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.
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The methods, applications, and benefits of the Kemtrak DCP007 include the following:
• ICUMSA Methods GS 1/3-7, GS 2/3-10 and GS 2/3-10 • Determination of the solution colour of white, brown, and raw sugars as well as coloured syrups • Decolourisation of glucose syrups • A charcoal filter alarm • Manufacturing quality and control • Real time in-line continuous measurement • A zero maintenance LED light source that never needs replacing. What is the ICUMSA Colour Scale and how is it used in sugar production? The International Commission for Uniform Methods of Sugar Analysis (ICUMSA) describes a range of methods for the colorimetric determination of filtered sugar suspensions at known concentrations (Brix values). The ICUMSA colour scale is used to measure the grade and quality of the sugar. The colour of sugar directly relates to the degree of refining – raw sugars being dark brown in colour whilst highly refined sugars are white in colour. The ICUMSA colour scale is a measurement of the yellowness of the sugar resulting from residual molasses not removed in the refining process and can be used to monitor and control the manufacturing process. How can the Kemtrak DCP007 be applied to this process? The Kemtrak DCP007 process photometer has a high-performance long-life LED light source, precision optical filters and robust fibre optics that results in an ICUMSA colour analyser with outstanding performance and reliability. The Kemtrak DCP007 process photometer is recommended to accurately measure ICUMSA colour. The Kemtrak DCP007 employs proprietary dichromatic measurement technology that compensates for particulates, allowing accurate colour measurement without the need for filtration. Due to the proprietary dichromatic four channel measurement technology, particulates in the process media can be compensated for in real time providing an accurate measure of colour without the need for filtration. A primary “absorbing” wavelength then accurately measures colour changes in the process medium, while a second reference wavelength, which is not absorbed by the process medium, compensates for particulates and/or fouling of the optical windows. Since optic fibres are used to transfer light to the measurement point and back, the measurement cell contains no electronics, moving parts or sources of heat that result in condensation on the optical surfaces. Standard measurement cells are machined in sanitary grade stainless steel with sapphire windows. How should I set up and configure the Kemtrak DCP007 for my own work? ICUMSA recommend the absorption of light at 420nm for white and light-coloured sugars and 560nm for darker sugars. 720nm is recommended for the reference wavelength to measure and compensate for the turbidity of the solution. The Kemtrak DCP007 process photometer will accurately measure ICUMSA colour for a known concentration sample using the Brix value (degrees Brix or °Bx). ICUMSA colour is calculated as follows: ICUMSA Colour = 1000 × As/b c Where: As = absorbency of the solution (DCP007 primary measurement) b = the optical path-length (cm) c = concentration (g/mL) (using the Brix value). Colour score is expressed in RBU (reference base units) per ICUMSA standard method(s). Where an end user specific base reference is used, the DCP007 can be adjusted accordingly. The process Brix value, measured using a separate density or refractive index analyser, is input into the Kemtrak analyser through a 4-20mA analogue input to correct for differences in sugar concentration. As an alternative where a live density measurement is not available, process Brix values can be manually entered into the analyser. The Kemtrak DCP007 should be configured for the desired measurement range for maximum resolution and accuracy. An optical path-length of at least 10 cm or more is recommended for low colour white sugars, whilst shorter path-lengths are necessary for darker sugars. Please contact us directly for specific configuration details regarding measurement wavelength and selection of optical path-length for the desired measurement range. Where can I find out more about the Kemtrak DCP007? Head over to our dedicated webpage on the Kemtrak DCP007 for more information on this popular instrument. To discuss your requirements or for a free, no-obligation quote, please call us on: 01442 876777 or email: [email protected]. |
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