If All Your Rfs Were Too Large, What Would You Do to Your Solvent System to Bring Them Down
Thin Layer Chromatography
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Sparse layer chromatography (TLC) is a chromatographic technique used to separate the components of a mixture using a sparse stationary phase supported past an inert bankroll. Information technology may be performed on the belittling calibration equally a ways of monitoring the progress of a reaction, or on the preparative scale to purify small amounts of a chemical compound. TLC is an analytical tool widely used because of its simplicity, relative low cost, high sensitivity, and speed of separation.TLC functions on the same principle as all chromatography: a compound will accept dissimilar affinities for the mobile and stationary phases, and this affects the speed at which it migrates. The goal of TLC is to obtain well divers, well separated spots.
Retention Factor
After a separation is complete, individual compounds appear as spots separated vertically. Each spot has a retention factor (Rf) which is equal to the distance migrated over the total altitude covered by the solvent. The \( R_f\) formula is
\[ R_f= \dfrac{\text{altitude traveled by sample}}{\text{distance traveled by solvent}} \]
The \( R_f\) value can exist used to identify compounds due to their uniqueness to each compound. When comparison two dissimilar compounds under the same weather, the compound with the larger \( R_f\) value is less polar because it does not stick to the stationary stage as long every bit the polar compound, which would have a lower \( R_f\) value.
\( R_f\) values and reproducibility can exist afflicted by a number of different factors such as layer thickness, moisture on the TLC plate, vessel saturation, temperature, depth of mobile phase, nature of the TLC plate, sample size, and solvent parameters. These effects normally cause an increase in \( R_f\) values. Nevertheless, in the instance of layer thickness, the \( R_f\) value would subtract because the mobile phase moves slower up the plate.
If it is desired to express positions relative to the position of some other substance, x, the \( R_x\) (relative retentivity value) can be calculated:
\[ R_x= \dfrac{\text{distance of chemical compound from origin}}{\text{altitude of compound ten from origin}} \]
While \(R_f\) can never exist greater than 1, \( R_x\) can exist (i.e., faster than the reference compound \(x\).
Apparatus
Plates (Stationary Phase)
As stated earlier, TLC plates (besides known as chromatoplates) can be prepared in the lab, but are most ordinarily purchased. Silica gel and alumina are among the almost common stationary phases, but others are available as well. Many plates incorporate a compound which fluoresces nether short-wave UV (254 nm). The backing of TLC plates is often composed of glass, aluminum, or plastic. Glass plates are chemically inert and best withstand reactive stains and oestrus, but are brittle and can be difficult to cutting. Aluminum and plastic plates can be cut with scissors, but aluminum may not withstand strongly acidic or oxidizing stains, and plastic does not withstand the loftier heat required to develop many stains. Aluminum and plastic plates are besides flexible, which may result in flaking of the stationary stage. Never under whatsoever circumstances touch the face up of a TLC plate with your fingers as contamination from skin oils or residues on gloves can obscure results. Instead, always handle them past the edges, or with forceps.
The properties of your sample should be considered when selecting the stationary stage. Every bit shown below in Table \(\PageIndex{1}\), silica gel tin can exist exclusively used for amino acids and hydrocarbons. It is as well important to notation that silica gel is acidic. Therefore, silica gel offers poor separation of bones samples and tin can cause a deterioration of acid-labile molecules. This would be true for alumina plates in acidic solutions as well. It is of import to notation that in that location are differences between silica gel and alumina. Alumina is basic and it will non separate sample sizes as big every bit silica gel would at a given layer thickness. As well, alumina is more chemically reactive than silica gel and every bit a result, would require more care of compounds and compound classes. This care would avert decomposition and rearrangement of the sample.
| Stationary Stage | Chromatographic Mechanism | Typical 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 | sectionalisation | 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 practiced reference to learn more than about the unlike types of columns and stationary phases.
Solvent (Mobile Stage)
Proper solvent selection is perhaps the well-nigh of import attribute of TLC, and determining the best solvent may require a degree of trial and error. As with plate selection, keep in mind the chemical properties of the analytes. A common starting solvent is one:one hexane:ethyl acetate. Varying the ratio can have a pronounced effect of \(R_f\). \(R_f\) values range from 0 to 1 with 0 indicating that the solvent polarity is very low and 1 indicating that the solvent polarity is very high. When performing your experiment, you do not desire your values to exist 0 or 1 because your components that you are separating have different polarities. If the value is 0, you need to increase your solvent polarity because the sample is not moving and sticking to the stationary stage. If the value is i, you need to subtract your solvent polarity because the compound was not able to carve up.
If you lot know that one component of a mixture is insoluble in a given solvent, but another component is freely soluble in it, it oft gives good separations. 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 stage. If the chemical compound likes the stationary phase, information technology volition stick to it, which volition cause it to non move very far on the chromatogram.
Y'all should be able to determine which by looking at the \(R_f\) value.
