Page 28 - Application Notebook - Solution for Food Development

Application No.L471
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n Quantitation of Ethyl Gallate, Rutin, and Tyrosol
in Red Wine
The analytical conditions are shown in Table 1. We membrane filter, and then injected. Fig. 2 shows the
attempted to conduct separation by reversed-phase × calibration curve for ethyl gallate. Also, Table 2 shows
reversed phase using a neutral phosphate buffer the repeatability of the total and 2D retention times,
solution in the 1st dimension, and an acidic phosphate respectively, for the three analytes, the blob area
buffer solution in the 2nd dimension. repeatability values corresponding to the peak areas,
The calibration curve for each substance was generated the calibration curve coefficient of determination (R )
2
using 6 concentrations in the range of 5 - 250 mg/L. values, and the respective quantitation values.
The red wine sample was filtered through a 0.22 µm

Table 1 Analytical Conditions
1D Column : Shim-pack XR-ODS Ⅱ (100 mm L. × 1.5 mm I.D., 2.2 µm)
Mobile Phase : A : 10 mM (sodium) phosphate buffer pH = 6.8
B : acetonitrile
Flowrate : 0.05 mL/min
Time Program : B Conc. 5 % (0 min) → 30 % (70 min) → 90 % (80 min) → 90 % (90 min)
→ 5 % (90.1 min) → STOP (110 min)
Column Temp. : 40 °C
Injection Vol. : 3 µL
Loop Vol. : 50 µL (Modulation time: 60 sec)

2D Column : Kinetex XB-C18 (50 mm L. × 3 mm I.D., 2.6 µm)
Mobile Phase : A : 10 mM (sodium) phosphate buffer pH = 2.6
B : acetnitrile
Flowrate : 2 mL/min
Time Program : Auto-gradient: Initial. B Conc. 5 % (0 min) → 30 % (0.75 min) → 5 % (0.76 min)
→ STOP (1 min)
Final. B Conc. 30 % (0 min) → 40 % (0.75 min) → 30 % (0.76 min) → STOP (1 min)
The initial and final B Conc. were changed in a stepwise manner.
Detector : SPD-M30A photodiode array detector (high sensitivity cell 1 µL, wavelength =
270 nm, 278 nm, 354 nm)

Area

100000000.0

75000000.0

50000000.0

25000000.0

0.0
0 25 50 75 100 125 150 175 200 225 250
Conc. (mg/L)

Fig. 2 Calibration Curve for Ethyl Gallate

Table 2 Repeatability of Retention Time and Blob Area (%RSD, n = 5), R Value and Concentration (mg/L)
2
Compound Total retention time Retention time (2D) Area R squared Concentration
Tyrosol 0.007 0.159 2.7 0.999804 101.5
Ethyl gallate 0.007 0.49 3.8 0.999864 15.1
Rutin 0.007 0.52 4.1 0.998805 14.2

First Edition: Nov. 2014

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