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NAPA VALLEY VINTNERS ASSOCIATION TEACHING WINERY NAPA VALLEY COLLEGE WINE & MUST ANALYSES MANUAL
Prepared by the Students of Laboratory Analysis of Musts and Wines Fall 1997, 2001, 2002, 2003
Acknowledgements: Thank you to: The Napa Valley Vintners Association for generously donating the money to build the teaching winery. The many wineries, suppliers and individuals who have willingly donated equipment, cash, knowledge and time. Stacy Hitchcock for helping with some of the collation of the manual
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
Napa Valley College, Viticulture and Winery Technology Standard Operating Procedure
Table of Contents
Vineyard Sampling.3 Soluble Solids by Refractometry...4 Soluble Solids by Hydrometry...... 7 Titratable Acidity (TA) by Indicator Titration Quick & Dirty...10 Titratable Acidity (TA) by pH Titration....11 pH ......13 Formol Titration.......15 Nitrogen by Ammonia (NH4+) Electrode17 Alpha Amino Acid Estimation Using the NOPA Procedure.20 Clinitest / Dextrocheck..25 Determination of Reducing Sugar by Rebelein Method...27 Total SO2 (TS) by the Ripper Method....29 Free SO2 (TS) by the Ripper Method 30 Free SO2 (TS) using a Platinum Electrode31 Free SO2 (TS) Aspiration Oxidation..33 Malic Acid by Paper Chromatography.35 Alcohol by Ebulliometry.37 Spectral Measures for estimating Wine Color and Phenols40 Volatile Acidity (VA) using a Cash Still46 Fining Bentonite Fining...49 Fining - Other Than Bentonite...52 Tartrate Stability.60 Dissolved Oxygen by Orion 820 Dissolved Oxygen meter...62
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
Napa Valley College, Viticulture and Enology Department Standard Operating Procedure
Vineyard Sampling
Developed by Michelle Bowen Date: December 1997 Introduction Berry sampling can provide an accurate and economical technique with which to gauge grape maturity. However, the following variables can affect the integrity of the sample: 1. The composition of berries can differ with their position on the rachis. 2. Variability due to microclimate and soil changes in a very large vineyard (the location of the vine in the vineyard, irrigation practices, etc.) 3. The location of the cluster on the vine. 4. The degree of sun exposure (variation in leaf cover) 5. The natural tendency to select samples based on eye appeal. Preparation Sampling and measurements should be taken once or twice a week, depending on the rate of changes occurring in the grapes development. Daily sampling is recommended when the grapes are almost mature. Each sampling should be done at the same time of day in order to maintain accuracy. While sampling, keep in mind that the distribution of sugar in a bunch of grapes or on bunches growing on the same stem is not regular. The internal composition of the grape is also not homogeneous: the pulp from just under the skin has the highest sugar and lowest acid; the intermediary area is more acid and sometimes more sugary; and the pulp in the center of the berry nearest the seeds has a much lower sugar count and much more acid. Therefore, a varied sample must be obtained in order to create a homogenous sample of the vineyard. Materials Ice chest, quart-size plastic zip-lock bags, grape knife or clippers. Procedure Collect 250-500 berries, each one from different cluster locations on the vine, lighted and shadowed, and from both sides of the rows, and from different vines in the area. Berries should be picked as whole as possible in order not to lose their juice. Leave berries whole until ready to perform refractometry and titratable acidity. At that time, gently mash berries while still in the bag, taking care not to break the skins and seeds up too much. Pour juice through a strainer or filter into a beaker and test immediately.
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
Napa Valley College, Viticulture and Enology Department Standard Operating Procedure
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
Calculations and Units of Reporting For juice analysis a scale reading directly in % (w/w) sugar (Brix) is used. Readability is normally 0.1-0.2 Brix, depending on the instrument and on the preparation of the sample. Temperature correction (approximate) for every C above or below 20C add or subtract 0.07 Brix respectively to or from the indicated value. Temperature Correction Table for Brix by Refractometry Above and Below 20C
Temp C Minus 12 13 14 15 16 17 18 19 21 22 23 24 25 26 27 28 Brix 0 0.42 0.37 0.33 0.27 0.22 0.17 0.12 0.06 0.06 0.13 0.19 0.26 0.33 0.40 0.48 0.56 Brix 5 0.45 0.40 0.35 0.29 0.24 0.18 0.13 0.06 0.07 0.13 0.20 0.27 0.35 0.42 0.50 0.57 Brix 10 0.48 0.42 0.37 0.31 0.25 0.19 0.13 0.06 0.07 0.14 0.21 0.28 0.36 0.43 0.52 0.60 Brix 15 0.50 0.44 0.39 0.33 0.26 0.20 0.14 0.07 0.07 0.14 0.22 0.29 0.37 0.44 0.53 0.61 Brix 20 0.52 0.46 0.40 0.34 0.27 0.21 0.14 0.07 0.07 0.15 0.22 0.30 0.38 0.45 0.54 0.62 Brix 25 0.54 0.48 0.41 0.34 0.28 0.21 0.14 0.07 0.08 0.15 0.23 0.30 0.38 0.46 0.55 0.63 Brix 30 0.56 0.49 0.42 0.35 0.28 0.21 0.14 0.07 0.08 0.15 0.23 0.31 0.39 0.47 0.55 0.63 Brix 35 0.57 0.50 0.43 0.36 0.29 0.22 0.15 0.08 0.08 0.15 0.23 0.31 0.40 0.48 0.56 0.64
Plus
References Zoecklein, B.W., Fugelsang, K.C., Gump, B.H., Nury, F.S. 1995. Wine Analysis and Production. Chapman & Hall, New York. 2. Iland, P., Ewart, A., sitters, J. 1993. Techniques for Chemical Analysis and Stability Tests of Grape Juice and Wine. Patrick Iland Wine Promotions, Campbelltown, South Australia.
1.
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
Napa Valley College, Viticulture and Enology Department Standard Operating Procedure
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
Iland, P., Ewart, A., sitters, J. 1993. Techniques for Chemical Analysis and Stability Tests of Grape Juice and Wine. Patrick Iland Wine Promotions, Campbelltown, South Australia. Determination of Total Soluble Solids by Hydrometry
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
Temperature Correction Table for Various Hydrometers Temperature of Measurement (C) 15 16 17 18 19 20 21 22 23 24 25 Indicated Hydrometer Reading X X X X X X X X X X X Corrected Value (at 20C) Baume X 0.25 X 0.20 X 0.15 X 0.10 X 0.05 X X + 0.05 X + 0.10 X + 0.15 X + 0.20 X + 0.25 Corrected Value (at 20C) Brix X 0.30 X 0.24 X 0.18 X 0.12 X 0.06 X X + 0.06 X + 0.12 X + 0.18 X + 0.24 X + 0.30 Corrected Value (at 20C) SG X 0.0010 X 0.0008 X 0.0006 X 0.0004 X 0.0002 X X + 0.0002 X + 0.0004 X + 0.0006 X + 0.0008 X + 0.0010
Techniques for Chemical Analysis and Stability Tests of Grape Juice and Wine
References Zoecklein, B.W., Fugelsang, K.C., Gump, B.H., Nury, F.S. 1995. Wine Analysis and Production. Chapman & Hall, New York. 2. Iland, P., Ewart, A., sitters, J. 1993. Techniques for Chemical Analysis and Stability Tests of Grape Juice and Wine. Patrick Iland Wine Promotions, Campbelltown, South Australia.
1.
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
Napa Valley College, Viticulture and Enology Department Standard Operating Procedure
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
Napa Valley College, Viticulture and Enology Department Standard Operating Procedure
1. Standardize pH meter per operators manual. 2. Degas about 20-30ml wine for 4min using vacuum pump. 3. Shake occasionally during de-gassing 4. Pipette 5ml into a 100ml beaker, add stirrer bar. 5. Add 50ml DI water. 6. Begin stirring. 7. Put 0.067N NaOH in burette using squeezy bottle. Record the initial burette reading. 8. Place burette over beaker with tip 1/3 above level of liquid. Procedure The following describes how to add the alkali from the burette to achieve a pH of 8.2 in the beaker. 1. Add NaOH from burette until pH is about 5 and then close tap. 2. Wait until the pH has stabilized or starts to decrease. 3. Add more NaOH until the pH is 5.5 (or 6.0 if the pH was >5.5 after waiting for it to stabilize or to start to decrease). 4. Wait until the pH has stabilized. 5. Add more NaOH until the pH is 6.0 (or 6.5 if the pH was >6.0 after waiting for it to stabilize or start to decrease.
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
6. Wait until the pH has stabilized. 7. Repeat this process until pH is 7.00. 8. Add NaOH more slowly by rotating the tap through a complete circle once and waiting for pH to stabilize / decrease. 9. Continue until the pH is 8.2 +/- 0.3. 10. Record volume of 0.067N NaOH added. NB It takes less than one drop of alkali to increase the pH of the de-ionized water to 8.2 and therefore it is considered a negligible amount. Calculations and Units of Reporting Titratable Acidity in Musts and Wines TA (g/L Tartaric Acid) = (ml NaOH) (N NaOH) (0.075) (1,000) ml wine sample In this case: TA (g/L) = volume (mls) of 0.067N NaOH added.
