January 2001   

Roger Harris
Monitoring MLF by TLC Ė an update
R.L.N. Harris 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 satisfactory.

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 carefully followed:

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 migrate.

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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