Skip to content

High-Performance Liquid Chromatography (HPLC) for Pigment Analysis Of Red Wine

Red wine obtains its characteristic red color from the presence of anthocyanin pigments extracted from the grape skins during winemaking. However, the color is also influenced by reactions between anthocyanins and other phenolic compounds like tannins and acids throughout wine aging, which leads to the formation of more stable pigmented polymers. The composition and concentration of these pigments evolve during wine maturation and affect the resulting color properties of the finished red wine.

Chromatographic Analysis of Red Wine Pigments

High-performance liquid chromatography (HPLC) is an essential analytical technique for separating and quantifying the complex mixture of phenolic compounds that contribute to the characteristic color of red wine.

It allows sensitive measurement of the individual anthocyanins as well as derived pigments that contribute to wine color.

The typical HPLC setup uses a reverse-phase C18 column to separate compounds based on hydrophobicity. Anthocyanins and other polar phenolics elute early in the chromatogram while more nonpolar polymeric pigments elute later. Detection is performed with a photodiode array detector to acquire visible absorbance spectra of eluting peaks.

Comparison of spectra and retention times to reference standards allows the identification of individual anthocyanin and pigment compounds. Quantification is achieved by calibration with external standards.

HPLC Studies on Red Wine Pigment Composition

Numerous HPLC studies have examined the anthocyanin and pigment profile of wines made from different grape varieties and production regions.1 For example, Pinot Noir wines are characterized by high proportions of pigmented polymers compared to other red varietals, correlating with their lighter red hues.

Common Pinot noir pigments identified by HPLC include pyranoanthocyanins formed from reactions between anthocyanins and fermentation-derived metabolites like acetaldehyde, vitamins derived from reactions with pyruvic acid, and polymeric pigments formed by condensation reactions between anthocyanins and procyanidins.

In contrast, young Cabernet Sauvignon grape wines contain high proportions of individual anthocyanin pigments like malvidin-3-O-glucoside and have high ratios of monomeric to polymeric pigments.2 During maturation, the anthocyanin concentration decreases while polymeric pigments increase, resulting in the incremental development of a brick-red hue. HPLC analysis consistently demonstrates greater extraction and retention of anthocyanins in Cabernet wines compared to Pinot noir.

Advantages of HPLC Pigment Analysis

The main advantages of using HPLC analysis for characterization of wine pigments are:

  • The high chromatographic resolution allows the separation of over 50 individual anthocyanins and derived pigment compounds in a single run.
  • Sensitive and precise quantification of pigment classes including monomeric anthocyanins, proanthocyanins, direct flavanol-anthocyanin condensation products, acetaldehyde-mediated, and other polymeric pigments.
  • Spectral analysis helps in more accurate identification and confirmation of known pigments.
  • Minimal pre-analysis sample preparation is required compared to other techniques.
  • Automated sampling and analysis enable high sample throughput.

Limitations and Future Directions

One limitation of HPLC is the lack of commercially available standards for many derived wine pigment compounds, which precludes precise quantification. There is also debate about the appropriate methodology for quantifying polymeric pigment fractions.

Future applications could incorporate mass spectrometry detection for further structural confirmation of unknown pigments. Two-dimensional LC or hydrophilic interaction liquid chromatography (HILIC) systems may provide even greater separation power for characterizing pigment diversity in wines. Overall, however, HPLC remains an indispensable tool for understanding red wine pigment chemistry.

Role of Co-pigmentation Reactions

In addition to direct condensation reactions, co-pigmentation between anthocyanins and other phenolics like flavonols and hydroxycinnamic acids can stabilize color in young red wines.

These reversible non-covalent associations enhance anthocyanin stability and color expression. HPLC enables the detection and quantification of the various co-pigments and their contribution to wine color properties.

Monitoring Pigment Evolution During Winemaking

HPLC analysis of grape, must, and wine samples at different stages from harvest through fermentation and aging provides detailed insights into pigment evolution during winemaking. The anthocyanin extraction kinetics, concentration effects, reactions, and retention can be monitored to evaluate how winemaking techniques influence final wine color outcomes. This allows the refinement of protocols to optimize color stability in the finished wine style.

Frequently Asked Questions

What are the main individual anthocyanin pigments in red wine?

The five principal anthocyanin pigments found in all red wines are malvidin, peonidin, petunidin, delphinidin, and cyanidin glycosides. Malvidin-3-glucoside is typically the predominant anthocyanin.

What causes the color change in red wines from purple to brick red hues during aging?

The progressive loss of monomeric anthocyanins coupled with the increasing formation of more stable red-orange polymeric pigments causes red wines to take on brick-red hues over time rather than the initial vibrant purple color.

How does HPLC analysis help winemakers evaluate grape maturity?

HPLC provides precise quantification of not only sugars and acids but also anthocyanins and other phenolics which are indicators of physiological maturity. The anthocyanin profile can reveal information about grape ripeness and color potential.

Can white wines contain anthocyanins?

Yes, certain white wine styles like orange wines can retain low levels of anthocyanins from brief skin contact during fermentation. HPLC analysis is sensitive enough to detect traces of anthocyanins in such wines.


  1. Bimpilas, A., Panagopoulou, M., Tsimogiannis, D., & Oreopoulou, V. (2016). Anthocyanin co-pigmentation and color of wine: The effect of naturally obtained hydroxycinnamic acids as cofactors. Food Chemistry ↩︎
  2. Schwarz, M., Picazo-Bacete, J. J., Winterhalter, P., & Hermosín-Gutiérrez, I. (2005). Effect of co-pigments and grape cultivars on the color of red wines fermented after the addition of co-pigments. Journal of agricultural and food chemistry ↩︎