An external pH gradient delivered using conventional gradient systems, in which bottled buffers with adjusted pH are mixed using a gradient pump, was replaced by a chromatography system equipped with an isocratic pump and an electrolytic eluent generator (EG). The EG is capable of generating high purity base or acid required for online titration of the buffer at the point of use utilising only deionised water as feed. In one approach, an equimolar (5 mM) solution of CAPS (N-cyclohexyl-3-aminopropanesulfonic acid) and TAPS (N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid) was used as the buffer, which was delivered isocratically into a T-piece mixer, where the buffer solution was titrated to the required pH with the hydroxide ions generated in the EG. Alternatively, an equimolar (10 mM) of diethanolamine (DEA) and triethanolamine (TEA) was used as the buffer and titrated with the acid generated through a methanesulfonic acid (MSA) EG. The required pH gradient is therefore conveniently generated in the T-piece by applying a gradient of electrical current to the EG. Given the pH is adjusted at the point of use, this approach also offers gain in throughput in terms of eluent preparation time and labour, with more reproducibility of the pH profile. The performance of the system was demonstrated by running pH gradients over a polymer monolith cation-exchange column for high throughput profiling of the charge heterogeneity of some therapeutic monoclonal antibodies. High resolution of closely related antibody species was achieved for the studied antibodies. By offering a high degree of flexibility in controlling the pH gradient slope through fine tuning of the operating pH range, this system was demonstrated to be widely applicable, enabling implementation of very shallow pH profiles over a specified pH ranges for each individual antibody for further resolving of heterogeneous species.