Student Comments Fall 2008
TLC Lab
Try several different mixtures of solvents to see what effects solvents have on Rf values. The more practice with choosing the right solvent you have, the better it will come out. MS
Try not to move the TLC chamber much after you have placed the gel slide into the solvent. You could splash solvent further up the TLC slide and hurt your results.CA
When spotting the TLC plates, be sure to keep track of what position/order (i.e. first, second, third) that you spot each solvent/compound and the identity of solvent/compound because after you spot the plates and develop them, it is very hard to know what is what after you develop the plates. AG
Make sure you don't use the same capillary for spotting the slides with different compounds. This will save you time and work. PO
Come prepared to the first lab knowing what exactly you are doing and what do the results mean to save you struggle of figuring out what is the next step you need to do. PO
During the TLC lab, be sure to not write on the shiny side of the silica gel.KM
Use only pencil as ink will spread through the slide.KM
Do not use too much solvent for the TLC chamber. KM
To speed up the experiment, you can get two plates ready, and put them into their separate chambersall atonce. Even if these are going slow, you can get another ready to put into another chamber. I would recommend not mixing solvents in the beginning, but seeing howeach solvent reacts withthe compound first, then finding a combination of two that will give you the results you desire. RR
Remember to always use a pencil and not a pen when writing on the silica plate, and as another already stated, label with your pencil on the bottom of the platewhat compoundyou put where. It would be easy to use just one or two letters to differentiate. RR
Try not to put a big dot of compound on the slide, this will result in a larger spot when looking at the end results through the UV light. The results are much more precise and clear when a small dot is placed on the slide. RR
Make sure to have a very small amount of the liquid inside your capillary tubes before spotting your plate because the liquid comes out very fast and it will be hard to make the dot not too large or not too small. I suggest using a piece of paper to get most of the liquid out of the capillary tube before actually using it to spot your plate. NT
The solvent front disappears really quickly, therefore, once you are ready to take out the TLC plate from the developing chamber make sure to have a pencil nearby to mark it or you might need to redo the trial. BL
Do not write too hard with a pencil on the TLC plate becausethe silica will wear out.NNM
Also, use the dull side and not the shiny side of the TLC plate. NNM
When you're looking at your TLC plate under UV light, circle your spots accurately and very lightly (don't press too hard or you will scrape away the ceramic on the slide) in order to get the most accurate measurement of how far your spot moved which will in turn give you a more accurate Rf value. JW
Be careful with the amount of TLC solution at the bottom of the chamber. Some of the containers are not straight at the bottom and you do not want the TLC solution to be above the spotting of the solutions.JSub
Take your time and be patient. Do not take out the TLC plate before it is finished developing or your results will be inadequate. JSub
When doing TLC, remember you can also use Iodine to develop the slide. This is a good way to mark the blots even when you have forgotten to do it earlier. You can keep the slides in a drawer for a permanent record as well. BES
When waiting for your TLC slide to develop do not move the chamber too much, this not only doesn't really speed things up much, but it creates a problem later when you look under UV light at the spots. In my case they were pretty messy and difficult thus to extrapolate an Rf value. DF
When you want to check how far up the solvent front has moved up the slide, put a piece of paper behind the jar, it allows you to see how far up the plate the solvent has moved. JVar
Make sure to put the lid on you developing chamber or the solvent will evaporate. LC
It does not take much solution to fill the capillary tubes. Gently placing the capillary tubeagainst a small amount of solution on a watch plate will put enough solution into the capillary tube for TLC plate spotting. If youdon'tfeel like youwere able to get enough solution onto the TLC plate the first time you spot, you can alwaysgo back for more. Better to spot the plate a couple of times then make the solution spot toolarge the first time!KV
When drawing the starting line you should put it far enough away from the end of the plate. If you put it too close to the end, the solvent may be raised above the line when placing it in the jar. Also it is much easier to put the slide in with the tweezers rather than just putting it in with your fingers. BR
When spotting your plate, be careful not to spot your samples to close together. This will cause the samples to blot under the UV-light, and it will be difficult to obtain an accurate reading of you Rf values. KJ
Make sure that the lid on the TLC developing chamber jar is closed. Also, you should place only one slide at a time in the chamber. It is easier to see the solvent front if only one slide is in the chamber. LM
Part of the difficulty in performing TLC is finding a solvent with the correct polarity to show a measurable separation between different solutes. A solvent that is too polar will cause all of the solutes to travel along the solvent front, and there will be no separation. A solvent that is not polar enough will cause the solvents to remain at the starting line and will also show no separation. It was found that maximum separation was achieved with a solvent of moderate polarity that didn't drastically vary from the polarity of the solutes. AM
Be sure to keep track of the time that the plate is in the chamber otherwise the solvent will run completely up the plate and you'll have to re-do your TLC. Also, wait until the plate completely dries before observing under the UV light. When dotting, be sure to wait until the dots completely dry before dotting over the pre-existing dot on the plate. Don't forget to sketch pictures of your slides with Rf values and record colors of solutions used. SF
Be sure to use a pencil when marking the TLC plate (slide). The use of a pen when marking your starting and finishing line can disrupt your results. i.e. the ink from the pen actually slides on the plate and is observed under the UV lamp. Thereby, causing the chance of it mixing with any solvents on the slide. NMR
Carefully label candidate 1, your assigned unknown number, and candidate 2. Make sure that you remember which is which when you plate them as well. It is easy to forget which sample was spotting, so write very small corresponding letters or numbers next to them very small. KJ
Review the prelab order of polarity chart before class, because you will have to determine your experimental order of polarity. If you are unsure exactly how to arrange in order of polarity from experimental data, the lab will be long drawn out and difficult. KJ
When you take your plates out of the solvent make sure to grab the plate by the side and be gentle with the side that has the powder. You don't want to accidentally scratch you plate. Also remember to pay attention to where your solvent front stops, if you don't this can mess up your Rf calculations and values. LC
A good thing to note is to not to spot your sample more than a few times. If you spot it more then when you observe silical gel slide under UV the spot runs into the spot with standard and the result cannot be comprehended. VP
The smaller developing chambers work better than the larger ones. Also, only a very small volume of solvent is actually needed- if its too deep in the chamber it can cover the spotted area of the slide. A brief note in the lab instructions noting this would be helpful. MS
What does Rf stand for? JBF
After performing a Google search and doing a bit of online reading I found that Rf stands for "Retention factor."
References:
NL
Does Thin Layer Chromatography distinguish compounds by their varying polarities only, or are there other characteristics that may also be important? JBF
What provoked the caffeine travel up the TLC plate? JVil