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    TLC Staining Procedure TLC Stain Recipe Stain Chemistry / Physics Comments

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    Lara Dias
    TLC staining provides a method to visualize compounds separated on a TLC plate. There are several common staining techniques: 1. Permanganate stain is a universal oxidative stain that oxidizes most functional groups like alcohols, ethers, alkenes, and aromatics, causing them to appear as brown-yellow spots on the plate. 2. Phosphomolybdic acid (PMA) stain is also oxidative and universal, causing reducing compounds to form blue or green spots, though it is less sensitive than permanganate stain. 3. Ninhydrin stain specifically detects amino acids and primary amines, causing them to appear as blue-pink spots.

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    TLC Staining Procedure TLC Stain Recipe Stain Chemistry / Physics Comments

    Uploaded by

    Lara Dias
    383 views3 pages
    TLC staining provides a method to visualize compounds separated on a TLC plate. There are several common staining techniques: 1. Permanganate stain is a universal oxidative stain that oxidizes most functional groups like alcohols, ethers, alkenes, and aromatics, causing them to appear as brown-yellow spots on the plate. 2. Phosphomolybdic acid (PMA) stain is also oxidative and universal, causing reducing compounds to form blue or green spots, though it is less sensitive than permanganate stain. 3. Ninhydrin stain specifically detects amino acids and primary amines, causing them to appear as blue-pink spots.

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    TLC Stains

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    TLC staining provides a method to visualize compounds separated on a TLC plate. There are several common staining techniques: 1. Permanganate stain is a universal oxidative stain that oxidizes most functional groups like alcohols, ethers, alkenes, and aromatics, causing them to appear as brown-yellow spots on the plate. 2. Phosphomolybdic acid (PMA) stain is also oxidative and universal, causing reducing compounds to form blue or green spots, though it is less sensitive than permanganate stain. 3. Ninhydrin stain specifically detects amino acids and primary amines, causing them to appear as blue-pink spots.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    383 views3 pages

    TLC Staining Procedure TLC Stain Recipe Stain Chemistry / Physics Comments

    Uploaded by

    Lara Dias
    TLC staining provides a method to visualize compounds separated on a TLC plate. There are several common staining techniques: 1. Permanganate stain is a universal oxidative stain that oxidizes most functional groups like alcohols, ethers, alkenes, and aromatics, causing them to appear as brown-yellow spots on the plate. 2. Phosphomolybdic acid (PMA) stain is also oxidative and universal, causing reducing compounds to form blue or green spots, though it is less sensitive than permanganate stain. 3. Ninhydrin stain specifically detects amino acids and primary amines, causing them to appear as blue-pink spots.

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    © All Rights Reserved
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    TLC Staining

    TLC Stain Recipe Stain chemistry / physics Comments


    procedure
    UV light excites a
    fluorescent additive in TLC A compound must have a
    plate. Compounds screen chromophore. Even then, this
    254nm UV lamp with filter.
    some of the UV, making technique is NOT sensitive for
    Darker spots on light green if
    fluorescence weaker. isolated alkenes and benzyl ethers.
    254nm UV light plates with VU indicator are
    Sometimes, visible Fluorescence is often excited in
    used. Occasionally brighter
    fluorescence is excited by polyconjugated compounds
    spots (typically blue)
    UV making a spot brighter, (benzophenone, antracene and so
    and so is colored differently on).
    than the background
    KMnO4
    K2CO3
    Water
    The most universal stain. Harsh
    Carefully dry the plate from oxidizer. Any oxidizable compound
    elutant with a heat gun. If all such as alcohol, ether, ester, alkene,
    residual elutant is not removed, alkyne, alkyl aromatic, ketone,
    Oxidative stain. carboxylic acid, amine, amide and
    there will be smears and
    Strong heating almost all the rest will show up as
    streaks. Do not overheat! The
    plate must be hot, but required. brown-yellow spots (MnO2). Strongly
    Permanganate touchable (50-60C). Dip in reductive compounds such as thiols,
    stain stain briefly, wipe up excess phosphines and even dienes will
    (soak with a paper towel from show up as white spots (Mn+2)
    the side) and heat up with a BEFORE heating. Alkanes and
    heat gun on high setting (move pyridine won't show-up at all. This
    the plate, heat uniformly) until stain is NOT compatible with elutants
    the pink color of plate just starts containing triethylamine - the entire
    to become yellowish. Material plate will become yellow before
    develops as yellow-brown spots develop.
    spots. If overheated all plate
    goes brown.
    Prepare a well-closed chamber Different absorption of
    with a few crystals of elemental brown colored iodine on
    iodine. Let it sit for 30 min to clean silica as opposed to
    equilibrate. Put a TLC plate silica loaded with analytes. Hit and miss stain. Works in about
    inside, keep the plate for 2 sec Spots may be darker or 50% of all cases. Works with
    Iodine chamber
    to 10 min inside (trial and error) lighter than background. alkanes! Thiols and phosphines will
    and then take the plate out. Thiols will oxidize to immediately show up as white spots.
    Watch the color change. Pick disulfides:
    the best moment to circle spots
    - they might soon disappear. 2R-SH + I2 = R-S-S-R + 2HI
    Oxidative stain. Strong
    heating required.

