January 2018 Issue of Wines & Vines

Putting Modern Lab Equipment to the Test

A comparison of analytical equipment for small to medium-sized wineries

by Richard Carey

Laboratory analysis for the wine industry has seen many innovations in equipment during the past decade. The technology has developed to the point that the traditional wet chemistry winery lab of the past is becoming the equivalent of a buggy whip. Advances in equipment allow anyone who can follow a cooking recipe to become proficient in a wine laboratory.

For this article, Wines & Vines obtained laboratory equipment from some of the major suppliers for small to medium-sized wineries. This equipment offers a variety of ways to modernize a winery lab and increase the value of the analytical data while still fitting into a winery’s budget. In many cases, using some modern lab techniques and equipment will reduce the total average cost per analysis compared to wet chemistry methods, especially when the time per analysis is taken into consideration.

After narrowing down the most important analyses a winery should be performing on a regular basis, we conducted these tests on each piece of equipment, using the same wine sample for a given analysis. ETS Laboratories provided a third-party reference to determine analytical baselines.

Laboratory instruments and how they work

Agápi Lab Solutions – Ebulliometer
A lower cost alternative to electronic instruments
The Agápi ebulliometer was developed for the measure of alcohol; it is similar to Dujardin-Salleron’s electronic instrument but costs between 25% and 30% less. The unit has glass replacement parts that, if broken, are easy and relatively inexpensive to replace. Temperature is measured by a National Institute of Standards traceable digital thermometer, which is easier to read than standard mercury thermometers. However, they also have a less expensive mercury thermometer model. 

Agápi Lab Solutions – Sartorius Biosart
Plating bottled wine made simple
Agápi Lab Solutions is a Sartorius distributor. The Sartorius Biosart uses an integrated system of equipment that makes it easy for the average person to plate bottle samples and detect microorganisms. The user pours a wine sample into a sterile funnel/membrane unit that fits onto a manifold, which collects all microorganisms onto a sterile membrane. The membrane is transferred onto a sterile nutrient pad that is then incubated in a warm location, allowing any microbes to grow. 

The system requires a simple enclosure to protect against false-positive results in the sampling environment. The Agápi Lab enclosure is inexpensive and can be disassembled easily for storage when not in use. 

CDR WineLab
An all-in-one lab instrument with a straightforward principle
The CDR WineLab, a new entrant in laboratory equipment, has two models: CDR WineLab (used for this article) and CDR WineLab Jr. The CDR WineLab lists 25 separate analyses applicable for the wine industry, whereas the Jr. does 16 tests. The supplier declined to disclose prices for this equipment.

The instrument is built on a spectrometer-based technology that uses specific wavelength LED emitters. This technique has allowed them to expand the measurable Absorbance Units (A) in the linear range to 6A. There are four separate analytical bays using six different wavelengths that perform the tests. The instrument has 16 heated slots (Jr. has three) where the sample cuvettes used for analysis reside in preparation for analysis. 

The test is run using a touch-screen LCD. The menu lists the analyses that the instrument will run. There are several ways to store and retrieve data. First, the instrument itself can store thousands of analytical results. Second, there is a USB Type 2 port for technical service to the instrument and connection to a PC as well as a direct Ethernet LAN port and two USB 2.0 ports for database transfer of the performed tests and software updates and configuration. The instrument can conduct more than one test at a time and has an 80-mm graphic printer (the Jr. does not have these features). 

The CDR WineLab has a straightforward testing principle. Most tests kits have one or more reagent bottles assembled in a pack of 10 or 10 packs of 10 tests. A few tests have no additional reagent bottles; the user simply adds the sample to analyze. The shelf life of most tests is about 18 months from manufacture (travel time to the winery reduces the shelf life to about 12 months). Since the kits are mostly sold in 10-test packs, wineries should able to use a pack within the fresh timeframe. Most test packs require storage at refrigerator temperature. 

When performing a test, the LCD screen prompts direct every step. Once each sample is analyzed, a printout pops up with the necessary information. If the direct-to-computer cables are installed, data can be saved to a computer file. 

