The comparison of nanospray and microspray ionizations for detecting mixtures of compounds by ion mobility spectrometry has been investigated for sensitivity, ion transmission through a drift tube, and ion suppression effects when used as an ionization source for ambient pressure ion mobility spectrometry (IMS). Several articles have demonstrated that nano-electrospray ionization mass spectrometry (n-ESI-MS) has improved sensitivity, provides less background noise, and lower limits of detection than micro-electrospray ionization (μ-ESI) for IMS. Most importantly, data from n-ESI-MS is concentration-sensitive. Our laboratory previously published an article that observed a striking result when μ-ESI-IMS was investigated for a single compound in the positive ion mode. The data reported was mass-sensitive. In this new investigation, we have investigated mixtures, and experiments were designed to evaluate the effect of sensitivity, ion transmission and ion suppressions in μ-ESI-IMS and n-ESI-IMS. At an electrospray flow rate in the μL min−1 range, compounds with higher proton affinities responded best while at the nanospray flow rates of nL min−1, relative responses were more equal. This study observed that a decreased ESI flow rate resulted in a decreased ion signal. These trends demonstrated less sensitivity for ESI-IMS at reduced flow rates but suggest better quantification. At higher flow rates, relative ionization efficiencies were still uniform for all the components studied individually and in mixtures and sensitivity improved by about 78%. Concentration studies showed that at high concentrations, ion detection efficiencies were uniform at about 33% for all compounds studied individually and in mixtures. At low concentrations, the detection efficiency varied from 31% to 86%, depending on the proton affinity of the component in the mixture. Ion transmission through the IMS tube measured with a segmented Faraday detector that was incorporated into the IMS design indicated that most of the ion current for mixtures was transported through the IMS tube with a radius of less than 18 mm for both positive and negative ion modes.