Monitoring MLF by TLC Ė an update
Brindabella Hills Winery
Via Hall, ACT, 2618
In 1993 (The Australian Grapegrower & Winemaker, April)
we described a simple, rapid procedure using thin layer
chromatography (TLC) to monitor the progress of malo-lactic
fermentation (MLF) in wine. The advantages of this procedure
over paper chromatography were that it was rapid, requiring
less than 15 minutes to carry out, and that it distinguished
between succinic and lactic acids, allowing the appearance of
the latter to be seen, indicating the onset of MLF, as well as the
disappearance of malic acid, indicating completion of MLF.
The TLC procedure, as published, was specifically
developed for Schleicher and Schull TLC films, which gave
excellent results. Unfortunately, these
films are no longer available in
Australia. Substitution with readily-available
Merck Silica gel 60 TLC
films (plastic or aluminium backed) in
the original procedure is not
We now report a modification of the
original procedure using Macherey-Nagel
Sigel films. These are available
in packets of 25 from Selby Biolab
(phone 132991) or in smaller
quantities from Winery Supplies
(phone 03 9764 9547). For successful
monitoring of MLF the following procedure should be
1. The M-N films as supplied may be cut with sharp
scissors into small strips. Apiece 50 x 25mm is sufficient
to examine three wine samples as well as a reference
solution (malic + lactic, 2g/L each in water).
2. The wine samples are applied to the TLC film by means
of a fine glass capillary. This may be made by drawing
out the tip of a pasteur pipette using a gas flame. The
sample is spotted once, allowing the spot to grow to
about 2mm diameter. Donít overload the film! Samples
are spotted in a straight line about 7mm from the end of
the TLC strip. All water is then removed by a stream of
hot air from a hairdryer held about 1-2cm from the strip.
The strip is held against the light and drying continued
until the reference spot (originally grey) is invisible
(about 1 minute). Insufficient drying results in streaking
is particularly obvious when red pigment is seen to
3. The solvent mixture (toluene + acetic acid + n-butyl
acetate 2:1:1) is placed in a small jar to a depth < 5mm
and the TLC strip placed in the jar (using tweezers) so
that the base is in the solvent but the spots are just above
it. A lid is placed on the jar and the solvent allowed to
ascend the strip for a distance of approximately 40mm
(about 5 minutes).
4. The strip is removed with tweezers and immediately
dried as before with a hairdryer until the solvent has been
removed (about 1 minute). This should be done in a well-ventilated
area and inhalation of solvent fumes avoided.
5. The strip is then immersed in an indicator solution
comprising bromocresol green water soluble form in
methylated spirits (2mg/ml) in another glass jar. Dipping
should be in one motion to avoid lap marks on the strip.
The strip is dried again but this time with cold air from the hairdryer.
6. The organic wine acids will appear as bright yellow spots against a
background, which is initially orange but changes slowly
to olive green. (Note: using hot air to evaporate the
indicator solvent causes the background colour to revert
to orange. Cold air changes the colour back to olive
green). The development of the contrast between the
spots and the background can be accelerated by briefly
holding the strip above a solution of DAP in water which
has been made alkaline with a little 1M NaOH. The
ammonia vapours produced rapidly change the indicator
colour towards green (exposure for too long may
neutralise the wine acid spots also!).
Lactic acid is visible from very early in the onset of MLF.
Sometimes a weak acid spot appears between malic and lactic
very early in the life of the wine, but disappears after a few
weeks. This compound has not been identified, but may be
ethyl hydrogen tartrate (which subsequently hydrolyses).
Remember that TLC is a procedure for monitoring the
progress of MLF, and not for quantitative measurement of
organic acids in wine. The latter is possible by enzymatic
analysis or HPLC services available from analytical
laboratories in the industry.
Roger Harris can be contacted at Brindabella Hills, 4
Woodgrove Close, via Wallaroo Road, Hall, ACT 2618, phone
(02) 6230 2583, fax (02) 6230 2023.
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