TLC plates do not have long stationary phases. Therefore, the length of separation is limited compared to other chromatographic techniques. Also, the detection limit is a lot higher. If you would need a lower detection limit, one would have to use other chromatographic techniques. TLC operates as an open system, so factors such as humidity and temperature can be consequences to the results of your chromatogram.
Retention Factor After a separation is complete, individual compounds appear as spots separated vertically. Apparatus Plates Stationary Phase As stated earlier, TLC plates also known as chromatoplates can be prepared in the lab, but are most commonly purchased. Solvent Mobile Phase Proper solvent selection is perhaps the most important aspect of TLC, and determining the best solvent may require a degree of trial and error.
How fast the compounds travel up the plate depends on two things: If the compound is soluble in the solvent, it will travel further up the TLC plate How well the compound likes the stationary phase. If the compound likes the stationary phase, it will stick to it, which will cause it to not move very far on the chromatogram. Useful Solvent Mixtures A solvent that can be used for separating mixtures of strongly polar compounds is ethyl acetate : butanol : acetic acid : water, Pipettes Spots are applied to the plate using very thin glass pipettes.
The capillary should be thin enough to apply a neat spot, but not so thin as to prevent the uptake of an adequate quantity of analyte. Here is a popular method of producing TLC pipettes. Heat a glass capillary in the very tip of a Bunsen burner flame just until it becomes pliable and then pull the ends apart until the center of the capillary is significantly narrower. Snap this in half and use the thin end to apply spots. Cut the plate to the correct size and using a pencil never ever use a pen , gently draw a straight line across the plate approximately 1 cm from the bottom.
Do not use excessive forces when writing on a TLC plate as this will remove the stationary phase. It is important to use a pencil rather than a pen because inks commonly travel up the plate with the solvent. An example of how black ink separates is shown in the section labeled "examples". Using TLC pipettes, apply spots of analyte to the line.
Make sure enough sample is spotted on the plate. This can be done by using the short-wave UV. A purple spot should be seen. If the spot is not visible, more sample needs to be applied to the plate. If a standard of the target compound is available, it is good practice to produce a co-spot by spotting the standard onto a spot of the unknown mixture. This ensures the identity of the target compound.
Place the plate into the chamber as evenly as possible and lean it against the side. Never allow the bulk solvent to rise above the line you drew. Allow capillary action to draw the solvent up the plate until it is approximately 1 cm from the end. Never allow the solvent to migrate all the way to the end of the plate. Remove the plate and immediately draw a pencil line across the solvent front. Use a short-wave UV light and circle the components shown with a pencil.
The sequence involved in TLC. Visualizing If fluorescent plates are used, a number of compounds can be seen by illuminating the plate with short-wave UV. Over-large Spot s: Spotting sizes of your sample should be not be larger than mm in diameter. The component spots will never be larger than or smaller than your sample origin spot. If overlapping occurs, it would prove difficult to resolve the different components. Consequences would be inaccurate R f values due to the uneven advance of sample origin spots.
The different compounds in the sample mixture move through the stationary phase at different rates, due to the different attractions for the mobile an stationary phases. Thus, individual compounds in the mixture separate as they move through the stationary phase. The separate compounds can be collected or detected, depending on the particular chromatographic technique involved.
In, TLC, capillary action allows a liquid mobile phase to ascent a solid stationary phase coated on a support plate. A sample of the compound mixture is applied near the bottom of a dry TLC plate, as shown in Figure 1 a.
The plate is placed into a developing chamber, a covered container with a shallow layer of mobile phase liquid in the bottom see Figure 2. As the mobile phase ascends the plate, the mixture compounds dissolve in the mobile phase to different extents, due to the differences in their relative attractions for the mobile and stationary phases.
After the separation si complete, the TLC plate is called a chromatogram, as shown in Figure 1 b. Figure 2. Developing Chambers. During the TLC process, the solid stationary phase, called the adsorbent, adsorbs the mixture compounds.