    PMA (CAS:12026-57-2)
    H3[P(Mo3O10)4] * xH2O is
    Ethanol Expensive. Fairly universal stain.
    Application technique is the Mo6+ compound, light
    PMA stain Some amines, amides and
    same as for Permanganate yellow. Upon exposure to
    (Phosphomolybdic oxidation-resistant aromatics won't
    acid stain) stain. Upon heating, dark green reducing organics Mo5+ and
    show up. Stain solution is somewhat
    spots appear on light green Mo4+ compounds are light-sensitive.
    background formed (molybdenum blue,
    so called Keggin structure)
    which are green or blue
    colored
    Some heating may be
    Dinitrophenyl 2,4-dinitrophenylhydrazine required. Developed mainly for aldehydes and
    Conc. Sulfuric acid
    hydrazine (DNP) Water ketones, which are usually yellow to
    ethanol Formation of correspondent orange.
    hydrazones.
    Ammonium molybdate tetrahydrate
    CAM stain (CAS:12054-85-2) 2.5g
    Oxidative stain.
    Cerium ammonium sulfate dihydrate
    (Hanessian's (CAS:10378-47-9) Strong
    1g heating required. Very universal stain. More sensitive
    Stain; Cerium Sulfuric acid 10mL than PMA stain.
    Ammonium Water Procedure
    90mL same as PMA
    Molybdate Application technique same as
    stain) for the Permanganate stain.
    Upon heating, dark blue spots
    appear on light blue
    background
    Some heating
    required.
    p-Anisaldehyde (CAS:123-11-5) Not as universal as oxidative stains.
    Sulfuric Acid
    Chemistry of this stain is Works for allylic alcohols (green
    Acetic Acid spots), phenols (violet spots)
    Ethanol unknown. Probably
    something along the amines, aldehydes, ketones,
    p-Anisaldehyde Application technique same as
    following lines: carbohydrates, esters like
    Stain for the Permanganate stain. alkylphtalates (blue/red spots) and
    Upon heating spots of varying some other compounds. Alkenes,
    color appear on light pink alkynes and aromatic compounds
    background will NOT show up.

    Gentle warming
    required.
    ninhydrin (CAS:485-47-2)
    Acetic Acid Specific stain for amino acids (see
    Acetone
    Blue-pink color is due to the group TLC below) and primary
    Dip plate into stain, let acetone formation of Ruhemann's amines. Secondary amines stains
    Ninhydrin Stain evaporate and then gently purple: light yellow which is difficult to see.
    warm up the plate. Usually, a Tertiary amines do not stain.
    light pink coloration may (An interesting review:Friedman, M. J.
    develop on a background. Agric. Food Chem. 2004, 52, 385-406.)

    Vanillin Some heating may be


    Very good general stain, giving a
    Vanillin Ethanol required.
    Conc. Sulfuric acid range of colors for different spots.
    PERMANGANATE STAIN
    (KMnO4)

    IODINE

    PMA STAIN
    (Phosphomolybdic acid stain)

    PMA STAIN
    (Phosphomolybdic acid stain)

    DNP STAIN
    (Dinitrophenyl hydrazine)

    DNP STAIN
    (Dinitrophenyl hydrazine)

    CAM STAIN
    (Cerium Ammonium Molybdate Stain)

    CAM STAIN
    (Cerium Ammonium Molybdate Stain)

    p-ANISALDEHYDE STAIN

    p-ANISALDEHYDE STAIN

    NINHYDRIN STAIN

    VANILLIN

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