Method of separation of anthocyanins in red wines by HPLC

Abstract

The selective and reproducible separation of anthocyanins is important not only from the analytical but also from the practical points of view, for judgment the origin of anthocyanins in products of the most frequently occurring cultures of Vitis uinifera.This work deals with the selection of the suitable conditions for the separation of anthocyanins in red wines by HPLC using gradient elution to determine the optimal pH of the mobil phase to improve the detection in the visible range and the selectivity of the column.In the works published up to now, tartaric acid, acetic acid, phosphoric acid and perchloric acid have been used in different systems to adjust the pH value, discussing the formation of a suitable elution strength and the aggressivity of the acids [ 1 -51. Material and methods Results and discussionIn the first stage, the influence of pH in the range of 1.5 to 2.2 on the separation and detection of the pigments has been studied. For the gradient elution, solvent A (HCIO, in water) and solvent B (methanol) have been used, beginning with 25% in A at 1% B/min increase rate and ending with 15% B in A.On the basis of the areas of the peak as a function of pH and of the separative efficiency Ri, j , the range of pH from 1.5 to 1.8 was shown to be the most suitable. For testing the aggressivity of the acids and their influence on the column life, three acids have been used (15% acetic acid solution had a pH value 2.17, what was outside of the optimal detection; the same, the separative efficiency at this value of pH was not satisfied). It was shown that with regard to the elution time, capacity factors k , selectivity a, separation of two components eluting one after another, corrosion (including the detector), HC10, was the most suitable acid to adjust the pH.The determination of the most suitable gradient has been made parallel for H,P04 and HCIO, selecting the composition of the mobile phase at the beginning and the slope of the gradient. The choise of suitable gradient was carried out empirically in two steps: selection of the mobile phase at the beginning and adjustation of a profile of a gradient. The optimal composition of mobile phase was evaluated on the base of both capacity factors and selectivities for peaks No. 4 and 5 in the system studied. A great difference of concentrations of methanol in solvents A and B brought about the deterioration of the separation. The last searched types of gradients are shown in Table 1. The suitability of the selected types of gradient have been judged on the basis of the capacity factors k and the selectivity coefficients a (Table 2).

Natural Food Colorants

“…The technique is nondestructive and, therefore, separated peaks are readily collected for subsequent analyses. With appropriate selection of eluent, column type and length, flow rate and temperature, HPLC can be used to resolve and quantitate microgram quantities of anthocyanin without the need for preliminary purification of extracts [351][352][353][354][355]. A major application of HPLC is, therefore, the simultaneous qualitative and quantitative analysis of anthocyanins [356].…”

Analysis of anthocyanins in red wine by HPLC using alkylamines

“…Fig. 1 shows the chromatographic record for the analysis of anthocyanins in the Cabernet Sauvignon wine with the identified peaks (1) delphidin-3-glucoside, (2) cyanidin-3-glucoside, (3) petunidin-3glucoside, (4) peonidin-3-glucoside, ( 5 ) malvidin-3-glucoside, (6) unidentified, (7) delphidin-3-glucoside acetate, (8) cyanidin-3-glucoside acetate, (9) petunidin-3-glucoside acetate, (10) peonidin-3-glucoside acetate, (1 1) malvidin-3-glucoside acetate, (12) delphidin-3-glucoside p-coumarate, (13) peonidin-3glucoside p-coumarate and (14) malvidin-3-glucoside p-coumarate.…”

“…The method of separation of anthocyanins with addition of alkylamines and the previous recommended method for separation using gradient elution [7] have been compared. This comparison was made on the basis of the time analysis, separation efficiency (selectivity, differentiation factor, capacity factor) and reproducibility of the gradient.…”

“…Fig. 1 shows the chromatographic record for the analysis of anthocyanins in the Cabernet Sauvignon wine with the identified peaks (1) delphidin-3-glucoside, (2) cyanidin-3-glucoside, (3) petunidin-3-glucoside, (4) peonidin-3-glucoside, ( 5 ) malvidin-3-glucoside, (6) unidentified, (7) delphidin-3-glucoside acetate, (8) cyanidin-3-glucoside acetate, (9) petunidin-3-glucoside acetate, (10) peonidin-3-glucoside acetate, (1 1) malvidin-3-glucoside acetate, (12) delphidin-3-glucoside p-coumarate, (13) peonidin-3-glucoside p-coumarate and (14) malvidin-3-glucoside p-coumarate.The method of separation of anthocyanins with addition of alkylamines and the previous recommended method for separation using gradient elution [7] have been compared. This comparison was made on the basis of the time analysis, separation efficiency (selectivity, differentiation factor, capacity factor) and reproducibility of the gradient.…”

Comparison of some commercial pectic enzyme preparations applicable in wine technology

“…All samples were clarified by filtration and centrifugation before the measurements. Anthocyanins were separated by LC 10AD Shimadzu Liquid Chromatogram with SPD-M10 AVP diode array detector under the following analysis conditions: column: Biosphere SIC 18, 150 mm × 4.6 mm, wavelength 520 nm (DRDÁK 1992). Helios α and δ spectrophotometers were used for measuring of A 520 .…”