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References
1. 2. 3. 4. 5. McMaster, M.C. HPLC A Practical Users Guide, VCH Publishers, Inc.: New York, NY, 1994; 85. Poole, C.F. and Poole, S.K. Chromatography Today, Elsevier Science: Amsterdam, The Netherlands, 1991; 431. Analytix, Five-part series on Mobile Phase Additives for LC-MS, Issue 3, 2006 (www.sigma-aldrich.com/analytix). Temesi, D., Law, B., 1999, The E ect of LC Eluent Composition on MS Response Using Electrospray Ionization, LC-GC, 17:626. Ap el, A. et. al. 1995. Enhanced Sensitivity for Peptide Mapping with Electrospray Liquid Chromatography-Mass Spectrometry in the Presence of Signal Suppression Due to Tri uoroacetic Acid-Containing Mobile Phases, J. Chrom. A. 712:177.
Bu er Selection
The typical pH range for reversed-phase on silica-based packing is pH 2 to 8. Choice of bu er is typically governed by the desired pH. It is important that the bu er has a pKa close to the desired pH since bu ers control pH best at their pKa. A rule of thumb is to choose a bu er with a pKa value <2 units of the desired mobile phase pH (see Table 1). Table 1. HPLC Bu ers, pKa Values and Useful pH Range
Bu er TFA Sulfonate Phosphate Chloroacetate Formate Acetate Sulfonate Phosphate Ammonia Phosphate pKa (25C) 0.5 1.8 2.1 2.9 3.8 4.8 6.9 7.2 9.2 12.3 Useful pH Range <1.5 <1-2.8 1.1-3.1 1.9-3.9 2.8-4.8 3.8-5.8 5.9-7.9 6.2-8.2 8.2-10.2 11.3-13.3
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Description
Bu er Concentration: Generally, a bu er concentration of 10-50 mM is adequate for small molecules. Bu er Solubility: A general rule is no more than 50% organic should be used with a bu er. This will depend on the speci c bu er as well as its concentration.
HPLC-grade Bu ers and Additives from Sigma-Aldrich/Fluka Ammonium acetate 50 g, 250 g Ammonium formate 50 g, 250 g Ammonium hydroxide solution in water 100 mL, 1 L Ammonium phosphate monobasic 250 g Ammonium tri uoroacetate 10 g, 50 g Potassium phosphate dibasic anhydrous 250 g Sodium formate 50 g, 250 g Sodium phosphate dibasic dehydrate 250 g Sodium phosphate monobasic anhydrous 50 g, 250 g Sodium tri uoroacetate 10 g Tri uoroacetic acid:Triethylamine 2M:1M 500 mL Tri uoroacetic acid:Triethylamine 2M:2M 100 mL
For a complete list of HPLC bu ers and additives, please refer to our online product catalog: sigma-aldrich.com
Reporter 28.5
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