The vanillin assay

An alternative colorimetric method relies on the reaction with vanillin under acidic conditions. A 2-mL aliquot of a freshly prepared solution of vanillin (1 g/100 mL) in 70% sulfuric acid is added to 1 mL of aqueous plant extract. The mixture is incubated in a 20°C-waterbath and after exactly 15 min. the absorbance at 500 nm read. The concentration of proanthocyanidins is expressed as (+)-catechin equivalents (used for the standard curve). This assay is specific for flavonols. As a consequence, when using this assay to determine the concentration of condensed tannins, widely distributed monomeric flavonols, such as catechin (1.39) and epicatechin (1.90), can interfere (Hagerman and Butler, 1989). 

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The use of HPLC for quantification of phenols is often limited to a single class of phenolics and then often only to low-molecular weight compounds that are available as standards. It is, therefore, often necessary to use colorimetric assays such as the Folin-Ciocalteau assay which rely on the reducing ability of phenols to quantify the amount of total phenolics in a sample (Waterman and Mole, 1994 Singleton et al, 1999 Schofield et al, 2001). The degree of condensation of polyphenols can be quantified by colorimetric assays such as the acid-butanol assay and the vanillin assay (Waterman and Mole, 1994 Schofield et al, 2001). 

The reaction kinetic of the vanillin assay depends on the chemical structure. For (+)-catechin the reaction was terminated after 7 minutes,... 

Conventional assays in hydrochloric acid work best, if moisture is rigorously excluded. Since procyanidins are most effectively extracted with aqueous organic solvents, this is a major drawback. For this reason Makkar and Becker [143] developed a procedure which also allows the performance of the vanillin assay in aqueous methanol or acetone. The use of aqueous acetone is delicate, since it is known that acetone reacts with acidified vanillin to produce a chromogen exhibiting a A,max at 548 nm which may interfere determinations substanially [143]. 

The vanillin procedure involves reaction of an aromatic aldehyde, vanillin, with the meta-substituted ring of flavonols to yield a red adduct. Although the vanillin assay is used for the estimation of condensed tannins (proanthocyanidins), the... 

The colorimetric assay of sulphadiazine is based on the acid-catalysed equilibrium reaction that occurs between vanillin (an aldehyde) and sulphadiazine (an aiylamine). The chemical species that forms as shown below is known as the Schiff s Base and is yellow in colour. 

The following functional group assays are used proanthocyanidin assay, vanillin assay and dimethylaminocinnamaldehyde (DMACA) assay. Details to the experimental designs are listed in Tab. (3.1) to (3.3). 

Although, during the last twenty years numerous attempts have been made to improve vanillin assays with respect to reproducibility and accuracy, resulting in a series of different protocols, adaptions are still necessary for certain matrices [144]. 

Several methods have been described in the literature to quantify proanthocyanidins (condensed tannins) however, the most common are the acid-butanol assay and vanillin assay. 

Once a Type 1 proanthocyanidin polymer has been isolated by the above methods, the purity of a preparation may be gauged by the vanillin-hydrochloric acid assay (17, 25) or by the value in the A ax 270-280 region in water or methanol (25). This procedure has been shown to be effective for the isolation of polymers from a wide range of plant sources (25, 29, 37), and it is usually reasonable to assume that the constitution of the polymer so isolated is an average representation of the condensed tannins as they exist in that particular plant tissue. 

Although the vanillin reaction has been widely used to estimate condensed tannins (proanthocyanidins), the reaction is not specific for this kind of compound. Any appropriately substituted flavanol reacts in the assay as does the monomeric unit of condensed tannins, catechin, that reacts with vanillin to yield a red colored adduct. Moreover, the subunits of the polymeric condensed tannins show variable reactivity with vanillin. In this sense, a modified vanillin method has been proposed to estimate the molecular weight on condensed tannins instead of using it with quantitative purposes. 

Hplc techniques are used to routinely separate and quantify less volatile compounds. The hplc columns used to affect this separation are selected based on the constituents of interest. They are typically reverse phase or anion exchange in nature. The constituents routinely assayed in this type of analysis are those high in molecular weight or low in volatility. Specific compounds of interest include wood sugars, vanillin, and tannin complexes. The most common types of hplc detectors employed in the analysis of distilled spirits are the refractive index detector and the ultraviolet detector. Additionally, the recent introduction of the photodiode array detector is making a significant impact in the analysis of distilled spirits. 

Affinity of MIP towards the target analyte should be examined prior to fabrication of the chemosensor. Batch binding assays are used to test selectivity of suitable MIPs. Especially, affinity of MIP to compounds, which are structurally related to the target analyte, should be tested. If MIP binds similarly with these compounds as the template, then cross-reactivity is manifested [156], This effect was exploited for determination of adenine and its derivatives with the use of MIP templated with 9-ethyladenine. Nevertheless, the cross-reactivity, if undesired, can be avoided by suitable sample pretreatment, e.g. by interferant extraction with a supported liquid membrane (SLM) coupled to the MIP-PZ chemosensor. The Fluoropore membrane filter of submicrometre porosity can serve that purpose. That way, this membrane holds interferants, thus eliminating the matrix effect. The SLM-involving determination procedure is cheaper than traditional laborious sample pretreatment used to remove the interfering substances. For instance, caffeine [143] and vanillin [157] in food samples have been determined using this procedure. 

Several attempts have been made to obtain a more complete picture of the phenolic composition of a sample. For this purpose some researchers propose the conductance of several complementary assays [106]. More popular is the formation of ratios which are claimed to correlate with the relative degree of polymerization. Considering the complex reaction scheme of these assays, interpretations of such ratios must be performed very carefully. In our opinion, some of them more likely reflect the specific proanthocyanidin structures than actual relative degrees of polymerization. In the last few years the following ratios have been described in the literature dimethylaminocinnamaldehyde / proanthocyanidin ratio for wine and grape tissue [149], vanillin / dimethylaminocinnamaldehyde ratio for wine and purified standards [96] and proanthocyanidin / vanillin ratio for plums [95] as well as purified proanthocyanidins from various plant sources [155]. 

Hygroscopic, dark red crystals. When exposed to air, may absorb about 12% water. The hydrated crystals are stable to air. Darkens at 210-220 . Not melted at 300 . [a] — 59 9" (dil aq soln). Absorption max (water) 278, 361. 550 nm (A12, 115. 204, 64). Odorless and tasteless. One gram dissolves in about 80 ml water. Aq solns are neutral, maximum stability in the pH range 4.5-5. Solns in this pH range can be autoclaved for 20 min at 120 . Soluble in ale. Insol In acetone, CHCl3, ether. Aq solns decomp in the presence of acacia, aldehydes, ascorbic acid, ferrous gluconate, ferrous sulfate, vanillin are stabilized hy the addn of ammonium sulfate. Talc has a tenacious affinity for vitamin B1t although this is not an incompatibility, it precludes the use of talc as a filter aid or lubricant for tablets, particularly in view of possible assay difficulties. 

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