THIN LAYER CHROMATOGRAPH1.docx

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THIN LAYER CHROMATOGRAPHY

This page is an introduction to chromatography using thin layerchromatography as an example. Although if you are a beginneryou may be more familiar with paper chromatography, thin layerchromatography is equally easy to describe and morestraightforward to explain.

Note: I'm taking a simple view of the way that thin layerchromatography works in terms of adsorption (see below)which should be adequate for students doing courses for 16- 18 year olds. The reality is more complicated and theexplanation will vary depending on what sort of solvent orsolvent mixture you are using. Some similar problems are

discussed on the page about paper chromatography, but Iam unwilling to do the same thing on this page which isintended as a fairly gentle introduction to chromatography.

Carrying out thin layer chromatography

Background

Chromatography is used to separate mixtures of substances intotheir components. All forms of chromatography work on thesame principle.

They all have a s ta t ionary phase (a solid, or a liquid supportedon a solid) and a mob i le phase (a liquid or a gas). The mobilephase flows through the stationary phase and carries thecomponents of the mixture with it. Different components travel atdifferent rates. We'll look at the reasons for this further down thepage.

Thin layer chromatography is done exactly as it says - using athin, uniform layer of silica gel or alumina coated onto a piece ofglass, metal or rigid plastic.

The silica gel (or the alumina) is the stationary phase. Thestationary phase for thin layer chromatography also oftencontains a substance which fluoresces in UV light - for reasonsyou will see later. The mobile phase is a suitable liquid solvent


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or mixture of solvents.

Producing the chromatogram

We'll start with a very simple case - just trying to show that aparticular dye is in fact a mixture of simpler dyes.

Note: The chromatography plate will in fact be pure white -not pale grey. I'm forced to show it as off-white because of

the way I construct the diagrams. Anything I draw as purewhite allows the background colour of the page to showthrough.

A pencil line is drawn near the bottom of the plate and a smalldrop of a solution of the dye mixture is placed on it. Any labellingon the plate to show the original position of the drop must alsobe in pencil. If any of this was done in ink, dyes from the inkwould also move as the chromatogram developed.

When the spot of mixture is dry, the plate is stood in a shallowlayer of solvent in a covered beaker. It is important that thesolvent level is below the line with the spot on it.

The reason for covering the beaker is to make sure that theatmosphere in the beaker is saturated with solvent vapour. Tohelp this, the beaker is often lined with some filter paper soakedin solvent. Saturating the atmosphere in the beaker with vapour


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stops the solvent from evaporating as it rises up the plate.

As the solvent slowly travels up the plate, the differentcomponents of the dye mixture travel at different rates and themixture is separated into different coloured spots.

The diagram shows the plate after the solvent has moved abouthalf way up it.

The solvent is allowed to rise until it almost reaches the top ofthe plate. That will give the maximum separation of the dyecomponents for this particular combination of solvent andstationary phase.

Measuring R f values

If all you wanted to know is how many different dyes made upthe mixture, you could just stop there. However, measurementsare often taken from the plate in order to help identify thecompounds present. These measurements are the distancetravelled by the solvent, and the distance travelled by individualspots.

When the solvent front gets close to the top of the plate, theplate is removed from the beaker and the position of the solventis marked with another line before it has a chance to evaporate.

These measurements are then taken:


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The R f value for each dye is then worked out using the formula:

For example, if the red component travelled 1.7 cm from thebase line while the solvent had travelled 5.0 cm, then the R f value for the red dye is:

If you could repeat this experiment under exactly the sameconditions, then the R f values for each dye would always be thesame. For example, the R f value for the red dye would alwaysbe 0.34. However, if anything changes (the temperature, theexact composition of the solvent, and so on), that is no longertrue. You have to bear this in mind if you want to use thistechnique to identify a particular dye. We'll look at how you canuse thin layer chromatography for analysis further down thepage.

What if the substances you are interested in are colourless?


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There are two simple ways of getting around this problem.

Using f luorescence

You may remember that I mentioned that the stationary phaseon a thin layer plate often has a substance added to it which willfluoresce when exposed to UV light. That means that if youshine UV light on it, it will glow.