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
Napa Valley College, Viticulture and Enology Department Standard Operating Procedure
pH
Developed by: Jonathan Grant and Janet McDonald Date: October 1997 Introduction The pH is important in winemaking as it directly affects microbial stability (bacterial growth), wine oxidation, wine color, SO2 activity, flavor, aroma and chemical stability. pH is a measure of the concentration of hydrogen ions in a solution. pH = -log [H+]. Errors may occur due to incorrect calibration of the pH meter or worn or insensitive electrode. If adjustment of the sensitivity control in setting the pH 4.00 cannot achieve the correct setting or is less than 90% accurate, this indicates that the electrode has lost sensitivity. Another common cause of error in pH measurement is temperature. Some electrodes have automatic temperature correction or some pH meters have temperature compensation probes that correct for temperature. On the other hand, some set ups have neither and correction needs to be achieved by measuring the temperature and using tables of correction factors. The temperature of the sample and standard buffers needs to be the same, even if the electrode assembly has a temperature compensation probe. To ensure a quick response and free-flowing liquid junction, the sensing bulb on the electrode must not be allowed to dry out. Always store in the pH electrode storage solution. Materials pH Meter & electrode, 50ml beakers for each sample, pH 7.00 buffer solution, pH 4.00 buffer solution, juice/wine sample, Pasteur pipette, distilled water, magnetic mixer and stir bar. Procedure 1. The buffers are stored in containers that the electrode can be immersed in directly. 2. Turn on the meter, slide the rubber sleeve off of the vent hold in the electrode (if need be), check the level of internal filling KCl solution (to inch below the hole), add if needed. Calibration of the meter: You must first calibrate the meter with two buffers before proceeding with the sample measurement. This must be done every time the meter is switched on. Once calibrated, do not turn the machine off until finished for the day. If you are measuring a lot of pHs, recalibrate after 4hrs. Place the pH 7.00 buffer solution container (yellow solution) on a magnetic stirrer. Immerse electrode in the container (there is a magnetic stirrer bar already in the container). Switch on the stirrer carefully and stir slowly, making sure that the stirrer bar does not hit the glass bulb. 4. Follow the instructions for the calibration of the pH meter that are on the wall above the pH meter. 5. When moving from the the pH7 buffer to the pH 4 buffer, rinse the electrode with a few milliliters of deionized water from a squeezy bottle, touch the tip with a tissue to remove excess water. and then place the electrode in the pH 4.00 buffer (red solution) Stir slowly as before and complete the calibrations as described in the instructions.
3.
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
Measurement of samples: Place approximately 50 mL of sample in a small beaker with a stir bar and set on magnetic stirrer. If necessary, wine and juice samples should be clarified prior to measurement either by natural settling or centrifuging. 7. Remove electrode from buffer and rinse with deionized water. Place electrode into the beaker with the 50 mL sample and start stirring, making sure that stirring bar does not contact the tip of the electrode. 8. .Follow the instructions for pH measurement on the wall above the pH meter 9. After recording the pH value, rinse the electrode with distilled water and return the electrode to the storage solution.
6.
VERY IMPORTANT: When not in use, store electrode immersed in storage solution. Do not leave electrode immersed in grape juice or wine longer than is necessary. Storage Solution: Either the pH 4.00 buffer solution, a saturated solution of potassium hydrogen tartrate or as per manufacturers recommendations.
Calculations and Units of Reporting for pH Readings Temperature Correction Table: Variation of pH value with temperature pH Value pH Value pH value pH value Temperature () Phosphate Buffer Phosphate Buffer Phthalate Buffer Saturated (commercially (lab prepared) Solution of KHT prepared) 10 7.12 6.92 4.00 -15 7.06 6.90 4.00 -20 7.02 6.88 4.00 3.56 25 7.00 6.86 4.01 3.56 30 6.99 6.85 4.02 3.55
Electrode Storage Short-term Storage (up to one week): Soak electrode in pH electrode storage solution. If there is none available use 200 ml. pH 7 buffer to which about 1 gram of KCl has been added, as a temporary substitute. Long-term Storage: The reference chamber should be filled and the filling hold securely covered. Cover the sensing element and reference junction with its protective cap containing a few drops of storage solution. Before returning the electrode to use, prepare it as a new electrode (See manual for preparing new electrode). Electrodes should be periodically cleaned to ensure proper operation. Immerse them in a solution of 75% Methanol followed by several rinses in Distilled Water. References Orion Laboratory Products Division, 720A pH/ISE Meter, Part No. 216616-001 Rev. A reference manual. Techniques for Chemical Analysis and Stability Tests of Grape Juice & Wine, Determination of pH, page 22-25.
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
Napa Valley College, Viticulture and Wine Technology Standard Operating Procedures
Formol Titration
Developed by Keith Behlmer, Roger Low and Ralph White December 2002 Introduction Yeast available nitrogen (YAN) is required for yeast to complete the fermentation of sugars to alcohol. It is needed for cell division, growth and synthesis of cell parts. YAN is made up of two forms of Nitrogen: Ammonium ion NH4 2. Free alpha amino acids (yeasts will only utilize some of the amino acids)
1.
The Formol titration is a method used to estimate the YAN in musts. Formaldehyde is used to react with free amino groups and the released hydrogen is titrated with an alkali (sodium hydroxide). Ammonium ions are also titrated by the sodium hydroxide Errors pH adjustment is not done correctly Titration should be done slowly due to high buffering of the solution in the presence of formaldehyde. Equipment Centrifuge Filtration system for 5 micron filter papers pH meter magnetic stirrer and stirring bars 10ml pipette 10ml burette and stand 100ml beakers Chemicals pH 4 and pH 7 buffer 1N NaOH 0.067N NaOH Formaldehyde (37%) at pH 8 De-ionized water
Preparation Clarify the must using a centrifuge and filtering equipment 1. Centrifuge for 10 minutes at 4000 rpm 2. Filter through a 5 micron swinnex cartridge. Procedure 1. Pipette 10ml sample of clarified and filtered must into a 100ml beaker containing a stirring bar. 2. Add 50ml of distilled water and immerse pH electrode in the solution. 3. Start stirring the solution. 4. Adjust pH to 7.0 by adding 1 N NaOH from a Pasteur pipette. 5. Adjust pH to 8 by adding 0.067N NaOH from a Pasteur pipette. 6. Add 5 ml of 37% formaldehyde (also adjusted to pH 8) and wait 5 minutes 7. Titrate to a pH of 8.0 using 0.067 N NaOH 8. Repeat with water blank if necessary.
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
Calculation: mg YAN /L = (mls NaOH for sample mls for blank) x (Normality of NaOH) x Dilution factor x (1000/sample volume) x At. Wt. Of N =(mls NaOH for sample-mls NaOH for blank) x 93.8 Notes on the Calculation 1 mole of OH- in the alkali reacts with 1 mole of hydrogen released from an amino acid of an ammonium ion 1 mole of OH- added in titration = 1 mole of nitrogen Dilution factor = final volume/initial volume (NB dilution occurs before pipetting the 10 ml sample) Grams of Nitrogen in 1 mole or equivalent = Atomic weight of N = 14 mg/L = ppm
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
Napa Valley College, Viticulture and Winery Technology Standard Operating Procedure
Preparation of Juice / Must sample 1. Centrifuge four 30ml centrifuge tubes of each sample for 10mins at 4000rpm
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
Procedure A calibration curve is constructed from standard solutions of NH3 which is then used to estimate the concentration of ammonium N in musts or juice. Determination of NH3 in standard solutions of known concentration 1. Using a measuring cylinder, add 40mls of the 500ppm NH3 standard solution to a 50ml beaker. 2. Rinse the ammonia electrode with DI water and submerge into beaker. 3. Using the 5ml micro pipette, add 2mls of 10N NaOH. 4. Place stirring bar in beaker and place on magnetic stirrer. 5. Record the mV reading after the electrode has stabilized. 6. Repeat steps 1-5 using 40mls of 200ppm NH3. 7. Repeat steps 1-5 using 40mls of 50ppm NH3. Determination of NH3 in juice / must 1. Repeat steps 1-5 above using 40mls of must or juice. 2. .If the mV reading of must sample falls outside the range covered by the known standard solutions, the sample needs to be diluted. Pipette 20mls of must sample into a 100ml volumetric flask and fill to the mark with DI water. You now have a sample with a dilution factor of 5 (100/20=5). Record the mV of the diluted sample as described in steps 1-5 above. Calculation Determine the concentration of NH3 in the must sample using the following steps: 1. Draw the calibration graph. Plot a graph of mV reading on the vertical (y) axis vs. concentration of standard solution on the horizontal (x) axis. If doing this by hand, use semi log paper. If using a computer, use the log of the concentration on the horizontal axis 2. Fit a straight line through the points. You can either a. use regression analysis to fit a straight line through the three points given by the standard solutions b. draw a straight line through the points by eye 3. Estimate the concentration: a. By regression analysis: mg/L of NH3 = mV-(intercept/slope) b. By eye: find the mV equivalent to that for the unknown must sample on the vertical (y) axis of the calibration curve. Draw a horizontal line from that point across to the calibration curve. At the point that this horizontal line touches the calibration curve, draw a vertical line down to the horizontal (x) axis. The value on the x axis is the log of the concentration of NH3 in the must sample. 4. If the samples was diluted, multiply the concentration estimated from the calibration curve by the dilution factor (DF = Final Volume / Initial volume)
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
Chart Example
Ammonium Electrode Maintenance and Storage To ensure accurate electrode readings, refer to the electrode owners handbook and follow proper procedures for short-term and long-term electrode storage. Common Reasons for Errors If inconsistent readings are being observed: 1. Ensure there are no bubbles of air trapped on the membrane when taking a measurement 2. Ensure that the pH is between 11 and 14. 3. If the above to possibilities have been discounted, an unclean or damaged membrane may be the cause erroneous readings. Follow the electrode owners handbook instructions for membrane replacement.
