Instrument contributions to resolution and sensitivity in ultra high performance liquid chromatography using small...

UHPLC with DAD-UV detection or in combination with mass spectrometry (MS) has proven to be a robust and widely applicable platform for high sensitivity analyses of many types of chemical compounds. The majority of users employ narrow bore columns with 2.1mm internal diameter (ID) typically exhibiting very high efficiencies (>200,000 plates/m). This ultimately sets stringent demands upon the chromatographic system as the separation efficiency can be compromised by external contributions to dispersion caused by connection capillaries, auto-sampler and/or the detection device. Sample limited applications often use reduced column diameters down to capillary- or even nano-column format. Capillary (ID≤0.5mm) or small-bore columns (ID≤1mm) can be a good compromise between system robustness and enhanced sensitivity. Yet in this case, extra-column dispersion gains additional importance due to reduced peak volumes. To design an optimized system configuration for specific column dimensions and applications it is crucial to understand the dispersion contributions of individual extra-column components. This was subject to many studies done within our group and by others. Here, we employed a fully optimized UHPLC/UV system to investigate the contribution to peak dispersion obtained from columns ranging from capillary to narrow bore (0.3, 0.5, 1, 2.1mm) using a set of small molecules that were analyzed in gradient mode. Further UV detection was replaced by a triple quadrupole (QQQ) MS in order to evaluate its contribution to band broadening. In this context the impact of column-ID upon MS sensitivity when interfaced with an Agilent Jet Stream source was investigated. Data obtained from our test suite of compounds shows mostly mass-sensitive behavior of this advanced electrospray technology.