The Fundamentals of Chromatography
In this free online course, learn the interactions and solutions involved in the fundamentals in chromatography.Publisher: NPTEL
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When you study the fundamentals of chromatography, you will explore the fact that it is a technique that allows scientists to separate molecules of interest from a crude mixture. The purpose of chromatography is to separate a complex mixture into individual components exploiting the partition effect which distributes the molecules into different phases, will also be discussed. You will find out that in cation exchange chromatography, the matric has a negatively charged functional group with an affinity towards positively charged molecules. You will be introduced to the concept of hydrophobic interaction chromatography (HIC), which exploits the ability of strong interactions between the hydrophobic group attached to the matrix and hydrophobic patches present on an analyte such as a protein. You will learn that in the presence of a high amount of salt, the water molecule shielding the protein side chains, are displaced completely with an exposure of hydrophobic patches on the protein surface to induce protein precipitation or a reduction in protein solubility. You will discover that the addition of a low amount of salt to the protein solution results in the displacement of bonded water molecules with an increase in protein solubility. Demonstrations, problems, examples and assessment questions in this instructor-led video-based course, are designed to provide you with a comprehensive foundation in chromatography.
The material introduces you to the mechanisms of separation in chromatography and the different physical properties that can be used to separate molecules. Then, the proteins will be discussed, which have minimum solubility in solutions with a pH that corresponds to their Pi and they often precipitate out of solution. You will explore the fact that the molecular weight and size of a protein is related to the shape of the molecule and the relationship between its molecular weight and the radius of gyration. You will discover the fact that the retention time refers to the time it takes for an analyte to come out from the column. It will be clarified that the two major forms of chromatography are partition chromatography and adsorption chromatography. You will discover that in column chromatography, a stationary phase is filled into a cylindrical tube made up of glass or steel. You will also learn that the plot of the elution volume, along with the absorbance, is known as a chromatogram. You will find out that the term resolution, refers to the ability of a chromatography column to separate two analyte peaks from one another.
Next, you will learn that for the fast protein liquid chromatography (FPLC) instrument the plastic columns are the most appropriate to use, whilst for the high-performance liquid chromatography (HPLC) instrument the stainless still columns are the most appropriate to use. You will gain insight into the fact the size of the loop depends on how much protein you want to inject and the column capacity. You will then discover that the four properties of a protein molecule are the charge, surface area, affinity and its hydrophobicity. You will be taught that the different chromatography techniques that you can use to exploit the properties of a protein molecule are ion-exchange chromatography (IEX), HIC, gel filtration chromatography and affinity chromatography. You will discover that when preparing matrix beads for chromatography, the first step is to swell the medium, followed by the removal of very small particles and the last step is the equilibration with counter ions. Finally, you will study chromatography techniques, column connections, cation exchange chromatography buffers, and methods of analyzing eluents. This course will be of interest to biotechnology students, or those professionals in experimental biotechnology who want to hone their technical knowledge.Start Course Now
Upon successful completion of this course, you should be able to:
- Discuss the mechanism of separation in chromatography
- Explain the different precautions to take while using the chromatography machine
- Describe the properties of a protein molecule
- Outline the ion-exchange chromatography purification steps
- Explain the popular hydrophobic interaction column matrices
- Describe the two methods of analyzing eluents
- Explain the different ways of eluting an analyte from the hydrophobic interaction column
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