ApparatusProblems and Solutions

Thin layer color layer analyzer (TLC) is a chromatographic technique used to different the contents of a mixture utilizing a slim stationary phase supported by an inert backing. It might be carry out on the analytical scale as a way of monitoring the progression of a reaction, or ~ above the preparative scale to purify tiny amounts that a compound. TLC is an analytical tool widely used since of that is simplicity, relative low cost, high sensitivity, and speed of separation.TLC functions on the same principle together all chromatography: a compound will certainly have different affinities for the mobile and stationary phases, and also this affects the rate at which that migrates. The goal of TLC is to achieve well defined, well separated spots.

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Retention Factor

After a separation is complete, individual compounds appear as point out separated vertically. Every spot has actually a retention factor (Rf) which is same to the distance moved over the complete distance covered by the solvent. The \( R_f\) formula is

\< R_f= \dfrac\textdistance travel by sample\textdistance travel by solvent \>

The \( R_f\) value can be supplied to identify compounds because of their uniqueness to each compound. When comparing two various compounds under the very same conditions, the compound v the bigger \( R_f\) value is less polar due to the fact that it does not stick come the stationary step as long as the polar compound, which would have a lower \( R_f\) value.

\( R_f\) values and reproducibility deserve to be influenced by a variety of different components such as layer thickness, humidity on the TLC plate, courage saturation, temperature, depth of mobile phase, nature of the TLC plate, sample size, and solvent parameters. These results normally cause rise in \( R_f\) values. However, in the instance of layer thickness, the \( R_f\) value would decrease since the mobile step moves slow up the plate.

If it is desired to express positions family member to the place of an additional substance, x, the \( R_x\) (relative retention value) deserve to be calculated:

\< R_x= \dfrac\textdistance of link from origin\textdistance of compound x from origin \>

While \(R_f\) have the right to never be higher than 1, \( R_x\) can be (i.e., much faster than the recommendation compound \(x\).


Plates (Stationary Phase)

As declared earlier, TLC key (also known as chromatoplates) have the right to be prepared in the lab, however are most frequently purchased. Silica gel and also alumina are amongst the most usual stationary phases, however others are obtainable as well. Plenty of plates incorporate a compound which fluoresces under short-wave UV (254 nm). The backing that TLC plates is often composed that glass, aluminum, or plastic. Glass plates room smashville247.netically inert and best withstand reactive stains and heat, yet are brittle and can be difficult to cut. Aluminum and also plastic plates deserve to be reduced with scissors, but aluminum might not withstand strongly acidic or oxidizing stains, and also plastic does not resist the high heat required to build many stains. Aluminum and also plastic bowl are also flexible, i m sorry may result in flaking of the stationary phase. Never ever under any type of circumstances touch the face of a TLC plate with your fingers as air pollution from skin oils or residues on gloves have the right to obscure results. Instead, constantly handle castle by the edges, or through forceps.

The properties of her sample should be thought about when choosing the stationary phase. As shown listed below in Table \(\PageIndex1\), silica gel deserve to be exclusively used for amino acids and also hydrocarbons. The is likewise important to note that silica gelatin is acidic. Therefore, silica gelatin offers negative separation of simple samples and can reason a deterioration of acid-labile molecules. This would be true because that alumina bowl in acidic options as well. It is crucial to note that there are differences between silica gel and alumina. Alumina is straightforward and it will not separate sample size as large as silica gel would at a offered layer thickness. Also, alumina is much more smashville247.netically reactive than silica gel and as a result, would certainly require much more care the compounds and compound classes. This care would avoid decomposition and rearrangement that the sample.

Table \(\PageIndex1\): Stationary phase and also mode the separation Stationary PhaseChromatographic MechanismTypical Application
Silica Gel adsorption steroids, amino acids, alcohols, hydrocarbons, lipids, aflaxtoxin, bile, acids, vitamins, alkaloids
Silica gel RP reversed phase fatty acids, vitamins, steroids, hormones, carotenoids
Cellulose, kieselguhr partition carbohydrates, sugars, alcohols, amino acids, carboxylic acids, fatty acids
Aluminum oxide adsorption amines, alcohols, steroids, lipids, aflatoxins, bile acids, vitamins, alkaloids
PEI cellulose ion exchange nucleic acids, nucleotides, nucelosides, purines, pyrimidines
Magnesium silicate adsorption steroids, pesticides, lipids, alkaloids

Chromatographic Columns is a an excellent reference to learn an ext about the different species of columns and stationary phases.


