Method 21.1 High Pressure Liquid Chromatography (HPLC)

Purpose: To determine pungency levels in capsicums and their oleoresins.

 A. Apparatus: 
  1. High pressure liquid chromatograph, pump flow rate (0.6 to 1.8 ml/min).
  2. Detector
  1. UV at 280 nm, flow cell path (1 x 10 mm), greater than 700 ppm capsaicin.
  2. Fluorescence at 288 excitation, 320 emission, flow cell path of 10 x 1 micron, less than 700 ppm capsaicin.
  1. Column: C18, 10 micron spherical, 4.6 mm x250 mm.
  2. Rheodyne syringe loading sample injector with 20 microliter loop.
  3. 100 ml volumetric flask.

B. Reagents:

  1. Acetonitrile, HPLC grade.
  2. Dioxane, HPLC grade.
  3. HPLC grade water.
  4. HPLC grade methanol.
  5. 2% perchloric acid.
  6. 95% ethanol, saturated with sodium acetate.
  7. HPLC grade sodium acetate.
  8. Capsaicin. (Pharmaceutical quality).

C. Procedure:

  1.  Weigh sample into 100 ml. volumetric flask:
    1. 10.00 g for pungencies from 0 to 1000 ASTA Heat Units
    2. 1.00 g for pungencies from 1,000 to 10,000 ASTA Heat Units
    3. 0.10 g for pungencies greater than 10,000 ASTA Heat Units
  2. Bring to 100 ml. with 95% ethanol saturated with sodium acetate.
  3. Cap and place on hot plate at 60 C or water bath for three hours, swirling occasionally.
  4. Swirl and allow solids to settle.
  5. Fill sample loop and elute with a mix* of: 48.4% methanol, 30.2% HPLC grade water, 13.3% dioxane, 7.9% acetonitrile, 0.2% of 2% perchloric acid.
  6. The first peak is the pigment peak, the second peak is capsaicin, the third peak is capsaicinoid.
  7. Prepare capsaicin standards at 0.01, 0.10, 1.0 mg./ml. (label 10, 100 and 1,000 ppm capsaicin) in 95% ethanol, saturated with sodium acetate.

*For low pungency capsicums (less than 700 ppm capsaicin):

  1. Prepare solvent mixture A (5.4% acetonitrile, 3.6% dioxane, 0.7% perchloric acid, 90.3% water) and B (9.0% acetonitrile, 18.0% dioxane, 73.0% methanol).
  2. Program gradient separation: At 0 min. (0% solvent B); at 2.5 min. (increase B from 0 to 60% in 5 min.); 7.5 to 12.5 min. (isocratic at 60%); 12.5 min. (60% to 100% B).

Calculation:  

  1. Total capsaicin area=Area peak 2+.82 Area peak 3.

  2. Capsaicin (ppm) = Total capsaicin area of sample x ppm standard x 100 / Total capsaicin area of standard xg.

  3. Sample ASTA Pungency=ppm capsaicin

  4. Pungency (Scoville) =ppm capsaicin x 15 (assume Capsaicin standard at 15,000,000 Scoville)

ppm capsaicin x 15 = Scoville units (assumes capsaicin = 15,000,000 Scoville)