Dissolved oxygen meters give variable numbers. Photo by Vernier Optical

In the March 2014 issue of Wines & Vines, I described the basic wine laboratory and discussed the minimum laboratory equipment and analytical procedures for soluble solids, total acidity, pH, ethanol, sulfur dioxide and sample preparation. In this article, I will examine more complex testing procedures, the equipment needed to run those tests and different strategies to get your best analysis “bang for the buck.”

Bottled wine integrity
One of the most challenging aspects of winemaking, but absolutely one of the most important, is ensuring the integrity of the final packaged product. To maintain the public’s trust, wineries must be certain that the wines they release are stable. Microbiological testing of the wine is essential to having that assurance. This is an area where most winemakers and many laboratory personnel are out of their element. What looks clean is not necessarily so from a microbiological perspective. To gain that assurance, many wineries send samples to an outside service laboratory.

The full micro-analytical set-up can be quite costly, and it includes acquisition of some way to sterilize equipment. Some basic supplies are also necessary, such as transfer hoods, and the media and plates to measure the bottles in question.

However, there is a testing approach where many difficulties can be reduced, if not eliminated, for those wanting a more immediate, hands-on understanding of their bottle integrity. One of the better systems has been developed by Sartorius. They have a prepackaged filter unit called Biosart that comes with a grid. They also have a wide selection of media that helps select the microbes that might be present in a bottle.

With just a little bit of preparation and practice, it is possible to get a good idea of the integrity of a bottling run about four days after bottling. If this system is used as a screening test, only those samples that fail the testing procedure need to be sent out for analysis and validation. When a winery adopts this type of system, that winery can sample more frequently and know from the start that a particular bottling was clean.

Amino and ammonium nitrogen
The easiest and least expensive test for fermentable nitrogen uses the Formol test. It is a simple titration of the ammonia ion in the free form and amino acid-derived ammonia. This test is conducted by titration with formaldehyde. If a winery laboratory does not have a spectrometric instrument, this test is the best way to determine the fermentable nitrogen level of juice.

Dr. Barry Gump of Florida International University developed a new version of this test that combines the analysis of fermentable nitrogen with total acidity analysis (reference). By first running total acidity, the result is a sample titrated to pH 8.2. Then by adding formaldehyde to that sample and re-titrating, it is possible to determine fermentable nitrogen. Even more important, if a winery has one of the auto-titrators such as the Hanna 901, all it takes is to switch the program from TA to Formol, add the formaldehyde and push the button.

The most complete solution is to purchase one of the multi-purpose enzymatic titrators such as the Randox Monza. With one of these instruments it is possible to analyze many different substances in a wine. The enzymatic method allows discrimination between free ammonium ions and those associated with amino acid-derived ammonia. This will tell you more about the overall health of your grapes, which will help inform your vineyards on best practices for their grapes.

It is important to measure fermentable nitrogen and not just add diammonium phosphate (DAP) proactively. Amounts exceeding 500 ppm can lead to the formation of carcinogenic compounds such as carbamate in the wine. These compounds are regulated by many jurisdictions including Canada and the European Union for wines imported into those countries, and limits are also set by TTB for United States wine sales.

Heat stability
Heat stability is a test that is absolutely important. There are two ways to conduct this test: the right way and the other way. Too many winemakers gamble with physics and hope that a wine will sell before there is a haze caused by unstable proteins. As an analogy, it isn’t a case of whether an egg left at room temperature will have the albumin (egg white) solidify, but a case of when it will solidify. The same is true with unstable proteins in wine.

One of the important devices for a winery laboratory is a nephelometer. This device measures turbidity of a liquid. You can spend a lot of money for a nephelometer, but the one sold by Hanna is a good value for the money.

The test for unstable protein is simple. Filter a wine to 0.45µ and then place in boiling water for 10 minutes. A wine is heat stable if the beginning turbidity complies with the ending turbidity using a formula. The formula I use is X=T2-(T1/2) and the wine is stable when X is less than five. If the result is greater than five, the wine is not stable and bentonite must be added to correct the problem.

The other way is to let your eye determine heat stability. I have found through hard experience that the eye is not as good as a nephelometer.

