Mobile phase organic solvents used

In GPC, the solvent in which the standards and sample are dissolved should be identical to the mobile-phase solvent in which the analysis will be performed. In most cases filtration is the only step needed to prepare the mobile phase. Organic solvents should be vacuum filtered through a 0.45-pm fluorocarbon filter, while acetate-type filters are used with aqueous mobile phases. In some cases mobile-phase additives are required. When polar solvents such as N,N-dimethyformamide, N,N-dimethylacetamide, and -methyl pyrrolidone are used to analyze polar polymers such as poly-... 

Anionic and neutral polymers are usually analyzed successfully on Syn-Chropak GPC columns because they have minimal interaction with the appropriate mobile-phase selection however, cationic polymers adsorb to these columns, often irreversibly. Mobile-phase selection for hydrophilic polymers is similar to that for proteins but the solubilities are of primary importance. Organic solvents can be added to the mobile phase to increase solubility. In polymer analysis, ionic strength and pH can change the shape of the solute from mostly linear to globular therefore, it is very important to use the same conditions during calibration and analysis of unknowns (8). Many mobile phases have been used, but 0.05-0.2 M sodium sulfate or sodium nitrate is common. 

The organic solvent used to elute the compound must be adequately strong (polar for the adsorbent silica gel) and a good solvent for the component. Absolute methanol should be avoided as a siugle solvent because silica gel itself and some of its common impurities (Fe, Na, SO4) are soluble iu this solvent and will contaminate the isolated material. Solvent containing less than 30% methanol is recommended, or ethanol, acetone, chloroform, dichloromethane, or the mobile phase originally used for PLC are other frequently nsed choices for solnte recovery. Water is not recommended because it is so difficult to remove by evaporation during the concentration step (removal by lyophilization is necessary). A formula that has been used to calculate the volume of solvent needed when the PLC mobile phase is chosen for elution is ... 

The mobile phase does not vary and does not require disposal and, even if helium is used as a carrier gas, is cheap compared to the organic solvents used in HPLC. 

For analysis of basic compounds, silica gel which has been sprayed with a solution of KOH in methanol, may be used. Treating the plate with base ensures that basic compounds chromatograph as their free bases rather than as their salts. The salts of the amines have very low mobility in organic solvent-based mobile phases since basic compounds tend to interact strongly with silanol groups on the surface of the silica the presence of KOH in the stationary phase suppresses this interaction. 

Mobile phase as sample solvent. If possible, always use mobile phase as the sample solvent. This ensures the composition (e.g., percent organic, pH) of sample solution matches that of mobile phase and reduces the chance of any problem due to incompatibility of sample solvent and mobile phase. Alternatively, always use sample solvent weaker than that of the mobile phase to ensure that the chromatography is not deteriorated. For example, in reversed-phase HPLC, use less organic solvent in the sample solvent than in the mobile phase. 

Evaporation and redissolving. The solvent of the combined upper layer is evaporated under nitrogen flow or low-temperature vacuum distillation. An oily material appears after it is dried. A precisely measured aliquot of mobile phase is normally used to redissolve theextract. These procedures are intended to not only increase the concentration of tocopherols and tocotrienols to the measurable level of the detector, but also to avoid uncertain volume change of organic layer during extraction, which results in inaccurate results. The redissolved sample is transferred to a vial for HPLC analysis. 

The polymeric chips require special attention to their surface properties due to their poor compatibility with many samples and organic solvents used in forming a coating and in the composition of the mobile phases. Lee et al. 

Gradients of aqueous and organic mobile phases are typically used for LC-MS/MS analysis of drug compounds and metabolites. The most common aqueous solvents are water with 0.1 % formic acid or 0.1 % acetic acid (v/v) or volatile buffers like 5 mM ammonium-acetate or ammonium-formate. Often adjusted to a certain pH value with the corresponding acid or base (the pH of the eluents will have to be optimized with respect to the polarity of the analytes, since ionic species will have very low or no retention on the reversed pahse LC-columns). Other volatile buffers can be used as well. Phosphate buffers should be avoided, since they will cause suppression of the ionization and thus lead to very bad analytical performance (Venn 2000). Reagents like triethyl-amine should also be avoided as mobile phase or as part of mobile phases. They induce ion suppression as well. In terms of the organic solvents, methanol and acetonitrile are very widely used and they are very well suitable for LC-MS. Other solvents can be used as well, as long as they are compatible with the materials used in the LC-MS system. 

Docetaxel and paclitaxel were obtained from Hande Tech Development Co. (Houston TX, USA) with a purity of 99%. Methanol and acetonitrile were used in HPLC grade quality from Fisher Scientific (Houston TX, USA). Organic solvents have been filtered using 0.2 xM nylon membrane filters from Whatman (Maidstone UK) with a diameter of 47 mm. Milli-Q deionised water was used in this study. All water based mobile phases were filtered using 0.22 pM GP Express plus membranes from Millipore (Bedford, USA). Formic acid (95 % or higher) from Sigma (St. Louis USA) was used. 

In the real world, the interactions with the stationary phase contribute more or less to the retention. Theoretically, alkylsilica stationary phases are similar to liquid alkanes immobilised on a solid support, but the bonded alkyl chains differ from the free molecules of liquid hydrocarbons by limited mobility, which may affect the retention. Further, the retention behaviour can be more or less complicated by specific polar interactions of unreacted silanol groups in silica-based bonded phases, especially with basic solutes. Finally, organic solvents used as the components of the mobile phases in reversed-phase systems can be preferentially adsorbed by the stationary phase and modify its properties 159,60]. 

Both the solvent-interaction model (Scott and Kucera, 1979) and the solvent-competition model (Snyder, 1968, 1983) have been used to describe the effects of mobile-phase composition on retention in normal-phase liquid chromatography. The solvent interaction model on the one hand provides a convenient mathematical model for describing the relationship between retention and mobile phase composition. The solvent competition model on the other hand provides a more complete, quantitative description of the relative strengths of adsorbents and organic solvents used in normal-phase chromatography. 

A further problem associated with syringe pumps arises from the fact that most of the fluids used as mobile phases in HPLC can be compressed, to some degree. This is especially the case with the purely organic solvents used in normal phase HPLC, some of which are reduced in volume by up to 0.015% for each bar of pressure that is applied. So for example, 200 ml of a mobile phase having a compressibility of 0.01% per bar, being driven at 100 bar to give a flow rate of 1 cm min will require at least 10 minutes to reach its nominal flow rate. The initial displacement of the plunger only serves to compress the mobile phase. 

---