Most tests use one to three injections of 10 to 100µl. This process is easy to learn (you can practice with water to develop your technique). This is probably the most difficult part of learning the new lab procedures, but necessary as the goal is to get consistent results. 

The cost of the tests ranges from about $3.50 to $6 per test. The common tests are usually in stock, and when not, the delay is usually not long. Advance arrangements should be made for the lesser used test kits. 

Proprietary software for computer display of results can be downloaded during setup. 

Megazyme MegaQuant Wave
Simplified enzymatic testing using a defined wavelength spectrophotometer
The MegaQuant Wave, introduced by Megazyme, was one of the first spectrophotometers to offer a simplified way to perform enzymatic testing. Their instrument uses a tungsten lamp light source and comes equipped with six interference filters providing wavelengths of 340, 405, 505, 545, 580 and 630 nm as standard. The linear range is 0 to 3 absorbance units that easily accommodate the range of most enzymatic tests. The MegaQuant Wave accommodates 12 sample tubes in the incubation block. This block only accepts round 12 mm diameter tubes. However, the actual measuring cell accepts both 10 mm cuvettes as well as the 1 mm x 10 mm cuvettes in addition to the round tubes. The accuracy of a reading is 0.005A, or about 1% of the reading. 

On startup, the onboard touchscreen presents tabs including utilities, settings, tests and manage tests, and the user enters specific information to customize the printer output. Most of the time the screen input centers on the list of tests, and you select the one to run. The MegaQuant Wave has a repertoire of 24 separate test kits for the wine industry. The average test kit has three to five reagents that are added to the test tube or cuvette using micro-pipettes. The instrument provides step-by-step instructions for processes such as necessary dilutions, sample insertions, and when to insert or remove a tube. On completion of the test, the instrument calculates and prints out the results or sends those results to external software, such as the SF Capture software available from Megazyme.

Megazyme test kits come in different configurations of reagents. The stability of their reagents is generally two years. This is important since the number of tests in any one kit ranges from 50 to 100 tests. The cost per test is $3 to $5 per full test. However, if the user runs the half-volume test, the lab can get twice the number of tests out of one test kit, reducing all reagent costs by half.

The MegaQuant Wave test kits often require both refrigerated and freezer storage to get the two-year shelf life out of the kit. If you use the tests in six to 12 months, freezer storage may not be required. 

The MegaQuant Wave protocol for analysis and the reagents used gives the instrument greater sensitivity at low concentrations. 

Hanna Mini Titrator Titratable Acidity
Step motor precision increases ease of use and accuracy
The Hanna Mini Titrator automates what the wet chemistry burettes have done for decades. The principle of operation is the same, but modified to specific standards, so more repeatable and accurate results can be obtained. The instrument is driven by a step motor. It drives a plastic syringe that is attached to a base platform and, in precise steps, raises the platform, pumping a titrant into a reaction beaker. A pH probe measures the amount of standardized base that is added to the wine sample until the endpoint is determined. The probe output is sent to the stored algorithm in the meter and is then converted into a digital display. A USB port can connect to a thumb drive or, via a cable, to a PC. The exported log files allow further investigation of the analyzed data. 

In the setup menu, the user selects the endpoint (pH 7.0 or 8.2) as well as temperature unit choices (C, K or F), date, time, the number of days until a notice for recalibration and a choice of resolution (0.0 or 0.00) that the meter shows when returning the results.

Calibration of the pH probe and the pump is done after setup. This task is necessary when any component changes, such as the titrant, syringe or probe. One quirk of the meter in calibration mode is the short time between the endpoint calibration and at least one other calibration point. The LCD screen makes it appear as if the wrong buffer was chosen. Simply putting the next buffer in allows the meter to recognize the new buffer and calibrate to that one. The pump is calibrated by adding a calibration standard to the analysis beaker and setting the meter first at high range (4 to 25g/L) and then calibrating again at low range (0.1 to 5 g/L).

The meter can log a total of 400 results, 200 pH/mV and 200 titration results. The cost for a complete system is $825, and the meter can double as a pH meter. 

