In this week’s blog post, we discuss how Kemtrak’s popular photometer can be used to improve the efficiency of chromatographic separation processes.
What are chromatographic techniques?
Chromatography is widely used in bioprocessing to separate protein molecules from concentrated process fluids. The centre of a chromatography system is a column, filled with a media specific to the separation being carried out.
The versatility and efficacy of chromatographic techniques have made them a crucial part of both large scale and analytical separations.
The current methods of chromatographic separation are as follows:
Gel filtration – refers to sorting the material by molecular size. The physical dimensions of the molecule determine the separation because proteins are naturally spherical. This means the molecular weight of the protein will be proportional to its size, allowing for separation based on this property. Larger molecules pass more slowly through the column media, while smaller molecules pass through more quickly.
Ion exchange – refers to sorting the material by electrical charge. Separation works by opposite charge attraction and like charge repulsion. Using a specific charge within the column media allows for attraction and binding of the molecule(s) of interest.
Hydrophobic interaction – refers to sorting the material by degree of Hydrophobicity. Separation relies on polar (hydrophilic) molecules “sticking” together and repelling those which are non-polar (hydrophobic).
Affinity - this method uses a bio-specific binding site. This is a section of a molecule in which the shape and distribution of charged and hydrophobic groups allow for highly specific binding to a corresponding site on another molecule. The fit between the two sites is analogous to a lock and key.
How does chromatography work?
Chromatography is a process for separating components of a mixture. To start the process, the mixture is dissolved in a substance called the mobile phase. This then carries it through a second substance called the stationary phase.
The different components of the mixture travel through the stationary phase at different speeds, causing them to separate from one another. The nature of the specific mobile and stationary phases determines which substances travel more quickly or slowly, and this is how they are separated. These different travel times are known as retention times.
A chromatography gel is designed to have one half of this lock and key (the ligand), making it stationary within the column. As the product solution is passed through column, the specific mating molecules are bound in place until they are eluted from the column.
Examples of affinity interactions include the binding between antibodies and antigens, and enzymes and substrates.
The molecular properties utilised in chromatographic separation can be seen in the figure below:
Separation through chromatography requires highly specialized equipment to ensure maximum yield and purity. A system may include a variety of instruments and sensors for pre - and post - column control.
Nearly all proteins absorb UV light at 280 nm, the primary reason for this is due to the aromatic amino acids, e.g. phenylalanine, tryptophan, tyrosine, and histidine.
How does installing a Kemtrak DCP007 help improve chromatographic processes?
Installing a Kemtrak DCP007-UV photometer, measurement cell at the column outlet, allows the presence of proteins to be detected, and therefore collection/pooling to begin.
It is vital that any UV analyser has no dead or hold up volume to ensure crisp, sharp peaks are detected. UV analysers utilizing measurement cells with internal hold up volumes can lower the purity of the collected protein because of dilution. Dilution blurs sharp peak detection lines, and can cause lower yields.
Using a Kemtrak DCP007-NIR photometer, mounted pre-column to determine solvent concentration/composition, allows for a feedback control of the solvent “mobile media” feed through the column, and increases the accuracy and repeatability of the chromatography system.
With the unique zero dead volume Kemtrak measurement cell installed, the Kemtrak DCP007 analyser can provide single or dual wavelength absorbance analysis, in real time, for improved control of the chromatographic separation processes. Therefore, the Kemtrak DCP007 is the recommended analyser for chromatographic separations.
To find out more about the Kemtrak DCP007 (UV or NIR) Photometer, please visit our website: or call: 01442 87677 to discuss your requirements further. We will also be happy to provide a free, no obligation quote.
October 2016 Fullbrook Systems Ltd move to new premises in Hemel Hempstead. After being in the same offices for many years the company moved to more suitable premises