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-Differences in Preparation Method May Result in Different Chromatograms A water-based solvent, organic solvent, or a mixture of the two is mainly used as the mobile phase for HPLC. A buffer solution is often used as the aqueous solvent. LCtalk38 LAB Preparing Buffer Solutions describes the actual preparation methods for typical buffer solutions used with HPLC. However, it seems that many people have a vague understanding of buffer solutions. If the preparation method is not the same as the method described in the material for the analysis method, differences can occur in the mobile phase that affect the chromatograms and analysis results. Apart from the buffer solution, many other unforeseen factors affect the mobile phase preparation, such as the method of mixing the solvent. Here, we use actual examples to consider the effects of the mobile phase preparation method on the analysis results.
Shim-pack VP-ODS (150 4.6 mm I.D.) Buffer solution (pH 2.5) / acetonitrile = 9 / 1 Top row A 20 mM phosphoric acid (sodium) buffer solution (pH 2.5)
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Middle row B Lower row C Flowrate Temperature Detection 1.0 mL/minute 40 C 210 nm
20 mM sodium dihydrogen phosphate buffer solution (pH2.5); phosphoric acid added to adjust the pH 20 mM sodium dihydrogen phosphate buffer solution (pH 2.5); perchloric acid added to adjust the pH
Shim-pack VP-ODS (1504.6 mm I.D.) Top row A Lower row B 9:1 (v/v) mixture of buffer solution and acetonitrile Make up to 10 % (v/v) acetonitrile with buffer solution. The buffer solution is 20 mM sodium dihydrogen phosphate adjusted to pH 2.5 by the addition of phosphoric acid.
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