 The versatility and efficacy of chromatographic techniques have made them essential in both large scale and analytical separation.
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. How is the Kemtrak DCP007 Photometer used for chromatography product detection? Chromatography is used for protein separation and purification in biotechnology and can require one or more chromatography steps during downstream processing. Separation through chromatography requires specialized equipment to ensure maximum yield and purity. A Kemtrak DCP007-UV photometer is optimally designed for in-line real time measurement and control, allowing the detection of a protein rich solution to be diverted for collection. With an optional NIST Validation accessory mounted it will allow the user to do a validation of instrument in-line in a simple way. How does the Kemtrak DCP007 Photometer help with measuring protein levels? Advances in bioprocessing methods have led to protein products being expressed and concentrated to higher and higher levels. Higher protein concentrations mean higher absorbance and therefore, UV analysers need the ability to measure accurately at these higher absorbance levels. While absorbance measurement is essentially linear, many traditional instruments use light sources that generate a broad spectrum of wavelengths that cause stray light to enter the measurement cell resulting in a non-linear response to protein concentration, particularly at high levels. Also, traditional light sources are sensitive to temperature, generating a lot of thermal energy, and constantly drift due to deterioration. This makes them unsuitable, particularly for fragile protein molecule work. How do I install the Kemtrak DCP007 for post-column chromatography monitoring? The Kemtrak DCP007-UV analyser will deliver accurate, reliable, and repeatable post column UV and are a minimum requirement during chromatographic separation to ensure good protein fraction purity and maximum yields. With the unique Kemtrak zero dead volume measurement cell installed at the column outlet, the Kemtrak DCP007-UV analyser can provide single or dual wavelength UV absorbance analysis, in real time, for improved control of the separation process. With this, the need for offline testing and manual analyses is greatly reduced. Furthermore, the use of solid-state light sources provides the ability to measure up to 4.5 Au at 280 nm and to monitor high concentration processes up to 90 OD linearly without “peak clipping” and the undesirable effects of “hot” and powerful UV light sources on the product. Therefore, the Kemtrak DCP007-UV is the best analyser for chromatographic separation. For more information about how the Kemtrak DCP007 can assist with your chromatography, please contact us at: [email protected] or call: 01442 876777.
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Recently, we looked at how the Kemtrak DCP007-NIR can be used to improve chromatographic separations. In this week’s post, we analyse how this popular and diverse instrument can also be used to improve solvent concentration control.
The versatility and efficacy of chromatographic techniques have made them essential in both large scale, and analytical separations. Chromatography is widely used in bio-processing to separate protein molecules from concentrated process fluids. The centre of a chromatography system is the column, filled with a media specific to the separation to be carried out. How is the technique applied? Solvent gradient control can significantly enhance peak separation and the process economy of chromatography applications. An in-line quantitative sensor permits on-line mobile phase assessment, which with controlled feedback can guarantee accurate and reproducible results. Controlled gradients minimize the effects of changing properties of the stock solvents. How was the instrument installed? Pre-Column chromatography monitoring: The Kemtrak DCP007-NIR mounted in-line before the chromatography column, continuously monitoring the solvent concentration, gives the user control over the composition delivered to the chromatography column. The online monitoring of solvent composition is more significant than measuring physical parameters like mass flow alone. As It allows the generation of batch reports that confirm the actual gradient composition and inhibits the incorrect use of a solvent. The Kemtrak hygienic measurement cells available with FDA and UPC VI approved materials, convenient zero dead volume design; contain no electronics or moving parts for ease of use. There are two versions of the Kemtrak DCP007-NIR currently available: DCP007-NIRL (850 – 1550 nm) for measurement of 0 - 100% water and solvent gradients. DCP007-NIRH (850 – 2000 nm) for trace water and hydrocarbon detection. This model incorporates a multiple stage Peltier cooled and temperature regulated photodiodes and NIR LED light source for the very best in stability and performance. For more information about this instrument and how it can help you with your chromatographic processes, please call us on: 01442 87677 or email: [email protected], where we will also be happy to provide you with a free, no obligation quote. 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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