Q5. How do I prepare the mobile phase?

Please refer to Technical Information, Preparation of Mobile Phase for HPLC (PDF 582 KB)

Caution:

1. Prepare mobile phase consistently each time because concentration and pH may affect separation performance.
2. Degas the solvent after mixing.

Q6. What solvent grade should I use for the mobile phase?

We recommend using HPLC grade solvents. GR grade solvents are not suitable for gradient analysis or micro-scale analysis because they contain impurities that have ultraviolet absorption, causing unstable baseline or inaccurate detection. This is especially problematic in the short wavelength range (210-220nm). GR grade solvents with antioxidants (e.g.. tetrahydrofuran, chloroform) may produce ghost peaks. GR grade trifluoroacetic acid may be chemically unstable and is not recommended for HPLC.

Q7. Should I use acetonitrile or methanol for the mobile phase?

The table below shows some differences between acetonitrile and methanol when used for HPLC mobile phases.

	Acetonitrile (for HPLC)	Methanol (for HPLC)
Pressure	Backpressure differs depending on species of organic solvents and mixing ratio. The backpressure of acetonitrile is lower than that of methanol at the same concentration.
Elution Strength	The elution strength of acetonitrile/water is stronger than that of methanol/water at the same concentration. Within the range of about 30-80% organic solvent, the elution strength of acetonitrile/water is almost equivalent to that of methanol/water with about 10% higher concentration (e.g., acetonitrile : water = 60 : 40 →methanol : water = 70 : 30)
Absorbance	Acetonitrile has a lower UV absorbance in far UV region (less than 250 nm) .	Methanol has a higher UV absorbance than acetonitrile in far UV region (less than 250 nm).
Degas of Mobile Phase	When acetonitrile is mixed with water, it is endothermic. It is difficult to degas.	When methanol is mixed with water, it is exothermic. It is easy to degas.