Issue link: http://winesandvines.uberflip.com/i/742839
November 2016 WINES&VINES 85 WINEMAKING important wavelength to measure was 340 nm, right at the hairy edge of its ability. This wave- length, which falls at the transition to the UV portion of the spectrum, has the best absor- bance of light that can uniquely identify the degree of the enzymatic reactions used by the wine industry. Unfortunately for the less expensive spec- trophotometers, this wavelength is a difficult one to measure because it falls at this transi- tion point. There is not a lot of intensity of that wavelength in the less expensive light sources used in this type of instrument. Couple this fact with the need for a precision diffraction grating to single out only the wavelength needed, and the cost of an instrument dramatically in- creases to do the job. As a result, the lower cost instruments were a constant fiddle for winemakers to get good consistent answers. During the past several years, however, new technology has provided solutions that have solved the analysis of the wavelength measurement by designing special purpose-built instruments that maximize ac- curacy and reliability for those wavelengths. Another factor that is as important as wave- length is enzymatic purity. The higher the quality of the enzymatic materials, the more stable and accurate the results, and the longer the shelf life will be for the enzymatic kits used in the analyses. This latter fact is an important consideration when a winery is choosing a company to be their reagent supplier, as is the formulation in which the kit components are supplied. In some cases, the reagents must be used within a few days to a week of prepara- tion. This can be extremely wasteful for a small laboratory analyzing just a few samples each day or week. The Megazyme kits are formu- lated so the reagents are stable and can be used for at least a year after preparation. Megazyme MegaQuant Wave In order to lessen instrument cost and increase reliability and accuracy, Megazyme changed the diffraction grating to a series of filters in the MegaQuant Wave. These filters allow only a narrow band of light centered on the target wavelength. In the case of the MegaQuant Wave, there are six standard wavelengths in- cluded (see table "Standard Wavelengths") to accommodate all of the available tests for wine analysis, with an option of adding two more. The filters are important because they pro- vide a simple mechanism for the instrument's software to measure two wavelengths at the same time. As a result, this permits an internal standardization of the light intensity transmit- ted through the sample, and this standardiza- tion helps to determine a true sample absorbance more accurately. It also accounts for the slight perturbations due to imperfec- tions in the borosilicate glass tubes used for testing. In the past, the alternative was to use quartz crystal cuvettes at hundreds of times the cost of "regular" glass tubes. Another important addition to this instru- ment, which spectrophotometers do not have, is the ability to automate the results analyses needed to calculate the concentration of the target analyte accurately. These analyses are calculated by recording the change in the ab- sorbance of the light passing through the reac- tion solution. This change in absorbance (increasing or decreasing) is a direct function of a specific enzyme reaction acting on the analyte being measured and is proportional to the concentration of this analyte. At low con- centrations, up to a given upper concentration, the reaction is linear and it is important to determine when the reaction starts to deviate from linearity. It is at this point where the quality of the assay reagents and the quality of the light signature interacts with the software of the instrument to give the widest measuring range. The MegaQuant Wave software increases the range of linearity of the absorbance curve from a normal 1.5 absorbance units to 3.0 units. In the first half of the range, the linearity is within 1%; in the second half, it is 2%. In combination with high quality assay reagents, MegaQuant Wave accommodates a wider linear dynamic range, meaning fewer dilutions are needed to analyze a single sample accurately. The next criterion is an instrument's lowest detectable absorbance. The lower limit of de- tection varies depending on the assay. If lower limits are important to a winery, the buyer should be sure that the instrument being con- sidered measures at or below the lowest con- centration expected in a sample. The basic concept of detection limits applies across all enzymatic testing procedures. For the first example, I will use the analysis of sulfites in wine, since that is one of the enzymatic analyses not usually discussed when talking about enzymatic analytical protocols. This analysis is useful to the winery and should be an important factor in evaluating whether to purchase this type of instrument. The smallest differentiating absorbance is defined as the lowest detectable absorbance change. However, this is dependent on the quality of the spectrophotometer, quality of assay reagents and skill level of the analyst and, for improved accuracy, a higher absor- bance change may be recommended. For the sulfite reaction, the smallest differentiating absorbance is 0.01 abs units. Therefore, if the test is performed with a standard sample size of 0.1 ml, the smallest differentiating absor- bance corresponds to 3.4 mg/L, and the recom- mended lower limit of detection is 6.8 mg/L using an absorbance difference of 0.02. How- ever, if the maximum recommended sample size of 2 ml is used, then the lower limit of detection is reduced to 0.34 mg/L using an STANDARD WAVELENGTHS Monza Megaquant Wave ChemWell (rAPID-T) Astoria and Astoria2 340 340 340 410 – 1000 nm Interference Filters 415 405 405 510 505 420 546 545 480 578 580 505 600 630 540 660 570 700 660 Customer Selected KEY POINTS For many years, small to medium-sized wineries that wanted to analyze wine com- ponents using enzymes had to work with spectrophotometers. Now, small-format enzymatic-analysis instruments are avail- able to maximize accuracy and reliability at the appropriate wavelength for analyses needed by the wine industry. Megazyme's MegaQuant Wave includes a series of filters that allow only a narrow band of light for six standard wavelengths, which accommodate all of the available tests for wine analysis. This instrument also has the ability to automate the results needed to calculate the concentration of the sample accurately. The Randox Monza is similar to the Mega- Quant Wave, but with some differences in the number of analyses and equipment in- cluded and price. The Astoria from Astoria- Pacific uses auto sampling and is capable of running 200 tests or more per hour. Different wavelengths allow wineries to perform a variety of tests.