If you are using a method with a buffer such as 16 mM ammonium acetate in the mobile phase and then stored the column in pure Acetonitrile immediately afterwards. You may observe peak splitting when using your Ammonium Acetate Method.

It is possible that Ammonium Acetate has precipitated in the Column since it is insoluble in pure Acetonitrile, so it is always advisable to first wash the Ammonium Acetate out of the Column before introducing it to a pure Organic Solvent to prevent this.

If it has already occurred, try washing the Column for 2 hours with 70:30 Water/Acetonitrile and retest the Column for peak shape.

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