On-line on-chip post-column derivatisation reactions for pre-ionisation of analytes and cluster analysis in gradient μ-liquid chromatography/electrospray mass spectrometry

A system is presented that demonstrates the principle of on-line and on-chip post-column derivatisation reactions in micro-high-performance liquid chromatography (μ-HPLC) hyphenated to electrospray time-of-flight mass spectrometry (ESI-TOFMS). In this μ-HPLC-chip-MS set-up, the analytes are separated using gradient μ-HPLC and subsequently derivatised on-chip and detected. One of the major limitations of MS detection is its dependency on the degree of ionisation, which is widely variable and compound-specific. Optimising and controlling the degree of ionisation in a simple manner would allow MS detection to be truly generic. One way of achieving this is by preionisation of analytes using simple derivatisation procedures that are both rapid and quantitative. Performing this in situ on the system described here overcomes issues of sample handling and efficiency losses when time-consuming "bench chemistry" is necessary prior to analysis. The power of the system is demonstrated by the separation of primary and secondary amines, which are subsequently derivatised with a positively charged phosphonium complex and detected in an enhanced manner. Typically, molecular cations (M⁺) are detected showing that the ionisation process is dominated by the phosphonium species, leading to more constant ionisation for a variety of compounds. In addition, stable isotopically labelled (¹²C/¹³C)-phosphonium reagent is used for the reactions, allowing for inherent signal/noise (S/N) improvement and automated data processing using cluster analysis. A similar reaction scheme is used for the derivatisation of ketones and aldehydes, also demonstrating dramatic increases in sensitivity, especially with increasing temperature. Minimal loss in chromatographic fidelity in terms of retention times is observed by the introduction of the micromixer chip into the system. Optimal flow rates in μ-HPLC and ESI-MS are compatible with flow rates for the chip as well as a multitude of in-line optical detectors including UV and fluorescence. In addition, the micromixer chip can be positioned pre-column if preferred. The system is robust, easily fully automated and applicable to a wide variety of reactions. The system has a major advantage in its simple robust connection to the "normal scale" outside world.