Size Exclusion Chromatography (SEC) is a very convenient tool to measure the molecular weight of proteins and it enables preparative chromatography. it On the other hand, this method using calibration curves of protein Mw markers can?t always provide the absolute Mw due to the difference in protein structure, ionicity and hydrophobicity. To make use of this advantage and compensate the disadvantage, a combined technology SEC/MALS analysis was conducted, using multi angle light scattering (MALS) as a detector. In order to confirm its effect, several protein Mw markers of given Mw are used and their detection was compared between two different types of columns (PROTEIN KW-804 and Asahipak GS-620 HQ).
Fig. 1 shows a chromatogram of the absolute Mw by KW-804 and RI detector, and Fig. 2 is by GS-620 HQ and RI detector. Peaks using KW-804 are eluted according to the order of their Mw, but the peaks using GS-620 HQ are not according to their Mw orders. The reason is that the separation principle of KW-804 is just SEC mode that has no interaction with proteins, and GS-620 HQ has a multi-mode that has interactions in addition to SEC mode. Even if the elution volumes by SEC change, it won’t affect on the MALS detection to measure the absolute Mw.
| Sample Protein molecular weight standards Kit (Mw-GF-1000 KIT) | |
| 1. Carbonic Anhydrase(Bovine Erythrocytes) | 1.5mg/mL 100µL |
| 2. Bovine serum albumin, BSA | 1.5mg/mL 100µL |
| 3. Alcohol Dehydrogenase, (Yeast) | 1.5mg/mL 100µL |
| 4. β-Amylase,(Sweet Potato) | 1.5mg/mL 100µL |
| 5. Thyroglobulin,(Bovine) | 1.5mg/mL 100µL |
Column : Shodex PROTEIN KW-804 (8.0mmID*300mm)(Fig. 1) Shodex Asahipak GS-620 HQ (7.5mmID*300mm)(Fig. 2) Eluent : 0.1M Phosphate buffer(pH7.0) + 0.3M NaCl Flow rate : 1.0mL/min (Fig. 1) 0.6mL/min (Fig. 2) Detector : MALS (Multi angle light scattering), Shodex RI Column temp. : 26°C
