Measuring Alcohol - Part III
-by Gary Spedding, Alcohol Beverage Chemist-
Density and or Specific Gravity Measurements for Alcohol Determination
Historically, alcohol measurements were grounded in physical measurements of mass and volume through density or mass per unit volume intensive proper ties. Through density and specific gravity relationships, instruments and devices such as density bottles, hydrometers, densitometers, refractometers and pycnometers were used to establish a recognized and officially accepted body of work. This extensive research effort culminated in the derivation of algorithms and tables which define the relationships between density values and specific gravity readings and alcohol by weight and by volume.
Densitometers
Modern densitometers have largely replaced the classic methods based on prior distillation of samples and either the use of refractometers or hydrometers for measuring extracts and alcohol in samples. These new density measuring devices are “Oscillating U-tube” density meters and these units are highly sophisticated and expensive instruments for measuring density; they are accurate to 5 or 6 decimal places and can be used to measure the density and specific gravities (SG ’s) of unfermented and fermented samples and distillates, to obtain original extract (OE ) values, final SG (apparent extract) values or the alcohol content of the samples respectively (the latter via alcohol value specific gravities and reference to tables).
Oscillating U-tube densitometers work on the principle of electronic excitation of a measuring cell - the U-tube filled with a solution to be measured. Based on a fixed volume within the cell, similar to the older instruments such as the pycnometer (or density bottle), and defining the density as mass per unit volume, an increase in mass within the same volume leads to an increase in density. The harmonics or oscillation of the tube (frequency of resonance) is affected by solutions of different density and the density is then calculated from the oscillation period. Such instruments will report density and specific gravities of a beverage or, if distillates are used, the percent alcohol by weight at 20 °C based on official tables generated by the Organization of Legal Metrology (The “OIML tables”).
The percent alcohol by volume may also be reported based on OIML tables at 20 °C and percent alcohol by volume based on tables by the Association of Official Agricultural Chemists (AOAC). The AOAC tables were also later adopted by the American Society of Brewing Chemists (ASBC) and these tables record the alcohol contents as they would be at 60°F (15.56 °C). (AOAC, 1995, ASBC, 1940, OIML, 2015). These units are easy to maintain and need only to be calibrated on dry-air and pure water (a fluid for which densities are known to the desired degree of accuracy).
Enzymatically Measuring Alcohol (including detection via U/V Spectroscopic Methods)
Enzymatic assays have proven popular and very useful for alcohol determinations. Essentially these assays are invitro (“ in the test tube”) biochemical assays relying on the natural enzymes and coenzymes (factors) involved in ethanol metabolism in living organisms. In living cells -and in the laboratory- ethanol can be readily oxidized to yield ethanol (acetaldehyde) or completely oxidized to acetic acid (ethanoic acid) [CH3CH2OH -> CH3CHO -> CH3CO2H].
In the sensitive and specific enzyme assay the ethanol present from the added sample is first oxidized to ethanal (acetaldehyde – CH3CHO ) using the biochemical compound nicotinamide adenine dinucleotide (NAD) in the presence of the alcohol dehydrogenase enzyme instead of a chemical oxidizing agent (used in earlier test tube assays). As the reverse reaction is the thermodynamically favored process, the overall reaction is driven to completion by removing the acetaldehyde. This is done in a second step in the presence of aldehyde dehydrogenase which involves the quantitative oxidation of the acetaldehyde (CH3CHO ) to acetic acid (CH3CO2H), again with NAD involved.
The NAD is reduced to NADH (and a proton: H+) in each reaction (two molecules of NAD are consumed for ever y ethanol molecule oxidized to acetic acid) which then affords the quantitation of the alcohol spectrally via NADH ’s absorbance of energy from wavelengths within the uv/visible spectrum; 334, 340, or 365 nanometers (nm).
The use of natural biochemical catalysts and cofactors and the right temperature and pH conditions gives rise to the exquisite sensitivity and precision for measuring ethanol in well -prepared and diluted samples. Full test details and limitations to the assay being described in kit manufacturer specification sheets.
Such assays were originally developed to test for the presence of alcohol in alcohol free or low alcohol products (with anything below 0.5% alcohol by volume considered non-alcoholic).'
Such assays are often used in such cases due to their extreme sensitivity, specificity and accuracy. However, with suitable and careful dilution, higher alcohol containing beverages and foods can be tested via these very sensitive enzymatic assays.
Again, it is to be noted that in order to obtain the true alcohol by volume from such tests, the sample specific gravity must be known. The methods supplied with test kits make no assumption in this regard and do not give true alcohol by volume values unless a separate sample of the product is tested for its current specific gravity.
Stay tuned for Part IV of this informative series, to be continued next month.