Spring, 1999 excerpt

TFA is often preferred over other ionic modifiers because its volatility permits easy removal from preparative fractions. The UV absorbance spectrum of TFA peaks below 200 nm and thus creates minimal interference with detection of peptides at low wavelengths (Ref. 2).

Altering TFA concentration changes reversed-phase selectivity for peptides in subtle ways (also reported in Ref. 1). These changes can be exploited to optimize separations or increase the information obtained from complex chromatograms, for example, peptide fingerprints.

TFA is most often added to mobile phases at a concentration of 0.1%. This concentration produces good peak shapes with most reversed-phase columns, whereas TFA concentrations much below that level produce noticeable peak broadening and tailing.

LC/MS

New Low-TFA Columns

Figure 1 shows a separation of four peptides on the 218MS54 Low-TFA C18 column. Note that peak shapes and symmetry are maintained over a 10-fold reduction in TFA concentration. Reductions in retention times occur as a result of less activity of TFA in bringing polar groups on sample molecules to the bonded C18 phase at lower TFA concentrations. The effect is more pronounced for angiotensin II by virtue of its positive-charged arginine side chain, which results in different selectivity and, in fact, a reversal of elution order with oxytocin at 0.01% TFA.

Figure 1. Separation of peptides on Low-TFA C18 column with various TFA concentrations. Column: Vydac 218MS54 C18 300Å 5µm 4.6mmID x 250mmL. Detection: UV absorption, 220 nm. Mobile Phase: A = 5% acetonitrile in water with TFA as indicated (v/v). B = 95% acetonitrile in water containing same TFA concentration as in A. Flow: 1.5 mL/min. Gradient: Linear from 0 to 20%B over 20 minutes, then to 100%B in 5 minutes.

Figure 2. Separation of two peptides and two proteins on Low-TFA C4 column with various TFA concentrations. Column: Vydac 214MS54 C4 300Å 5µm 4.6mmID x 250mmL. Detection: UV absorption, 220 nm. Mobile Phase: A = 5% acetonitrile in water with TFA as indicated (v/v). B = 95% acetonitrile in water containing same TFA concentration as in A. Flow: 1.0 mL/min. Gradient: Linear from 0 to 10%B over 15 minutes, then to 25%B in 30 minutes, and finally to 100%B in 5 minutes.

Figure 2 shows a separation of two peptides and two proteins on the 214MS54 Low-TFA C4 column. Peak shapes for the peptides are maintained down to 0.01%TFA for the peptides, and down to 0.02% TFA for bovine and human insulin, with some noticeable broadening at 0.01%. A change in selectivity with reversal of elution order is seen for oxytocin and angiotensin II, similar to that seen on the C18 column. The basis for greater dependency of the protein peaks on TFA is not known, but one could speculate that, in addition to intermolecular effects, TFA may have effects on molecular conformation that make it more difficult for smaller peptide molecules to access multiple sites on the adsorbent surface.

Small-Diameter and Microbore Columns Available

References

1. What is 0.1% TFA? Vydac Advances, Spring 1997, pages 2-3.
2. Vydac Handbook of Analysis and Purification of Peptides and Proteins

by Reversed-Phase HPLC, page 7.
3. A. Apffel, S. Fischer, G. Goldberg, P.C. Goodley, and F.E. Kuhlmann, "Enhanced sensitivity for peptide mapping with electrospray liquid chromatography - mass spectrometry in the presence of signal suppression due to trifluoroacetic acid-containing mobile phases." J. Chrom. A, 712 (1995) 177-190.

LC/MS Column Information:

Other column sizes are available for analytical and preparative applications.