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
Napa Valley College, Viticulture and Enology Department Standard Operating Procedure
Spectrophotometer capable of detection at 335nm 4.5mL methyl acrylate cuvettes 100uL pipette tips 1000uL pipette tips 3mL dispenser 100 1000uL micro pipette 10 100uL micro pipette 2 X 1L vol. flasks Weighing Scale or analytical balance to 0.000g Syringe & 5um swinnex cartridges for clarification Tissues Beakers for waste
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
Chemicals
N-acetyl-L-cysteine (NAC) Ortho-phthaldialdehyde (OPA) L-isoleucine Boric Acid Sodium Hydroxide Denatured ethanol De-ionized water
Solution Preparation OPA/NAC Reagent Preparation Reagent Solution A (1000mLs) 1. Dissolve 0.671g of OPA in 100mL of 95% ethanol. 2. Dissolve: 3.837 g NAOH (s), 8.468 g ortho boric acid (s), and 0.816g NAC (s) in approximately 500mL de-ionized (DI)water in a 1000mL volumetric flask. 3. Add the alcohol/OPA brew to the flask and mix well. 4. Add de-ionized water to make 1000mL of solution Reagent Solution B (1000mLs) 1. Dissolve 3.837 g NaOH (s), 8.468 g ortho-boric acid, and 0.816 NAC in Approximately 500mL of de-ionized (DI) water in a 1000mL volumetric flask. 2. Add 100mL 95% ethanol to the flask and mix well. 3. Add de-ionized (DI) water to make 1000mL of solution. Note: Reagents can be stored in the refrigerator for 2 weeks and should be used at room temperature. Amino Acid Stock Solution Preparation 1. Dissolve 0.328 g of isoleucine (s) in de-ionized water in a 250mL volumetric flask. 2. 2 mL aliquots of this solution can be frozen for use later. Juice / Must Preparation 1. Clarify juice sample by centrifuging for 10min at 4000rpm 2. Filter through a 5um swinnex cartridge.
Procedure
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
A calibration curve is constructed by measuring the absorbance, at 335nm, of four different standards whose mg N/L is known. Once the curve is plotted, the absorbance of the juice sample is measured and the corresponding mg N/L is estimated using the calibration curve. Calibration Curve 1. Prepare the following standard solutions (i.e. solutions that contain a known concentration of amino acids) by pipetting the volumes of stock solution, reagents and DI water given in the table into a 4.5ml cuvette. ml = milliliters (one thousandth of a liter) uL = L = microliters (one millionth of a liter) 1 2 3 4 28 56 84 112
Cuvette Code Amino Acid Concentration (mg/L) uL of Stock Solution uL of DI water uL of OPA (Reagent A) 2. 3. 4. 5.
Blank 0
5 140
0 50 3000
10 40 3000
20 30 3000
30 20 3000
40 10 3000
50 0 3000
Mix thoroughly and wait 10mins. Zero the spectrophotometer using the blank cuvette. Measure the absorbance of each cuvette containing a standard solution. Construct a calibration curve by plotting the absorbance on the vertical axis (y axis) versus the known mg/L of amino acids in each standard on the horizontal axis (x axis)
Determination of Amino Acid N in Unknown Sample A. Sample 1. Pipette 50uL of clarified must or juice into a cuvette. Juice may need to be diluted if its nitrogen concentration greatly exceeds the range of the calibration curve (>200Nmg/L). 2. Using pipette, add 3000l of Reagent A. 3. Mix thoroughly and wait 10mins. 4. Measure the absorbance of light at a wavelength of 335nm. B. Juice Blank 1. 2. 3. 4. 5. Pipette 50uL of the clarified must or juice (diluted if necessary) into cuvette. Using pipette, add 3000uL of Reagent B to the cuvette. Mix thoroughly and wait 10mins Measure the absorbance of light at a wavelength of 335nm. Calculate Net Absorbance = sample absorbance juice blank absorbance
Calculation Determine the concentration of amino acids in the juice / must sample using the following steps:
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
1. Draw the calibration graph. Plot a graph of Absorbance reading on the vertical (y) axis vs. concentration of each standard solution on the horizontal (x) axis. 2. Fit a straight line through the points. You can either a. use regression analysis to fit a straight line through the three points given by the standard solutions (Excel has this capability) b. draw a straight line through the points by eye. 3. If you are using regression analysis, you will need to know the slope and intercept of the fitted line. Regression analysis should do this for you automatically. If not: c. The intercept is the value on the vertical axis where the fitted line passes through that axis. d. The slope is calculated from: slope = y/x select any y value on the vertical axis and find its corresponding x value on the horizontal axis and divide the former number by the latter 4. Estimate the concentration: net absorbance of N in must = sample abs-juice blank abs a. By regression analysis: mg/L of amino N = (net abs-intercept)/slope b. By eye: find the point on the vertical axis that is equivalent to the net absorbance for the unknown sample. Draw a horizontal line from that point across to the calibration curve. At the point that this horizontal line touches the calibration curve, draw a vertical line down to the horizontal (x) axis. The value on the x axis is the concentration of amino acids in the unknown sample. 5. If the samples was diluted, multiply the concentration estimated from the calibration curve by the dilution factor (DF = Final Volume / Initial volume) Chart Example
POTENTIAL ERRORS Very small amounts (in the magnitude of micro liters) of juice samples, standards and reagents are used. Micro pipetting correct amounts of reagent and samples is critical and care should be taken when using digital micro pipettes.
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
Potential for miss-tracking the cuvettes (i.e.-keep track of the samples). Each cuvette will contain the same total amount of solution, with varying amounts of stock solution and de-ionized water. When filed, all the cuvettes will look the same! Spectrophotometer needs to be zeroed with blank sample. Sample may need to be diluted if nitrogen concentration exceeds 140 mg Nitrogen/L. Regression equation needs to be correctly calculated. Juice needs to be clarified by centrifugation or cold settling. Care must be taken when preparing the reagent solutions. Solutions need to be brought to room temperature before using. Calibration curve should be plotted each time a set of new sample is measured. Duplicate determinations are suggested.
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
Napa Valley College, Viticulture and Enology Department Standard Operating Procedure
Clinitest/Dextrocheck
Developed by Pat Collins, Mona Huisman and Gerry Ritchie Date: November 2002 & 2003 Introduction The Clinitest tablet is a standardized self heating method for quantitative determination of sugar by copper reduction. The Clinitest Reagent Tablets contain copper sulfate which reacts with reducing substances in wine converting cupric sulfate to cuprous oxide. This results in a color change which varies with the amount of reducing substances present. The Clinitest Reagent Tablet reacts with all reducing sugars and is not specific to glucose. Clinitest is a useful tool for monitoring fermentation progress but will react with phenolic content in wine from red pigmentation to barrel phenolics. Fermentable sugars, glucose and fructose, determined by enzymatic analysis will confirm dryness. Clinitest Reagent Tablets are the identical tablets previously sold as Dextrocheck for testing reducing sugar. Red Wines must be de-colorized before carrying out the Clinitest once the sugar concentration reaches 0.4%. Equipment De-colorizing: Filter Funnel 250ml conical flask 15cm diameter qualitative filter paper Teaspoon Activated Carbon Clinitest: 1 mL pipette Test Tube Clinitest Tablet Color Chart (supplied with test kit) Procedure De-colorizing of red wines: 1. Place the filter funnel in the neck of the 250ml conical flask 2. Flute the filter paper and put it in the filter funnel 3. Put teaspoon of Activated Carbon in the filter paper. 4. Pour sufficient wine into the filter paper to fill it completely 5. Wait until about 2-5mls has filtered through the paper and then discard the rest. Clinitest: 1. Pipette 0.5 mL of (decolorized)sample into test tube 2. Drop a sugar tablet (Clinitest) into the test tube. Note: Clinitest tablets are sensitive to moisture, so packets should be kept sealed until used
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
3.
4. 5. 6. 7.