That glow is masked at the position where the spots are on thefinal chromatogram - even if those spots are invisible to the eye.That means that if you shine UV light on the plate, it will all glowapart from where the spots are. The spots show up as darkerpatches.

While the UV is still shining on the plate, you obviously have tomark the positions of the spots by drawing a pencil circle aroundthem. As soon as you switch off the UV source, the spots willdisappear again.

Sh o win g th e sp o t s u p ch emica lly

In some cases, it may be possible to make the spots visible byreacting them with something which produces a colouredproduct. A good example of this is in chromatograms producedfrom amino acid mixtures.

The chromatogram is allowed to dry and is then sprayed with asolution of n in h y d r in . Ninhydrin reacts with amino acids to give


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coloured compounds, mainly brown or purple.

In another method, the chromatogram is again allowed to dryand then placed in an enclosed container (such as anotherbeaker covered with a watch glass) along with a few i o d in e crys ta l s .

The iodine vapour in the container may either react with thespots on the chromatogram, or simply stick more to the spotsthan to the rest of the plate. Either way, the substances you areinterested in may show up as brownish spots.

Using thin layer chromatography to identify compounds

Suppose you had a mixture of amino acids and wanted to findout which particular amino acids the mixture contained. Forsimplicity we'll assume that you know the mixture can onlypossibly contain five of the common amino acids.

A small drop of the mixture is placed on the base line of the thinlayer plate, and similar small spots of the known amino acids are

placed alongside it. The plate is then stood in a suitable solventand left to develop as before. In the diagram, the mixture is M,and the known amino acids are labelled 1 to 5.

The left-hand diagram shows the plate after the solvent front hasalmost reached the top. The spots are still invisible. The seconddiagram shows what it might look like after spraying with


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ninhydrin.

There is no need to measure the R f values because you caneasily compare the spots in the mixture with those of the knownamino acids - both from their positions and their colours.

In this example, the mixture contains the amino acids labelled as1, 4 and 5.

And what if the mixture contained amino acids other than theones we have used for comparison? There would be spots inthe mixture which didn't match those from the known amino

acids. You would have to re-run the experiment using otheramino acids for comparison.

How does thin layer chromatography work?

The stationary phase - silica gel

Silica gel is a form of silicon dioxide (silica). The silicon atomsare joined via oxygen atoms in a giant covalent structure.

However, at the surface of the silica gel, the silicon atoms areattached to -OH groups.

Note: If you aren't sure about it, you will find one possiblestructure of silicon dioxide towards the bottom of the pageyou will get to by following this link.

Use the BACK button on your browser to return quickly to
http://www.chemguide.co.uk/atoms/structures/giantcov.html#tophttp://www.chemguide.co.uk/atoms/structures/giantcov.html#tophttp://www.chemguide.co.uk/atoms/structures/giantcov.html#top


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develops?

As the solvent begins to soak up the plate, it first dissolves thecompounds in the spot that you have put on the base line. Thecompounds present will then tend to get carried up the

chromatography plate as the solvent continues to moveupwards.

How fast the compounds get carried up the plate depends ontwo things:

How soluble the compound is in the solvent. This willdepend on how much attraction there is between themolecules of the compound and those of the solvent.

How much the compound sticks to the stationary phase -the silica gel, for example. This will depend on how much

attraction there is between the molecules of thecompound and the silica gel.

Suppose the original spot contained two compounds - one ofwhich can form hydrogen bonds, and one of which can only takepart in weaker van der Waals interactions.

The one which can hydrogen bond will stick to the surface of thesilica gel more firmly than the other one. We say that one isadsorbed more strongly than the other. Adsorption is the namegiven to one substance forming some sort of bonds to the

surface of another one.

Adsorption isn't permanent - there is a constant movement of amolecule between being adsorbed onto the silica gel surfaceand going back into solution in the solvent.

Obviously the compound can only travel up the plate during thetime that it is dissolved in the solvent. While it is adsorbed on thesilica gel, it is temporarily stopped - the solvent is moving onwithout it. That means that the more strongly a compound isadsorbed, the less distance it can travel up the plate.

