Considerations for Mixing of HPLC Mobile Phase Solvents Off-line

你会遇到一个非常罕见的人HPLCmethod 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.

溶剂混合是作为程序偏差可以影响色谱图和分析的关键步骤。

溶剂的混溶性

溶剂的混溶性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.

Temperature effects

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.

Purity

应使用HPLC级溶剂。该等级可从Ar级溶剂的蒸馏商业制备，然后过滤。水应具有高纯度，并且可以使用基于逆转渗透的市售系统获得。See link on Water Requirements of HPLC.

UV cut-off wavelength

UV截止波长是在使用UV检测器时决定可以混合的HPLC溶剂的重要考虑因素。UV截止指定UV检测器可用于特定溶剂的最低波长。这里提供了一些普通溶剂的截止值：

Solvent	UV Cut off (nm)
甲醇	205.
水	190
Acetonitrile	190
Tetrahydrofuran	212.
Hexane	195

Refractive index

在使用RI探测器时，应在混合溶剂之前考虑溶剂的折射率。样品的折射率应尽可能不同地从流动相的折射率进行显着反应

粘度

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.

Compressibility

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.

Hazardous solvents

Safety of use should be your prime concern. Try to avoid as far as possible solvents which are poisonous, highly flammable or carcinogenic.

在本文中，涵盖了手动混合移动阶段的注意事项。随后的物品将介绍使用低压混合选项在系统中的移动阶段混合。

We shall welcome your comments, suggestions and experience on the topic covered.