Watch the test tube as a boiling reaction proceeds. Caution: make sure you are not holding the test tube as the boiling reaction occurs Wait 15 seconds after boiling stops, then compare the color in the tube with color chart included with the test kit Estimate % residual sugar from chart (% approx. = g/100 mL; for g/L multiply by 10) If color goes past orange to brown the sample is too sweet and must be diluted before retesting For samples of 1 to 5% sugar, use 0.1 mL (2 drops) of wine and 0.4 mL (8 drops) of water. Multiply % sugar reading from color chart x5 to compensate for dilution
Interpretation Use color chart for 2 Drop Method with following % reducing sugar scale: 0 Blue 0.05% Blue/Green 0.10% Green 0.20% Olive Green 0.40% Brown 0.60% Brown/ Orange 1.00% Orange
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
Napa Valley College, Viticulture and Enology Department Standard Operating Procedure
Amount required 5mL 2.5mL 5mL 5mL 5mL Fill burette 1mL 1mL 1mL
Reagent Preparation Accurate Accurate Accurate Accurate Accurate Accurate Decolorized for reds Diluted Accurate
Reagent Addition Accurate Approximate Approximate Approximate Approximate Accurate Accurate Accurate Accurate
Preparation Before analysis of the wine sample, the wine sample must be prepared. If the wine sample is red it must be decolorized (procedure below). Decolorization Place a funnel in a 250 ml Erlenmeyer flask and line the funnel with filter paper (Whatman Qualitative will work). Add teaspoon of carbon into the filter paper. Add 10 ml of wine sample into carbon filled filter paper
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
and stir gently. When 10 ml wine sample has filtered through; discard the paper and carbon and repeat until enough wine sample has been obtained. Dilution Wines containing more than 20 g/L reducing sugars require dilution so that accurate volume of Na Thiosulfate may be used as the titrate. Check that wine is less than 20 g/L by using Clinitest. If the sugar level is more than 20 g/L the wine sample must be diluted. Dilution factor (DF) can be calculated as follows: DF = (volume wine + volume distilled water)/volume wine Once you have decolorized wine sample (if necessary) and determined whether or not to do a dilution factor, two analyses must be completed: blank analysis (using distilled water) and analysis of the wine sample. Procedure Blank Analysis: 1. Pipette 5ml of Z1 (copper sulfate) and add 2.5ml of Z2 (NaK Tartrate) into a conical flask. Touch pipette to side of flask to release last drops into flask. 2. Add 3 boiling chips 3. Pipette 1ml of distilled water (Blank) into flask. 4. Turn hot plate on to 10. Place flask on hot plate until a boil is observed. Pick up flask using the oven mitt and hold (swirl) over hot plate so that a slow boil is maintained for 30 seconds. (heating of solution speeds up copper reaction) 5. Run the side of the flask under cool water for a few seconds and then place in an ice bath until bottom of flask is moderately cool to the touch. 6. Now add 5ml each of Z3 (sodium iodide), Z4 (sulfuric acid) and Z5 (starch), in this order, stirring after each addition. 7. Fill burette with Z6 (Na thiosulfate) and record the initial burette reading. 8. Titrate the mixture in the conical flask with Na thiosulfate, swirling the flask throughout the titration. Solution goes from a blue/green/black color to gray then to a cream color (end point). Initial tinges of yellow indicate there is Cu still to titrate. 9. Record the final burette reading and calculate the difference between the final and initial burette readings. The Blank Titre should be in the range of 29 to 31 mL and will vary slightly for each set of reagents prepared. Wine/juice Analysis: 1. Repeat the above procedure, but instead replace distilled water with the same amount of decolorized (and if necessary, diluted) wine sample at step 3. 2. Note a red/brown color while titrating indicates too much sugar and the wine/juice must be diluted first. See Preparation section above. 3. Record the final burette reading and calculate the difference between this final burette reading and the corresponding initial reading. This is called the Sample Titre. Calculation: Reducing Sugars (g/L) = Dilution factor x [Blank Titre (mL) Sample Titre (mL)] Source of Errors Incorrect judgment of the end point take as the first obvious change to cream. Failure to decolorize red wines.
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
Napa Valley College, Viticulture and Enology Department Standard Operating Procedure
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
Napa Valley College, Viticulture and Enology Department Standard Operating Procedure
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
Napa Valley College, Viticulture and Enology Department Standard Operating Procedure
End Point
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
Volume added at endpoint = 5.65-3.35 FS = 2.3 x 9.92 = 22.8ppm 10. After each titration, clean the Platinum electrode by rubbing the platinum with cardboard (as if using sand paper). 11. Rinse with water. 12. Store in electrode solution when finished. Calculation Ppm FS = titre x molarity of iodate (0.0155) x 32000 / (volume of sample (50ml)) = titre x 9.92 Example curves of the titration
Redox titration
4 1 0
00
00
5 .
5 .
5 .
5 .
l u
( m
l s)
Redox titration
420 410 400 390 380 370 360 350 340 330 320 7.00 8.00 9.00 10.00 11.00
mV
Volume (mls)
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
Napa Valley College, Viticulture and Enology Department Standard Operating Procedure
2.
3. 4.
5.
Secure the round-bottom, wine flask (4) to the stand. Fill the 10ml burette with 0.01 NaOH Put 0.3% hydrogen peroxide solution up to the mark on the side of the impinger flask(3) and add one drop of the SO2 indicator solution. If the peroxide turns violet, add 0.01 N NaOH drop wise from the 10ml burette, until the solution just turns from the original violet color to a green color. (note the shade of green, as it should be the same shade as the final end point of your titrations in Step 8) Attach the impinger flask (3) to the stand and insert the impinger (2) securely into the flask
Procedure 1. Pipette 20mls of wine into the round bottom flask (4) using the top opening. 2. .Take the plastic bottle containing the 1+3 Phosphoric acid and squeeze the bottle until the plastic cup is about two thirds full. When the bottle is released, the level in the cup will self adjust to 10mls. Carefully add this to the round bottom flask, using the top opening. 3. Insert the Stopper/Glass tubing (7) securely into the top opening of the round bottom flask. 4. Put the Bubbler /Stopper (6) securely into the side port of the round bottom flask.
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
Turn on vacuum pump and aspirate the sample for 10 minutes. In the presence of SO2, the peroxide in the impinger flask should turn violet during this period. 6. After 10 minutes, turn off the vacuum pump by unplugging it. 7. Lift the impinger (2) to just above the peroxide solution and blow into the Bubbler/Stopper (6) to remove any liquid remaining in the impinger 8. Titrate the contents of the impinger flask to the endpoint described in step 4 of the preparation, above. Read the titration volume to the nearest 0.05 ml.
5.
Calculations Free SO2 (ppm) = ml NaOH x N NaOH x 32 x 1000 20 ml (sample size) If one uses 0.01N NaOH, then the above equation becomes: SO2 (ppm) = ml NaOH x 16 Report the Free sulfur dioxide concentration to the nearest +/- 0.5 ppm (mg/l) Example calculation: a 1.5 ml titration with 0.01N NaOH will have a free SO2 content of 24.0 ppm Examples of Aspiration-Oxidation Apparatus
Support Stand Impinger Impinger flask for sample collection Round bottom wine flask Flask stand Bubbler and stopper Stopper & glass tubing adaptor Vacuum system Quick disconnect Latex tubing Tygon tubing Flow meter Clamps Green joint clamp
Bubbler / Stopper
Sample flask
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
Napa Valley College, Viticulture and Enology Department Standard Operating Procedure
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
lactic spot should indicate completion of the malolactic fermentation. If there is an indication of any malic spot the fermentation is not complete.
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
Napa Valley College, Viticulture and Enology Department Standard Operating Procedures
Alcohol by Ebulliometry
Developed by: Martin Garcia, Matt Gardner, and Paul Jenkins December 1997 Introduction Ebulliometry is a quantitative analysis for measuring the alcohol content of a given wine sample. Ebulliometric analysis is a quick and easy method for general purposes, and is accurate to + 0.5% v/v. The ebulliometric procedure is based on the fact that wine has a lower boiling point relative to the boiling point of pure water at 1 atmosphere. Alcohol (Ethanol) is a byproduct of fermentation, and should be tested for tax/legal purposes as well as winemaking practices. A wine containing alcohol of 14% (by volume) and greater is taxed at a higher rate than wine containing less than 14% alcohol. In addition, alcohol plays an important role in the structure of a wine; high alcohol can cause hot characteristics within a wine, and relatively low alcohol content can cause a wine to be flabby and unbalanced. The major interference involved in this test is residual sugar. Ebulliometric analysis will not give an accurate measurement of alcohol content for wines containing more than 2% residual sugar. Ebulliometric results for samples that contain residual sugar between 0.2% and 2.0% must be corrected for in accordance with the formula given in the Calculations section of this analysis. In testing, ice-cold water must be placed in the condenser to prevent premature alcohol evaporation; this water should be emptied and refilled before each new sample is placed in the ebulliometer. Due to changes in barometric pressure, the boiling point of water should be determined at least once every two hours. If a gas burner will be used instead of the alcohol lamp, the ebulliometer should be elevated away from the flame to prevent overheating which would lead to improper boiling point readings (bumping). Record the temperature when it reaches the first stable reading on the thermometer. Make sure to inspect the thermometer to ensure that the mercury tube is not broken (separated). Debris may coat the boiling chamber with a boiling solution of 1% NaOH. Materials 1. DuJardin-Salleron Ebulliometer 2. Alcohol or gas fueled burner 3. Magic Dial (supplied with most Ebulliometers)/alcohol scale 4. Ice water 5. Deionized/distilled water 6. 50 or 100 mL graduated cylinder 7. A chart summarizing the Churward formula is needed if measuring samples of alcohol content greater than 16%. The chart is shown in figure 2. Procedure See diagram for ebulliometer components (fig.1). A. Determine the boiling point of water: 1. Rinse boiling chamber twice with approximately 20 mL of deionized water. 2. Add 20 mL of deionized water to the boiling chamber. Do not place water in condenser.
