The Extended H&A Assay
MEASURING FAMILIES OF PHENOLIC COMPOUNDS PRESENT IN RED WINE
To measure the concentrations of selected families of phenolic compounds present in a red wine, Wine X Ray uses a proprietary, extended version of the original Harbertson & Adams Tannin Assay which has been detailed in a series of publications, starting with Harbertson et al in 2002.
This assay relies on four fundamental characteristics of the behavior of wine phenolic compounds when they come in contact with salivary proteins, sulfur dioxide, and ferric chloride. These underlying principles include the pH behavior of anthocyanins in solution in the acidic region, the ability of a solution of potassium metabisulfite to bleach anthocyanins, the capability of proteins to form complexes with tannins and precipitate from solution, and the reactivity of ferric chloride with phenolic compounds that possess vicinal dihydroxyl groups.
The shift of the wine sample to the very low pH region exposes the anthocyanins’ flavylium cation species, and in this form it allows the determination of the total anthocyanins content. Once the tannins are precipitated from solution by the protein, the resulting complex is re-suspended and reacted with the ferric chloride reagent. The ensuing color change in the visible region of the light spectrum is proportional to the level of tannins present. Free anthocyanins and complexed anthocyanins behave differently when exposed to a solution of potassium metabisulfite: bleaching of the free anthocyanins is essentially complete, while bleaching of the complexed anthocyanins is partial and variable. This differential is reflected in absorbance readings in the visible region, and is used to determine the levels of free anthocyanins and Bound Anthocyanins© present. Under very high pH conditions, anthocyanins do not react with ferric chloride, but all phenolics containing vicinal dihydroxyls do. Changing the wine sample’s pH and allowing its phenolic compounds to react with the ferric chloride reagent then, the wine’s total level of iron-reactive phenolics is measured.
Since 2005, we have run tens of thousands of wine samples in the winery production laboratory environment using various forms of the assay protocol. Using this experience base, we have developed and validated the proprietary standard operating procedure (SOP) for the assay that we run routinely in our laboratory today.
This proprietary standard operating procedure has normalized the quantities of buffers, wine samples, and reagents that are used in the four individual parts of the assay; it has streamlined the incubation periods and reaction times that are necessary to attain the necessary chemical equilibrium states; it has restructured the assay’s internal calculations; and it now reports the following assay parameters: "Total Anthocyanins" in ppm ME, "Free Anthocyanins" in ppm ME, "Bound Anthocyanins©" in ppm ME, “(protein-precipitable) Tannins” in ppm CE, and "Total (iron-reactive) Phenolics" (in short "Total Phenolics") in ppm CE. ME and CE denote malvidin equivalents and catechin equivalents, respectively.
Using this SOP, Wine X Ray is able to reduce its wine sample preparation and assay execution time by one-third when running sets of twenty-four samples. Moreover, following the standard Six Sigma Quality Systems Methodology, we have conducted a number of Gage Repeatability & Reproducibility tests over the last three years of operation that have consistently shown single-digit precision error results. Today, Wine X Ray operates at levels of repeatability and reproducibility of over 95% precision.