Hanna Mini Titrator SO2
Step motor precision dials in your results on levels of SO2
The SO2 mini titrator is similar in design to the titratable acidity meter above. The differences are specific to the analysis (free or total sulfur) run with the instrument. The same step motor function controls the syringe delivery of the titrant, and there are similar functions to setup date, time, etc. Once a worker masters one meter, the differences are in the reagents and their amounts that are specific to the meter. 

The only real difference between the two meters is a function in the SO2 meter that damps noise in some titrations. In my experience, it generally is not necessary for most titrations, and this function can be turned off. The basic theory is to dampen the signal by taking a percent of the first derivative of the signal. Fortunately, this number is calculated during calibration. The SO2 mini titrator also allows the user to adjust their threshold which allows them to adapt the method to better analyze their wine. 

Hanna Edge pH, EC and DO Meter
Slim design and multiple probes increase lab functionality
The Edge meter combines the ability to have three different probes used with one base meter. It is similar to a tablet device and has two different mounting platforms: a benchtop meter or a wall-mounted device. It is powered by a USB cable connected to a wall outlet; when fully charged, it can operate off an internal battery for several hours. This meter can accept pH, electrical conductivity (EC) and dissolved oxygen (DO) probes, but only the pH and EC probes are examined in this study. According to a Hanna representative, many wineries needed oxygen levels to be measured below 0.01 ppm ± 1.5%, and that is the lowest amount that this probe can accurately measure. If your tolerance for oxygen is not that critical, and you want to know about oxygen in your wines, this meter is one of the most cost-effective ways of having that functionality in your winery. 

The meter has a straightforward calibration procedure: Hanna Instruments includes the pH 4.0 calibration buffer as well as a pH 3.0 buffer for the wine industry. That assures the best linear range in the region most wine pH readings are found. 

The screen icons on the meter give the probe function and condition, and there are reminders for calibration and an extensive logging function. The probes have an internal temperature sensor for temperature compensation freeing space in the test beaker. 

Electrical conductivity provides one of the easiest means for a small to medium-sized winery to perform a better analysis of cold stability. The EC probe is immersed in the wine during the cold stability trial and a reading is taken at specific times.  

Hanna Turbidity Meter
One of the most cost-effective solutions for heat stability testing

In the turbidity meter, a spectrometer measures the amount of light compensated for color that pass through a liquid for a specific distance. Light scatters due to suspended particles in the wine or must sample (such as glucan, tannin, yeast, etc.) The measurement is taken 90º from the starting point by a photodetector. The amount of light scatter due to particles in a liquid or gas is measured in nephelometric turbidity units (NTU).

The Hanna turbidity meter comes in a case with many components for measuring the turbidity in liquids. The most common use in a winery is to check for heat stability to provide an accurate measure of protein haze in a wine subjected to increased temperatures. It can also be used for measuring the turbidity in liquids from settling ponds, soil samples and floor drain water clarity.

The meter comes with NTU calibration standards in a stabilized solution that range in increments of four from less than 0.1 to 500. After the meter is calibrated, a sample is placed in the meter and its NTU measurement can be read.

Hanna Refractometer
Simple, easy and cost effective
A refractometer measures the degree of refraction of the light through a solution. The degree of refraction combines the effects of all solutes of that particular solution. In grape juice, the majority of the compounds contributing to this measurement are the primary sugars of the fruit, and any errors introduced from other compounds are insignificant. An automatic temperature-compensation algorithm corrects for the ambient temperature in the instrument’s operation and calculation. The Hanna refractometer can measure percentage Brix from 0 to 85% with a resolution of 0.1% Brix and
+/- 0.2% Brix.

Rudolph Research Analytics Alcohol and Refractive Index
State of the art for high-volume alcohol determinations

Rudolph Research Analytics offers a highly accurate refractometer, the J457, and has developed a protocol called Alcotest that combines refractometry with densitometry and can quickly and accurately measure the percent alcohol in many alcoholic beverages. The original system was validated for spirits, but wine is a more complex beverage.

Alcotest is now available with a wine scale. Its operation is based on a combination of refractometry and densitometry. Like the Foss system of infrared analysis, complex systems can be accurately measured once you know the system matrix. Then it is up to the algorithm of the analysis to calculate the compound or compounds from the complex mixture presented. 

