A,779(1), 29-71 (1997). Table II lists the common mobile phase additives with their respective pKa and UV cutoffs. Figure 5: A case study illustrating the potential issue of a ghost peak caused by dipping the pH electrode into mobile-phase A during pH adjustment. Column: 150 mm x 3.0 mm, 3.5-m Waters XTerra MS C18; mobile-phase A: 20 mM ammonium bicarbonate, pH 9.1; mobile-phase B: acetonitrile; gradient: 350% B in 40 min, 5080% B in 3 min; flow rate: 1 mL/min; temperature: 50 C. Fischer, G. Goldberg, and P.C. For this mAb, mobile phases using formic acid exhibit poor retention and peak shape, compared to either trifluoroacetic acid or difluoroacetic acid mobile phases. 2, C. Horvth, Ed. The stationary phase provides retention, and, in conjunction with the mobile phase, differential migration of the solutes. Squire and J.M. Adapted with permission from reference 17. An acidic pH of 24 is used for most pharmaceutical applications. It is UV-transparent to 200 nm, but it is not volatile, and thereby not MS-compatible. In this installment, we present a brief overview of mobile phase fundamentals in reversed-phase LC and describe modern trends in their selection, as summarized in Table I. Volatile buffers are labeled with an asterisk are used for LCMS applications. Direct correspondence to: [emailprotected], Chromatography Simplified: Maximizing the Power of PDA Data via Fully Integrated CDS Spectral Processing Tools, Combustion Ion Chromatography: The What, How, Why, & Where, How to Do More with Less: All Biopharmaceutical Batch Processes Can Benefit from Multi-Column Chromatography, Improved Performance of UHPLCMS Hyphenated Systems, Trends and Developments in Liquid Chromatography, http://www.chromacademy.com/essential_guide_webcast/mobile_phase_optimization_strategies_for_reversed_phase_hplc/mobile_phase_optimization_strategies_for_reversed_phase_hplc.pdf. This approach has worked well for acetate and formate buffers. In such cases, periodic checks of the pH and blank injections can verify the integrity and cleanliness of the mobile phase preparations. (6) W.R. Melander and C. Horvth, in HPLC, Advances and Perspectives Vol. Iraneta, and D. Morrison, Waters Corporation, Milford, Massachusetts, 720003720EN, Sep. 2010. Boyes, and R.C. Most analytical methods for purity assessments, quality control, and stability testing of pharmaceuticals employ reversed-phase LC with UV detection because of the excellent precision and reliability of the technique. Pharm. Methanol has substantial UV end-absorbance below 210 nm. Another approach is to use an advanced coating technique to reduce the activities of the residual silanols. Column: 50 mm x 3.0 mm, 1.7-m Waters BEH C18. The example shown is for a mixture of 5 synthetic peptides, resolved in 10 mM of each acid or acid plus salt (in the case of ammonium formate). Many laboratories have eliminated the filtration process with 0.2 or 0.5 m membrane filters by using high-purity reagents (such as 99.995% ammonium formate from Aldrich, Cat# 516961), and HPLC-grade solvents and water (22). The development of hybrid particles and improved bonding chemistries for silica particles has extended the usable pH range of these specific columns to and range of 112 (210 at column temperatures >40 C) (3,15-16). The use of a scavenger column (for example, C18, 33 4.6 mm id, 10 m) (24) can provide excellent protection against mobile-phase-borne particulates and contaminants). (9) M.W. Figure 1: A chromatogram of a cocktail of two water-soluble basic active pharmaceutical ingredients (opioids) and potential impurities and degradation product for a stability-indicating method of a combination drug product. Adapted with permission from reference 3. (22) M.W. Adapted with permission from reference 3. Commonly used acids are trifluoroacetic acid, formic acid, and acetic acid at concentrations of 0.05 to 0.1% v/v. Column: 50 mm x 3.0 mm, 2.5-m Waters XSelect CSH C18; mobile-phase A: 0.1% formic acid or 10 mM ammonium hydroxide; mobile-phase B: methanol; gradient: 595% B in 8.22 min; flow rate: 0.83 mL/min; detection: UV absorbance at 254 nm. Each peak is designated by its code name, retention time in minutes and [M+H]+ parent ion. This elimination of the filtration step can reduce potential mobile phase contaminations from the filtration process. Longer chain aliphatic alcohols such as ethanol, propanol, and butanol are not generally used because of their higher viscosities, with notable exceptions in separations of chiral compounds by normal-phase chromatography or in reversed-phase LC of larger proteins. The order of eluotropic strengths are methanol Hoont Solar Powered Motion Activated Ultrasonic,
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