As the mobile phase, called the eluent, travels up over the adsorbent, the compounds within the mixture move at different rates. A reversible and continuous competitive attraction between the eluent and the adsorbent for the mixture compounds causes this rate difference.
Compounds with less attraction for the adsorbent move rapidly with the eluent. Compounds with the more attraction for the adsorbent move slowly with the eluent. Because TLC adsorbents are typically very polar, the more polar is a compound in the mixture, the more strongly it adheres to the adsorbent and the more slowly it moves.
Similarly, intermolecular attractions between the eluent and the compounds determine the solubility of the compounds in the mobile phase. In general, the more polar the eluent, the more rapidly agiven compound moves. FAQ : Do I have to use two solvents for my eluent? FAQ : Which solvent s do you choose for your eluent? What do I do now? FAQ : Which is the developing solvent? During chromatography, a mobile phase eluent distributes the compounds present in a mixture over a stationary phase adsorbent.
Thin Layer Chromatography TLC is one type of chromatography, where: - the mobile phase is a solvent or mixture of solvents eluent , - the stationary phase is a solid adsorbent e. First, the TLC plate is prepared. The unknown solid is applied as a solution by dissolving it in a solvent. Some solution is drawn up into a micropipette, then applied to the adsorbent near one end of the TLC plate baseline by allowing the solution to wick out onto the adsorbent.
This process is generally referred to as "spotting your TLC plate". The chromatogram is then developed by placing the TLC plate into a TLC chamber a beaker containing the mobile phase, i. The eluent will travel up the adsorbent by capillary action to the top of the TLC plate known as the solvent front , carrying the sample with it.
This process is generally referred to as "running your TLC plate". Depending on the relative polarity of the sample and the eluent, the sample either adsorbs to the solid phase or is eluted by the mobile phase eluent.
If the eluent is very polar relative to your compound, it will dissolve your sample and the sample will move with the mobile phase. Overall, the eluent and your sample will compete for a space an active site on the adsorbent stationary phase coated on the TLC plate. The more polar compound will win this competition and adhere adsorb to the adsorbent, while the rest of the mixture will move. The more polar the compound, the more it will adhere to the adsorbent and the smaller the distance it will travel from the baseline, and the lower its Rf value.
Eluent : the solvent or mixture of solvents mobile phase used to develop a TLC chromatogram plate. Elution : the overall process of developing a TLC plate. Rf retention or retardation factor : is a measure of the distance travelled by the compound spot in relation to the distance travelled by the eluent.
A desirable Rf value lies between 0. First a TLC plate is prepared by spotting the purified unknown and an authentic sample of each possible compound. Then the TLC plate is developed.
For the next step co-spotting , an authentic sample of the compound closest in Rf value to the unknown is chosen. TLC co-spotting of a second plate allows for preliminary identification of your compound. Three spots are applied to the adsorbent on the baseline of the TLC plate: the purified unknown, an authentic sample, and a co-spot of unknown and authentic sample.
If the developed TLC plate shows only one row of spots, it can be concluded that the unknown has been purifed, and that the unknown is possibly the same compound as the authentic sample. However, because Rf values are relative, not absolute, some compounds may have very similar Rf values. A mixed melting point measurement is needed to unambiguously identify the unknown compound. It is primarily used to determine the purity of a compound.
In simple terms, this value is an indication of how far up a TLC-plate a compound has wandered. A high R f -value indicates that the compound has travelled far up the plate and is less polar, while a lower R f -value indicates that the compound has not travelled far, and is more polar.
This value is easily calculated by measuring the distance the spot has wandered, and dividing this by the distance the solvent has traveled. The R f -value is dependent on both the compound and the solvent used for development. If we analyze two compounds and they give the same R f -value with the same solvent system, the two compounds are most likely identical.
The apparatus for doing a TLC-experiment is very simple. We need a developing chamber where the mobile phase is kept. The chamber should be sealed, and although there are special TLC-chambers one can use, a beaker covered by a watch glass to create a seal often suffices. We will discuss two common ways to use a TLC- analysis.
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