Mobile phase robustness

Solid-phase microextraction (SPME) consists of dipping a fiber into an aqueous sample to adsorb the analytes followed by thermal desorption into the carrier stream for GC, or, if the analytes are thermally labile, they can be desorbed into the mobile phase for LC. Examples of commercially available fibers include 100-qm PDMS, 65-qm Carbowax-divinylbenzene (CW-DVB), 75-qm Carboxen-polydimethylsiloxane (CX-PDMS), and 85-qm polyacrylate, the last being more suitable for the determination of triazines. The LCDs can be as low as 0.1 qgL Since the quantity of analyte adsorbed on the fiber is based on equilibrium rather than extraction, procedural recovery cannot be assessed on the basis of percentage extraction. The robustness and sensitivity of the technique were demonstrated in an inter-laboratory validation study for several parent triazines and DEA and DIA. A 65-qm CW-DVB fiber was employed for analyte adsorption followed by desorption into the injection port (split/splitless) of a gas chromatograph. The sample was adjusted to neutral pH, and sodium chloride was added to obtain a concentration of 0.3 g During continuous... 

Applications High-temperature liquid chromatography with packed-capillary columns, nonaqueous mobile phases, and ELSD and ICP-MS detection, has been developed specifically as a robust analytical tool for the analysis of high-MW polymer additives [731,738]. Dissolving such moderately polar, heavy compounds with low water solubility at ambient temperature usually... 

Robustness is defined as a measure of how well the method will remain unaffected by small variations in the parameters. Robustness can be assured by setting appropriate system suitability. However, it is important that these parameters be set properly. Some parameters which could be used to demonstrate robustness are the varying age of columns, column brands, temperature, pH of mobile phase, and the different amounts of mobile phase modifiers. 

Cation-exchange resin and C g-bonded silica columns have lower sample capacities than amine-modihed silica-gel columns, but they are more robust, do not covalently interact with sample components, and can be eluted with pure water at low flow-rates. Water is an ideal mobile-phase, because of low cost, solute solubility, and ability to be completely removed from collected samples by evaporation. All mobile phases should have these characteristics—including buffers, which should contain volatile components. ... 

A typical example of HPLC method development and validation was provided by Boneschans et al. [9]. They developed an HPLC method for piroxicam benzoate and its major hydrolytic degradation products, piroxicam and benzoic acid. The authors utilised a robust stationary phase (Phenomenex Luna, Cig), with an optimised mobile phase comprising of acetonitrile/water/acetic acid (45/7/8 v/v), and a flow rate of 1.5 ml/min. The operating pH of the mobile phase (pH 2.45) was selected on the basis that it is ca. 2 pH units from the pKa of the drug, and hence reasonably insensitive to changes in mobile-phase preparation. The injection volume was 20 pi with a detection wavelength of 254 nm. They utihsed... 

The instruments for polymer HPLC except for the columns (Section 16.8.1) and for some detectors are in principle the same as for the HPLC of small molecules. Due to sensitivity of particular detectors to the pressure variations (Section 16.9.1) the pumping systems should be equipped with the efficient dampeners to suppress the rest pulsation of pressure and flow rate of mobile phase. In most methods of polymer HPLC, and especially in SEC, the retention volume of sample (fraction) is the parameter of the same importance as the sample concentration. The conventional volumeters— siphons, drop counters, heat pulse counters—do not exhibit necessary robustness and precision [270]. Therefore the timescale is utilized and the eluent flow rate has to be very constant even when rather viscous samples are introduced into column. The problems with the constant eluent flow rate may be caused by the poor resettability of some pumping systems. Therefore, it is advisable to carefully check the actual flow rate after each restarting of instrument and in the course of the long-time experiments. A continuous operation— 24h a day and 7 days a week—is advisable for the high-precision SEC measurements. THE or other eluent is continuously distilled and recycled. 