The refractometer and densitometer instruments require highly accurate, temperature-controlled environments to carry out the calculations necessary for accurate results. For example, the refractometer is accurate to 0.015° Brix and has a resolution of 0.01° Brix. The densitometer is accurate to 0.0001 g/cm3 and has a repeatability of 0.00005 g/cm3. These instruments are rugged, splash-proof and designed for high-volume production environments. 
Editor’s note: The author’s firm, Tamanend Wine Consulting, worked with Rudolph to help validate its system for wine.

Using the equipment: analyses and results
The equipment described above was used to conduct the appropriate analyses using the same wine, and each section of results that follow had triplicate analysis of the same sample.

When reviewing these results, it is important to understand the difference between precision and accuracy. Precision is the measure of the degree of variance in the results. A very low degree in variance has a high precision. However, high precision does not necessarily provide high accuracy. Great precision off the target is worse than larger variance on or near the target. The user needs to know what the target (most accurate) is in order to decide if a triplicate measure of +/- 1 is better than +/- 3. For this study, ETS Laboratories provided the target. 

Another consideration is tolerance. A highly accurate measurement with great precision is likely the best solution, but the cost of that equipment may be too high. The user must then determine their degree of tolerance—what is good enough—to obtain an affordable result. 

The third factor is the time needed to get the result. New technology has streamlined analytical procedures, reducing the time needed to get an answer. When compared with traditional equipment, new methods are faster, more repeatable, and many times more accurate. In addition, maintenance and calibration is much easier. Consequently, a lab can often run tests in a more timely manner and therefore help you make better wine.

Acetic acid: two ways to test
Acetic acid is analyzed using enzymes, and the technique does not analyze for anything else. As long as you don’t smell the nail polish aroma of ethyl acetate, this reading will be an essentially accurate measure of volatile acidity. The smell of ethyl acetate indicates it has reached the noticeable threshold of 100 to 150 ppm or more in the wine. At that point, the true volatile acidity will be slightly higher, due to the ethyl acetate present. 

For the CDR WineLab, the procedure adds two reagents to the sample addition. One reagent is added with a wait time, then the second reagent is added with additional wait time, after which the result is printed. Total time is six minutes for the first test in a series. 

For the MegaQuant Wave, water, two buffers, the sample and two enzymes are added sequentially to the test tube, which is then inserted for the first measurement. The last enzyme with a wait is added for the last measurement. The total time is six minutes for the first test in a series. The lowest amount the MegaQuant Wave can detect is 30 ppm; for the CDR WineLab it is 50 ppm.

For both instruments, add about 30 seconds for each additional analysis in a series of analyses. The same basic procedural steps outlined here are followed with these two instruments when conducting other analyses. The only differences are the particular enzymes or other reagents, the time of the tests and the amounts of reagents. Once you learn the procedure, you can run all of their tests.

Alcohol: ebulliometry goes digital
The most common way to analyze for ethanol in wine is by ebulliometry, which is based on the boiling points of water and wine. With the right equipment, the results are accurate enough for regulators for label determinations. For dry wines, accuracy is about +/- 0.1% v/v. This in-house analysis should not be relied upon for decisions where alcohol tax levels change if your wine is within twice the average error. 

Setup for the first ebulliometer measurement takes 15 to 20 minutes for a dry wine with less than 1% residual sugar. Subsequent tests take about seven to eight minutes. As residual sugar levels rise, this test becomes less accurate. Dilution to less than 1% residual sugar helps, but errors increase due to dilution inaccuracy. These errors can only be somewhat tolerated to about 3% residual sugar. Above that, a two-step process is required to obtain a reasonable result: 1) distillation of all alcohol, and 2) reanalyzing with the ebulliometer. This procedure can add 45 to 60 minutes to the test. 

Enzymatic analysis by CDR and Megazyme can make these analyses whether with or without residual sugar. As simple as these tests appear, analysis of ethanol by these systems is the one test with the greatest degree of difference from the traditional means of measuring ethanol in wine. 

