In HPLC, a solvent is the solution used to prepare the mobile phase, dissolve and transport the sample. The mobile phase is the main factor affecting the performance of HPLC, and its properties must meet the strict requirements of HPLC testing.
01
UV detector
Under the detection wavelength of the sample, the absorbance of the selected mobile phase is preferably A<0.2AU, especially in gradient elution, lower absorbance means better detection accuracy and results can be obtained. However, solvents with slightly higher absorbance in isocratic elutions are also acceptable. It is rare to use a solvent with a UV detection wavelength below 200nm, except for the detection of substances with low absorbance above 200nm.
Because water has no UV absorption at wavelengths greater than or equal to 200nm, the absorbance of the aqueous mobile phase is equal to the absorbance of the pure solvent multiplied by the volume fraction φ of the mobile phase B solvent. For example, at a wavelength of 215nm, when B is the following different solvents, the absorption value A of the mobile phase containing 25% B solvent corresponds to: acetonitrile, 0.00AU; methanol, 0.09AU; degassed methanol, 0.05AU; tetrahydrofuran, 0.22AU; Isopropanol, 0.07AU.
Be careful that degassing methanol reduces the concentration of oxygen in the pure solvent, resulting in a nearly 1/3 reduction in absorbance at 200–240 nm. When the mobile phase is degassed by filling helium, the absorbance value of the B solvent in the mobile phase will also decrease due to the decrease in the proportion of oxygen content in the solvent. Oxygen is more soluble in less polar solvents such as tetrahydrofuran and isopropanol, so, in practice, absorbance values for less polar solvents may be higher than theoretical. (For theoretical values, see <Selection of UV Absorption Wavelength in Liquid Chromatography Method Development>)
Water has no absorption at a wavelength ≥ 200nm, which assumes that the water is strictly purified. HPLC grade water is specified in ASTM D1193. Detection in low wavelength sometimes not only requires the total organic carbon (TOC) in water to be lower than 50ppb, but also has a sufficiently high resistivity.
02
RI detector
In isocratic elution, the choice of mobile phase is generally not limited by the RI detector. The detection sensitivity can be improved by selecting a mobile phase whose refractive index is quite different from that of the sample components. Detectors based on differential measurements (eg RI) cannot be used for gradient elution. Because usually the response values between A and B solvents are very different.
03
MS detector
The mobile phase is evaporated at the MS interface, and the mobile phase is generally composed of water, organic solvents, and the use of volatile additives is also common (additives cannot be non-volatile buffers or salts). Since the mobile phase has been removed, there is no UV absorption of the mobile phase in LC-MS, but there must be no particles in the solvent.
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