Malolactic Chromatography Testing

In Vintners Corner by Admin2 Comments

Testing for Malolactic Completion (Chromatography Test)

Don Graves presented the following information at the October 2002 Club meeting. For an understanding of the advantage and disadvantages of malolactic fermentation, and the when-to and when-not-to, he recommends “Malolactic Fermentation” by Bill Collings, on the BCAWA website.  http://www.bcawa.ca/winemaking/ml.htm

Background

Some wine experts claim that the accurate monitoring of Malo-Lactic Fermentation (MLF) is one of the most critical parameters in red wine making but one that is most commonly overlooked. Paper Chromatography is a simple qualitative analytical procedure that has been used for many years in the wine industry to assess the progress of MLF. Knowing that your wine has completed MLF gives peace of mind that the process will not occur after bottling and potentially spoil your wine.

Paper Chromatography is not the only procedure for monitoring MLF. Other procedures, such as Thin Layer Chromatograph, High Pressure Liquid Chromatography, and Enzymatic Chromatography, are used by industry and are more precise than Paper Chromatography. But they are more complex, more costly, and more suited to large winery operations.

Paper Chromatography method

Paper Chromatography uses simple filter paper, a special solvent, and an indicator to separate complex mixtures into individual components. In the case of MLF monitoring in wine, the procedure simply allows one to identify the presence of tartaric, citric, malic and lactic acids in a qualitative (or semi-quantitative) manner. Since citric acid is not a major issue in wine of MLF, it is not normally monitored or tested. Through capillary action, the acids move up the filter paper with help from the solvent.

Each acid, with its unique molecular properties, moves a characteristic distance up the paper and, with the help of acid standards, you can readily identify the acids in the wine sample. A colour indicator is used to change the acid spot to yellow distinguishing it from the blue/green background colour developed on the paper.

Malic acid will show as a large spot on the paper before MLF but as the bacteria fermentation progresses, the malic spot diminishes and the lactic acid spot increases in size. At some point the malic acid spot is difficult to distinguish and MLF is nearing completion. However, since the paper chromatography procedure can only detect down to 100 mg/L (100 ppm), there could still be trace amounts of malic acid in the wine. As little as 30 mg/L can cause bubbling in your bottled wine. Therefore, one should allow the fermentation to continue until no more characteristic MLF bubbles appear in the neck of the carboy. This may take another week or so.

Chromatography Kit Components

Most commercially available kits consist of the following:

  • 30 sheets Whatman #1 chromatography paper (size approx. 19cm x 23cm)
  • 250 mls solvent
  • 100 capillary pipettes
  • Acid standards (30 mls tartaric, malic and lactic)
  • 1 gal wide mouth jar

The solvent is made from water, n-butanol, concentrated formic acid (very dangerous) and bromocresol green indicator. It has a limited shelf life of 3-6 months if stored properly, preferably in a refrigerator in a dark bottle. Several suppliers of kits are available in Canada and the USA. The following were contacted (prices do not include taxes or shipping):

  • Funk Winemaking Supplies, Ontario ($61.00 – no jar.)
  • Watson’s, Ontario ($40.25.)
  • Presque Isle, Phil. USA (Can$69.00. Reduced to $54.00 for multiple kits.)
  • The Wine Lab, Napa, USA ($81.00.)
  • Duncan McBarley’s, Duncan ($165.00.)

Paper Chromatography Test Procedure

Each kit comes with a testing procedure. Basically it consists of the following steps:

  1. Prepare paper. Draw pencil line approx. 2 cm from bottom to sheet and place ‘x’ marks on the line approx 2 cm apart
  2. Draw a small sample into capillary, approx 1 cm or less
  3. Touch capillary on paper at the ’x’ mark – (identify sample at bottom of paper). Malic and lactic standards can also be spotted if you wish. Not necessary on a routine basis.
  4. After all samples are on paper, air dry for approx 30 minutes.
  5. Roll paper into cylinder shape and staple, top & bottom.
  6. Add 2 cm of solvent to jar and place paper in jar—replace lid.
  7. Allow the solvent to make its way up to the top of the paper. This can take 6-8 hours.
  8. Attach a clothespin and string to the top of the paper. Remove from jar. Hang paper in a well-ventilated area and allow to dry/develop. This can take 6-8 hours or longer depending on air conditions.
  9. Tartaric, malic and lactic acids will show up as yellow spots if present in the wine.

The sample spot should be kept as small as possible. To achieve this, simply add less sample but repeat twice with air drying in between. Toothpicks also work in place of the capillary. (A sample chromatogram was submitted indicating lacitc, malic and tartaric acids. Unfortunately I was unable to reproduce the sample electronically.)

Drafted by Don Graves, October 2002

Comments

  1. My entire paper came out yellow, making it impossible to detect the acid spots.

    1. Hi Clayton,
      This may happen with old chromatography solution. Store your chromatography solution in the fridge and make sure to shake it before pouring into the jar. Also be sure to store your chromatography paper in an envelope where light cannot get to it. You can try the steps below once the paper is dry – I have been able to use chromatography solution well beyond the expiration date with this tip.

      * Put a small amount of ammonia in a dish or pan and set at the bottom of a 23L pail
      * Suspend the chromatography paper in the pail close to the top (I use a coat hanger bent to shape and hook onto the lip of the pail on each side)
      * Give it 15 minutes or so and you should see the yellow go to blue accept where the acid blobs are located

      Notes:
      * The above is applicable once the paper has dried by the normal process
      * This will enhance the readability of any test by providing more contrast
      * DO NOT LET ANY LIQUID AMMONIA TOUCH YOU PAPER – it will ruin your test
      * If you don’t have liquid ammonia – try Windex

      Let me know if this works for you
      -Dave

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