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
3. Insert thermometer into its respective position as shown in diagram (fig. 1). 4. Place burner under the projecting tube and ignite burner. 5. Watch the mercury column rise, and note when it reaches a stable point for 30 seconds. Record this temperature reading (T1). 6. Set the magic dial so that T1 is opposite the 0% alcohol mark. 7. Drain the ebulliometer. B. Determine the boiling point of the sample: 1. Flush the boiling chamber with 2 rinses of approximately 25 mL each of the wine sample. Close the outlet tap. 2. Measure 50 mL of the wine sample into the boiling chamber. 3. Fill the condenser with ice water. 4. Insert the thermometer. 5. Place burner under the projecting tube and ignite burner. 6. Watch mercury column rise. Note when the temperature remains constant for 30 seconds. Record this temperature (T2). 7. Find the % alcohol that corresponds to T2 on the magic dial. This is the % alcohol of the sample. Calculations and Units of Reporting 1. The % alcohol should be calculated as follows for samples greater than 16% alcohol: a. Find the difference between T1 and T2 (T1 T2), this is the ebulliometer degree;. b. Enter this number into the table (fig. 2) to find corresponding % alcohol. 2. The % alcohol should be calculated as follows for samples with Baume > 0.5. a. True Alcohol % (v/v) = Apparent Alcohol [%(v/v)] x [1 Baume level x 0.015] b. Note, Correct Baume to 20C. 3. Alcohol is measured and reported on a percent by volume basis.
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
Ebull Deg.
6.15 6.20 6.25 6.30 6.35 6.40 6.45 6.50 6.55 6.60 6.65 6.70 6.75 6.80 6.85 6.90 6.95 7.00 7.05 7.10 7.15 7.20 7.25 7.30 7.35 7.40 7.45 7.50 7.55 7.60 7.65 7.70 7.75 7.80 7.85 7.90 7.95 8.00 8.05 8.10 8.15 8.20 8.25 8.30 8.35 8.40 8.45
% v/v Alcohol
7.4 7.5 7.6 7.6 7.7 7.8 7.9 7.9 8.0 8.1 8.2 8.3 8.3 8.4 8.5 8.6 8.7 8.8 8.8 8.9 9.0 9.1 9.2 9.3 9.3 9.4 9.5 9.6 9.7 9.8 9.8 10.0 10.0 10.1 10.2 10.2 10.4 10.5 10.6 10.7 10.8 10.9 10.9 11.0 11.1 11.2 11.3
Ebull Deg
12.76 12.78 12.80 12.82 12.84 12.86 12.88 12.90 12.92 12.94 12.96 12.98 13.00 13.02 13.04 13.06 13.08 13.10 13.12 13.14 13.16 13.18 13.20 13.22 13.24 13.26 13.28 13.30 13.32 13.34 13.36 13.38 13.40 13.44 13.46 13.48 13.50 13.52 13.54 13.56 13.58 13.60 13.62 13.64 13.66 13.68
% v/v Alcohol
22.0 22.0 22.1 22.2 22.2 22.3 22.4 22.4 22.5 22.5 22.6 22.7 22.8 22.8 22.9 22.9 23.0 23.1 23.2 23.2 23.3 23.3 23.4 23.5 23.6 23.6 23.7 23.8 23.8 23.9 23.9 24.1 24.2 24.2 24.3 24.4 24.5 24.5 24.6 24.7 24.7 24.8 24.9 25.0 25.0 25.1
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
Napa Valley College Viticulture and Enology Department Standard Operating Procedure
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
Preparation of Buffer Solution Add 24ml of pure ethanol to 176ml of de-ionized water. Dissolve 0.5g of KHT into the solution. Adjust pH to 3.6 with HCl or NaOH. Procedure Measure the pH of the wine. Sample Preparation 1. Take 50ml of wine and filter through <0.45micron using a swinnex cartridge 2. Adjust pH to 3.6 by adding dropwise and mixing with either 1 M NaOH or 1 M HCl depending on the initial pH of the wine.. Use this wine on all the measurements below. 3. For each wine sample, set up five, 10mm cuvettes labeled as shown in the first column of the table below 4. Add the samples / solutions given in Table 1 and leave for the designated time period. Table 1 Cuvette # 1 2 Measurement Code Wine at pH 3.6 AHCl Contents Wine Wine HCl (Hydrochloric Acid) Wine 10% CH3CHO (Acetaldehyde) wine SO2 Wine Buffer Quantities Fill cuvette
20L
2ml
2ml
20L
AAcet
45mins
4 5
ASO2 A20
2ml
160L 100L 1900L
NA 5 min
5. Make a blank of de-ionized water in both a 10mm and a 1mm cuvette cuvette. 6. Run blank in the Spectrophotometer. Run blank every time you change cuvette size. 7. Following the instructions in the table below, transfer (if necessary) an aliquot of the prepared sample to the correct cuvette size (see column 3 in Table 2) and measure the absorbance of light at the wavelengths indicated in the fourth column of Table 2. 8. Use Table 3 to record your results.
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
Table 2 Cuvette # 1 2 3 4 5 Measurement Code Wine at pH 3.6 AHCl AAcet ASO2 A20 Cuvette size for Measurement 1mm 10mm 1mm 1mm 10mm Wavelengths of measurement 420 520 620 520 520 520 280 520 365 Number to multiply readings by: 10
101
10 10.8
20
Table 3 Cuvette # Measurement Code Wavelengths of measurement Actual Reading (Absorbance units): Number to multiply readings by: 10 10 10
101
True Value
1 1 1 2 3 4 5 5 5
Wine pH 3.6 Wine pH 3.6 Wine pH 3.6 AHCl AAcet ASO2 A20 A20 A20
10 10.8
20 20 20
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
Calculations 1. Use Tables 3 and 4 to calculate and record the spectral measures. Explanations of the Spectral Measures are given after the table. Table 4 Cuvette # and Wavelength 1 /420 1 / 520 1 / 620 Measurement Code A420 A520 A620 A420 + A520 A420/ A520 2 / 520 3 / 520 4 / 520 5 / 280 AHCl AAcet ASO2 A20280 - 4 Spectral measure Yellow / brown Red Blue Color Density Color Hue TA TC Polymerized Color (P) Total Phenols A+P FlavonePhenols Free Colored Anthocyanin s (A) Co-pigment Color (C) Degree of Coloration Value
5 / 520 5 / 365
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
Interpretation of the Results Assumptions Diluting the wine x 20 breaks down all the co-pigments to non colored anthocyanins and leaving behind colored, free anthocyanins and colored, polymerized anthocyanins Adding SO2 bleaches away free anthocyanins and co-pigments leaving behind the colored, polymerized anthocyanins Adding acetaldehyde reacts with SO2 preventing it from bleaching any color compounds and hence gives an estimate of the total color possible at a particular pH. Measures of Color Wine Color Density relates to the color intensity of the red pigments (A520) plus the yellow/brown pigments (A420): = (A420 + A520) Wine Color Hue relates to the color yellow/brown pigments (A420 ) divided by the red pigments (A520): = (A420/ A520) Brilliance of red is determined by the shape of the spectrum from 350nm 700nm. Wine is more brilliant when the peak of the curve is at 520nm. Free Colored Anthocyanins (A) at pH 3.6: A = A 20 ASO2 Co-Pigmented color (C) at pH 3.6 C=A acet - A 20 Polymerized Color (P) at pH 3.6: P = ASO2 Total Colored Anthocyanins at pH 3.6 gives an estimate of the concentration of free colored anthocyanins (A) plus co-pigmented, colored anthocyanins (C) plus polymerized, colored anthocyanins (P): (TC) = (A + C + P) = A acet Total Colored and Uncolored Anthocyanins gives an estimate of all the anthocyanins in the wine irrespective of their color: (TA) = AHCl Degree of Coloration of Anthocyanins gives the percentage of all the anthocyanins that are in the colored form in the natural wine: = (TC / TA) x 100 = (AAcet / AHCl) x 100 Measures of Phenols Total Phenolics measures the concentration of all phenolic material present in the wine. The subtraction of the (-4) in the formula allows for absorbance of non-phenolic materials: = A20280 4
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
Napa Valley College, Viticulture and Enology Department Standard Operating Procedure
Procedure
Set up and cleaning
1. Using the small plastic funnel connected to the bottom of the cash still by latex tubing, fill the boiling chamber with DI water so that the water level is approx. 1 above the heating coil. 2. Attach condenser tubing to the sink faucet and switch on cold water so that the flow is not too fast or too slow. 3. Empty any liquid in the sample (inner) chamber by turning Stopcock B to a horizontal position and Stopcock A to a vertical position, arrow up (see Photo 1). If the liquid will not flow out, slightly adjust the flow of water using the faucet. 4. After the sample chamber has emptied, rinse it by completely filling the glass sample delivery funnel above stopcock A. with DI water. Let it drain into the sample chamber and then evacuate. Repeat the process two more times.