In the example we started with, the compound which canhydrogen bond will adsorb more strongly than the onedependent on van der Waals interactions, and so won't travel sofar up the plate.


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What if both components of the mixture can hydrogen bond?

It is very unlikely that both will hydrogen bond to exactly thesame extent, and be soluble in the solvent to exactly the sameextent. It isn't just the attraction of the compound for the silica

gel which matters. Attractions between the compound and thesolvent are also important - they will affect how easily thecompound is pulled back into solution away from the surface ofthe silica.

However, it may be that the compounds don't separate out verywell when you make the chromatogram. In that case, changingthe solvent may well help - including perhaps changing the pH ofthe solvent.

This is to some extent just a matter of trial and error - if one

solvent or solvent mixture doesn't work very well, you try anotherone. (Or, more likely, given the level you are probably workingat, someone else has already done all the hard work for you,and you just use the solvent mixture you are given andeverything will work perfectly!)


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OrganicChemistry at CU Boulder

Lecture Courses o 3311-100 (Richardson)o 3311-200 (Asirvatham)o 3331-100 (Ellison)o 3351-100 (Yin) Exam Archives

o 3311 (OChem I)o 3331 (OChem II)o 3351 (OChem I for Majors)o 3371 (OChem II for Majors) Lab Courses

o 3321 (OChem I Lab)o 3341 (OChem II Lab)o 3361 (Majors OChem I Lab)o Cleanup points Lab Technique

o Lab Safetyo Chemical Informationo Lab Equipmento Procedures and Techniques Spectroscopy

o IR Theoryo NMR Theory
http://orgchem.colorado.edu/Lectures/Lectures.htmlhttp://orgchem.colorado.edu/Lectures/3311-100.htmlhttp://orgchem.colorado.edu/Lectures/3311-200.htmlhttp://orgchem.colorado.edu/Lectures/3331-100.htmlhttp://orgchem.colorado.edu/Lectures/3351-100.htmlhttp://orgchem.colorado.edu/Examarchives/Examarchives.htmlhttp://orgchem.colorado.edu/Examarchives/3311.htmlhttp://orgchem.colorado.edu/Examarchives/3331.htmlhttp://orgchem.colorado.edu/Examarchives/3351.htmlhttp://orgchem.colorado.edu/Examarchives/3371.htmlhttp://orgchem.colorado.edu/Labs/Labs.htmlhttp://orgchem.colorado.edu/Labs/3321.htmlhttp://orgchem.colorado.edu/Labs/3341.htmlhttp://orgchem.colorado.edu/Labs/3361.htmlhttp://orgchem.colorado.edu/Labs/Cleanup.htmlhttp://orgchem.colorado.edu/Technique/Technique.htmlhttp://orgchem.colorado.edu/Technique/Safety/Safety.htmlhttp://orgchem.colorado.edu/Technique/Cheminfo/Cheminfo.htmlhttp://orgchem.colorado.edu/Technique/Equipment/Equipment.htmlhttp://orgchem.colorado.edu/Technique/Procedures/Procedures.htmlhttp://orgchem.colorado.edu/Spectroscopy/Spectroscopy.htmlhttp://orgchem.colorado.edu/Spectroscopy/IR/IR.htmlhttp://orgchem.colorado.edu/Spectroscopy/NMR/NMR.htmlhttp://orgchem.colorado.edu/Spectroscopy/NMR/NMR.htmlhttp://orgchem.colorado.edu/Spectroscopy/IR/IR.htmlhttp://orgchem.colorado.edu/Spectroscopy/Spectroscopy.htmlhttp://orgchem.colorado.edu/Technique/Procedures/Procedures.htmlhttp://orgchem.colorado.edu/Technique/Equipment/Equipment.htmlhttp://orgchem.colorado.edu/Technique/Cheminfo/Cheminfo.htmlhttp://orgchem.colorado.edu/Technique/Safety/Safety.htmlhttp://orgchem.colorado.edu/Technique/Technique.htmlhttp://orgchem.colorado.edu/Labs/Cleanup.htmlhttp://orgchem.colorado.edu/Labs/3361.htmlhttp://orgchem.colorado.edu/Labs/3341.htmlhttp://orgchem.colorado.edu/Labs/3321.htmlhttp://orgchem.colorado.edu/Labs/Labs.htmlhttp://orgchem.colorado.edu/Examarchives/3371.htmlhttp://orgchem.colorado.edu/Examarchives/3351.htmlhttp://orgchem.colorado.edu/Examarchives/3331.htmlhttp://orgchem.colorado.edu/Examarchives/3311.htmlhttp://orgchem.colorado.edu/Examarchives/Examarchives.htmlhttp://orgchem.colorado.edu/Lectures/3351-100.htmlhttp://orgchem.colorado.edu/Lectures/3331-100.htmlhttp://orgchem.colorado.edu/Lectures/3311-200.htmlhttp://orgchem.colorado.edu/Lectures/3311-100.htmlhttp://orgchem.colorado.edu/Lectures/Lectures.html


