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Stains
for Developing TLC Plates
Once a TLC has been developed, it is frequently necessary to aid in the visualization of the components of a reaction mixture. This is true primarily because most organic compounds are colorless. Frequently, the organic compounds of interest contain a chromophore which may be visualized by employing either a short or a long wave UV lamp. These lamps may be found as part of a standard organic chemistry research or teaching lab. Typical examples of functional groups which may be visualized through this method are aromatic groups, a,b-unsaturated carbonyls, and any other compounds containing extensively p-conjugated systems. While exposing these TLC plates to UV light, you will notice that the silica gel will fluoresce, while any organic molecule which absorbs UV light will appear as a dark blue spot. Circling these spots gently with a dull pencil will permit an initial method for visualization. Fortunately, there are a number of permanent or semi-permanent methods for visualization which will not only allow one to see these compounds but also provide a method for determining what functional groups are contained within the molecule. This method is referred to as staining the TLC plate, and experience will allow you to determine what functional groups will appear as what color upon visualization. Following is a listing of the most commonly employed stains, the kind of compounds for which they're usually employed, and a typical recipe. A Note on TLC Plates Although it should be obvious, be sure that the kind of TLC plate you are using is compatible with the stain or the conditions for its development. For instance, the inexpensive plates using a plastic polymer backing cannot be used for stains requiring heat for development. Glass backing is fine for this, but the silica gel is typically not tightly bonded to the glass, and tends to be inadvertantly scraped off very easily; thus, these are not suitable for storage following development. In our group, we use aluminum-backed plates, which are less expensive than glass, are heat-impervious, the silica gel is very tightly bound to the backing, and are so thin that, if desired, a particularly spectacular plate can be taped into your lab notebook. The Stain List
Iodine
The staining of a TLC plate with iodine
vapor is among the oldest methods for the visualization of organic
compounds.
It is based upon the observation that iodine has a high affinity for
both
unsaturated and aromatic compounds.
Recipe
A chamber may be assembled as follows:
To 100 mL wide mouth jar (with cap) is added a piece of filter paper
and
few crystals of iodine. Iodine has a high vapor pressure for a solid
and
the chamber will rapidly become saturated with iodine vapor. Insert
your
TLC plate and allow it to remain within the chamber until it develops a
light brown color over the entire plate. Commonly, if your compound has
an affinity for iodine, it will appear as a dark brown spot on a
lighter
brown background. Carefully remove the TLC plate at this point and
gently
circle the spots with a dull pencil. The iodine will not remain on the
TLC plate for long periods of time so circling these spots is necessary
if one wishes to refer to these TLC's at a later date.
Ultraviolet Light Good for
visualizing any compounds which
are UV-active, particularly those with extended conjugation, aromatic
rings, etc. Spot(s) must be lightly traced with a pencil while
visible, since when the UV light is removed, the spots disappear.
Ceric Ammonium Sulfate Specifically developed for vinca alkaloids (aspidospermas). Recipe Prepare a 1% solution of of cerium (IV) ammonium sulfate in 50% phosphoric acid. Cerium Sulfate General stain, particularly effective for alkaloids. Recipe Prepare an aqueous solution of 10% cerium (IV) sulfate and 15% sulfuric acid. Ferric Chloride Excellent for phenols. Recipe Prepare a solution of 1% ferric (III) chloride in 50% aqueous methanol. Morin Hydrate General stain (morin is a hydroxy flavone), is fluorescently active. Recipe Prepare a 0.1% solution of morin hydrate (by weight) in methanol. Ninhydrin Excellent for amino acids Recipe Dissolve 1.5g ninhydrin in 100mL of n-butanol and then add 3.0mL acetic acid. Dinitrophenylhydrazine (DNP)
Developed mainly for aldehydes and ketones; forms the corresponding
hydrazones, which are usually yellow to orange and thus easily
visualized.
Recipe Dissolve 12g
of 2,4-dinitrophenylhydrazine, 60mL of conc. sulfuric acid, and 80mL of
water in 200mL of 95% ethanol.
Vanillin Very good general stain, giving a range of colors for different spots. Recipe Prepare a solution of 15g vanillin in 250mL ethanol and 2.5mL conc. sulfuric acid. Potassium Permanganate This particular stain is excellent for functional groups which are sensitive to oxidation. Alkenes and alkynes will appear readily on a TLC plate following immersion into the stain and will appear as a bright yellow spot on a bright purple background. Alcohols, amines, sulfides, mercaptans and other oxidizable functional groups may also be visualized, however it will be necessary to gently heat the TLC plate following immersion into the stain. These spots will appear as either yellow or light brown on a light purple or pink background. Again it would be advantageous to circle such spots following visualization as eventually the TLC will take on a light brown color upon standing for prolonged periods of time. Recipe
Dissolve 1.5g of KMnO4, 10g K2CO3,
and 1.25mL 10% NaOH in 200mL water. A typical
lifetime
for this stain is approximately 3 months.