Collecting the distillate
5. Turn Stopcock B to the vertical position and stopcock A to the vertical position, arrow up (Photo 2).
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
6. Pipette 10ml of wine into the sample chamber from the sample delivery funnel. 7. Add 0.5ml of 0.3% hydrogen peroxide using a Pasteur pipette. 8. Rinse the sample funnel with about 10ml distilled water using a squeezy bottle 9. Place a 250ml Erlenmeyer flask under the condenser outlet to collect the distillate 10. Switch on heater. 11. When water boils, let steam escape for ~10 seconds and then close stopcock A (horizontal position-Photo 3). 12. Collect 100ml of distillate. This should take ~7 minutes 13. SWITCH OFF HEATER. 14. Remove Erlenmeyer flask from below the condenser. 15. Evacuate the wine sample and rinse the sample chamber by repeating steps 3 and 4 above 16. Repeat step 5-14 for other wine samples Titrating the distillate 1. Add 2-3 drops of phenolphthalein to the distillate in the Erlenmeyer flask. 2. Titrate with NaOH to a pink endpoint that lasts 10-15 seconds. Calculation VA (g/L) = (mL NaOH) (N NaOH) (0.060) 1,000) mL wine VA (g/L) = mls NaOH x N NaOH x 6 VA is usually reported in g/100ml in the US, so the above figure needs to be divided by 10. Using 0.01N NaOH, Using 0.067N NaOH, VA (g/100ml) = mls NaOH x 0.006 VA (g/100ml) = mls NaOH x 0.0402
Cleaning the Still Weekly or as needed, mix ~10mls of 0.01N NaOH with 30mls of DI water. Put into the inner chamber and boil for 5 minutes. Evacuate the NaOH mixture then boil DI water for 2 minutes to flush the inner chamber.
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
Alternative Sulfur dioxide correction Governmentally regulated limits for the VA content of a wine are listed as exclusive of SO2. When the VA approaches legal limits (1.2 g/L for reds, 1.1 g/L for whites in California) it is necessary to correct for the contribution of SO2. 1. Immediately upon completion of the VA titration, cool the sample. 2. Add approximately 1 mL of starch indicator and 1 mL of 1 + 3 sulfuric acid. 3. Titrate with standardized iodine solution to a faint blue-green end point. 4. Calculate the free sulfurous acid equivalent according to the following equation: F.S.A.E. (g acetic acid / L Equivalent to SO2 present) = (mL I2) (N2) 32) (2) (60) (1,000) (1,000) (sample vol) (64)
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
Napa Valley College, Viticulture and Enology Department Standard Operating Procedure
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
Reagents and Equipment 5 % bentonite slurry (*see preparation instructions below) 150ml beaker Wine or juice samples 6 x 100ml-graduated cylinders several 1ml graduated wide-bore pipettes syringe with tubing on end 6 disposable 0.45um swinnex cartridges (or re-usable swinnex cartridge and 0.45um filters) 12 x 35ml nalgene centrifuge tubes 6 x 20ml test tubes 2 x 100ml beakers saran wrap marker pen test tube and centrifuge tube racks glass rod stir bar magnetic stirrer/hot plate pin-point light source (i.e. pen light)
*5% Bentonite slurry preparation: Weigh out 5 grams of bentonite. Measure 85ml of tap water in a graduated cylinder. Add the water to a 150ml beaker, place beaker on a hotplate/stirrer and heat up to 60 C. Place a stir bar in the beaker and adjust the stir speed to a medium rate. Slowly sprinkle the bentonite until all of the clay has rehydrated. Turn off the heat and allow to cool overnight while continuing to stir. The bentonite will swell considerably during this period. Add more tap water until the final volume of the slurry is 100ml and mix well. Procedure: Turn on hot water bath and set control about 6 to reach 80C. Sample Preparation 1. Label each of six 100ml graduated cylinders with the code # 1-6. 2. Mix the bentonite slurry thoroughly and add the appropriate quantity to each cylinder, using a graduated, wide-bore 1ml pipette, according to the following table: Cylinder # 1 2 3 4 5 6 mls of 5% Bentonite slurry 0 0.125 0.25 0.50 0.75 1.0 lbs.Bentonite/1000 gal 0 0.5 1 2 3 4
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
3. Add 100 ml of wine or juice to cylinder #1 , cap it immediately and immediately mix after each addition by inverting several times before proceeding to next cylinder. 4. Let cylinders settle for about 30 minutes, until solution starts to clear. Carefully decant about 35 ml from each cylinder into 6 individual 35ml centrifuge tubes, numbering each tube the same as the respective cylinders. 5. Place tubes in centrifuge and spin at 4000 rpm for 10 minutes. 6. Attach plastic tubing to the end of a 60ml syringe (in order to reach the bottom of the centrifuge tubes) and carefully suck up as much wine as possible from centrifuge tube #1. Then hold syringe vertically upward and suck the wine from the tubing into the body of the syringe. 7. Remove the tubing and attach a swinnex filter over the syringe and filter all the wine into a clean 35ml centrifuge tube, labeled according to the sample number. 8. Pour the filtered wine into a 20ml test tube (also labeled) as near to the top as possible. 9. Repeat this procedure for samples #2 through 6. Assess the protein stability of each trial sample utilizing the heat stability test (see below.) Heat Stability Test: 1. Cover each 20ml test tube with saran wrap and place tubes upright in 100ml beakers, 3 tubes per beaker. 2. Set beakers in the 80C water bath and leave for about six hours. 3. At the end of this period, remove test tubes from bath and allow cooling to room temperature. 4. Mix the contents and observe each sample for clarity and haze. The presence of a haze can be estimated by shining a strong pin-point light source diagonally through the sample. Any internal reflection indicates the presence of a haze. Results and Interpretations: The solution corresponding to the lowest fining level which produces no haze after the heat stability test is the bentonite addition rate at which the wine is considered heat stable. References: 1. Zoecklein, B.W., Fugelsang, K.C., Gump, B.H., Nury, F.S. 1995. Wine Analysis and Production. Chapman & Hall, New York. Illand, P., Ewart, A., Sitters, J. 1993. Techniques for Chemical Analysis and Stability Tests of Grape Juice and Wine. Patrick Iland Wine Promotions, Campbelltown, South Australia.