Cold stability
Cold stability measures the degree to which tartaric acid is in supersaturation in the wine. As in heat stability, it is a simple test but not as easy to conduct. The easiest test, and one that is fairly rigorous, is to freeze a sample in a regular freezer for precisely 12 h ours. To the degree that there are no crystals at the bottom, the wine is cold stable. As with the heat stability test indicated above, evaluating the test result using your eyes can lead to an erroneous conclusion. This method is highly subjective, especially at the margins of cold stability.

There are several ways to set up a routine test environment that is much more accurate than looking for crystals with your eyes. The instrument of choice is a conductivity meter, which is a relatively inexpensive meter that evaluates cold stability reasonably accurately.

Procedurally, the steps that are required involve chilling a wine to 28oF, dropping in cream of tartar and then measuring the fall in conductivity. If the fall is less than 5% of the initial reading, your wine is cold stable. To run this test, one needs to make a reservoir that can use the winery’s existing glycol system and hold a liter of wine at 28oF. Then add the cream of tartar. Keep the temperature within 0.5o of at 28oF during the test. This will make a good approximation of cold stability to about 95% + assurance that it has achieved cold stability.

For greater accuracy (at a cost of about $12,000 to $15,000) you can get a system from Ameridia or Alpine Scientific that uses a commercial glycol unit and a computer formula to calculate the conductivity drop to infinity. In 15 to 20 minutes, the program projects conductivity so that there is a virtual guarantee of cold stability.

Glucose fructose
Megazyme has developed a simple meter that can take the place of a spectrometer to measure malic acid and glucose fructose. This hand-held device has a simplified pipetting method that is easier to use than a full-blown spectrophotometer. The same 20µl pipette is used for all the small additions. If you are stepping into more sophisticated analysis, this several hundred dollar instrument will allow these two enzymatic analysis to be conducted.

However, if a winery wants to expand the laboratory into a more self-sustaining unit, it would be wise to consider the Randox Monza or similar instrument since this test is just one of many that this type of instrument can analyze. While the steps for each of these instruments are slightly different, this type of instrument can be run by technicians trained to follow the procedures carefully.

Malic acid
The original, simple way to test for the completion of malolactic fermentation (ML) in a wine has been paper chromatography. There are many problems with this method of testing. First, most users of this test do not change the chromatography solutions often enough—once a week is recommended. Second, while the sensitivity of the test is about 100ppm, completion of ML is defined as less than 30ppm.

The most common way to assure that ML is complete is with an enzymatic test. Megazyme uses the same instrument used for glucose/fructose to measure malic acid, and this test follows the same procedures as with the glucose/fructose test. Megazyme’s instrument is not a spectrometer. The company takes advantage of an enzymatic “trick” of taking the traditional end point that is measured at 340nm wavelength and converting it to one that measures in the visible light range of 520nm. This effect makes the instrument performing the analysis much less expensive to produce with nearly the same accuracy of a traditional spectrometer.

The Randox Monza type of instrument provides a more precise analysis than either of the previous two procedures and is more useful for a wider variety of tests.

Volatile acidity
The traditional method of analyzing volatile acidity (VA) uses the cash still. This device extracts the volatile acids in wine using steam distillation. In addition to acetic acid and ethyl acetate, the main non-target acids this procedure also detects include CO2, SO2 and sorbic acid. Lactic acid, proprionic acid and a few others also can contribute to the total volatile acid number. To the extent that all of these acids are present, techniques must be used to account for them so an artificially high value isn’t reported for VA, especially if the VA level is approaching the legal limit.

The cash still is a rather crude method for determining VA. Whereas one can account for the major contributors to the VA number, the most difficult issue with the cash still is running the tests. It is a clumsy procedure that takes about 30 minutes per sample and requires the full attention of the technician. Since it is a glass still and there is a nichrome wire heating the water in the still, all it takes is a few minutes of inattention and the nichrome cracks the glass bowl and the still is ruined. I know, I’ve done it!

While enzymatic tests are easier to set up and run, their difficulty lies in accounting for ethyl acetate. I have found from experience that as long as ethyl acetate is not organoleptically present, its contribution to the final VA concentration is less than 10%. This means that unless the wine is near or over the legal limit, it is not a serious contributor to the problem from a legal standpoint. If ethyl acetate is noticeably present, it can be a significant percentage of the total VA. This becomes an issue if the VA needs to be removed by reverse osmosis. Removing this volatile acid is more difficult than removing acetic acid and can significantly raise the cost of VA removal.

