Column tips for successful SEC-MALS

Using a MALS system together with size exclusion chromatography (SEC)? Not performing as expected? Tap into these column tips for how to solve this and other issues.

Measuring mass of macromolecules

Size exclusion chromatography (SEC) separates molecules based on hydrodynamic volume. If you need to identify the molecules, multi-angle static light scattering (MALS) is a common technique to use in combination with SEC.

MALS is commonly used in-line in a combined SEC-MALS setup. The weight average molecular mass is determined for molecules passing a detector cell. The principle is based on reading the intensity and the angular dependence of the scattered light. 

SEC column tips for great MALS results

MALS is a very precise measurement, but difficulties might arise when working with a SEC-MALS setup. I have listed four possible issues and tips for how you can overcome these.

1. Problem: high mass species knocked off the column by the injection itself (by a pressure pulse, by detergents in the buffer, etc.).
To diagnose: perform a blank run prior to running samples. 
Tip: do not increase the flow too rapidly.

2. Problem: nonspecific binding to the SEC resin resulting in the proteins eluting later than expected.
Tip: screen buffer conditions.

3. Problem: high mass species co-elutes with the target mass. This results in a much higher mass than expected.
Tip: screen buffer conditions.

4. Problem: difficulties detecting components present in small quantities. The concentration is just too low to get good MALS data.
Tip: increase separation efficiency and concentrate.

I also recommend having a dedicated SEC-MALS setup for molecular mass determinations. This will ensure an effective station for identification of target molecules. Never turn the flow off. A system that runs on a low flow (re-circulation) does not suffer from pressure-induced signals in the SEC-MALS setup.

To get a good baseline, you should wash the column before the first injection with several column volumes of water and buffer (often overnight with a low flow rate). Do not use the SEC column at the maximum flow rate, though.

Recent SEC-MALS applications

Interested in recent applications where GE’s Superdex 200 Increase 10/300 GL columns have been used for SEC-MALS? Start reading here:


Do you have other tips for successful SEC-MALS analysis? Share them in the comments field.

2 Comments

GE\dansome's profile image

dansome

Hi Pia, those are great tips. Here are some more basic tips for improving SEC-MALS measurements: 1. Pre-filter buffers with 0.1 µm sterile filters into sterile bottles, and refresh buffers regularly. Always discard the first 50-100 mL of buffer (depending on the filter diameter) which contain particles from the dry filter. Buffers should be prepared with HPLC-grade reagents. 2. Pre-filter proteins, if possible, with 0.02 µm Anotop syringe-tip filters. If you wish to see larger species, pre-filter with 0.1 µm syringe-tip filters. Always discard the first few drops from the filtrate, which contain particles from the dry filter (or else pre-wash the filter with buffer) 3. Add an inline filter of 0.1 µm between the buffer pump and injector, and replace the filter membrane regularly. 4. Bypass anything you don't need for MALS analysis, e.g. the conductivity meter. Don't use the same injector for crude samples and analytical samples. 5. All tubing between the column and the detectors should be replaced with 0.25 mm i.d. tubing. 6. Periodically flush the detectors off-line with a protease detergent. If you have an integrated ultrasonic flow cell cleaner (COMET), program it to run after each run or sequence. Hope all the readers get great SEC-MALS data! There's nothing like having absolute measurements of oligomer molecular weight or protein glycosylation to really understand what you have expressed and purified.

September 05, 2017 Reply
GE\dansome's profile image

dansome

Hi Pia, those are great tips. Here are some more basic tips for improving SEC-MALS measurements: 1. Pre-filter buffers with 0.1 µm sterile filters into sterile bottles, and refresh buffers regularly. Always discard the first 50-100 mL of buffer (depending on the filter diameter) which contain particles from the dry filter. Buffers should be prepared with HPLC-grade reagents. 2. Pre-filter proteins, if possible, with 0.02 µm Anotop syringe-tip filters. If you wish to see larger species, pre-filter with 0.1 µm syringe-tip filters. Always discard the first few drops from the filtrate, which contain particles from the dry filter (or else pre-wash the filter with buffer) 3. Add an inline filter of 0.1 µm between the buffer pump and injector, and replace the filter membrane regularly. 4. Bypass anything you don't need for MALS analysis, e.g. the conductivity meter. Don't use the same injector for crude samples and analytical samples. 5. All tubing between the column and the detectors should be replaced with 0.25 mm i.d. tubing. 6. Periodically flush the detectors off-line with a protease detergent. If you have an integrated ultrasonic flow cell cleaner (COMET), program it to run after each run or sequence. Hope all the readers get great SEC-MALS data! There's nothing like having absolute measurements of oligomer molecular weight or protein glycosylation to really understand what you have expressed and purified.

September 05, 2017 Reply

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