Residual Sugar Assay Manual
Contents
1. 4 of assay Hope 5 25 100 if over 100 g L Volume of pH d 0 20 mL wine sample to aaa 1 0 mL 0 40 mL take 0 10 mL Volume of diluent 9 8 mL to add ae 4 0 mL 9 6 mL 9 9 mL Wine type designations and corresponding residual sugar values are taken from EU regulation 753 2002 Residual Sugar Assay Manual 8 version 32. NEGLIGENCE STRICT LIABILITY OR OTHER TORT WITH RESPECT TO THE MATERIALS OR ANY SERVICES IN CONNECTION WITH THE MATERIALS IS LIMITED TO AN AMOUNT NOT TO EXCEED THE PRICE OF THE MATERIALS IN NO EVENT SHALL VINMETRICA BE LIABLE TO BUYER FOR ANY INCIDENTAL CONSEQUENTIAL OR SPECIAL DAMAGES INCLUDING WITHOUT LIMITATION LOST REVENUES AND PROFITS HAZARDS AND TOXICITY All Materials offered by Vinmetrica are intended for use by individuals who are familiar with laboratory procedures and their potential hazards The Materials contain chemicals which may be harmful if misused Due care should be exercised with all Materials to prevent direct human contact Glassware can break and chemicals can splash during experiments always use safety glasses We strongly recommend using nitrile or latex gloves and wearing long pants long sleeves and closed toed shoes Keep out of reach of children Vumetrica 1945 Camino Vida Roble Suite I Carlsbad CA 92008 www vinmetrica com 760 494 0597 info vinmetrica com Technical assistance info vinmetrica com tel 760 494 0597 Copyright 2014 15 Sportsman Consulting LLC DBA Vinmetrica All rights reserved Residual Sugar Assay Manual 6 version 3 Appendix A Measuring sucrose Although sucrose is not normally present in unsweetened grape musts and wines it is sometimes added for various reasons You can measure sucrose as a combined value with all fermentable sugars or as a separate value from other s
3. Number RS 10 and follow one of the two possible sucrose methods in the Procedure see steps 12 15 and Appendix A e Plastic vials may be cleaned with DI water after completion of assay and reused Residual Sugar Assay Manual 2 version 3 Setup 1 To prepare for this assay you will need to have your pH meter probe calibrated and ready for measurement We recommend that you follow your meter s procedure to calibrate at pH 7 00 and pH 4 01 Take all reagents out of cold storage and allow them to come to room temperature Prepare Reaction Buffer NOTE skip this step if your Reaction Buffer came in the form of a liquid Remove the crimp seal and rubber cap from the bottle of Reaction Buffer Powder With a pipette or other accurate measuring device dispense exactly 25 mL of DI water into the bottle this amount fills the bottle nearly to level of the neck Replace the rubber septum and swirl gently several times until all solids are dissolved to give a clear orange tinted solution Note any unused portion of this solution may be stored up to 1 month in the freezer but it is im portant to re freeze it as soon as possible after use in steps 6 through 9 below Procedures 1 With the 5 mL serological pipette measure exactly 3 0 mL of wine that is to be tested and place this in the 100 mL plastic beaker Rinse the inside and outside of the 5 mL pipette with dH2O into a waste container and drain thoroughly Then use the pipette to m
4. rinsing Rinse bottle available from Vinmetrica Part number SC 100 17 Optional Invertase Powder Vials for measuring sucrose levels Available from Vinmetrica Part Number RS 10 Optional Additional plastic vials Available from Vinmetrica Part Number RS 8 Optional 25 mL Sampling Pipette for reconstituting Reaction Buffer powder Part number SC 100 4 Residual Sugar Assay Manual 1 version 3 Why Test for Residual Sugar Residual sugar RS refers to any significant concentration of sugar that is contained in wine at the end of fermentation Winemakers are typically most interested in knowing the concentration of the fermentable hexoses glucose and fructose in some cases sucrose may be present as well These determine the level of sweetness of the wine see Appendix B for more information on sweetness characteristics At residual sugar levels above around 2 g L 0 2 your wine can spontaneously restart fermentation unless it has been properly stabilized How it works The enzymes present in Vinmetrica s Enzymatic Powder convert residual sugars in wine into their 6 phosphate forms This reaction gives off a hydrogen ion thereby lowering the pH of the test solution Therefore the more residual sugar in the wine the greater the drop in pH By measuring the drop in pH of your wine sample and comparing that value to a known glucose standard you can reliably calculate a residual sugar value for your wine If you want to measur
