بسم الله الرحمن الرحيم
طريقة الـTLC Chromatography هي من أسهل الطرق للتحليل.
وبما أن هذه الطريقة مطلوبة منا في التحليلية و جزء الNarcotics .
فأنا جايب لكم شرح وجده في أحد المواقع العلمية للإستفادة.
Procedure for TLC
1- Prepare the developing container
The developing container for TLC can be a specially designed chamber, a jar with a lid, or a beaker with a watch glass on the top
In the teaching labs, we use a beaker with a watch glass on top
Pour solvent into the beaker to a depth of just less than 0.5 cm
To aid in the saturation of the TLC chamber with solvent vapors, line part of the inside of the beaker with filter paper
Cover the beaker with a watch glass, swirl it gently, and allow it to stand while you prepare your TLC plate
2-Prepare the TLC plate
TLC plates used in the organic chem teaching labs are purchased as 5 cm x 20 cm sheets. Each large sheet is cut horizontally into plates which are 5 cm tall by various widths; the more samples you plan to run on a plate, the wider it needs to be
Shown in the photo a box of TLC plates, a large un-cut TLC sheet, and a small TLC plate which has been cut to a convenient size.
Plates will usually be cut and ready for you when you come to lab
Handle the plates carefully so that you do not disturb the coating of adsorbent or get them dirty
Measure 0.5 cm from the bottom of the plate. Take care not to press so hard with the pencil that you disturb the adsorbent
Using a pencil, draw a line across the plate at the 0.5 cm mark. This is the
origin: the line on which you will "spot" the plate
It's kind of hard to see the pencil line in the above photos, so here is a close-up of how the plate looks after the line has been drawn
Under the line, mark lightly the name of the samples you will spot on the plate, or mark numbers for time points. Leave enough space between the samples so that they do not run together, about 4 samples on a 5 cm wide plate is advised
Use a pencil and do not press down so hard that you disturb the surface of the plate. A close-up of a plate labeled "1 2 3" is shown above
3-Spot the TLC plate
The sample to be analyzed is added to the plate in a process called spotting
If the sample is not already in solution, dissolve about 1 mg in a few drops of a volatile solvent such as hexanes, ethyl acetate, or methylene chloride. As a rule of thumb, a concentration of "1%" or "1 gram in 100 mL" usually works well for TLC analysis. If the sample is too concentrated, it will run as a smear or streak; if it is not concentrated enough, you will see nothing on the plate. The "rule of thumb" above is usually a good estimate, however, sometimes only a process trial and error (as in, do it over) will result in well-sized, easy to read spots
add a few drops of solvent
swirl until dissolved
The solution is applied to the TLC plate with a 1µL microcap
Microcaps come in plastic vials inside red-and-white boxes. If you are opening a new vial, you will need to take off the silver cap, remove the white styrofoam plug, and put the silver cap back on. A small hole in the silver cap allows you to shake out one microcap at a time. Microcaps are very tiny; the arrow points to one, and it is hard to see in the photo
Take a microcap and dip it into the solution of the sample to be spotted. Then, touch the end of the microcap gently to the adsorbent on the origin in the place which you have marked for the sample. Let all of the contents of the microcap run onto the plate. Be careful not to disturb the coating of adsorbent
dip the microcap into solution - the arrow points to the microcap, it is tiny and hard to see
make sure it is filled - hold it up to the light if necessary
touch the filled microcap to TLC plate to spot it - make sure you watch to see that all the liquid has drained from the microcap
touch the filled microcap to TLC plate to spot it - make sure you watch to see that all the liquid has drained from the microcap
do this rinse process 3 times
and then draining it by touching it to a paper towel
If the microcap breaks or clogs, you may obtain a new one. The microcaps should be cleaned and re-used whenever possible both because this is an environmentally sound practice and because they are relatively expensive
here's the TLC plate, spotted and ready to be developed
4-Develop the plate
Place the prepared TLC plate in the developing beaker, cover the beaker with the watch glass, and leave it undisturbed on your bench top. Run until the solvent is about half a centimeter below the top of the plate see photos below
place the TLC plate in the developing container - make sure the solvent is not too deep
TLC: Make sure that the solvent does not rise above the level of the origin, or the spots will dissolve off the plate into the solvent
photo shows how the yellow compound is running into the solvent when lifted from the developing jar
The solvent will rise up the TLC plate by capillary action. In this photo, it is not quite halfway up the
plate
In this photo, it is about 3/4 of the way up the plate
The solvent front is about half a cm below the top of the plate - it is now ready to be removed
Remove the plate from the beaker
quickly mark a line across the plate at the solvent front with a pencil
Allow the solvent to evaporate completely from the plate. If the spots are colored, simply mark them with a pencil
5-Visualize the spots
If your samples are colored, mark them before they fade by circling them lightly with a pencil see photo above
Most samples are not colored and need to be visualized with a UV lamp. Hold a UV lamp over the plate and mark any spots which you see lightly with a pencil
Beware! UV light is damaging both to your eyes and to your skin! Make sure you are wearing your goggles and do not look directly into the lamp. Protect your skin by wearing gloves
If the TLC plate runs samples which are too concentrated, the spots will be streaked and/or run together. If this happens, you will have to start over with a more dilute sample to spot and run on a TLC plate
this is a UV lamp
here are two proper sized spots, viewed under a UV lamp
you would circle these while viewing them
The plate to the left shows three compounds run at three different concentrations. The middle and right plate show reasonable spots; the left plate is run too concentrated and the spots are running together, making it difficult to get a good
Here's what overloaded plates look like compared to well-spotted plates. The plate on the left has a large yellow smear; this smear contains the same two compounds which are nicely resolved on the plate next to it. The plate to the far right is a UV visualization of the same overloaded plate
أخر حاجة إزاي نعين الـ(Rf Retention Factor)
The retention factor, or Rf, is defined as the distance traveled by the compound divided by the
For example, if a distance traveled by the solvent compound travels 2.1 cm and the solvent front travels 2.8 cm, the Rf is 0.75
The Rf for a compound is a constant from one experiment to the next only if the chromatography conditions below are also constant
solvent system
adsorbent
thickness of the adsorbent
amount of material spotted
temperature
Since these factors are difficult to keep constant from experiment to experiment, relative Rf values are generally considered. “Relative Rf” means that the values are reported relative to a standard, or it means that you compare the Rf values of compounds run on the same plate at the same time
شرح مبسط بالصور و الفيديو لطريقة الـTLC Chromatography
