I am using Biotage Isolera one. There are different columns available based on amount of stationary phase. Does anyone know/have a chart on maximum limit of crude sample that can be loaded into the columns?
Hi!
Competitor disclosure: Please note that I work for Teledyne Isco, which also makes flash chromatography equipment and columns. However, the information is the same for all flash chromatography equipment. I will give you a lengthy reply, please forgive me.
The answer is it depends on the resolution of the compound you want from the nearest impurity. As you load more sample, the peaks tend to get wider. A simple explanation for this is that the first molecules that go onto the column get adsorbed at the top of the column; eventually, all the "active sites" are occupied and compounds that are loaded on the column later have to go further down the column to get adsorbed. This causes the peaks to get wider. If the peak gets wide enough, it overlaps another peak. There are other factors that affect peak loading, too that will be mentioned later.
The easiest way to determine sample loading is to run a TLC plate. After running the TLC plate, you have to determine the difference in column volumes between your desired compound, and the nearest impurity. Calculate the retention factor for each spot (Rf). To calculate the column volume (CV), take the inverse of the Rf (CV = 1/Rf). Subtract one CV from the other. The number of CVs calculated is the time it takes for a sample to move down a column with the same solvent composition used for the TLC plate. An CV = 1 (Rf = 1, spot at the TLC solvent front) means the compound is not retained, and will elute at the column "void volume); an Rf of 0.2 means the compound elutes at 5 CV. Once you calculate the difference in CV between the spots, this link will show you a loading table-use the percent loading to determine the loading for whatever columns you use (10% loading on a 10 g column is 1 g sample)
https://www.teledyneisco.com/en-us/liquidChromatography/Chromatography%20Documents/Datasheets/Flash%20Column%20Load%20Card.pdf
Note that the sample loading technique will affect loading too. These links below show how the sample loading solvent and loading technique affects compound resolution in flash chromatography. Although written for Teledyne Isco products, it applies ot Biotage as well:
https://www.teledyneisco.com/en-us/liquidChromatography/Chromatography%20Documents/Application%20Notes/RediSep%20Rf%20C18%20Flash%20Column%20Loading%20Techniques%20App%20Note.pdf
https://www.teledyneisco.com/en-us/liquidChromatography/Chromatography%20Documents/Application%20Notes/Overview%20of%20Silica%20Column%20Sample%20Loading%20Techniques%20App%20Note.pdf
Please look at the following below links which may help you in your analysis:
https://biochemmack.ru/upload/iblock/45f/45f1c2b05c9993d4e1c3c7e73df62dee.pdf
https://www.biotage.com/product-page/biotage-flash-75-system
Thanks
Jack Silver I really appreciate your kind efforts in explaining the concept. It's easy to understand and to the point. However, I have one question regarding calculation of CV. Let's say the Rf1=0.3 and Rf2= 0.2. So the difference in CV i.e. ΔCV will be 1.67. In the given chart, to which category it falls into? (I mean easy, difficult etc.) Because all the columns in the chart mentioned say ΔCV=6. Thats quite confusing for me.
Once again thanks for your detailed answer.
Isam Eldin Hussein Elgailani Thanks for your inputs. The links you posted here seems to be related to industrial category cartridges (probably for large scale purifications?).
Vikram Thimaradka - thanks for noticing that; I've notified the people who run our web site.
1.67 CV isn't a large amount of resolution for flash chromatography, so I would limit the loading to around 2% for the example that you gave me.
Jack Silver Could you please update the link for sample loading table?
- Badges
- Science method