Solvent (Mobile Phase)

Proper solvent selection is possibly the most important aspect of TLC, and determining the best solvent may require a level of trial and error. Similar to plate selection, store in psychic the smashville247.netical nature of the analytes. A common beginning solvent is 1:1 hexane:ethyl acetate. Differing the ratio have the right to have a pronounced effect of \(R_f\). \(R_f\) values variety from 0 to 1 v 0 indicating that the solvent polarity is very low and 1 indicating the the solvent polarity is really high. Once performing her experiment, you carry out not desire your worths to be 0 or 1 since your materials that you are separating have different polarities. If the worth is 0, you require to increase your solvent polarity due to the fact that the sample is no moving and sticking come the stationary phase. If the worth is 1, you need to decrease her solvent polarity due to the fact that the link was not able come separate.

If you understand that one ingredient of a mixture is insoluble in a offered solvent, but an additional component is openly soluble in it, it often gives good separations. How rapid the compounds travel up the plate depends on 2 things:

If the compound is soluble in the solvent, it will certainly travel further up the TLC plate exactly how well the compound likes the stationary phase. If the link likes the stationary phase, it will certainly stick come it, i beg your pardon will cause it to not move an extremely far top top the chromatogram.

You should be able to determine which by looking in ~ the \(R_f\) value.

Acids, bases, and also strongly polar link often develop streaks quite than point out in neutral solvents. Streaks do it challenging to calculation an \(R_f\) and also may occlude various other spots. Including a couple of percent that acetic or formic mountain to the solvent have the right to correct streaking with acids. Similarly for bases, including a few percent triethylamine can improve results. Because that polar compounds adding a few percent methanol can also improve results.

The volatility of solvents should also be thought about when smashville247.netical stains space to be used. Any solvent left ~ above the plate may react v the stain and conceal spots. Many solvents can be gotten rid of by allowing them come sit on the bench for a couple of minutes, but very nonvolatile solvents may require time in a vacuum chamber. Volatile solvents need to only be used once. If the mobile step is supplied repeatedly, results will not be continual or reproducible.


Useful Solvent Mixtures

A solvent that can be supplied for separating mixtures of strongly polar link is ethyl acetate : butanol : acetic mountain : water, 80:10:5:5. To different strongly an easy components, make a mixture that 10% NH4OH in methanol, and then do a 1 to 10% mixture the this in dichlormethane. Mixtures of 10% methanol or less in DCM have the right to be advantageous for separating polar compounds.

Pipettes

point out are applied to the key using very thin glass pipettes. The capillary must be thin sufficient to apply a neat spot, but not so thin as to prevent the absorb of one adequate amount of analyte. Right here is a popular an approach of creating TLC pipettes. warmth a glass capillary in the really tip the a Bunsen burner flame simply until it becomes pliable and also then pull the ends apart until the facility of the capillary is considerably narrower. Snap this in fifty percent and use the thin end to use spots.

Spotting and also Developing

Developing a TLC plate requires a occurring chamber or vessel. This have the right to be as an easy as a wide-mouth jar, but more devoted pieces that glassware come accommodate big plates room available. The chamber have to contain enough solvent to just cover the bottom. That should also contain a piece of filter paper, or other absorbent product to saturation the atmosphere with solvent vapors. Finally, that should have a lid or various other covering to minimization evaporation.

reduced the plate to the exactly size and also using a pencil (never ever use a pen), gently attract a right line throughout the plate approximately 1 centimeter from the bottom. Do not use extreme forces once writing ~ above a TLC plate together this will eliminate the stationary phase. It is important to use a pencil rather than a pen due to the fact that inks commonly travel up the plate through the solvent. An example of just how black octopus separates is presented in the ar labeled "examples". Using TLC pipettes, use spots that analyte to the line. Make sure enough sample is spotted top top the plate. This have the right to be excellent by utilizing the short-wave UV. A violet spot must be seen. If the point out is not visible, more sample demands to be applied to the plate. If a typical of the target compound is available, that is great practice to develop a co-spot through spotting the traditional onto a point out of the unknown mixture. This ensures the identity of the target compound. Place the plate right into the chamber together evenly as possible and lean it versus the side. Never enable the bulk solvent come rise over the heat you drew. Permit capillary activity to attract the solvent increase the plate till it is roughly 1 centimeter from the end. Never permit the solvent to migrate all the method to the finish of the plate. Eliminate the plate and also immediately draw a pencil line across the solvent front. Use a short-wave UV light and also circle the components shown through a pencil.
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api/deki/files/138337/imageedit_7_3773618470.jpg?revision=1" />Five samples prior to elution. Image used v permission (Free for education and learning use, Nigel Baldwinviasmashville247.netligin).

The samples were liquified in ethanol for spotting ~ above the plate. The TLC bowl was run in an open beaker under brief wavelength u.v. Light using ethyl ethanoate as the eluting solvent.

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Separation of the samples. (Free for education use, Nigel Baldwinviasmashville247.netligin).

The motion of the dark purple spots (samples) during the to run of the plate can be it was observed in the animation. The initial movie deserve to be viewed here.

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Touchstone, Joseph C. Practice of slim layer chromatography. Third ed. Brand-new York: Wiley, 1992. Print.