Measuring Alcohol, Part II
-by Gary Spedding, Alcohol Beverage Chemist-
The various methods discussed below have all been used to determine alcohol in distillates or were, in fact, designed to circumvent the need for distillation altogether. It will, however, be seen below that most alcohol beverage chemists and the modern instrumentation they use will utilize or rely upon tables and algorithms relating distillate or alcohol -water mixture specific gravity values to their corresponding alcohol values as determined by extensive work done over a century ago. Some of the technologies as used in the laboratory have now also been extended to in- line instruments and measuring alcohol and solids present (extracts) during production of the product. These in- line units rely on the same principles of detection and measurement as the laboratory methods. An international collaboration resulted in the publication of standard tables relating to alcohol measurements by the Organization of Legal Metrology in the 1980’s.
The methods used to measure alcohol content and current applications:
Chromatographic methods for alcohol determination (Gas Chromatography and High Performance Liquid – HPLC - Chromatography)
Alcohol has been determined utilizing chromatographic methods, which separate and analyze mixtures of chemical components. These methods of ten rely on densitometry of distillates for calibration purposes and were initially more frequently used in wine and distilled spirits testing facilities rather than by those producers of lower alcohol –containing beverages such as beer. However, GC systems with a flame ionization type detector (FID , see below) have been used in many laboratories and by producers for several decades for alcoholic products and for non-alcoholic and dealcoholyzed products where target concentration is 0.5% alcohol by volume or less. Furthermore, government bodies around the world have relied upon GC/FID for routine ethanol analysis in wines, beers and spirits and for the testing of non-alcoholic and dealcoholized products.
This method relies on selective adsorption and desorption of volatile components on a stationary phase. Components are carried through a column by means of an inert gas and emerge, depending on their retention times on the column, and pass to a specific type of detector; for alcohol detection a flame ionization detector (FID), as noted above, is used. The detector is linked via an amplifier to record a profile of peaks corresponding to each compound that passes through the detector. Thus alcohol and other components are identified and quantitated by weight (wt/vol) (via calibration with known standard compounds) based on retention (residency) time in or on the column. Clear resolution of each compound of interest is possible, via partitioning between the carrier gas stream and liquid phase supported on an inert solid in the column. When a component is clearly separated from others in the sample the compound will be accurately quantified by the instrument after a calibration curve has been generated. The calibration curve being generated via the use of carefully calibrated amounts of that pure compound across the concentration range expected to be seen in typical samples. Described in some references as an AOAC (Association of Official Agricultural Chemists) approved method accurate to about +/- 0.2% v/v, gas chromatography has been described for measuring ethanol at low concentrations in beer distillates and in beer for example by the US agency TTB. In real world modern practice standard deviation on control samples is more like 0.05% by volume.
GC sees some application in evidential breath and blood analyses and in other forensic applications. Regulatory authorities may rely on GC determinations of alcohol based on both the method’s sensitivity and rapid turnaround. As a method in its own right it is fully adequate to the task of measuring most volatile compounds of interest. In this regard it is noted that gas chromatography may also be utilized with detectors other than f lame ionization, such as by electron capture, thermal conductivity, sulfur specific detectors and by mass spectroscopy.
Gas Chromatography/Mass Spectrometry.
Gas chromatography, through column separation of volatile compounds and detection via specific detectors (see above), is a powerful method when compounds are known, or suspected, to be present in a sample and when the instrument is calibrated to measure those compounds. Where it becomes a more powerful method (mainly for seeking out unknown compounds and identifying them through their mass proper ties) is when coupled to a mass spectrometer. Mass spectrometry can be used to obtain the formulae and structures of molecules. Yet is overkill for measuring known compounds in routine practice.
The use of GC/MS is actually an expensive proposition for the solution to a low resolution problem. If available to a facility it would be valid for use in alcohol measurements but the GC unit itself is all that is needed for routine alcohol beverage chemistry purposes. The GC portion chromatographically separates the species present in solution. A portion of the separated compounds are siphoned off in turn and introduced to an ionization chamber where the vapor is bombarded with high energy electrons. This generates positively charged ions which can be determined based upon both their mass and electrical charge. Each chemical species can be identified via a peak formed as a function of relative abundance of its formation and at its appropriate relative molar mass (its mass spectrum). Extra information is available regarding how molecules are fragmented (broken down to component parts) during the testing – with the appearance of new mass-spectral peaks.
Based on the charge and mass ratios of species produced compounds of interest can be identified and precisely quantitated. Further discussion on the molecular ions formed, the fragmentation pat terns and their interpretation which are mathematically complex is beyond the scope of this review. Moreover, the type of internal control compound (for instrument /process calibration) needs to be carefully selected for and understood.
Static headspace testing by GC can be quite tricky. If a headspace GC-MS system is used the partition coefficients of species will affect the subsequent separation and quantitation. Furthermore, the method is reliant on the use of the aforementioned adequate internal standard. It is important to match the matrix of the sample with standards; i.e. the standard should ideally be as close to the analyte of interest as possible (such as deuterated ethanol in this case) and absolutely must not be a compound present in the samples being analyzed. A method of additions technique is suggested as working the best. These latter factors are also topics beyond the scope of this present note. It is, as stated above, a complex and expensive means to quantifying known compounds. The advantage for routine analyses is selectivity in complex matrices where chromatographic interference is inevitable. GC coupled with mass spectroscopy (GC-MS ) is better suited for a rapid analysis of complicated mixtures of different compounds (or products from complex chemical reactions) rather than for monitoring a single analyte or two.
HPLC - High performance liquid chromatography
In general chromatographic techniques are suitable for specific types of separation processes since the principles they employ reflect the physical characteristics of the sample’s components. Thus any one technique is limited in its general applicability. However the advent of high performance liquid chromatography overcame many limitations based on the fact it can separate components based on many principles: selective adsorption, partitioning, ion-exchange, exclusion and affinity chromatography (simply put this means relying on different chemical principles and proper ties to separate groups of molecules). Making for an extremely versatile technique high performance liquid chromatography thus became a most powerful single chromatographic procedure. This chromatographic method uses a liquid mobile phase to transport compounds of a mixture, injected under high pressure, onto a packed column stationary phase. The mixture is resolved into its constituents via adsorption and release from the column matrix (using the principles alluded to above and depending on the desired group separation). The eluting molecules are then detected and quantified via a suitable detector; for ethanol this is usually a U/V or refractive index detector (see above for the physical chemical proper ties of alcohol that dictate how in fact it may be detected). Again (like for GC systems) the system must be calibrated using known standards. As HPLC is better suited to less volatile compounds, it is usually used to determine sugars and organic acids in fermentation samples or beverages with ethanol (and glycerol) also coming along for the ride. This method finds more frequent use in the distilling industry than the brewing industry for example but can give very accurate alcohol readings for those using the technology on a regular basis. Ethanol, by its nature can have a much “wider” window of accuracy due to the type of column used and/or the physical and chemical proper ties of sugars and organic acids vs. ethanol in a so called isocratic system (not further defined here). A much more precise reading can be obtained if the scientist is willing to give up resolution of some sugars and organic acids.
A neat feature of using both types of chromatography (GC and HPLC) is that different chemical proper ties and physical conditions are used to resolve the species present in mixtures and has certainly helped to confirm that true alcohol measurements can be made using either technology alone.
Stay tuned for Part III of this informative series, to be continued next month