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    TLC Stainingprocedure

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    17roopa2010
    The document discusses various techniques for staining thin layer chromatography plates to visualize compounds separated during TLC. It provides details on common stains including iodine vapor, potassium permanganate, cerium ammonium molybdate, p-anisaldehyde, and ninhydrin. Each stain works via a different chemical reaction and is suitable for visualizing certain classes of compounds like amines, lipids, carbohydrates, or amino acids depending on the stain's chemistry and conditions used. Proper staining technique and avoiding solubility in the stain solution are important for clear visualization without tailing of spots.

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    TLC Stainingprocedure

    Uploaded by

    17roopa2010
    315 views5 pages
    The document discusses various techniques for staining thin layer chromatography plates to visualize compounds separated during TLC. It provides details on common stains including iodine vapor, potassium permanganate, cerium ammonium molybdate, p-anisaldehyde, and ninhydrin. Each stain works via a different chemical reaction and is suitable for visualizing certain classes of compounds like amines, lipids, carbohydrates, or amino acids depending on the stain's chemistry and conditions used. Proper staining technique and avoiding solubility in the stain solution are important for clear visualization without tailing of spots.

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    Tlc Stainingprocedure

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    The document discusses various techniques for staining thin layer chromatography plates to visualize compounds separated during TLC. It provides details on common stains including iodine vapor, potassium permanganate, cerium ammonium molybdate, p-anisaldehyde, and ninhydrin. Each stain works via a different chemical reaction and is suitable for visualizing certain classes of compounds like amines, lipids, carbohydrates, or amino acids depending on the stain's chemistry and conditions used. Proper staining technique and avoiding solubility in the stain solution are important for clear visualization without tailing of spots.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as DOCX, PDF, TXT or read online from Scribd
    Download as docx, pdf, or txt
    315 views5 pages

    TLC Stainingprocedure

    Uploaded by

    17roopa2010
    The document discusses various techniques for staining thin layer chromatography plates to visualize compounds separated during TLC. It provides details on common stains including iodine vapor, potassium permanganate, cerium ammonium molybdate, p-anisaldehyde, and ninhydrin. Each stain works via a different chemical reaction and is suitable for visualizing certain classes of compounds like amines, lipids, carbohydrates, or amino acids depending on the stain's chemistry and conditions used. Proper staining technique and avoiding solubility in the stain solution are important for clear visualization without tailing of spots.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as DOCX, PDF, TXT or read online from Scribd
    Download as docx, pdf, or txt
    of 5

    http://www.reachdevices.com/TLC_stains.

    html

    Thin Layer Chromatography stains


    TLC Stains

    TLC Staining procedure

    TLC Stain Recipe

    Stain chemistry / physics UV light excites a fluorescent additive in TLC plate. Compounds screen some of the UV, making fluorescence weaker. Sometimes, visible fluorescence is exited by UV making a spot brighter, and so is colored differently than the background Oxidative stain. Strong heating required.

    Comments

    254nm UV light

    254nm UV lamp with filter. Darker spots on light green if plates with VU indicator are used. Occasionally brighter spots (typically blue)

    A compound must have a chromophore. Even then, this technique is NOT sensitive for isolated alkenes and benzyl ethers. Fluorescence is often excited in polyconjugated compounds (benzophenone, antracene and so on).

    Permanganate stain

    KMnO4 K2CO3

    3g 10g

    The most universal stain. Harsh oxidizer. Any oxidizable compound such as alcohol, ether, ester, alkene, alkyne, alkyl aromatic, ketone, carboxylic acid, amine, amide and

    Water

    300mL

    Carefully dry the plate from elutant with a heat gun. If all residual elutant is not removed, there will be smears and streaks. Do not overheat! The plate must be hot, but touchable (5060C). Dip in stain briefly, wipe up excess (soak with a paper towel from the side) and heat up with a heat gun on high setting (move the plate, heat uniformly) until the pink color of plate just starts to become yellowish. Material develops as yellow-brown spots. If overheated all plate goes brown. Prepare a well-closed chamber with a few crystals of elemental iodine. Let it sit for 30 min to equilibrate. Put a TLC Different absorption of brown colored iodine on clean silica as opposed to silica loaded with analytes. Spots may be darker or lighter than background. Thiols will

    almost all the rest will show up as brownyellow spots (MnO2). Strongly reductive compounds such as thiols, phosphines and even dienes will show up as white spots (Mn+2) BEFORE heating. Alkanes and pyridine won't show-up at all. This stain is NOT compatible with elutants containing triethylamine - the entire plate will became yellow before spots develop...