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o MS Theoryo Structural Determinationo Exampleso Problems

About Us

Thin Layer Chromatography (TLC)TLC is a simple, quick, and inexpensive procedure that gives the chemist aquick answer as to how many components are in a mixture. TLC is also usedto support the identity of a compound in a mixture when the R f of acompound is compared with the R f of a known compound (preferably bothrun on the same TLC plate).

A TLC plate is a sheet of glass, metal, or plastic which is coated with a thinlayer of a solid adsorbent (usually silica or alumina). A small amount of themixture to be analyzed is spotted near the bottom of this plate. The TLCplate is then placed in a shallow pool of a solvent in a developing chamberso that only the very bottom of the plate is in the liquid. This liquid, or theeluent, is the mobile phase, and it slowly rises up the TLC plate by capillaryaction.

As the solvent moves past the spot that was applied, an equilibrium isestablished for each component of the mixture between the molecules of

that component which are adsorbed on the solid and the molecules whichare in solution. In principle, the components will differ in solubility and in thestrength of their adsorption to the adsorbent and some components will becarried farther up the plate than others. When the solvent has reached thetop of the plate, the plate is removed from the developing chamber, dried,and the separated components of the mixture are visualized. If thecompounds are colored, visualization is straightforward. Usually thecompounds are not colored, so a UV lamp is used to visualize the plates.(The plate itself contains a fluorescent dye which glows everywhere except where an organic compound is on the plate.)

How To Run a TLC Plate
http://orgchem.colorado.edu/Spectroscopy/MS/MS.htmlhttp://orgchem.colorado.edu/Spectroscopy/Structure/Structure.htmlhttp://orgchem.colorado.edu/Spectroscopy/Examples/Examples.htmlhttp://orgchem.colorado.edu/Spectroscopy/Problems/Problems.htmlhttp://orgchem.colorado.edu/About/About.htmlhttp://orgchem.colorado.edu/About/About.htmlhttp://orgchem.colorado.edu/Spectroscopy/Problems/Problems.htmlhttp://orgchem.colorado.edu/Spectroscopy/Examples/Examples.htmlhttp://orgchem.colorado.edu/Spectroscopy/Structure/Structure.htmlhttp://orgchem.colorado.edu/Spectroscopy/MS/MS.html


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Step 1: Prepare thedeveloping container

The developing containerfor TLC can be a speciallydesigned chamber, a jar

with a lid, or a beakerwith a watch glass onthe top (the latter is

used in the undergradlabs at CU). Pour solvent

into the chamber to adepth of just less than

0.5 cm. To aid in thesaturation of the TLCchamber with solvent

vapors, you can line partof the inside of the

beaker with filter paper.Cover the beaker with a

watch glass, swirl itgently, and allow it to

stand while you prepareyour TLC plate.


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Measure 0.5 cm from thebottom of the plate.

Using a pencil, draw a

line across the plate atthe 0.5 cm mark. This is

the origin : the line onwhich you will spot theplate. Take care not topress so hard with thepencil that you disturb

the adsorbent. Under theline, mark lightly the

name of the samples youwill spot on the plate, ormark numbers for time

points. Leave enoughspace between the

samples so that they donot run together; about 4samples on a 5 cm wide

plate is advised.

