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Temperature gradient capillary electrophoresis was introduced to enhance separation selectivities for branched glycans of biotherapeutic interest. A mixture of afucosylated, fucosylated, and high mannose oligosaccharides was separated in the range of 15 to 45 °C at 5 °C temperature intervals. It was found that within this temperature range, the separation selectivity was carbohydrate structure dependent. The resolution between some glycan structures was greater at elevated temperatures, while others separated better at lower temperatures. More interestingly, the temperature of resolution maximum was different for most structures. On the basis of this observation, a temperature gradient was designed and optimized to fully resolve all the glycans in the mixture. Our results demonstrate how temperature is a critical separation parameter that can be utilized for selectivity manipulation in the analytical glycomics field.

High-Resolution Glycan Analysis by Temperature Gradient Capillary Electrophoresis