Robustness should be considered during the development phase, and should show the reliability of an analysis when deliberate variations are made in method parameters. For analytical methods, robustness depends on the type of analytical method under study. A method is only as robust as each of the components involved, and that includes the mobile phases. The method robustness was evaluated by altering the mobile phase composition +5 v/v, varying the column temperature by +2°C and adjusting the flow rate by +0.2 mL min −1. In robustness testing both mixture variables and process variables (e.g. The method validation guideline ICH Q2(R1) has clearly defined the type of variations expected for specific methods: In the case of liquid chromatography, examples of typical variations are: influence of variations of pH in a mobile phase; Other factors that will affect the reproducibility of separations include the effects of mobile phase composition and temperature. A ruggedness test showed that the method was not sensitive to minor variations in the mobile phase composition, post-column derivatization system or detection wavelength. With HILIC in particular, a very small change in the mobile phase composition can have a big impact. The need to document the sensitivity of an assay to small changes in environmental and operational conditions is particularly important if that method is submitted in support of a New Drug Application (NDA). In HPLC analysis the mobile phase can contain, besides the aqueous phase, one to three organic modifiers, yielding mixtures of two to four components. The evaluation of robustness should be conside-red during the method development phase. Mobile Phase Composition. View Show abstract Robustness is determined by using either an experimental design or one factor at a time (OFAT). The results were found to be within adequate limits as shown in Table IV. Preparing mobile phases may seem really simple, and often it is, but sometimes how you go about it really matters. mobile phase composition, pH and ionic strength, tem-perature and different lots or suppliers of columns (ICH, 1996; Brasil, 2003; The United States Pharmacopoeia, 2007). Modelled robustness testing was also performed, elucidating all important mobile phase and instrument parameters that could influence a method’s lifetime performance. flow, temperature, wavelength) need to … There is a rule that you can use to predict the effect of changing the composition of the mobile phase which applies to most small molecules in reversed phase conditions: ‘An increase of the organic solvent component in the mobile phase of approximately 10% will result in the retention time being reduced by a factor of 3.’ Robustness and Ruggedness Experimental Variables That Impact Resolution •Column —column lot* •Mobile Phase —buffer pH —buffer concentration —ionic strength —% organic modifier •Sample —injection volume —solvent strength •Instrument —column temperature —detector flow cell volume* •Gradient —dwell volume* pH in a mobile phase; Mobile phase composition; Different columns (lots/suppliers) Temperature and flow rate; How is robustness determined? Since at a fixed mobile phase composition, density is a driving factor in SFC retention, a method’s level of robustness will depend largely on the extent of the mobile phase compressibility. Robustness and ruggedness. In fact, the process of validating a method cannot be separated from ... — variations in mobile phase composition; — temperature; and — flow rate. Mobile phase compositions are 10mmol L−1 ammonium acetate–methanol (4:1) for determination of the d-epimer, and 10mmol L−1 phosphate buffer (pH 3.0)–methanol (9:1) containing 10mmol L−1 ammonium acetate and 10mmol L−1 pentanesulfonic acid for …