High Speed Separation of Isoflavones in Soybean Milk Using UHPLC

March 5, 2021


Isoflavones are polyphenolic compounds produced in Fabaceae such as soybeans. They act as phytoestrogens in mammals. They are also strong antioxidants and considered useful in treating cancer. Common isoflavones include daizin, genistin, glycitin, daidzein, genistein, and glycitein.

We have demonstrated the analysis of isoflavones using Ultra high-pressure liquid chromatography (UHPLC). The results were obtained using an X-PressPak C18S (2.1 mm I.D. x 50 mmL., 2 µm) column combined with a high-pressure gradient method described below. Total analysis time was approximately 4 minutes.

LC-4000 UHPLC system


Chromatographic conditions:
Column = X-PressPak C18S (2.1 mm I.D. x 50 mmL.)
Column temperature = 40°C
Mobile phase A = acetonitrile/water/acetic acid (10/90/0.1)
Mobile phase B = acetonitrile/water/acetic acid (40/60/0.1)
Gradient (A/B) = 0 min (100/0) —> 5 min (0/100) —> 5.5 min (0/100) —> 5.55 min (100/0)
Flow rate = 0.6 mL/min
Detection = UV detection (254 nm)
Injection volume = 1mL


Figure 1 shows the chromatogram of a standard mixture of isoflavones. Each component is sufficiently separated; the analysis time is 4 minutes and the time from injection to injection is 7.5 minutes.

Figure 1.X-LC chromatogram of a standard mixture of isoflavone.

Figure 2 shows the chromatogram of soymilk. Each component is well separated from unknown peaks.

Figure 2. Chromatogram of soybean milk.

1. Add methanol (2 mL) to soybean milk (2 mL)
2. Stand at 60°C for 1.5 hours
3. Add Hexane (1 mL) and mix
4. The hexane layer is filtered.

About the Author

Chromatography Group