5. Technical assistance info vinmetrica com tel 760 494 0597 Residual Sugar Assay Manual 5 version 3 WARRANTIES AND LIABILITIES l The materials provided in the kit as described on pages 1 and 2 above Materials are warranted as follows All reagents powders and non reagent accessories are warranted against defects in workmanship for 6 months from date of purchase The reagents are warranted to perform as described herein up until any stated expiration date or 6 months after purchase whichever is later provided storage recommendations are followed THE WARRANTIES IN THESE TERMS AND CONDITIONS ARE IN LIEU OF ALL OTHER WARRANTIES EXPRESS OR IMPLIED INCLUDING WITHOUT LIMITATION ANY WARRANTIES OF MERCHANTABILITY NONINFRINGEMENT OR FITNESS FOR A PARTICULAR PURPOSE SAID WARRANTIES BEING EXPRESSLY DISCLAIMED 2 Buyer agrees that its sole and exclusive remedy against Vinmetrica shall be limited to the repair and replacement of Materials or parts of Materials provided Vinmetrica is promptly notified in writing prior to the expiration of the warranty period specified above of any defect Vinmetrica s liability for any damages due Buyer shall be limited to the purchase price of the Materials 3 VINMETRICA S MAXIMUM LIABILITY FOR ALL DIRECT DAMAGES INCLUDING WITHOUT LIMITATION CONTRACT DAMAGES AND DAMAGES FOR INJURIES TO PERSONS OR PROPERTY WHETHER ARISING FROM VINMETRICA S BREACH OF THESE TERMS AND CONDITIONS BREACH OF WARRANTY
6. a Vinmetrica Residual Sugar Assay User Manual Oinmetnica The Vinmetrica Residual Sugar Assay Kit provides a simple accurate and affordable way to determine Residual Sugar glucose fructose concentrations in wine and other samples Materials provided in the kit s ar ey Buffer Solution Keep in Refrigerator Reaction Buffer Solution or Powder Keep in Freezer IN NaOH Store at room temperature Standard Solution Keep in Freezer Diluent Solution Keep in Refrigerator Enzymatic Powder Keep in Freezer Things you will need Figure 1 The Residual Sugar Reagent Kit 1 Vinmetrica Residual Sugar Assay Plastics Kit Available from Vinmetrica Part Number RS 2 It is highly recommended that the Plastics Kit be purchased with your first Residual Sugar Reagent Kit The plastics only need to be purchased once and replaced if necessary The Plastics Kit contains the following Measuring scoop for Enzymatic powder one 1 mL serological pipette one 10 mL serological pipette one 5 mL serological pipette one plastic transfer pipette one 100 mL plastic beaker and twelve plastic vials Vinmetrica SC 200 or SC 300 with pH electrode or comparable pH meter 0 01 pH resolution required Vinmetrica pH 4 01 and pH 7 00 Reference Solutions or equivalent solutions for pH calibration Distilled water DI water which usually can be found at your local grocery store It s handy to have a wash bottle for
7. e sucrose you can add Invertase Powder available separately Part Number RS 10 that will enable this measurement Assay Notes e The Reaction Buffer is usually provided as a freeze dried powder that must be dissolved in 25 mL of water see Setup section below In some cases the reaction buffer has been provided in liquid form ready to use e Vinmetrica s residual sugar assay has an incubation period of 20 minutes with approximately 10 20 minutes of sample preparation time e Most of the reagents of the kit must be stored in either a freezer 5 to 20C or refrigerator 4C please take note to store reagents properly e tis necessary to run one Residual Sugar Assay Standard and one Blank each time you use the kit whether for one sample or multiple samples e The kit provides enough Enzymatic Powder for over 24 tests This allows you to run up to 22 tests with at least one Residual Sugar Standard and one Blank Note that you must always run a blank and a standard each time you assay one or more samples With the 12 plastic vials pro vided you may run up to 10 tests at one time with 1 Standard and 1 Blank If you would like to run all 22 tests at once additional plastic reaction bottles will be needed Additional bottles are available Part Number RS 8 e Most wines made without added sucrose will not have measurable levels of this sugar in them If you want to measure sucrose in a sample obtain the Invertase Powder Vinmetrica Part
8. easure 3 0 mL of Buffer solution into the 100 mL beaker with wine sample This is your wine solution Note You may want to remove the cotton plug in the end of pipette for ease of rinsing Put your pH probe be sure it s calibrated in the wine solution and begin swirling or mixing Read pH and with plastic bulb pipette carefully add 1 N NaOH into the wine solution one drop at a time Continue to add 1 N NaOH until the pH of the wine solution is 8 20 0 03 i e 8 17 to 8 23 As you approach pH 7 5 it is important to only add one drop at a time as you may overshoot past 8 25 If you do go past 8 25 you may use the Acid Solution small drops that is provided as part of the SO gt kit to drop the pH back to 8 2 if you find it hard to hit pH 8 2 with the IN NaOH simply dilute the NaOH 5 to 10 fold with DI water and use this once you reach pH 7 5 If your wine sample is a dry wine proceed to step 5 If your wine is expected to be medium dry medium or sweet you may need to further dilute the wine sample as in step 4 See Appendix B for guidance From the wine solution that was just pH d to 8 2 use the 1 mL serological pipette to take ex actly the amount indicated in Appendix B into a second 100 mL beaker or other container With a 10 mL serological pipette add the exact amount of Diluent Appendix B to second container Mix well Reminder This step is only required if assaying a medium dry medium or sweet wine With the wine solutio
