Pretreatment | HPLC Q&A | Technical Notes & Support | HPLC

Pretreatment

	Q1	What is the common pretreatment method?
	Q2	When using CAPCELL PAK MF with a protein removal function, why is a filter of pore size 0.2 µm used for filtration before injection?
	Q3	We are thinking of using an MF column to remove protein but the protein peak will overlap the drug peak. What should we do?
	Q4	According to a brochure, the MF column can collect both protein-bound and protein-free drugs. Do protein-bound drugs go out with proteins?
	Q5	How should a sample containing surfactant be pretreated?

Q1	What is the common pretreatment method?

Pretreatment differs depending on the matrix, concentration, and impurity. If there is no particular problem, prepare an HPLC sample by dilution with a mobile phase and filtration. If necessary for preparing an HPLC sample, add extraction as well. The easiest way to perform extraction is to dissolve a target compound in an appropriate solvent by ultrasonic dispersion. After centrifugation, filter the supernatant, then dilute it with a mobile phase or re-dissolve it in a mobile phase after evaporation to prepare an HPLC sample.

Q2	When using CAPCELL PAK MF with a protein removal function, why is a filter of pore size 0.2 µm used for filtration before injection?

A2	The pore size of 0.22 µm can remove non-dissolved matter by filtration with no pressure. The pore size of 0.45 µm is not recommended because it shortens the column life. A smaller pore size makes the pressure too high for filtration.

Q3	We are thinking of using an MF column to remove protein but the protein peak will overlap the drug peak. What should we do?

If CAPCELL PAK MF Ph-1 is used for pretreatment, the drug is basically eluted in the same way as the C18 column because they are held by hydrophobic interaction. Overlapping with the protein peak may indicate inadequate retention by the MF column. Basically, when the concentration of organic solvent in the mobile phase is reduced, the drug will be retained more but that of protein will hardly change because it is eluted in size exclusion mode. Therefore, the conditions can be changed for achieving separation. Choosing a longer column will also improve separation.

Q4	According to a brochure, the MF column can collect both protein-bound and protein-free drugs. Do protein-bound drugs go out with proteins?

A4	Protein-bound drugs are basically measured as protein-free ones by cutting the protein bonding and so are not eluted with protein. However, the behavior differs depending on the mobile phase and the sample injection volume.

Q5	How should a sample containing surfactant be pretreated?

Even if the surfactant is not removed for analysis by liquid chromatography, the column has no problem of chemical stability. Depending on the conditions of the mobile phase or the surfactant type, the surfactant may not elute but remain in the column. This will increase the column pressure and accelerate column aging. The surfactant may be removed by solid-phase extraction or the analytical column may be protected by using a pre-column. Solid-phase extraction removes surfactant with a dedicated filter or cartridge, and can be applied to online removal by column switching in some cases and a pre-column attached to protect the analytical column can be replaced as required.

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