The CDR test takes 11 minutes, and the MegaQuant Wave takes seven minutes. Both tests require some form of sample dilution to get accurate results. Each one requires two measurement steps to obtain a final answer. 

The Rudolph Alcotest is the simplest test for alcohol measurement. A few drops of wine are placed on their refractometer from a 3-ml syringe. That syringe is then used to inject a 1+ ml of wine into the densitometer that is shown on the LCD screen. The screen allows one to be sure it is completely full. Push start and, in about one minute, a three X replication with a standard deviation result is displayed. In practice, the results will be very close to ebulliometry.  

Brix: spill-sensitive or splash proof
The Hanna refractometer has a 0.1% Brix Resolution and an accuracy of +/- 0.2%. The temperature compensation ranges from 0° to 40° C, and it takes 1.5 seconds to make an analysis. This instrument cannot withstand liquid spillage. 

Rudolph J457 returns a reading +/- 0.15° Brix at a resolution of 0.01° Brix. Instead of a compensation algorithm, it brings all samples to one temperature, so it takes 30 seconds or less per analysis. This unit is splash-proof and can withstand wet environments. It also has compensation software to detect the cleanliness of the sample chamber and can detect whether the sample is covering the prism properly. Its cover protects against evaporation and temperature swings during analysis. 

Copper test: reduce over-fining complications
This test is very useful when monitoring wines that have reductive aromas. Elevated copper in a wine and attempts to eliminate the reductive aromas can result in wine with copper casse (copper instability). CDR WineLab offers this test, which follows the pattern of adding a sample, a reagent, and then measuring. The range of this test covers from 0.05 to 1.2 mg/L. 

Cold stability: conductivity—a more precise measurement
Cold-stability tests are important for all wineries. The Edge meter from Hanna incorporates a conductivity probe as one of its options. With a marginal increase in the meter’s purchase price, a small winery can add this test to its repertoire. A winery will need to have a cooling source that will reduce the temperature of about 0.5 liters of the wine being tested. Desktop lab chillers are available and are the easiest and best way to do that. However, if the winery already has a glycol chiller to cold stabilize wine, a DIY cold-stabilization device can be created to move some glycol to chill a small amount of wine to run the test. 

The test is simple. Using the Davis Protocol, chill wine to 0° C, add 1.5 g/100 ml of cream of tartar, stir or bubble the wine for 30 minutes. Measure the conductivity of the wine at the beginning and end. If the conductivity goes down by more than 5% of the initial measure of conductivity, the wine is unstable.

Glucose/fructose: sweetness is knowing your sugar levels 
Both CDR WineLab and the MegaQuant Wave can measure glucose and fructose, either separately or combined, and can measure sucrose and other sugars. Most wineries only need combined glucose and fructose to determine sugar levels. Be sure to account for any sucrose additions and assure its complete hydrolysis before using these tests. From grapes to wine, sugar levels can be measured using grams or milligrams per liter. The same test is used to measure the sugars, which usually means dilutions are needed to keep the analysis in the linear range of measurement. The lowest amount that the MegaQuant Wave can measure is 40 ppm of sugars, and for the CDR WineLab it is 100 ppm.

Heat stability: NTU measurement is better than your eye 
There are many different methods for testing for heat stability. Use your preferred method for inducing heat stress into your wine. Prior to stressing the wine, filter the sample to 0.45 u, then measure the wine sample with the Hanna Turbidity meter and record that number. Hanna uses their Bentocheck reagent to denature protein, and their formula stipulates that if T2 < T1+2 the wine is stable; otherwise, bentonite needs to be added until the wine meets the formula. There are alternate ways to denature the protein and some minor variations in the formula to calculate results. 

Malic acid: enzyme analysis is easier and faster than chromatography 
The CDR WineLab and MegaQuant Wave follow their enzyme standard protocol for their two instruments. The test time for the CDR WineLab is four minutes, and for the MegaQuant Wave it is six minutes. The lowest amount detectable is 5 ppm by the MegaQuant Wave and 50 ppm by the CDR. 