Step 3: Spot the TLC plate

If the sample is notalready in solution,dissolve about 1 mg in 1mL of a volatile solventsuch as hexanes, ethyl


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the TLC plate. Don'tallow the spot to becometoo large - if necessary,

you can touch it to theplate, lift it off and blow

on the spot. If yourepeat these steps, the

wet area on the plate willstay small.

This example plate hasbeen spotted with three

different quantities of the same solution and isready to develop. If youare unsure of how muchsample to spot, you can

always spot multiplequantities and see which

looks best.

Step 4: Develop the plate

Place the prepared TLCplate in the developing

beaker, cover the beakerwith the watch glass,

and leave it undisturbedon your bench top. Thesolvent will rise up the


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When you need to determine the best solvent or mixture of solvents (a"solvent system") to develop a TLC plate or chromatography column loadedwith an unknown mixture, vary the polarity of the solvent in several trialruns: a process of trial and error. Carefully observe and record the results of the chromatography in each solvent system. You will find that as you

increase the polarity of the solvent system, all the components of themixture move faster (and vice versa with lowering the polarity). The idealsolvent system is simply the system that gives the best separation.

TLC elution patterns usually carry over to column chromatography elutionpatterns. Since TLC is a much faster procedure than columnchromatography, TLC is often used to determine the best solvent system forcolumn chromatography. For instance, in determining the solvent system fora flash chromatography procedure, the ideal system is the one that movesthe desired component of the mixture to a TLC R f of 0.25-0.35 and willseparate this component from its nearest neighbor by difference in TLC R f values of at least 0.20. Therefore a mixture is analyzed by TLC to determinethe ideal solvent(s) for a flash chromatography procedure.

Beginners often do not know where to start: What solvents should they pulloff the shelf to use to elute a TLC plate? Because of toxicity, cost, andflammability concerns, the common solvents are hexanes (or petroleumethers/ligroin) and ethyl acetate (an ester). Diethyl ether can be used, but itis very flammable and volatile. Alcohols (methanol, ethanol) can be used.Acetic acid (a carboxylic acid) can be used, usually as a small percentagecomponent of the system, since it is corrosive, non-volatile, very polar, and

has irritating vapors. Acetone (a ketone) can be used. Methylene chloride orand chloroform (halogenated hydrocarbons) are good solvents, but are toxicand should be avoided whenever possible. If two solvents are equal inperformance and toxicity, the more volatile solvent is preferred inchromatography because it will be easier to remove from the desiredcompound after isolation from a column chromatography procedure.

Ask the lab instructor what solvents are available and advisable. Then, mix anon-polar solvent (hexanes, a mixture of 6-carbon alkanes) with a polarsolvent (ethyl acetate or acetone) in varying percent combinations to makesolvent systems of greater and lesser polarity. The charts below should helpyou in your solvent selection. You can also download this pdf chart of elutionorder .
http://orgchem.colorado.edu/Technique/Procedures/Columnchrom/Columnchrom.htmlhttp://orgchem.colorado.edu/Technique/Procedures/Columnchrom/Columnchrom.htmlhttp://orgchem.colorado.edu/Technique/Procedures/Columnchrom/Columnchrom.htmlhttp://orgchem.colorado.edu/Technique/Procedures/TLC/Elution.pdfhttp://orgchem.colorado.edu/Technique/Procedures/TLC/Elution.pdfhttp://orgchem.colorado.edu/Technique/Procedures/TLC/Elution.pdfhttp://orgchem.colorado.edu/Technique/Procedures/TLC/Elution.pdfhttp://orgchem.colorado.edu/Technique/Procedures/TLC/Elution.pdfhttp://orgchem.colorado.edu/Technique/Procedures/TLC/Elution.pdfhttp://orgchem.colorado.edu/Technique/Procedures/Columnchrom/Columnchrom.html


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Interactions Between the Compound and the Adsorbent

The strength with which an organic compound binds to an adsorbentdepends on the strength of the following types of interactions: ion-dipole,dipole-dipole, hydrogen bonding, dipole induced dipole, and van der Waalsforces. With silica gel, the dominant interactive forces between theadsorbent and the materials to be separated are of the dipole-dipole type.Highly polar molecules interact fairly strongly with the polar SiOH groups atthe surface of these adsorbents, and will tend to stick or adsorb onto the fineparticles of the adsorbent while weakly polar molecules are held less tightly.Weakly polar molecules generally tend to move through the adsorbent morerapidly than the polar species. Roughly, the compounds follow the elutionorder given above.

