Chemical Forums
Chemistry Forums for Students => Organic Chemistry Forum => Organic Chemistry Forum for Graduate Students and Professionals => Topic started by: jvinlim on March 23, 2024, 10:46:22 AM
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Here’s the brief:
Firstly, I have never performed column chromatography by myself before. I need to synthesize a compound for my research in Bachelor's degree. After completing a reaction, I conducted TLC. On the TLC plate, before column chromatography, there were 3-4 spots, indicating the presence of 2-3 other compounds besides my target product.
So, I performed column chromatography to purify and separate my product from the mixture. I collected a total of 45 fractions, each with a volume of 2 mL. Here’s a summary of my fraction results:
- Fractions 1-11: Colorless
- Fractions 12-16: Light yellow
- Fractions 17-23: Colorless
- Fractions 24-45: Light yellow (slightly more yellow than fraction 12-16)
My questions:
- Given the distribution of my fractions, do I need to perform TLC on all of them? I want the result to be good but at the same time avoid using too many TLC plates. Is there an effective yet efficient method?
- How do you determine when to stop collecting fractions? I found myself stopping somewhat abruptly as I ran out of time towards the end of the 45 fractions (it was 4:45 PM, and my lab closed at 5:00 PM) and it seemed like the column would never finish.
- Is there anything wrong with my choice regarding column length, amount of silica gel, etc.?
Feel free to write other advice you've for me ;D
P.S:
- My mixture is about 0.39 grams
- My column length = 30 cm and the Inner Diameter = 1 cm
- The amount of silica gel that I used = 7.8 grams (I used the 20:1 method, where 20 is the amount of silica and 1 is the amount of my mixture)
- My mobile phase ---> Chloroform : Methanol = 100 : 1
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I would TLC every other fraction or every third and then combine the like fractions.
You stop collecting when nothing is showing up on the TLC plate. I tend to TLC as I go.
Hard to tell without seeing the TLCs if there is something wrong with what you are doing.
You can use a gradient, so I would have gone 100:1 chloroform methanol to 5% methanol as a gradient. It can speed up a column BUT it can also make your separation worse if you are not careful as it can make co-elution worse.
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When there are several spots by TLC and especially when the desired product is similar to another in Rf value, it is often necessary to increase the ratio of silica to crude product. A ratio of 40:1 or 50:1 is not uncommon. One way to cut down on the number of TLC plates is to change the size of the fractions. We often use fractions that are one-quarter of a column volume. For a very difficult separation, I might use one-fifth. If your column volume was 20 mL, then your fraction size could be chosen to be 4 or 5 mL.
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Sorry for maybe too late reply, I hope you were able to solve the column.
Anyway, here is my advice:
1) your column feels way too small for such amount. I prefer to use shorter but wider columns because its faster (you trade number of theoretical plates for their size and since the size has square relation to column diameter unlike the number of plates vs lenght which is linear). I would use 2 or 3 cm diameter and maybe 10-12 cm of silica in the column
2) for such amount, taking 2 ml fractions is overkill. 10 or 20 is good volume here in my opinion - cuts time, glassware use and number of TLCs to do.
3) you dont have to TLC all your fractions, I like to make a grid on TLC plate, check which fractions has SOMETHING and then elute only those containing something. I also run TLC during the column. This way you can figure out when to stop (you obviously stop when your compound of interest is eluted)
4) as one of my advisers told me commonly: "Color is five percent". It is SOME indication but hardly conclusive. Ofc, after you run the same column multiple times you can get more information from the color of fractions ("my compound is eluting AFTER this yellowish band so I can take all the stuff into a beaker before it starts eluting")
5) you can increase polarity of your mobile phase over time to get better separation
6) Based on by experinece, I feel you might need to run more fractions, run TLC on maybe each fifth fractions to get SOME idea but 90 ml of mobile phase doesnt seem to be enough in your case.
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Just some very general advice: We sometimes get fooled regarding which sample is our product and which is something else. My general principle is that if we see it, we pool and save it.
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Sorry for maybe too late reply, I hope you were able to solve the column.
Anyway, here is my advice:
1) your column feels way too small for such amount. I prefer to use shorter but wider columns because its faster (you trade number of theoretical plates for their size and since the size has square relation to column diameter unlike the number of plates vs lenght which is linear). I would use 2 or 3 cm diameter and maybe 10-12 cm of silica in the column
2) for such amount, taking 2 ml fractions is overkill. 10 or 20 is good volume here in my opinion - cuts time, glassware use and number of TLCs to do.
3) you dont have to TLC all your fractions, I like to make a grid on TLC plate, check which fractions has SOMETHING and then elute only those containing something. I also run TLC during the column. This way you can figure out when to stop (you obviously stop when your compound of interest is eluted)
4) as one of my advisers told me commonly: "Color is five percent". It is SOME indication but hardly conclusive. Ofc, after you run the same column multiple times you can get more information from the color of fractions ("my compound is eluting AFTER this yellowish band so I can take all the stuff into a beaker before it starts eluting")
5) you can increase polarity of your mobile phase over time to get better separation
6) Based on by experinece, I feel you might need to run more fractions, run TLC on maybe each fifth fractions to get SOME idea but 90 ml of mobile phase doesnt seem to be enough in your case.
I agree, I use a 30cm column (diameter) for this. But it is usually very nice to have TLC on all fractions, you dont need to speculate and guess what has happened. But its more work.