Hydro-organic mixtures

Mobile phases commonly used in reversed-phase HPLC are hydro-organic mixtures. The most common reversed-phase organic modifiers include methanol and acetonitrile and/or combinations of these two modifiers. Other mobile-phase modifiers such as tetrahydrofuran, IPA, and DMSO [32] have been also used for minor selectivity adjustment however, they are not common due to their high backpressure limitations and/or high background UV absorbance. 

The impact of the pH in hydro-organic mixtures on the analyte ionization and retention will be thoroughly discussed. The impact of pH on analyte UV absorbance will be discussed in the method development chapter. Chapter 8 (Section 8-6). 

Liquid chromatography has also been widely used for the determination of dissociation constants [88-92] since it only requires small quantity of compounds, compounds do not need to be pure, and solubility is not a serious concern. However, the effect of an organic eluent modifier on the analyte ionization needs to also be considered. It has been shown that increase of the organic content in hydro-organic mixture leads to suppression of the basic analyte pKa and leads to an increase in the acidic analyte pK compared to their potentiometric pKa values determined in pure water [74]. 

The k (retention factor) values are then plotted versus the s pH values, and the inflection point of this sigmoidal relationship could be taken as the sp of that particular compound at particular hydro-organic mixture. The ipKa determined at 30v/v% MeCN was determined to be 3.9 (using nonlinear regression analysis program MathCad 8). This corresponds well to our original estimation of IpKa 3.7. 

Note. The type and concentration of the organic eluent can cause a pH shift of the aqueous portion of the mobile phase as well as change the ionization state of the analyte in a particular hydro-organic mixture. Temperature can also lead to change in the ionization constants of analytes. 

At pH 2.5, the EOF is approximately 10 cm /V/s in 50 mM buffer. At pH 7, it is an order of magnitude higher. The EOF is inversely proportional to BGE viscosity and is proportional to temperature, up until the point where heat dissipation is inadequate. Organic modifiers such as methanol decrease the EOF because hydro-organic mixtures have higher viscosity compared to water alone. Acetonitrile does not strongly affect the EOF. Polymer additives such as methylcellu-lose derivatives increase viscosity as well as coat the capillary wall. 

Chromatographic peaks in MLC appear usually more evenly distributed with regard to those obtained with conventional hydro-organic mixtures. Owing to this compression effect, the use of gradients is less necessary. The above features have allowed the development of multiple applications that are competitive against conventional RPLC. 

Reversed phase Hydro-organic mixtures Organic modifiers acetonitrile, methanol, etc. Solvents, glycols, urea, acidic solutions... 

Numerous titrations of carboxylic acids have been proposed in the literature, in strictly aqueous solutions as well as in hydro-organic mixtures. Among those reported in strictly aqueous solutions are the titrations of maleic, malic, tartaric, citric, and sorbic acids (Fig. 11.1). Their equivalence points are detected with phenolphthalein. All of them, including the polyacids just listed, exhibit only one endpoint. The endpoint color change is detected for the titration stage at which the number of added equivalents of sodium hydroxide is equal to the number of acid functions the titrand possesses. We d like to point out the following facts about these titrations ... 

Other kinds of organic structures exhibit a sufficiently acidic character to be titrated with a sodium hydroxide solution in water and in hydro-organic mixtures examples include derivatives that bring a lactam function, acylureas, imides, some sulfamides, and some nitrogenous heterocycles. The pK of these derivatives may take any value along the entire pH range. Their acidic character would be due to the occurrence of enol or oxime forms. 

The commonly used sample solvent mixtures in RP-HPLC are hydro-organic mixtures of which methanol, acetonitrile, and/or combination of these two are the predominant organic solvents used. Other organic solvents such as tetrahydro-furan (THF), isopropyl alcohol (IPA), ethyl acetate, and acetone have also been used (see Table 4). However, the primary consideration for choice of solvents include miscibility of the solvents, compatibility with the RP-HPLC mobile phase, solubility of the API in the sample solvent, polarity, light transmission, viscosity, stability, and pH. ° Compatibility of the sample solvent used with the HPLC... 

The organic solvents most commonly used in the extraction-preconcentration step and sample dilution are hexane, methanol, ethyl ether, ethyl acetate and acetone. When determination involves the separation of compounds with different acid-base properties, i.e. acidic, basic and neutral analytes, separation occurs as a function of pH acidic and basic molecules are extracted making use of hydro-organic mixture also containing acids or bases while neutral species are dissolved in organic phases. 

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