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
Napa Valley College, Viticulture and Enology Department Standard Operating Procedure
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
benefit of Sparkalloid is that it has minimal effects on color or flavor and will, also, make the wine easier to filter. Proteinaceaous Materials: Proteinaceaous materials include gelatins, casein, egg whites and isinglass. These agents all have a positive charge when they are in a solution with a pH typical of wine (3-4) and react with phenols via hydrogen bonding. They favor reacting with larger phenols, such as those that cause astringency, because they have more potential bonding sites. Gelatins: Gelatins are created from collagen obtained from animal bones and skins. They are often used to soften red wines and reduce the phenol and brown color level in white wines. There is a large risk of over-fining when using gelatin. Kieselsol or tannin is commonly added to wine with gelatins as co-fining agents to improve the flocculation/precipitation of the gelatin. Casein: Casein is the principle protein in milk. Casein is nearly insoluble and must be dissolved at pH 11. Potassium caseinate is water-soluble and is preferred for this reason. Casein is a positively charged protein that flocculates in acidic media such as wine and absorbs and removes suspended materials. Casein is used to remove undesirable odors and bitterness, to bleach color and to clarify white wines. It is sometimes used as a substitute for carbon in color modification of juice and white wine and often used to remove the cooked character from sherries. Egg Whites: Albumen is a common fining agent for red wines that is found in egg whites. Albumen is colloidal in nature and has a positive charge that attracts negatively charged tannins. Albumen removes less fruit character and fewer phenols than gelatin. Fresh eggs contain 3 to 4 grams of active product per white and are preferred over frozen egg whites Isinglass: Isinglass is a positively charged agent derived from the air bladder of a sturgeon. Its flocculated form is easier to work with than the sheet form because it does not have to be rinsed to get rid of fishy smells. Isinglass is used mainly in still white wine and sparking wines to improve aroma and clarity and modify the finish. Polymers: Polymer fining agents are high molecular weight (HMW), cross-linked polymers that react with phenols via hydrogen bonding. Their molecules are inflexible, which tends to make them more useful at removing smaller molecules. Polyvinyl polypyrolidone (PVPP) is the most common type of polymer that is used as a fining agent. It is a HMW fining agent that binds with phenolic and polyphenolic molecules in wine by absorption and also attracts low molecular weight catechins through hydrogen bonding. It is most often used to remove bitter compounds, in both red and white wines, and browning precursors. This is a very fast acting fining agent and no preparation is required to use it. After fining, the wines must be filtered to remove the PVPP and these wines may, in the end, taste more astringent after the bitter compounds are taken out. Polyclar: Polyclar is a highly absorbent proprietary PVPP product used to remove polyphenolic compounds and oxidized melanoidins that, when used in a finished wine, can help remove haze-causing proteins. It can also remove oxidized flavor and aroma compounds and reduce tannins. Excessive amounts can strip melanoidins (color and flavor compounds) from a wine. Carbon: Activated carbons are nonspecific adsorptive agents made from wood. The sponge like carbon binds with weakly polar molecules, especially those containing benzene rings. Carbon effectively removes phenolic compounds, especially small phenolic compounds. Compounds larger than dimers are too large to be adsorbed. Stripping of wine color and odor is often a problem with carbon because of the low selectivity and great care has to be taken with its use. Carbon also contains
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
a large quantity of air, and oxidation sometimes follows carbon addition if the carbon is not quickly and thoroughly removed. The addition of carbon to juice, rather than wine, helps to diminish carbon induced oxidation. Co-fining agents: Compounds like silica oxides, tannins and gelatin are sometimes used in addition to proteinaceaous fining agents to either improve their performance or, as more often the case, to facilitate the removal of the fining agents from the wine. Co-fining agents are used mainly in white juices and wine. Tannin is used with proteinaceous fining agents, especially gelatin in white wines, to aid in the flocculation and precipitation of the fining agent. Silica oxides and gelatin are sometimes used together in a 7:1 ratio where the silica oxide is used as an aid in clarification of white wines and as a substitute for tannin in facilitating the settling of solids. Silica oxide is usually not necessary when fining red wines due to the naturally high level of tannin that they possess. Proteinaceaous fining agents should be added first and fining trials must be done to assure proper settling. Kieselsol: Kieselsol is a proprietary name for aqueous suspensions of silicon dioxide. The primary use of Kieselsol is for clarification and as a replacement for tannin during gelatin fining of white wines because some winemakers do not want to add any tannin to their white wine. Kieselsols are negatively charged and electrostatically bind to and adsorb positively charged proteins and initiate flocculation and settling. Several different Kieselsol formulations are available at a variety of pH levels. Fining Trials: Fining trials are commonly conducted to determine which fining agent and concentration has the desired effect upon the wine that is to be treated with minimum impact on flavor and aromas. Small samples of the target wine are treated with different agents at different concentrations and then examined and compared for clarity, aroma and taste. The fining agents used are selected by the winemaker based upon the type of problem(s) being experienced with the wine and the known efficacy of the agent for the problem(s). When trying to determine what would be the best fining agent(s) to be used on a particular wine, the winemaker must keep in mind: The smallest quantity of agent necessary should be used. Use pure fining agents free of off-odors and flavors. Contact time should be limited to that necessary for complete reaction. Lab trials should simulate as closely as possible the parameters to be used in the cellar, i.e. the agents should be prepared by the same method, and the temperature of the wine should be the same. The fining agent must be completely dehydrated and mixed thoroughly into the sample. The wine should be low in dissolved CO2. Gas will impede settling of the agent. Lower pH wines require less fining agents than higher pH wines, due to charges. Young wines are more forgiving than older wines to the actions of the presence of greater numbers/concentration of polymerized compounds. Be aware of the levels of use approved by the ATF before additions to the wines in the cellar.
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
MATERIALS For each fining agent to be tested, the following items will be required Stock solution of fining agent If filtering Wine Sample 4 100ml cylinder Syringe with tubing on end Wine (600ml) Swinnex cartridge 4 wine glasses Filters ranging in size from 2.0m Plastic food wrap to 0.45m Centrifuge 250 ml beaker Centrifuge sample containers Pasteur pipette Labels (or masking tape) METHOD General Methodology: 1. Prepare a stock solution of the fining agent. Instructions for mixing different stock solutions are provided in the following section of this procedure. 2. Mix the stock solution well. 3. Using a pipette, add appropriate volumes of the stock solution to each measuring cylinder to create samples with a range of fining agents and concentrations. a. Group the cylinders for each fining agent together on the lab bench. b. For each fining agent group, the cylinder furthest to your left should be the control sample (0 PPM of the agent) then add the appropriate amount of the agent so that the cylinders to the right of the control are in increasing concentration. c. Fill the pipette to the 0 mark and release the required volume of fining agent into the cylinders. 4. Measure 100ml of wine into each of four (4) 100ml measuring cylinders for every fining agent tested. 5. Cover and invert the cylinder several times to thoroughly mix the fining agent with the wine. 6. Allow time for the fining agent to react, normally less than five minutes 7. Either cover the cylinders with plastic food wrap and allow precipitate to settle out over night, centrifuge for 10 minutes at 4,000 rpm, or filter the samples. 8. If you choose to centrifuge the samples: a. Carefully decant equal volumes of wine/fining agent from a single 100ml cylinder into two centrifuge sample containers. The volume of liquid must be identical in each container, if necessary use a Pasteur pipette to equalize the volumes. Repeat for all samples. b. Cap the centrifuge sample containers and add labels of the same size/weight to each container; clearly identifying its contents. c. Place the two cylinders for each sample in the centrifuge so that they are directly opposite one another to ensure proper balance when the samples are spun. d. Run the centrifuge at 4000rpm for 10 minutes. e. Remove all samples from the centrifuge. f. Decant the wine carefully into labeled wine glasses. 9. If you choose to filter, for each sample: a. Remove the wine from the cylinder using a syringe with tubing on the end, retaining the liquid in the body of the syringe. b. Attach a Swinnex cartridge with a 2m filter to the end of the syringe. c. Press the syringes plunger to push the wine through the filter into a 250ml beaker. d. Repeat the above steps with progressively finer filters finishing with a 0.45m filter.
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
e. Pour samples into labeled wine glasses. 10. If wines are settled overnight, the following morning decant the wine carefully into wine glasses. 11. Carry out a blind sensory evaluation of clarity, aroma and flavor. 12. If the smallest concentration of fining agent tried produces satisfactory clarity, repeat above process with smaller concentrations of the agent. 13. Determine the appropriate quantity of the fining agent (i.e. balance between adequate removal of the problem without excessive stripping of positive attributes including taste and aroma) 14. Compare the selected samples for each fining agent with each other to determine the one that has the best clarity with the least effect on the wines positive attributes. PREPARING STOCK SOLUTIONS OF FINING AGENTS The following table shows stock solutions and suggested concentration ranges for fining trials. These are guidelines and may be adjusted based upon what works with the tested wine.
Fining Agent Egg White (1%) Gelatin (1%) Isinglass (0.5%) PVPP (1%) Sparkalloid/Klearmor (1%) Potassium Caseinate (1%) Carbon (1%) Treatment Range (mg/liter) 60 600 Whites: 15-120 Reds: 30 300 Whites: 10-100 Reds: 30 150 30 - 240 100-1000 50-250 Odor: 15 60 Color: 100-200 Preparation Notes 1 egg white/59 gallon barrel is approximately 120 mg/L
Procedures for Specific Fining Agents Equipment 100 ml volumetric flask for each fining agent stock solution prepared 500 ml beaker (for egg white stock solution) Weigh scale Deionized water Magnetic stirrer with stirring bar Egg Whites 1. Determine concentrations of egg whites to be tested. The following table contains suggested concentrations for the first trial.
Egg White Trial (Stock Solution Concentration 1%) Final Volume (ml) PPM of Fining Agent Mls Stock Solution Required of Wine Being Assessed 100 0 0 60 0.6 120 1.2 240 2.4
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
2. First make a 10% solution as follows: Carefully separate one egg white into a 500ml beaker, weigh it, and add deionized water adjusted to pH 7 along with 0.5% potassium chloride (to help dissolve it). For example, a 30 gram egg white would require 270 ml deionized water with 15 g KCl. Stir gently until dissolved, being careful not to denature the albumin by stirring too vigorously. Finally, in dilute this to a 1% stock solution by mixing 10 ml of the mixture with 90 ml deionized water in a 100ml volumetric flask. The solution should be made fresh daily. Gelatin 1. Determine concentrations of gelatin to be tested. The following table contains suggested concentrations for the first trial.
Gelatin Trial (Stock Solution Concentration 1%) Final Volume (ml) PPM of Fining Agent Mls Stock Solution Required of Wine Being Assessed 100 0 0 30 0.3 60 0.6 120 1.2
2. To prepare a 1 % stock solution of gelatin, use a liquid gelatin prepared especially for winemaking. Prepare the solution by noting the manufacturers figure for gelatin activity (e.g. 25%) and diluting with the appropriate volume of deionized water. If using powdered gelatin, add 1 gram of powder made up to 100 ml with deionized water in a 100ml volumetric flask. Stir gently while heating (but do not exceed 40C, to avoid denaturing the proteins). This solution should be made fresh every few days. Conduct trials as described above, using the table for the first trial. Note that Kieselsol is often used in conjunction with the gelatin fining of white wines, to help precipitate the gelatin. Isinglass 1. Determine concentrations of isinglass to be tested. The following table contains suggested concentrations for the first trial.
