Size Exclusion Chromatography (SEC or SEC-HPLC), also known as molecular sieve chromatography, is a technique of purity analysis and macromolecular separation based on molecule size. SEC is a major mode of HPLC that employs porous particles in the column. It is usually applied to determine molecular weight distributions of proteins or separate proteins and polymers used in a wide range of industries.



  • Mobile Phase: 150 mM Phosphate Buffered Saline, pH 7.0

Instruments and Consumables

  • Agilent 1200 Series
  • AdvanceBio SEC 300Å, 7.8 x 300 mm, 2.7 µm, LC column (PL1180-5301, Agilent)


Making the Mobile Phase

1. Dissolve Na2HPO4.12H2O and NaH2PO4.2H2O in ultrapure water to make 1000 mL. The resulting solution should have a pH between 6.8 to 7.0.

2. Filter the mobile phase through a 0.22 µm Nylon membrane filter under vacuum to degas the solution and to remove solids that could plug the chromatographic column.

3. Degas the mobile phase via ultrasonication to avoid having a bubble, which could either cause a void in the stationary phase at the inlet of the column or work its way into the detector cell, causing instability with the UV absorbance.

Balance AdvanceBio SEC Column

4. Put line in Mobile Phase solution and unscrew "Purge" valve, set flow rate to 5 mL/min for 3-5 min.

5. Adjusted flow rate to 0.5 mL/min and install chromatographic column, balance for 30-60 min until baseline is stable.

Preparing Sample

6. Adjusted concentration of sample to 1 mg/mL.

7. Filter the sample through a 0.22 µm Nylon membrane filter.

8. Place a volume (~20 µl) of protein solution into sample vials and put on the sample platform of Agilent 1200 Series.

9. Set up system parameters: Wash Location, Inject Location, Inject Volume, Sample Name, Column temperature and Wavelength.

10. Run the sequence.

11. Data analysis.

For research use only, not directly for clinical use.
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