    Iodine chamber

    Hit and miss stain. Works in about 50% of all cases. Works with alkanes! Thiols and phosphines will immediately show up as white spots.

    plate inside, keep the plate for 2 sec to 10 min inside (trial and error) and then take the plate out. Watch the color change. Pick the best moment to circle spots - they might soon disappear. PMA (CAS:1202657-2)

    oxidize to disulfides: 2R-SH + I2 = R-S-S-R + 2HI

    PMA stain (Phosphomoly bdic acid stain)

    Oxidative stain. Strong heating required. Ethanol 100mL H3[P(Mo3O10)4] * xH2O is Mo6+ compound, light yellow. Upon Application technique is exposure to reducing organics the same as for Mo5+ and Mo4+ compounds are Permanganate stain. Upon formed (molybdenum blue, so heating, dark green spots called Keggin structure) which are appear on light green green or blue colored background 10g Ammonium molybdate tetrahydrate (CAS:12054-85-2) Cerium ammonium sulfate

    Expensive. Fairly universal stain. Some amines, amides and oxidation-resistant aromatics won't show up. Stain solution is somewhat light-sensitive.

    CAM stain (Hanessian's Stain; Cerium Ammonium Molybdate stain)

    Oxidative stain. Strong heating required. 2.5g Procedure same as above 1g

    Very universal stain. More sensitive than PMA stain (above).

    dihydrate(CAS:1037 8-47-9) 10m L 90m L

    Sulfuric acid

    Water

    Application technique same as for the Permanganate stain. Upon heating, dark blue spots appear on light blue background p-Anisaldehyde (CAS:123-11-5) Sulfuric Acid pAnisaldehyde Stain Acetic Acid Ethanol Some heating required. Chemistry of this stain is unknown. Probably something along the following lines:

    4mL 5mL 2mL 150mL

    Application technique same as for the Permanganate stain. Upon heating spots of varying

    Not as universal as oxidative stains. Works for allylic alcohols (green spots), phenols (violet spots) amines, aldehydes, ketones, carbohydrates, esters like alkylphtalates (blue/red spots) and some other compounds. Alkenes, alkynes and aromatic compounds will NOT show up.

    color appear on light pink background ninhydrin (CAS:485-47-2) Acetic Acid Ninhydrin Stain Acetone Gentle warming required. Blue-pink color is due to the 0.5mL formation of Ruhemann's purple: 0.1g 100mL

    Dip plate into stain, let acetone evaporate and then gently warm up the plate. Usually a light pink coloration may develop on a background.

    Specific stain for amino acids (see group TLC below) and primary amines. Secondary amines stains light yellow which is difficult to see. Tertiary amines do not stain.(An interesting review:Friedman, M. J. Agric. Food Chem. 2004, 52, 385406.) See ninhydrin stain colors for all aminoacids!

    For colorless compounds, a visualizing technique is needed to observe TLC results. Stains can be applied by spraying or by dipping of a plate into solution. The latter is by far more convenient. However, in order to work, the right the stain solution SHOULD NOT dissolve analyte spots. For example, permangantate stain is perfect for most not-too-polar organic compounds while acetone-based nynhydrin stain is excellent for amino acids. If analyte solubility in stain solution is inevitable, try to dip the plate as quickly as possible, and then immediately wipe off an excess of stain. Still, there will be some artificial tails added to spots. Also, do not forget, if a compound that must be analyzed is volatile, it may evaporate before the stain visualizes it, especially if heating is required for visualization. The table below represents a few of these techniques:

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