Column Rinsing | HPLC Q&A | Technical Notes & Support | HPLC

Column Rinsing

	Q1	Is it better always to rinse a column after use?
	Q2	Which solvent is suitable for column rinsing?
	Q3	Which solvents can be used for rinsing and which cannot?
	Q4	Does the rinsing method differ with the particle size?
	Q5	How should a reversed phase column be rinsed?
	Q6	How should an NH2 column be rinsed?
	Q7	How should an MF column be rinsed?

Q1	Is it better always to rinse a column after use?

If the column pressure and peak shape are normal, avoid rinsing in order to use the column longer. If you are not using the column for one or two days, it is recommended to keep the flow at a reduced rate. For medium-term storage (several days to about one month), seal the column without rinsing unless the mobile phase is harsh acid or alkali and keep it where the temperature does not change greatly. For long-term storage (several months to about one year), store the column after replacing the mobile phase with one containing at least 50% organic solvent. (If the mobile phase is NH2, replace it 100% CH3CN when storing the column.)

Q2	Which solvent is suitable for column rinsing?

For rinsing, it is recommended to use solvent that easily dissolves a compound adsorbed on the column. A mixed solution of methanol and chloroform is also effective for extremely hydrophobic compounds. However, you need not rinse a column daily. If a sample contains a compound that are strongly adsorbed on the column, use a solvent that easily dissolves the compound but does not damage the packing materials.

Q3	Which solvents can be used for rinsing and which cannot?

For rinsing, use a solvent that easily dissolves the compound adsorbed on the column but does not damage the packing materials. The solvent should be pH2 to pH9, containing 10% or more organic solvent. The organic solvent may be CH3CN, CH3OH, THF, DMSO, chloroform, or other type that is commonly used for liquid chromatography. Solvents unsuitable for rinsing are H2O 100% or ones of below pH2 or over pH9.

Q4	Does the rinsing method differ with the particle size?

The rinsing method does not differ between the 3 µm and 5 µm columns. Column rinsing is usually not recommended except after alkali or harsh acid like TFA is used. Users should inject well-cleaned samples by filtration. For rinsing, use a solvent that dissolves the substance that may be remaining in the column. Note that the rinsing liquid differs depending on the properties of the substance to be rinsed away.

Q5	How should a reversed phase column be rinsed?

A reversed phase column may adsorb a very hydrophobic compound. Rinse the column by flowing one of the following overnight: 1.Solvent that includes all the components of the mobile phase but salt. 2. 100% CH3CN or 100% CH3OH, or 3. H2O: CH3CN = 1 : 1 or H2O : CH3OH = 1 : 1 If rinsing is still not enough, rinse the column with 90% H2O. However, column rinsing is not recommended. Rinse the column only as required if the peak shape or pressure has unacceptably changed.

Q6	How should an NH2 column be rinsed?

A6	If an NH2 column is rinsed with 80% CH3CN (pH11, ammonia water) for one or two hours, amino groups protonated by the use of acidic mobile phase can be returned to the original neutral amino groups.

Q7	How should an MF column be rinsed?

A7	It is not recommended to rinse a column with organic solvent. Alkali rinsing might work to some extent but will deteriorate the column more or less. Replace the column if the peak becomes broad.

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