction of the molecules with the stationary phase in order to control selectivity and thus enhance separations. Frequent conditions for peptide and protein separations include linear and shallow, low organic to high organic, LC gradients where the mobile phase is comprised of water and acetonitrile containing approximately 0.1% TFA. Typically, the analytes are detected with a UV detector at 210-220 nm for the peptide bonds, as well as at 280 nm for aromatic amino acid residues.
However, under these analytical LC conditions TFA shows some undesirable effects: TFA strongly absorbs UV light below 250 nm, depending on the water/acetonitrile ratio, resulting in a strong shift in baseline during gradient elution. In addition, TFA is retained on RP columns causing the TFA concentration of the mobile phase within the column to fluctuate with varying organic solvent concentration. In the case of incomplete mixing or fluctuating mobile phase content, the dynamics of TFA equilibrium in the column are disturbed causing a strong amplification of mixing noise. Because TFA absorbs 50-100 times stronger than water or acetonitrile in the UV range, significant baseline ripples are observed.
As a consequence, the TFA associated baseline ripples can significantly increase the limit of detection (LOD) for analytes. Baseline ripples can mask the detection of low concentrated impurities.
A solution to reduce baseline ripples is to use larger mixer volumes which also increase the gradient delay volume (GDV) of a LC system. However, by increasing the mixer volume, the separation is delayed which translates into longer LC run times and therefore limits sample throughput per day. When throughput is a concern, UHPLC systems with small GDVs, and therefore with small mixer configurations, are the preferred option.
When faced with a challenging TFA application that requires high throughput (small mixer volume required) and low LOD (large mixer volume required), a fundamental requirement can be the following: the pump flow must be extremely consistent to avoid fluctuations of TFA concentration.
To improve chromatographic separations for TFA related applications, three specific aspects of the pump contribute to baseline ripples: mixer volume, piston stroke-volume, and flow consistency. In his work, the three factors are evaluated and measures to minimize their effects are discussed.