Resolve C18, Nomura Devosil ODS-MG-5, YMC AQ312, Platinum EPS, Luna Phenyl C6 and Luna phenyl C8) were compared with slight modification in the mobile phase gradient and the Luna Phenyl C8 resulted the most selective and robust [609],... 

TLC experiment can be carried out in controlled conditions, but then the appealing characteristics of simplicity, cheapness and fastness disappear. In order to make the TLC method simple and robust against temperature and humidity changes, it is possible to select a mobile phase that minimizes the harmful effects of these changes. This is described in Chapter 6. 

The purpose of the optimization is to find mixture compositions which result in robust separations. Therefore the separation power of a mobile phase at all combinations of temperature and relative humidity should be examined. The question is how all these values should be combined. 

The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small but deliberate variations in the analytical procedure parameters. The robustness of the analytical procedure provides an indication of its reliability during normal use. The evaluation of robustness should be considered during development of the analytical procedure. If measurements are susceptible to variations in analytical conditions, the analytical conditions should be suitably controlled or a precautionary statement should be included in the procedure. For example, if the resolution of a critical pair of peaks was very sensitive to the percentage of organic composition in the mobile phase, that observation would have been observed during method development and should be stressed in the procedure. Common variations that are investigated for robustness include filter effect, stability of analytical solutions, extraction time during sample preparation, pH variations in the mobile-phase composition, variations in mobile-phase composition, columns, temperature effect, and flow rate. 

Other Considerations. Typically, the variations in robustness results are compared to the intermediate precision results to demonstrate that robustness is not affected significantly within normal day-to-day variation. When the related substance results are affected by some critical experimental parameters, a precautionary statement needs to be included in the procedure to ensure that this parameter is tightly controlled between experiments. For example, if percent organic of mobile phase affects the results significantly, the procedure should indicate the acceptable range for percent organic (e.g., 50% organic 2%)... 

System suitability. During the robustness testing of method validation, critical method parameters such as mobile phase composition and column temperature are varied to mimic the day-to-day variability. Therefore, the system suitability results from these robustness experiments should reflect the expected range for the system suitability results. As a result, system suitability results in these method validation experiments are very useful in determining the system suitability... 

Robustness for HPLC Analysis. The investigation of the effect of column, mobile phase, HPLC solution stability, and wavelength is performed in a manner similar to the HPLC potency/related substance assay. For solution stability, the... 

For determining the robustness of a method a number of parameters, such as extraction time, mobile-phase pH, mobile-phase composition, injection volume, source of column lots and/or suppliers, temperature, detection wavelength, and the flow rate, are varied within a realistic range and tlie quantitative influence of the variables is determined. If the influence of a parameter is within a previously specified tolerance, this parameter is said to be witliin the robustness range of the method. These method parameters may be evaluated one factor at a time or simultaneously as part of a factorial experiment. 

A rapid, sensitive, reversed-phase HPLC method for the separation of BHA isomers was described by Berridge et al. (117). Using a column packed with Hypersil ODS of 3-/nn particle size and with a mobile phase consisting of an acetonitrile-water mixture, it was possible to detect less than 0.5 ng of the isomers injected. The procedure is reliable and robust and compared to another HPLC method, claimed to have a more stable, longer-lasting column requiring only occasional maintenance (117). 

Robustness Systematically change chromatographic conditions (examples column temperature, flow rate, gradient composition, pH of mobile phase, detector wavelength). Check influence of parameters on separation and/or peak areas. 

Separation of five compounds (DL, 6-OH-DL, 3-OH-DL, 7V-OH-DL, and 1-pyridine-/V-oxide-DL) was achieved using an Alliance HPLC system (Waters Corp., Milford, CA) equipped with a 2690 model pump, an autoinjector, a Polaris Cl8-A guard column (Varian Inc., Lake Forest, CA), and a Luna Phenyl-Hexyl analytical column (Phenomenex, Inc., Torrance, CA) maintained at 40°C. For robust characterization of each isomeric compound, an online HDX LC-MS method was developed. The composition of regular and deuterated mobile phases is summarized below ... 

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