Paper Chromatography VEN124L Method

Paper Chromatography for Monitoring Malolactic Fermentations

(Note: Chromatography reagent will stain your hands and clothes.  Use gloves and a lab coat to avoid stains.  Always wear splash goggles when working with chemicals)

1        Preparation of the chromatography solvent:

  •  100 ml n-butanol (reagent grade)
  •  100 ml water
  •  10.7 ml formic acid (reagent grade)
  •        15 ml indicator solution (1 g bromocresol green in 100 ml water)
  •      Mix chemicals and place in separatory funnel.  Make sure stopper is securely closed. Mixture will separate into two layers; discard lower (aqueous) layer in proper waste container labeled “aqueous phase chromatography waste

 Store freshly made reagent in container labeled “new or rejuvenated chromatography reagent”.

2        Setting up chromatography paper (Figure 1):

  • Clean bench top; place paper towels down on surface
  • Take a sheet of chromatography paper and lay on towels
  •  Mark a pencil line across the bottom of the page, about 1 inch from the bottom
  • Make tic marks at 2 cm intervals along the pencil line
  • Label tic marks with standard or sample code using pencil
  •  Label sheet at top with initials, sample date and other information
  • Only use pencil for labeling.

(Note: Sweat contains lactic acid.  Handle only the edges of the paper with your fingers.  You may want to wear gloves to avoid lactic acid contamination of your test.)

3        Spotting of chromatography paper:

  • Use small (1.1-1.2 mm I.D.; 75 mm length) glass capillary tubes, one for

                  each sample or standard

  •  Draw liquid from sample in to the capillary and touch capillary to paper
  • Allow no more than a spot of 1 cm to form on paper
  • Allow spot to dry
  • Repeat spotting 4- 5 times

4        Running the Chromatogram

  • After all spots are completely dry, curve paper into a cylinder
  • Staple ends to hold cylinder together – do not overlap ends of paper
  • Transfer 70 ml of  new or rejuvenated solvent to jar (large mayonnaise type)
  • Carefully insert cylinder (Figure 2) spot side down
  • Close lid carefully

5        Development of the chromatogram:

  • After approximately 4 to 6 hours at room temperature the solvent will have ascended the chromatography paper carrying the spots with it.
  • Carefully remove chromatogram careful not to touch the wet portion
  • Carefully tear away edges from the staples
  • Hang chromatogram in a well-ventilated area (chemical hood) to dry
  • Yellow spots on a blue-green background should be visible and indicate the position of the acids
  • All chromatograms should have standards (solution of individual acids) spotted to identify position of the acids (Figure 3).

6        Cleaning up:

  • Carefully pour chromatography reagent into storage container marked “used chromatography solvent” (Chromatography reagent may be reused, but must be separated in a separatory funnel and occasionally reacidified before each use.)
  • Take the empty mayonnaise jar to the sink and rinse it thoroughly with warm water. The first rinse should go into a container labeled “acid waste”.
  • Place the cleaned jar upside down on clean paper towels for the next person to use.
Figure 1: Paper Chromatography Set up
Figure 1: Paper Chromatography Set up
Figure 2: Paper Chromatography Jar Set Up

Figure 2: Paper Chromatography Jar Set Up
Figure 3: Paper Chromatography: Spot Location

Figure 3: Paper Chromatography: Spot Location

Interpretation:  Samples 1, 4, and 5 have completed the malolactic conversion; there is no detectable malate spot and the presence of lactic spots.  Sample 2 has not started the malolactic conversion.  Sample 3 may be mid-way through the malolactic conversion  or the lactate may have derived from a different source.