GEL FILTRATION CHROMATOGRAPHY (Size Exclusion Chromatography OR Gel permeation chromatography)
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- čas přidán 8. 07. 2024
- 0:00-1:15 | What is size exclusion chromatography (SEC)?
1:15-2:28 | How does SEC work?
2:28-3:34 | Why is SEC useful?
Size exclusion chromatography can be shortened as SEC and is also known as gel filtration chromatography. It is used for protein purification and separates molecules based on their size by filtration through a gel. This gel consists of spherical beads made up of pores of specific size distribution. The separation depends on which molecules can enter the pores in these beads. Small molecules diffuse into the pores and their flow through the column is therefore slowed down, while larger molecules do not enter at all and are therefore able to move at a quicker pace through the column. To me at least, this was at first counterintuitive but this happens because of how these beads are constructed, forcing the molecules that enter to subsequently take a slower route. Similarly to how even a small maze will slow you down significantly due to how it prevents you from taking a straight route.
A good analogy is that of ants and beetles traveling through grass. Due to their size, ants need to travel around the grass making their path longer while beetles can just move through the grass by pushing it to the side. Subsequently, molecules are separated based on their size and are eluted in order of decreasing molecular weight (MW) or in huga-buga, BIG FIRST, SMALL LAST!
The workflow of size exclusion chromatography can be divided into 4 main steps:
First the sample is inserted into the column.
Second, all proteins present in the sample collect at the top.
Third, with time, larger proteins move quicker through the column while smaller proteins keep getting stuck inside the gel beads, slowing them down
Fourth and finally, the largest protein will exit first, followed by second largest, and so on and so forth. Then as they exit, it is simply a matter of collecting each protein as its exits the column.
So as I mentioned, size exclusion chromatography is used in protein purification, often as a second step, used after affinity chromatography. (Btw, if you want to check out my video on affinity chromatography it is linked after this one) Okay, getting back to the topic. This is why size exclusion chromatography is so useful. By separating proteins based on size, it can be utilized for desalting, simply meaning that we remove salts and other small molecules from the sample. In addition it can also be used for fractionation, again since molecules of varying weights are separated within the gel matrix.
If you have any questions, don't hesitate to ask me in the comments!!😇👍
It's so cool that I just discovered your channel, while I'm studying for my biochemical analytics exam, and all your new videos are about my exam topics :)
That's really great to hear! Let me know if there are other topics you want me to cover in the future😇👍
@@biotechlucas4126 sure, OUR, OTR, DO would be cool. In general fermentation, for extracellular and intracellular products.
I just had a course all about it! I found it quite challenging but I'll do my best to explain it in a video soon! 👍
Excellent video! I have a question regarding the techniques prior to SEC. Would it also be compatible to develop an HPLC-TOF-MS method to characterize an antibody and then purify it using SEC?
Yes, at least to me that sounds like a good idea!
The mass spectrometry will give you the relative mass values which I believe you can use to determine the "pore size" of the beads of the size exclusion chromatography.
However, I might be wrong as I'm not super familiar with the more practical aspects of size exclusion chromatography.
Will some of the small molecules still escape with the bigger one?
Interesting question. I must admit I don't know for certain but I'm sure as with almost all things in life that it's not a 100% perfect system.
So yes, I do believe that happens sometimes.
1:47 hahhahahah