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Mobile Phase Additives

The addition of TFA into the mobile phase provides a few vitally important benefits. At a simple level it takes the pH sufficiently low enough to ensure that the ionizable amino acids are in a single ionic form.

Essentially, it also lowers the pH sufficiently below the pKa of all residual surface silanol moieties in modern LC columns, this inhibits the unwanted secondary interactions between the protonated basic moiety of the N-terminus or residual basic moieties and the deprotonated silica surface, that can lead to peak tailing. Additionally, TFA as a mobile phase additive also has the significant benefit of aiding retention by way of its ability to ‘ion pair’. There are two commonly accepted routes by which ion pair reagents work:

  1. By complexing with the charged analyte and forming a neutral, or less charged, species in solution. This reduction in overall charge should increase hydrophobicity and therefore analyte retention

  2. The alternative theory, and more widely accepted, is that the hydrophobic portion of the ion pairing reagent, present in vast excess, binds onto the alkyl stationary phase ligand and permits enhanced retention by way of an ion exchange interaction via the charged ‘head’ of the ion pair reagent and the charged sites on the solute
For more information please refer to the Ion Pair Module of the Theory of HPLC Channel

The fact that TFA is a stronger acid, compared to formic acid (FA), but primarily due to the enhanced ion pairing properties afforded to TFA, means the resulting chromatography is superior in terms of chromatographic efficiency and resolution (Figure 1).

Trastuzumab peptide maps

Figure 1: Trastuzumab peptide maps using 0.05% TFA, 0.1% TFA and 0.1% FA as the mobile phase additives.


When different concentrations of TFA are added to the mobile phase, chromatographic efficiency is affected but selectivity is maintained (Figure 1 and Table 1). It is unclear whether the reduction in TFA strength has an overall negative or positive affect on chromatographic selectivity, as some critical peak pair separations are improved whilst others deteriorate.

However, as we shall discover in greater detail shortly, removing TFA, or at least reducing the concentration levels of TFA, may well be preferential. What is evident from the chromatograms is that using formic acid as the mobile phase additive severely hampers chromatographic efficiency.

Peptides 0.05% TFA 0.1% TFA
T5 and T7 unresolved resolved
T23 and T34 unresolved resolved
T1 and T22 resolved unresolved
T18 and T41 unresolved unresolved
T50 and T62 resolved resolved
T41,T18 and T62 resolved unresolved
T31 and T26 unresolved partially resolved
T8 and T59 unresolved resolved
T45 G2F and T27 resolved unresolved
T3 and T3’ flip

Table 1: Trastuzumab peptide critical peak pairs at differing TFA strengths.

TFA is the mobile phase additive of choice in routine environments where UV detection is the norm, however the benefit is not as great during protein characterization, or when MS is the detector of choice. This is because TFA causes the phenomenon of ion suppression in electrospray ionization. This occurs when TFA remains associated with solute ions after they transition from the liquid into the gaseous phase in the ion source, leading to a marked reduction in MS response compared to FA – see MS sensitivity values in Table 2 (FA responses are, on average, an order of magnitude greater than those for TFA). In contrast, FA dissociates from the solute ion as it crosses the phase boundary from liquid to gas.

It should be noted that although the MS sensitivity is far superior with FA, chromatographic peak widths are substantially larger.  Also, the sensitivities achieved in MS with TFA in the mobile phase are generally more than acceptable for the necessary levels of modified peptide required to be observed, so TFA is still often the modifier of choice. LC-MS instruments may become dedicated to using TFA however, as it can difficult to remove all traces of TFA from the source so will continue to suppress ionization even after removal from the mobile phase.

Peptide 0.05% TFA 0.1% TFA 0.1% FA
Peak width
(half height) *
MS Intensity ** Peak width
(half height) *
MS Intensity ** Peak width
(half height) *
MS Intensity **
T16 0.208 1.5x105 0.224 2.40x105 0.283 1.46x106
T14 0.258 3.82x105 0.233 4.11x105 0.349 3.08x106
T22 0.249 4.61x105 0.241 5.55x105 0.773 5.05x106
T58 0.233 2.04x105 0.208 2.34x105 0.416 2.54x106
T46 0.258 1.14x105 0.258 1.24x106 0.457 1.18x107
T41 0.308 5.15x105 0.224 6.06x105 0.815 5.70x106
T26 0.291 2.45x105 0.283 3.21x105 0.707 2.29x106

Table 2: Trastuzumab peptide MS responses and peak widths with TFA and FA as the mobile phase additive.


Additional examples of trastuzumab co- and post-translational chromatograms using 0.05% TFA, 0.1% TFA and 0.1% FA are shown in Figures 2, 3 and 4.

Trastuzumab peptide T62

Figure 2: Trastuzumab peptide T62 with lysine truncation PTM (+K) using 0.05% TFA, 0.1% TFA and 0.1% FA as the mobile phase additives.

 
Trastuzumab peptide T41

Figure 3: Trastuzumab peptide T41 with methionine oxidation PTM (ox) using 0.05% TFA, 0.1% TFA and 0.1% FA as the mobile phase additives.

 
Trastuzumab peptide T45

Figure 4: Trastuzumab peptide T45 with glycosylation using 0.05% TFA, 0.1% TFA and 0.1% FA as the mobile phase additives.


To prevent the commonly encountered increasing baseline observed when running a gradient with TFA in the mobile phase
(Figure 5), the amount of TFA in the organic solvent (MeCN) portion should be around 20% lower than in the aqueous portion. This is because TFA in MeCN increases the UV cut-off and interferes with the low wavelength employed (214 nm) when detecting the peptide bond.

Baseline effects when using equimolar amounts of TFA

Figure 5: Baseline affects when using equimolar amounts of TFA in aqueous and organic mobile phase portions.

 
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