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It appears that all the chromatographic conditions had a great effect on both the resolution and peak shape whereas the elution order was not affected. The baseline separation with symmetrical peaks of the seven tetracyclines can be obtained with a mobile phase containing either 5 mM phosphate buffer pH 2.5\/ACN (84\u00a0:\u00a016 v\/v) or 5 mM perchlorate buffer pH 2.5\/ACN (75\u00a0:\u00a025 v\/v) at a temperature not exceeding 20\u00b0C. 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