The R f value

The retention factor, or R f , is defined as the distance traveled by thecompound divided by the distance traveled by the solvent.

For example, if a compound travels 2.1 cm and the solvent front travels 2.8cm, the R f is 0.75:


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The R f 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 toexperiment, relative R f values are generally considered. "Relative R f " meansthat the values are reported relative to a standard, or it means that youcompare the R f values of compounds run on the same plate at the sametime.

The larger an R f of a compound, the larger the distance it travels on the TLCplate. When comparing two different compounds run under identicalchromatography conditions, the compound with the larger R f is less polarbecause it interacts less strongly with the polar adsorbent on the TLC plate.Conversely, if you know the structures of the compounds in a mixture, youcan predict that a compound of low polarity will have a larger R f value than apolar compound run on the same plate.

The R f can provide corroborative evidence as to the identity of a compound.If the identity of a compound is suspected but not yet proven, an authenticsample of the compound, or standard, is spotted and run on a TLC plate sideby side (or on top of each other) with the compound in question. If twosubstances have the same R f value, they are likely (but not necessarily) thesame compound. If they have different R f values, they are definitely differentcompounds. Note that this identity check must be performed on a singleplate, because it is difficult to duplicate all the factors which influence R f exactly from experiment to experiment.


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Troubleshooting TLC

All of the above (including the procedure page) might sound like TLC is quitean easy procedure. But what about the first time you run a TLC, and seespots everywhere and blurred, streaked spots? As with any technique, withpractice you get better. Examples of common problems encountered in TLC:

The compound runs as a streak rather than a spot: Thesample was overloaded. Run the TLC again afterdiluting your sample. Or, your sample might justcontain many components, creating many spotswhich run together and appear as a streak.Perhaps, the experiment did not go as well as

expected. The sample runs as a smear or a upward crescent:

Compounds which possess strongly acidic or basicgroups (amines or carboxylic acids) sometimesshow up on a TLC plate with this behavior. Add afew drops of ammonium hydroxide (amines) oracetic acid (carboxylic acids) to the eluting solventto obtain clearer plates.

The sample runs as a downward crescent: Likely, theadsorbent was disturbed during the spotting,causing the crescent shape.

The plate solvent front runs crookedly: Either theadsorbent has flaked off the sides of the plate orthe sides of the plate are touching the sides of thecontainer (or the paper used to saturate thecontainer) as the plate develops. Crooked platesmake it harder to measure R f values accurately.

Many random spots are seen on the plate: Make surethat you do not accidentally drop any organiccompound on the plate. If get a TLC plate and leaveit laying on your workbench as you do the


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experiment, you might drop or splash an organiccompound on the plate.

You see a blur of blue spots on the plate as it develops: Perhaps you used an ink pen instead of a pencil tomark the origin?

No spots are seen on the plate: You might not havespotted enough compound, perhaps because thesolution of the compound is too dilute. Tryconcentrating the solution, or spot it several timesin one place, allowing the solvent to dry betweenapplications. Some compounds do not show upunder UV light; try another method of visualizingthe plate (such as staining or exposing to iodinevapor). Or, perhaps you do not have any compoundbecause your experiment did not go as well asplanned. If the solvent level in the developing jar isdeeper than the origin (spotting line) of the TLCplate, the solvent will dissolve the compounds intothe solvent reservoir instead of allowing them to

move up the plate by capillary action. Thus, youwill not see spots after the plate is developed.These photos show how the yellow compound isrunning into the solvent when lifted from thedeveloping jar.


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