Acids, bases, and strongly polar compounds often produce streaks rather than spots in neutral solvents. Streaks make information technology difficult to calculate an \(R_f\) and may occlude other spots. Calculation a few percent of acetic or formic acrid to the solvent can correct streaking with acids. Similarly for bases, adding a few percent triethylamine can amend results. For polar compounds adding a few percent methanol can besides improve results.
The volatility of solvents should likewise be considered when chemic stains are to be used. Whatever solvent left on the plate may react with the stain and muffle spots. Many solvents tin can be removed by assuasive them to sit on the bench for a few minutes, but very nonvolatile solvents may require fourth dimension in a vacuum chamber. Volatile solvents should only exist used in one case. If the mobile stage is used repeatedly, results volition not be consistent or reproducible.
Useful Solvent Mixtures
- A solvent that can be used for separating mixtures of strongly polar compounds is ethyl acetate : butanol : acetic acid : water, lxxx:10:5:5.
- To divide strongly bones components, brand a mixture of ten% NHivOH in methanol, and and then brand a 1 to x% mixture of this in dichlormethane.
- Mixtures of 10% methanol or less in DCM can be useful for separating polar compounds.
Pipettes
- Spots are practical to the plate using very thin glass pipettes. The capillary should be thin plenty to apply a not bad spot, only non and so sparse equally to foreclose the uptake of an adequate quantity of analyte. Hither is a popular method of producing TLC pipettes.
- Estrus 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 apply the thin end to apply spots.
Spotting and Developing
Developing a TLC plate requires a developing sleeping room or vessel. This can exist equally simple as a wide-mouth jar, just more specialized pieces of glassware to adapt large plates are bachelor. The chamber should comprise enough solvent to only embrace the lesser. It should also contain a piece of filter newspaper, or other absorptive fabric to saturate the atmosphere with solvent vapors. Finally, it should have a lid or other roofing to minimize evaporation.
- Cut the plate to the right size and using a pencil (never ever use a pen), gently draw a straight line across the plate approximately 1 cm from the lesser. Exercise not apply excessive forces when writing on a TLC plate as this volition 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 department labeled "examples".
- Using TLC pipettes, utilize spots of analyte to the line. Make certain enough sample is spotted on the plate. This can exist washed by using the brusque-wave UV. A purple spot should exist seen. If the spot is not visible, more sample needs to be applied to the plate. If a standard of the target compound is bachelor, it is practiced practise 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 sleeping accommodation as evenly as possible and lean information technology against the side. Never allow the bulk solvent to ascent above the line you drew. Allow capillary action to draw the solvent upwards the plate until it is approximately 1 cm from the cease. Never allow the solvent to migrate all the fashion to the end of the plate.
- Remove the plate and immediately describe a pencil line across the solvent front.
- Utilize a short-wave UV light and circle the components shown with a pencil.
Visualizing
If fluorescent plates are used, a number of compounds tin can be seen by illuminating the plate with short-wave UV. Quenching causes nighttime spots on the surface of the plate. These nighttime patches should be circled with a pencil. For compounds which are not UV agile, a number of chemic stains can exist used. These tin exist very full general, or they can be specific for a item molecule or functional group.
Iodine is among the nearly common stains. Plates are placed in a jar containing iodine crystals, or covered in silica gel with iodine dispersed throughout, for approximately one minute. Most organic compounds will be temporarily stained brown. Some popular general utilize stains are Permanganate, ceric ammonium molybdate (CAM), and p-anisaldehyde. These tin be kept in jars which plates are dipped into, or in spray bottles.
To develop a plate with permanganate, spray or dip the plate and heat it with a oestrus-gun. Concur the plate face up 10 to 20 cm above the oestrus gun until the bulk h2o evaporates. Then move the plate to v to 10 cm above the heat gun and heat it until white/yellowish/dark-brown spots appear. Overheating will plough the entire plate chocolate-brown, obscuring the spots. If glass plates are used it is often easier to see spots through the backing because it is harder to overheat. CAM and p-anisaldehyde stained plates are developed similarly. Overheating CAM stained plates turns everything bluish.
Mutual Issues in TLC
There are common problems in TLC that should be avoided. Normally, these problems can be solved or avoided if taught proper techniques.
- Over-large Spotdue south: Spotting sizes of your sample should be non be larger than 1-two mm in diameter. The component spots will never be larger than or smaller than your sample origin spot. If you have an over-large spot, this could crusade overlapping of other component spots with similar \(R_f\) values on your TLC plate. If overlapping occurs, it would prove difficult to resolve the dissimilar components.
- Uneven Advance of Solvent Front: Uneven accelerate of the mobile stage is a common problem encountered in TLC. Consequences would be inaccurate Rf values due to the uneven advance of sample origin spots. This uneven advance can exist caused by a few factors listed below.
