UPLC peak separation

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Marga Pérez
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UPLC peak separation

Hi!We are trying to set a protocol for betaine. We are using a Waters UPLC with a Acquity UPLC BEH C18 1.7 micrometers column. TUV detector at 190nm. The comumn is a 2.1 x 100 mm column and we are using Potassium dihydrogen phosphate 0.05 M 95/5 water, as mobile phase. The volumen of injection is 35 microliters, 30* of column temperature and 0.08 ml of flow rate. The problem is that we have two peaks together at the same time (3'), betaine and proline. We have tried gradients, different temperatures, different phases, different flow rates... But we cannot separate both peaks. How could we separate them?please, kindly give us your suggestions,many thanks