Desalting and buffer exchange

Desalting and buffer exchange

Desalting refers to the separation of soluble macromolecules such as proteins and nucleic acids from salts and other small molecules. Buffer exchange is the replacement of buffer systems with appropriate ones to allow efficient downstream processing or use in the final application, such as an assay. Based on gel filtration chromatography techniques, the same resin can be used for both purposes. Buffer exchange and desalting involves the recovery of macromolecular components of samples in the buffer used to equilibrate the gel filtration matrix prior to loading the sample.

Common applications of desalting and buffer exchange in research and biologic manufacturing include:

• Removing salts from protein samples
• Removing unincorporated nucleotides or phenol from nucleic acids
• Removing excess labeling or crosslinking reagents from conjugated proteins

Desalting column buffer exchange transfer prepares samples for downstream applications, such as:

• Affinity chromatography
• Ion exchange (IEX) chromatography
• Electrophoresis

Desalting and buffer exchange principles

Buffer exchange and desalting techniques use exclusion limit and molecular weight cut-off (MWCO) for efficient molecule separation. Molecules that are smaller than the MWCO can diffuse into the resin pores, whereas large molecules remain in the void volume of the desalting column.

The chromatography columns have adequate length and volume, which allows the separation of large molecules from salts and small molecules. To ensure target molecules elute in the void volume, pack desalting columns with resins that have tiny pores. Typical buffer exchange and desalting applications require resins with MWCO ratings of 5 to 10 kDa. Other applications, like peptide separation, use resins with larger exclusion limits.

Desalting columns FAQs

Below are answers to some frequently asked questions about desalting columns.

What are desalting columns used for?

Desalting columns can be used with proteins and nucleic acids. They allow quick and easy purification of biomolecules from smaller molecules and salts. In research labs, disposable columns are often used for small sample volumes. One application is to remove excess nucleotides and enzymes after an enzymatic reaction followed by phenol extraction.

How do you use a desalting column?

When using a desalting column, you can recover the large molecule of interest in the buffer used to pre-equilibrate the gel filtration matrix. In research labs, spin columns that separate by centrifugation are common. Another option is to use gravity columns, sometimes called drip columns. For biopharmaceutical applications, an automated chromatography system and appropriate column are typically used.

What is the difference between desalting buffer exchange and dialysis?

Desalting buffer exchange and dialysis both separate based on size and are useful in similar applications. However, gel filtration only takes a few minutes and is faster than hours-long dialysis techniques. Desalting buffer exchange allows the removal of contaminants from relatively small-volume samples. Dialysis requires that a sample be placed inside tubing or cassette. Sometimes multiple buffer exchanges are needed in the container that houses the dialysis unit.

How do you spin a desalting column?

You can spin desalting columns using standard centrifugation or a microcentrifuge. Spin columns are available in varying sizes to accommodate different sample volumes.

What is gel filtration?

Gel filtration is a size-exclusion chromatography (SEC) technique that allows the separation of components based on their difference in molecular weight or size.

How do I optimize protein desalting?

This article offers some tips on protein desalting.