Micro testing: with this system, you can do integrity checks 
Microbiological testing is challenging for small wineries. Sartorius has developed a system called Biosart for cell counts that minimizes the difficulties of doing micro testing. The most important additional component that is needed to do integrity tests is a chamber in which to perform the test. These can be quite expensive. Agápi Lab Solutions offers an inexpensive chamber as a kit. Assemble when needed, then disassemble and store it in a box.

To conduct a test, put on disposable lab gloves, then spray the inside of the chamber liberally with grocery store sanitizing spray. Use 70% alcohol to sanitize the tips of tongs. Outside the chamber, flame the bottle top (after removing the capsule) and insert immediately into the chamber to protect from organisms in the air. 

Place a sterile Biosart-padded membrane funnel on a vacuum manifold. Remove its lid and turn on a vacuum source. Filter the desired amount from the bottle (note the quantity for later results). Follow with 3 ml sterile  water to rinse any residual sorbate. Open a Biosart Media ampoule and place on the filter disk. Immediately remove the funnel wall place the funnel top on the filter base and cap the bottom. Incubate in a warm spot for three days and check for growth. Biosart has photos to guide the procedure.

pH: a small footprint meter 
The most important factor in pH measurements is probe quality and its maintenance. Based on experience, accurate measurements from a probe that has been properly cared for is about 18 months, even if it is used infrequently. The Edge meter has calibration reminders and GLP (good laboratory practices) icons to remind you of maintenance times.

SO2: so simple to run 
With the Hanna mini-titrator, the free SO2 and total SO2 tests are conducted essentially in the same way, only using different reagents. The testing protocol for both tests is Ripper chemistry-based. The CDR WineLab and MegaQuant Wave use similar enzymatic based chemistry in their instruments. For CDR WineLab, different test kits are used to distinguish between free and total SO2. Megazyme’s MegaQuant Wave has three combinations of test kits for SO2. A combination kit that has both free and total analyses, one with only free and one for total. Each have the same number of tests per kit. 

For the Hanna titrator, the free SO2 takes a shorter time to run than total because the total test requires time to break the bound form into free before the analysis can be run. For CDR WineLab and the MegaQuant Wave, the timing is the reverse. Using the Hanna instrument for measuring free SO2, the acid reagent and potassium iodide are added to the sample and the test is begun. The titrant measures the inflection point in the titration curve and calculates the ppm of free SO2

For the MegaQuant Wave, the free SO2 test is based on fuschin and aldehyde binding. The tests using CDR are basically the same as the Megazyme tests. 

The lowest amount of free SO2 detectable by the Hanna titrator is 1 ppm, for the MegaQuant Wave 2 ppm and for CDR WineLab 1 ppm. 

For both MegaQuant Wave and CDR, the total test is based on thiol groups. The MegaQuant Wave uses Ellman’s reagent and CDR uses a proprietary reagent. The lowest amount of total SO2 for the Hanna titrator is 30 ppm, for the MegaQuant Wave it is 5 ppm and for CDR WineLab it is 15 ppm. 

Titratable acidity: step motor accuracy, and quick 
A defined function mini-titrator was one of the best advances in lab analysis when this particular instrument was released. The step motor advance was essential to this meter’s position in this arena. CDR has stepped up the game with this titrimetric method of analysis. 

The lowest amount of acidity that the Hanna titrator can measure is 0.1 g/L, and for the CDR WineLab it is 1 g/L.

Upgrade lab procedures along with lab technology
All small to medium-sized wineries should evaluate their laboratory, update their good laboratory procedures on a regular basis, and then upgrade the lab to incorporate the best analysis technology they can find for their needs.

It should be noted that no matter the size of the laboratory, all analyses should be under constant examination, whether wet chemistry-based or the advanced techniques described here. On a regular basis (once a quarter, semiannually or at least once per year) all tests should be verified that they are within a standard error by an outside source such as ETS Laboratories. 

Richard Carey, Ph.D., is a wine consultant in Lancaster, Pa., and owner of Tamanend Wine Inc. He wrote a software program to help small wineries keep track of their wine production records and results of laboratory analyses. He would like to thank the suppliers who provided the equipment that he used for this article.


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