Isinglass Trial (Stock Solution Concentration 0.5%) Final Volume (ml) PPM of Fining Agent Mls Stock Solution Required of Wine Being Assessed 100 0 0 15 0.3 30 0.6 60 1.2 2.
Isinglass is available in sheet or flocculated (powdered) form. The flocculated form is by far the easiest to work with. It is first made into a 0.5% stock solution by adding 0.5 grams to 100ml cold deionized water and stirring gently until completely dispersed.
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
PVPP/ (Polyvinylpolypyrrolidone) 1. Determine concentrations of PVPP to be tested. The following table contains suggested concentrations for the first trial.
PVPP Trial (Stock Solution Concentration 1%) Final Volume (ml) PPM of Fining Agent Mls Stock Solution Required of Wine Being Assessed 100 0 0 30 0.3 60 0.6 120 1.2
2. To make a 1% stock solution of PVPP, add 1 gram of PVPP made up to 100ml with deionized water in a 100ml volumetric flask. Stir to form thoroughly mixed slurry. Sparkalloid/Klearmor 1. Determine concentrations of Sparkalloid/Klearmor to be tested. The following table contains suggested concentrations for the first trial.
Sparkolloid / Klear-mor Trial (Stock Solution Concentration 1%) Final Volume (ml) PPM of Fining Agent Mls Stock Solution Required of Wine Being Assessed 100 0 0 60 0.6 120 1.2 240 2.4
2. To make a 1% stock solution of Sparkalloid or Klearmor, stir 1 gram of powder into 100ml of boiling water, and continue boiling and stirring for 20 minutes. The trials are conducted with the hot solution, without allowing it to cool. Potassium Caseinate 1. Determine concentrations of Potassium Caseinate to be tested. The following table contains suggested concentrations for the first trial.
Potassium Caseinate Trial (Stock Solution Concentration 1%) Final Volume (ml) PPM of Fining Agent Mls Stock Solution Required of Wine Being Assessed 100 0 0 20 0.2 40 0.4 80 0.8
2. Potassium caseinate is soluble in water. To make a 1% stock solution of potassium caseinate, stir 1 gram of potassium caseinate in 100ml of deionized water. Warm the solution but do not exceed 40C. Stirring may be required for several hours (up to 24) to completely mix the powder into solution. This solution should be used within a day or two.
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
If only casein is available, the preparation needs to be alkaline. Stir 1 gram of casein in 100ml of deionized water, which has been adjusted to about pH 8 by the addition of potassium carbonate. Carbon 1. Determine concentrations of carbon to be tested. The following table contains suggested concentrations for the first trial.
Carbon Trial (Stock Solution Concentration 1%) Final Volume (ml) PPM of Fining Agent of Wine Being Assessed 100 0 30 60 120 Mls Stock Solution Required 0 0.3 0.6 1.2
2. To make a 1% stock solution of carbon, add 1 gram of carbon made up to 100ml with deionized water in a 100ml volumetric flask. Stir to prepare thoroughly mixed slurry. Co-fining Agents 1. The following table shows suggested concentrations and treatment ranges for co-fining agents used with proteinaceous fining agents.
Co-Fining Agent Tannin (1%) Gelatin Kieselsol (15 or 30%) Treatment Range 40 200 mg/L 40-200 mg/L 40-300 mg/L
2. To make a 1% stock solution of tannin, add 1 gram of tannin made up to 100ml with deionized water in a 100ml volumetric flask and mix. The solution should be prepared fresh. 3. Kieselsol in normally supplied as a 15-30% SiO2 solution. Calculating the Required Addition of Selected Fining Agent to Wine General formula for calculating required volume:
Formula Example: Volume of Required rate Volume of wine (ml) stock solution = of addition X Conc. of stock solution of fining agent (ml) (mg/L) (mg/L) The volume (ml) of a 0.4% stock solution that must be added to a 100ml sample of wine to achieve a desired concentration 200 mg/L of the tested fining agent is: (Note: 0.4% solution = 0.4 grams in 100 ml = 4,000 milligrams in 1 liter) = 200 mg/L X (100 ml 4000 mg/L) = 5 ml
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
Napa Valley College, Viticulture and Enology Department Standard Operating Procedure
Tartrate Stability
Developed by: Barry Jan & Rick Smith Date: December 1997 Introduction Wine is a supersaturated solution of potassium bitartrate (KHT). Therefore, under certain conditions, especially at low temperatures, crystals of KHT will precipitate out. While having these crystals on the bottom of the bottled wines might not be objectionable for home winemakers, it is probably not desirable for commercial wines because of consumer resistance. In the case of sparkling wines, it is imperative that no crystals be formed because it is at these points where carbon dioxide will be released when the bottle is opened. This increased amount of released carbon dioxide will cause gushing of the wine out of the bottle, which is highly undesirable. The cold stability of the wine, based on the non-formation of crystals under normal storage conditions, can be determined by the five methods below:
1.
2.
3.
4. 5.
Conductivity Test. Roger Boulton of UC Davis designed this test. The test measures the electrical conductivity of a wine sample before and after the addition of KHT. A change of less than 5% during the test indicates a stable wine. This test is probably the most practical one for small wineries in terms of equipment and accuracy. A photograph of the equipment is shown in Appendix 1. Concentration Product Test. This test takes into account the concentration of the potassium, calcium, total tartrates, pH and alcohol. The potassium and calcium contents are determined by either Atomic Absorption Spectrophotometry or Flame Photometry. The estimated tartrates are obtained on a table based on the pH and alcohol of the wine. All these values are plugged into a formula to determine the CP and compared to the value which is considered stable for that particular wine type. Metavanadate Spectrometric Analysis. The metavanadate method can be used to determine the amount of tartaric acid. This method involves a colorimetric reaction between sodium (or ammonium) metavanadate and tartaric acid in acetic acid solution. One system uses activated carbon to decolorize the wine samples prior to the reaction with metavanadate. Another system is to subject the wine sample to an acetate-charged resin. Both systems use highperformance liquid chromatographic technique (HPLC) to determine results. Refrigerator Test. This test subjects a wine sample to 2 to 3C for 4 days. Absence of crystal indicates stability. Freezer Test. This test is similar to the Refrigerator Test except that temperature and time are changed to 10 to 20C and overnight, respectively.
The conductivity test involves the use of a cooling bath and an electrical conductivity meter. It is recommended for wineries because of its simplicity and reliability. The Concentration Product and Metavanadate Tests appear to be more accurate than the other three tests; however, they require more expensive equipment such as Atomic Absorption, spectrograph, and HPLC. The Refrigerator and Freezer Tests are simple tests and the results are rather subjective. They depend on the opinion of the tester on what is considered to be acceptable for a particular storage condition. These two tests should
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
be limited to home winemakers or small wineries where availability of equipment might be a problem. The procedures for running them are given in this document. Preparation and Interferences Finely ground KHT must be used in these tests. If the crystals are coarse, they can be prepared by grinding the KHT in a mortar and pestle. Some wineries run the stability tests without seeding the wine with finely powdered KHT; however, the seeds provide nuclei for immediate crystal growth. The size and growth of the crystal should be more pronounced by seeding. Procedure: Refrigerator Test 1. Place approximately 90 mL of a clear (but not filtered) sample of wine separately into each of two 100 mL clear glass screw cap bottles. 2. Add about 5mg of finely ground KHT to each bottle. Store one of the bottles in a refrigerator, set at 2 to 3C, for 4 days and leave the other bottle at room temperature. 3. Observe both bottles for the presence of crystals. Stability is indicated if the refrigerated sample does not show an appreciable increase in the amount of crystals compared to the control. Procedure: Freezer Test 1. Place approximately 90 mL of a clear (but not filtered) sample of wine separately into each of two 100 mL clear glass screw cap bottles. 2. Add about 5mg of finely ground KHT to each bottle. Store one of the bottles in the freezer compartment of a refrigerator, set at 10 to 20C, overnight and leave the other bottle at room temperature. 3. Remove the sample from the freezer and allow ice crystals to thaw. KHT crystals will not thaw or dissolve back into solution during the short period of this test. 4. Observe both bottles for the presence of crystals. Stability is indicated if the thawed sample does not show an appreciable increase in the amount of KHT crystals compared to the control. Calculations and Units of Reporting Report any increase of KHT crystals over that of the control sample.
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
Napa Valley College, Viticulture and Enology Department Standard Operating Procedure
VWT 172: Laboratory Analysis of Musts and Wines Standard Operating Procedures
Calibration 1. Saturate the sponge in the calibration sleeve and gently insert the probe as far as it will go. 2. Switch on the meter. 3. Depress and hold the Mode Key Pad until the display cursor is at Cal. As long as the Mode Key Pad is depressed, the display will cycle continuously between Cal, %, mg/L, and degrees C. 4. Depress quickly and release the Mode Key Pad. The display will show three dashed (---) followed by the slope of the membrane/electrode (good slope is 0.7 to 1.2). 5. Remove the Calibration sleeve. The probe is now ready to use. Measurement 1. Depress the Mode Key Pad to choose mg/L or %. 2. Immerse the probe in sample, making sure the stainless steel thermostat is submerged. Stir the sample or slowly move the probe through the sample. Take a reading when the value is stable.