21


phosphate buffer was nearly always better than the ammonium acetate (up to a 20% decrease in peak tailing was typically observed). As reported in Table 3, the analyte retention correlation at this pH is very good and indicates a smaller phosphate ion-pairing effect than at lower pH. The peak shape in this case can be rationalised through the significantly higher ionic strength of the mobile phase compared to the ammonium acetate mobile phase. In this instance it is believed that residual analyte-silanol interactions are decreased by mobile phase cation (K+


) competition for these sites. Baseline noise & artefacts


In this investigation, it was found that shifting to phosphate buffers not only increased analyte signal-to-noise, but also led to a decrease in baseline artefacts when comparing organic and phosphate buffer mobile phases at both low and mid-pH. An example of the improved baseline with phosphate compared to ammonium acetate at 210 nm is shown in Figure 6. It should be reiterated that phosphate buffer solubility is significantly lower in organic solvents than alternative organic additives. If the maximum organic solvent levels in the gradient are breached, this can lead to the phosphate salt ‘crashing- out’ in the instrument or column which in turn may lead to increased baseline noise and instrument back-pressure issues.


Additives at high pH The use of phosphate buffers at high pH is deleterious to silica based column stability which at high buffering pH’s (>11.3) leads to rapid silica dissolution and collapse of the phase [14,19]


. For this reason, the majority of mobile phases used in high pH Mobile phase


0.1% HCOOH (unadjusted pH 2.6) 10 mM NH4


OOCCH3


10 mM KH2PO4 with H3


PO4 PO4 )


10 mM K2HPO4 with H3


) Table 4. Ionic strengths of aqueous portion of low and mid-pH mobile phases used in this study. High pH additive


Ammonium hydroxide Triethylamine acetate Pyrrolidine


1-Methyl piperidine Ammonium acetate


Ammonium bicarbonate Ammonium formate CAPS


CAPSO Glycine


Concentration 0.1% (v/v)


0.1% (v/v) 0.1% (v/v) 0.1% (v/v) 10 mM 10 mM 10 mM 10 mM 10 mM 10 mM


Correlation coefficient (r2 N/A


0.8290 (0.9837) 0.5639 0.7786 0.6272 0.6860 0.6685 0.8992 0.8541 0.7466


Table 5. Correlation coefficients for test analytes comparing retention in the specified mobile phase with the ammonium hydroxide mobile phase. All solutions were titrated to pH 10.6 with ammonium hydroxide or acetic acid as required except ammonium hydroxide which was naturally pH 10.6 in aqueous solution at this concentration.


chromatography utilise organic based buffers which are less aggressive on the column. A ‘buffer’ commonly used for high pH work is ammonium hydroxide as it is both easy to prepare and MS compatible. However, in our experience ammonium hydroxide tends to degrade in aqueous solution quickly and this can be observed as baseline artefacts in the chromatogram. Additionally, at 0.1% (v/v) concentration, ammonium hydroxide offers


very limited buffering capacity which can lead to separation reproducibility issues (the pH of 0.1% ammonium hydroxide is around 10.6. The pKa


of the ammonium ion is 9.2).


One other point of note is that at high pH, on- column degradation of analyte molecules may be observed. This is usually confirmed by comparing a high pH separation with analysis of the analyte at low pH. In the higher pH separation, additional peaks may be observed which are not present in the low pH separation (e.g. identified by mass tracking using MS detection) and care must be taken not to misidentify these high pH impurities as originating from the sample under analysis.


A number of high pH buffers were therefore investigated as alternatives to ammonium hydroxide (see Experimental section for full details). The ammonium hydroxide mobile phase was chosen as a ‘baseline’ generic method having been previously found to provide a high degree of orthogonality [20]


) (adjusted to pH 6.8 0.026 (unadjusted pH 6.8) (adjusted to pH 2.6


Ionic Strength / mol dm-3 0.025


0.010 0.013


to


Figure 6. UV baselines of blank injections with the dipotassium phosphate buffer and ammonium acetate mobile phases for method 2. Clearly the ammonium acetate baseline (black trace) exhibits a greater number of baseline artefacts than the phosphate buffer (blue trace) which can lead to peak quantitation issues when low level analyte related impurities are present.


other generic methods employed in our department. Note that not all of the buffers investigated are MS compatible, but were chosen as potential alternative mobile phase additives that might exhibit improved sensitivity characteristics. The main criteria assessed was the ability to provide similar analyte retention to ammonium hydroxide


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