- No apartment bottom. When placing the TLC plate into the chamber, place the bottom of the plate on the edge of the chamber (normally drinking glass container (e.m. chalice)) and lean the top of the plate forth the other side of the sleeping accommodation. Also, make sure that the TLC plate is placed in the chamber evenly. Do not tilt the plate or sit information technology at an angle.
- Not enough solvent. There should exist enough solvent (depends on size of the chamber) to travel up the length of the TLC plate.
- Plate is not cutting evenly. It is recommended that a ruler is used so that the plate is cut evenly.
Rarely, water is used as a solvent considering it produces an uneven curve front which is mainly accounted for by its surface tension.
- Streaking : If the sample spot is as well concentrated, the substance volition travel up the stationary phase as a streak rather than a unmarried separated spot. In other words, the solvent can not handle the concentrated sample and in consequence, moves as much of the substance as it tin can up the stationary phase. The substance that it can non motion is left behind. This tin exist eliminated by diluting the sample solution. To ensure that you take enough solution, apply a brusk-moving ridge UV calorie-free to run into if the spot is visible (commonly purple in color), as stated earlier.
- Spotting : The sample should exist above the solvent level. If the solvent level covers the sample, the sample spot will exist done off into the solvent before it travels up the TLC plate. An example is shown below.
Instance: Analyzing Commercial Analgesics
Thin layer chromatography of 3 analgesics and caffeine under U.5. low-cal was carried out in society to show the separation taking identify. It is not a recommended technique in the laboratory. Due to the nature of the uv take a chance polycarbonate safety spectacles (which absorb brusque wavelength U.V. light) and rubber gloves were worn throughout.
V samples were run on a single TLC plate. The samples were (left to right on the plate):
- Ibuprofen (ICU)
- caffeine (CAF)
- u? = a commercial 'pain relief' medicine, used as an unknown
- Acetominophen (PAR)
- Aspirin (ASP)
The samples were dissolved in ethanol for spotting onto the plate. The TLC plate was run in an open up beaker under short wavelength u.five. light using ethyl ethanoate equally the eluting solvent.
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The movement of the nighttime purple spots (samples) during the running of the plate tin can exist observed in the animation. The original motion picture can be viewed here.
It is easy to encounter which are the two active ingredients in the unknown commercial pain relief medicine past comparison of the spots with the standard reference materials running on either side (caffeine and acetominophen).
Advantages and Disadvantages of TLC
TLC is very simple to employ and cheap. Undergraduates tin can exist taught this technique and use its similar principles to other chromatographic techniques. There are trivial materials needed for TLC (chamber, sentry glass, capillary, plate, solvent, pencil, and UV-low-cal). Therefore, once the best solvent is plant, it can be applied to other techniques such as High operation liquid chromatography. More than than one compound can be separated on a TLC plate as long as the mobile stage is preferred for each compound. The solvents for the TLC plate tin can exist inverse easily and it is possible to utilize several different solvents depending on your desired results. As stated earlier, TLC can be used to ensure purity of a compound. It is very easy to cheque the purity using a UV-lite. The identification of most compounds can be washed simply by checking \( R_f\) literature values. You can modify the chromatography conditions hands to increase the optimization for resolution of a specific component.
TLC plates do non have long stationary phases. Therefore, the length of separation is express compared to other chromatographic techniques. Also, the detection limit is a lot higher. If yous would demand a lower detection limit, one would take to use other chromatographic techniques. TLC operates equally an open system, so factors such equally humidity and temperature tin can be consequences to the results of your chromatogram.
References
- Touchstone, Joseph C. Practice of thin layer chromatography. 2d ed. New York: Wiley, 1983.Print.
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Geiss, Friedrich. Fundamentals of thin layer chromatography planar chromatography. Heidelberg: A. Hüthig, 1987. Print.
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Touchstone, Joseph C. Do of sparse layer chromatography. 3rd ed. New York: Wiley, 1992. Print.
Problems and Solutions
Figure iii: TLC plate under UV low-cal with values for altitude traveled of solvent and components.
Given:
#1=1.four cm
#ii= ane.5 cm
#3= 3.1 cm
#iv= 3.6 cm
Using only the given information and the to a higher place figure, answer the problems listed beneath.
- What is the Rf value for component #ii?
- What is the Rf value for component # three?
- What is the relative retention value for components #1 and # iv, with # 4 beingness chemical compound x?
- Using the answers from questions 1 and 2 and bold that components ii and 3 are unlike compounds, which component would exist considered more polar? Explain.
Answers
- i.5/4.four=0.34
- three.one/4.4=0.70
- one.4/3.vi=0.39
- Component # 2 would exist considered more polar because it has the lower Rf value, which means that it sticks to the stationary phase a lot stronger than component #iii and therefore moves slower in the mobile phase.
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Source: https://chem.libretexts.org/Ancillary_Materials/Demos_Techniques_and_Experiments/General_Lab_Techniques/Thin_Layer_Chromatography
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