Like the other enzymatic procedures, one can use any 340nm UV-capable spectrometer to analyze for VA. However, you can see a thread here: Most all of the enzymatic tests can be more simply done on one type of instrument such as the Randox Monza or the Mettler-Toledo version (which I have also used).

Phenolics and color in wine
Phenolic compounds are the most interesting yet most complex compounds we deal with in winemaking. Their very complexity makes it almost impossible for the basic winery laboratory to know much more about these compounds than measuring the intensity of the wine’s color and its hue. Anything more requires equipment that costs tens of thousands
of dollars and/or a laboratory setup managed by an experienced lab person with advanced degrees.

To run the intensity and hue test for wines, the sample is prepared by filtration to 0.45µ and then analyzed in a spectrometer at 420nm and 520nm. Adding the absorption units give intensity and the ratio of 420/520 gives the hue. It’s simple to run, but the spectrometer costs about $2,500. Adding a lot of labware and a UV/VIS spectrometer will supply more information about your phenoli c compounds, but it is probably better to send
a sample to your commercial lab partner for this information.

Dissolved oxygen
One of the important steps in winemaking is control of the oxygen in wine. I have used several dissolved oxygen probes; they are difficult to maintain and there is no easy way to standardize them. They are better than nothing, but they also can give widely variable numbers and so they have not proven that valuable to me.

That said, I saw a probe in Europe several years ago that uses a different technology than the traditional DO meters that are based on oxygen transmission through a membrane. I will soon have one of these probes for evaluation and will report on them in a later article. The element that I like in this new probe is that it is backwardly compatible with an existing DO meter. Whereas at $1,900 they are a bit pricey, the information provided could significantly improve your winemaking.

Fining and fining agents
The laboratory does not currently need to analyze for fining agents remaining in the wine, since the ideal agents are virtually completely removed in the course of normal processes. However, there is a move afoot to make wineries declare if any fining agents have been added. It is possible that we will have to analyze for these materials in the future.

The laboratory needs to be set up to prepare samples of wine using fining agents so that the wine can be judged effectively on the result of their addition. In general, that means accurately adding the agent, then removing it so wine can be evaluated.

The easiest method I have devised is to use a Becopad 550 filter pad cut to fit a 9-12cm Buchner funnel. Then place a glass fiber filter sheet on the bottom of the funnel with the filter pad on top. Attach the filter funnel to a vacuum flask 1 liter or larger and start the vacuum. Prepare an aliquot of diatomaceous earth (DE) slurry that has recently been washed with citric acid and then rinsed, and that is sufficient to coat the surface of the pad with about 2-4 mm of DE. Pour this onto the pad. Once the water is removed, remove the funnel and clean the vacuum flask. The flask is now ready for clarifying the sample.

This method allows you either to clarify a larger quantity of wine to which fining agents are added so they can be gravity-clarified sufficient to judge the effect of the agent organoleptically, or with a smaller flask setup, to filter the unfiltered wine to which the fining agent has been directly added for evaluation.

Conclusions
There are many other analyses that can be done on wine that have not been addressed in these two articles. However, these minimum levels of laboratory functions will make a significant contribution to wine quality and can be used by a winery to create a plan for progressing to a more fully functional laboratory that does not necessarily require a person with an advanced degree in either chemistry or biology to run the tests.

The analytical equipment described here is either owned by our laboratory or has been loaned to us for use in preparing this article. I am grateful for this support. Selecting the suppliers of lab equipment begins a partnership with that company. Validate and verify their ability to provide you with the level of support you will require. Whereas these sophisticated instruments are quite powerful, and the volume of data can be a valuable asset to making good wine, they usually take a person with good skills at attention to detail to get valuable answers. I’ve trained many people to use these types of instruments that have no science training. Remember, the more you know about your wine the better wine you will make.

Dr. Richard Carey is president of Vitis Wine Center and winemaker for Tamanend Winery in Lancaster, Pa. He has written numerous articles on new technologies for the grape and wine industry as well as a series of articles on laboratory analyses in Wine East magazine.