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This application note shows use of the ActiPix™ for determination of hydrodynamic radius of a protein. The method allows proteins to be used in their native form, without any labelling or denaturation. A plug of protein solution is injected into a fused silica capillary, driven through the capillary by application of pressure, and detected using UV area imaging as it passes windows at entrance to and exit from a loop in the capillary. The radius of the protein is determined by analysis of band broadening due to Taylor dispersion. The method is applicable over a wide concentration range and uses only nanolitres of sample.
This application note describes how the ActiPix™ UV area imaging detector can be used to test biocatalyst substrate specificity towards a mixture of UV active compounds using a continuous engagement electrophoretically mediated microanalysis (EMMA) assay method.
Substrate specificity screening with UV area imaging detector.pdf
AN004 lab-on-capillary systems
Quantum dots are typically fluorescent nanoparticles of high quantum efficiency (see Figure 1). They can be custom synthesized for a wide variety of applications ranging from medical imaging to next generation LCD displays. Paraytec have developed a new approach to determining the hydrodynamic radius of species in solution. This application note is a proof of principle study to demonstrate the use of the ActiPix™ HT Nano-Sizing System for determination of hydrodynamic radii of quantum dot and gold nanoparticle samples provided by the Physics Department at the University of Leeds. Excellent correlation was obtained between experimental and expected values with typical analysis times of less than 10 minutes. This approach is a significantly faster and more cost efficient approach compared to transmission electron microscopy (TEM) which typically takes several hours to perform this analysis. In addition, conventional particle measuring techniques cannot effectively measure down to sub 20 nanometre sizes.
Paraytec have developed a new approach for real-time measurement of the amount of Ca2+ released from the surface of a bovine tooth sample. This technique is significantly faster and more sensitive than other current techniques. Great potential exists for quick measurement of anti-erosion consumer products.
Paraytec have developed a new approach to measuring the amount of nicotine released from a sustained release dermal patch. Rate of release and insight into membrane transport information are explored.
Surface dissolution imaging offers a rapid means of determining intrinsic dissolution rates (IDRs) of active pharmaceuticals ingredients (APIs). In this study the IDR of griseofulvin was determined in less than 20 minutes.
This application note illustrates use of the ActiPix™ D100 for detection of three peptides (angiotensin II, Metenkephalin and Leu-enkephalin) following separation by nanoLC on a 75 m i.d. column. Using a nanoLC cartridge with detection capillary having i.d. 75 m, results are shown for loadings of 0.5 ng each peptide. The S/N for Leu-enkephalin is 63, suggesting that the limit of detection is below 0.05 ng (80 fmol). Detection is also demonstrated in the 20 m i.d. fused silica transfer line, and with loading of 1 ng each peptide on column the S/N for Leu-enkephalin is 4.4. Peak broadening due to Taylor dispersion in the wider bore capillary causes the peaks to be slightly wider than those measured directly in the transfer line.
This application note illustrates on-column detection using a monolithic capillary column 100 μm i.d./ 360 μm o.d. and isocratic elution. Flow resistance in monolithic columns is much lower than in conventional particle packed columns, and a syringe pump is used to drive the flow. For 1 pmol loading of caffeine and 0.5 s time constant, the signal-to-noise ratio is 23. This shows that the ActiPix™ D100 detector has femtomole sensitivity for on-column detection. A particular benefit of imaging through the column bed with the ActiPix™ D100 is that time-displaced averaging used in Paraytec’s proprietary software removes any random fluctuations in the signal due to non-uniformity of the stationary phase and scattering effects. As shown with a sharply fronting peak of caffeine under overload conditions, this occurs without any sacrifice of the 70 μm spatial resolution.
There is an increasing need within the biopharmaceutical industry for techniques which complement or enhance currently available techniques such as dynamic light scattering (DLS), size exclusion chromatography (SEC) and transmission electron microscopy (TEM) for monitoring the amount and extent of aggregation of therapeutic proteins and monoclonal antibodies. All of the techniques previously mentioned have limitations, e.g. it is not possible to determine size for proteins without dilution, they cannot be used in conjunction with in-line systems, e.g. GE AKTA explorers and also have limited application in the analysis of membrane proteins. This application note outlines results with a test protein, myoglobin, using a technique developed by Paraytec for determining the hydrodynamic radius of species in solution. Repeatability and intermediate precision are reported, and the key factor influencing the accuracy of the method is documented. The technique measures the extent of Taylor dispersion of a plug of sample moving through a buf
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