It will be very rare that you will come across an HPLC method which uses a single solvent throughout the run. Pure solvents kept in mobile phase reservoirs need to be mixed in required proportions under isocratic conditions.
Mixing of solvents is a critical step as procedural deviations can affect the chromatograms and analysis.
Miscibility of solvents
Miscibility of solvents is directly related to their polarities. Remember ‘Like dissolves like ‘. The solvents to be mixed should be miscible with each other in all proportions as immiscible or partly miscible solvents cannot be used in HPLC analysis. Commonly used solvents such as water, methanol, acetonitrile and tetrahydrofuran are fully miscible with each other and are commonly used in reverse phase separations.
The density of solvents varies with temperature which in turn affects the dispensed volume. For good reproducibility before mixing the solvent bottles should be kept for some time in the laboratory room so as to stabilize to laboratory temperature.
Solvents seldom mix stoichiometrically and on mixing occupy less volume than the sum total of their individual volumes prior to mixing. Additionally there can be temperature changes as a result of mixing which can lead to volumetric variations. Mixing of methanol with water results in heating whereas acetonitrile and water on mixing result in cooling. In order to compensate for the thermal changes on same mixing sequence should be followed every time. It is advisable to measure required volumes separately and mix them rather than taking a volume of one solvent in a graduated cylinder or volumetric flask and making up the volume to the mark with the other.
HPLC grade solvents should be used. This grade is commercially prepared from distillation of AR grade solvents followed by filtration. Water should be of high purity and can be obtained using reverse-osmosis based commercially available systems. See link on Water Requirements of HPLC.
UV cut-off wavelength
UV cut-off wavelength is important consideration for deciding which HPLC solvents can be mixed when using a UV detector. The UV cut-off specifies the lowest wavelength that UV detector can be used for a particular solvent.. The cut-off values of some common solvents are provided here :
|Solvent||UV Cut off (nm)|
Refractive index of solvents should be considered before mixing of solvents when using RI detector. The refractive index of sample should be as different as possible from the refractive index of the mobile phase for significant response
Solvents should have low viscosity for efficient flow and mass transfer of analytes in and out of stationary phase pores. Viscous solvents also lead to high column back pressures.
Some solvents are more compressible than others. HPLC operates under high pressures and flow rate deviations can result on account of solvent compressibilities. Modern pumping systems correct for solvent compressibilities and ensure consistent flow rates.
Safety of use should be your prime concern. Try to avoid as far as possible solvents which are poisonous, highly flammable or carcinogenic.
In this article considerations for manually mixing the mobile phase are covered. A subsequent article will cover mixing of mobile phases in system using low and high pressure mixing options.
We shall welcome your comments, suggestions and experience on the topic covered.