Bromocresol Green Stain This particular stain is excellent for functional groups whose pKa is approximately 5.0 and lower. Thus, this stain provides an excellent means of selectively visualizing carboxylic acids. These will appear as bright yellow spots on either a dark or light blue background and typically, it is not necessary to heat the TLC plate following immersion. This TLC visualization method has a fairly long lifetime (usually weeks) thus, it is not often necessary to circle such spots following activation by staining. Recipe
To 100 ml of absolute ethanol is added
0.04 g of bromocresol green. Then a 0.1 M solution of aqueous NaOH is
added
dropwise until a blue color just appears in solution (the solution
should
be colorless prior to addition). Ideally, these stains may be stored in
100 mL wide mouth jars. The lifetime of such a solution typically
depends
upon solvent evaporation. Thus, it would be advantageous to tightly
seal
such jars in-between uses.
Cerium Molybdate Stain (Hanessian's Stain) This stain is a highly sensitive, multipurpose (multifunctional group stain). One word of caution, very minor constituents may appear as significant impurities by employing this stain. To ensure accurate results when employing this stain, it is necessary to heat the treated TLC plate vigorously (a heat gun works well). Thus, this may not be a stain to employ if your sample is somewhat volatile. The TLC plate itself will appear as either light blue or light green upon treatment, while the color of the spots may vary (although they usually appear as a dark blue spot). Typically, functional groups will not be distinguishable based upon the color of their spots; however, it would be worth while to make a list of potential colors of various functional groups as you experience variations in colors. This may permit future correlations which may prove beneficial when performing similar chemistry on related substrates. Recipe
To 235 mL of distilled water was added
12 g of ammonium molybdate, 0.5 g of ceric ammonium molybdate, and 15
mL
of concentrated sulfuric acid. Storage is possible in a 250 mL wide
mouth
jar. This stain has a long shelf-life so long as solvent evaporation is
limited. It may also prove worth while to surround the jar with
aluminum
foil as the stain may be somewhat photo-sensitive and exposure to
direct
light may shorten the shelf-life of this reagent. It is worth while to
also mention that it would be beneficial to circle the observed spots
with
a dull pencil following heating as this stain will eventually fade on
the
TLC plate after a few days.
p-Anisaldehyde Stain #1 This stain is an excellent multipurpose visualization method for examining TLC plates. It is sensitive to most functional groups, especially those which are strongly and weakly nucleophilic. It tends to be insensitive to alkenes, alkynes, and aromatic compounds unless other functional groups are present in the molecules which are being analyzed. It tends to stain the TLC plate itself, upon mild heating, to a light pink color, while other functional groups tend to vary with respect to coloration. It is recommended that a record is kept of which functional group stains which color for future reference, although these types of comparisons may be misleading when attempting to ascertain which functional groups are present in a molecule (especially in complex molecules). The shelf-life of this stain tends to be quite long except when exposed to direct light or solvent is allowed to evaporate. It is recommended that the stain be stored in a 100 mL wide mouth jar wrapped with aluminum foil to ensure a long life time. Recipe
p-Anisaldehyde Stain #2 A more specialized stain than
#1 (above), used for terpenes, cineoles, withanolides, acronycine,
etc. As above, heating with a heat gun must be employed to effect
visualization.
Recipe Prepare solution as follows: anisaldehyde:HClO4:acetone:water (1:10:20:80)
Phosphomolybdic Acid (PMA) Stain Phosphomolybdic acid stain is a good "universal" stain which is fairly sensitive to low concentrated solutions. It will stain most functional groups, however it does not distinguish between different functional groups based upon the coloration of the spots on the TLC plate. Most often, TLC's treated with this stain will appear as a light green color, while compounds of interest will appear as much darker green spots. It is necessary to heat TLC plates treated with this solution in order to activate the stain for visualization. The shelf life of these solutions are typically quite long, provided solvent evaporation is kept to a minimum. Recipe
Dissolve 10 g of
phosphomolybdic acid
in 100 mL of absolute ethanol. Occasionally, if you find it
necessary
to develop or investigate other staining techniques, the following
references
may be helpful:
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