text: A comprehensive article on the modern trends and best practices of mobile phase selection in reversed-phase chromatography was published by Boyes and Dong. A mobile phase in reversed-phase chromatograpy consists of mixtures of water or aqueous buffers, to which organic solvents are added, to elute analytes from a reversed-phase column in a selective manner. The added organic solvents must be miscible with water, and the two most common organic solvents used are acetonitrile and methanol. Other solvents can also be used such as ethanol or 2-propanol (isopropyl alcohol) and tetrahydrofuran (THF). The organic solvent is called also a modifier, since it is added to the aqueous solution in the mobile phase in order to modify the polarity of the mobile phase. Water is the most polar solvent in the reversed phase mobile phase; therefore, lowering the polarity of the mobile phase by adding modifiers enhances its elution strength. The two most widely used organic modifiers are acetonitrile and methanol, although acetonitrile is the more popular choice. Isopropanol (2-propanol) can also be used, because of its strong eluting properties, but its use is limited by its high viscosity, which results in higher backpressures. Both acetonitrile and methanol are less viscous than isopropanol, although a mixture of 50:50 percent of methanol:water is also very viscous and causes high backpressures. 
  All three solvents are essentially UV transparent. This is a crucial property for common reversed phase chromatography since sample components are typically detected by UV detectors. Acetonitrile is more transparent than the others in low UV wavelengths range, therefore it is used almost exclusively when separating molecules with weak or no chromophores (UV-VIS absorbing groups), such as peptides. Most peptides only absorb at low wavelengths in the ultra-violet spectrum (typically less than 225 nm) and acetonitrile provides much lower background absorbance at low wavelengths than the other common solvents.
  The pH of the mobile phase can have an important role on the retention of an analyte and can change the selectivity of certain analytes. For samples containing solutes with ionized functional groups, such as amines, carboxyls, phosphates, phosphonates, sulfates, and sulfonates, the ionization of these groups can be controlled using mobile phase buffers. 
  For example, carboxylic groups in solutes become increasingly negatively charged as the pH of the mobile phase rises above their pKa, hence the whole molecule becomes more polar and less retained on the a-polar stationary phase. In this case, raising the pH of the phase mobile above 4–5 = pH (which is the typical pKa range for carboxylic groups) increases their ionization, hence decreases their retention. Conversely, using a mobile phase at a pH lower than 4 will increase their retention, because it will decrease their ionization degree, rendering them less polar. 
  The same considerations apply to substances containing basic functional groups, such as amines, whose pKa ranges are around 8 and above, are retained more, as the pH of the mobile phase increases, approaching 8 and above, because they are less ionized, hence less polar. However, in the case of high pH mobile phases, most of the traditional silica gel based Reversed Phase columns are generally limited for use with mobile phases at pH 8 and above, therefore, control over the retention of amines in this range is limited.
  The choice of buffer type is an important factor in RP-LC method development, as it can affect the retention, selectivity, and resolution of the analytes of interest. When selecting a buffer for RP-HPLC, there are a number of factors to consider, including: 
  * The desired pH of the mobile phase: Buffers are most effective around their pKa value, so it is important to choose a buffer with a pKa that is close to the desired mobile phase pH needed.
  * The solubility of the buffer in the organic solvent: The buffer must be compatible with the organic solvent that is being used in the mobile phase, mostly with the common organic solvents mentioned above, acetonitrile, methanol, and isopropanol.
  * The UV cut-off of the buffer: In case of UV detection, the buffer should have a UV absorption that is below the detection wavelength of the analytes of interest. This will prevent the buffer from interfering with the detection of that analytes.
  * The compatibility of the buffer with the detector: If mass spectrometry (MS) is being used for detection, the buffer must be compatible with the mass spectrometry (MS) instrument. Some buffers, such as those containing phosphate salts, cannot be used with the MS detectors, as they are not volatile as needed, and they interfere with the MS detection by suppressing the analytes ionization, making them undetected by MS.
  Some of the most common buffers used in RP-HPLC include:
  * Phosphate buffers: Phosphate buffer is versatile and can be used to achieve a wide range of pH values, thanks to 3 pKa values. They also have very low UV background for UV detection. However, they are not appropriate for MS detection.
  * Acetate buffers: Acetate buffers are also versatile and can be used to achieve range of pH values typically used in RP-LC. In terms of UV detection at sub 220 nm wavelength, it is not so favorable. The ammonium acetate buffer is compatible with MS.
  * Formate buffers: Formate buffers is similar to the acetate buffer in terms of range of pHs used and limited UV detection under 225 nm. Its ammonium acetate is also compatible with MS.
  * Ammonium buffers: Ammonium buffers are volatile and are often used in LC-MS methods. They also are limited for low UV detection.
  Charged analytes can be separated on a reversed-phase column by the use of ion-pairing (also called ion-interaction). This technique is known as reversed-phase ion-pairing chromatography.
  Elution can be performed isocratically (the water-solvent composition does not change during the separation process) or by using a solution gradient (the water-solvent composition changes during the separation process, usually by decreasing the polarity).