9. le includ ing standard and blank Try to recover all the liquid off of the pH probe back into the sample bottle by gently flicking the probe a few times above the liquid level Remove then rinse the pH electrode thoroughly with DI water between each sample and remove excess water from the electrode tip by gently shaking or blotting it away With the initial pH recorded you are now ready to begin your assay Add 20 mg of Enzymatic Powder to each vial This is the amount delivered by a level fill of the measuring scoop provid ed in the Plastics kit see Figure 2 Immediately swirl the vial for 5 seconds to get good mixing Repeat this step for each sample including Standard and Blank If you are measuring sucrose as a combined value add one scoop of invertase powder now See Appendix A Figure 2 Measuring scoop with level fill about 20 mg of Enzymatic Powder Allow vials to sit at room temperature for 20 minutes agitate gently at least once over this time At 20 minutes measure pH of sample and record the value as in step 11 Repeat this step for all samples including the Standard and the Blank The Standard and any wine samples containing residual sugar will see a drop in pH Skip this step if you are not measuring sucrose as a separate value see Appendix A If you are measuring sucrose as a separate value add invertase powder now mix well and allow to react 60 minutes Meanwhile proceed with the calculations in step 16 18 below fo
10. n at pH 8 2 and further diluted if necessary use the 1 mL serological pi pette to add 0 5 mL of diluted wine sample to a clean plastic vial Rinse 1 mL serological pipette with dH2O into a waste container drain thoroughly then meas ure exactly 1 0 mL of Reaction Buffer and add to the plastic vial Residual Sugar Assay Manual 3 version 3 10 11 12 13 14 15 Repeat steps 2 6 for all wine samples We recommend that you label each individual wine sam ple To prepare the Standard use the clean 1 mL serological pipette and measure exactly 0 50 mL of Residual Sugar Standard add this to a clean plastic vial Rinse and drain the pipette thoroughly then add exactly 1 0 mL of Reaction Buffer to this vial Label this bottle with Standard For the blank measure exactly 0 50 mL of Diluent and add this to a clean plastic vial Rinse and drain the pipette thoroughly then add exactly 1 0 mL of Reaction Buffer to this bottle La bel this bottle Blank You should now have at least three 3 plastic vials a blank a standard and at least one sample You may have more if you are running more wine samples With all vials lined up and labeled measure and record the initial pH Note Electrode will not be completely submerged in solu tion By gently swirling solution with electrode contacting it you should be able to obtain a stable pH value This value should be between 8 10 and 8 30 Record this measurement for each samp
11. r the non sucrose Residual Sugar Assay Manual 4 version 3 16 17 18 sugars At the end of this second 60 minute period take additional pH readings as in step 14 Refer to Appendix A to calculate the separate value for sucrose Now use the values you recorded and subtract them from each other For example Initial pH 8 27 60 min pH 7 88 A 8 27 7 88 0 39 Repeat this subtraction step for all samples including standard and blank Call these values Aws wine sample Asrp standard and Ag blank Plug these values into this equation residual sugar g L 2AC1 25 Aws Ag Astp Ap This gives your residual sugar concentration in g L For example if Aws 0 11 Astrp 0 39 Ag 0 07 then residual sugar 2 1 25 0 11 0 07 0 39 0 07 0 3 g L If you further diluted your wine sample as in step 4 simply multiply your answer by the dilu tion factor In the example above if you had diluted the sample by a dilution factor of 100 your concentration of residual sugar would be 30 g L Typically in sweet wines the residual sugar ranges between 50 150 g L this was used for example only Finishing Up Turn off your pH meter and rinse and store the electrode properly If you have unused solutions or Enzymatic Powder vials store back immediately as directed on bottle Especially be sure to place any unused Reaction Buffer in the freezer as soon as possible Rinse all plastic ware with DI water
12. ugars Adding the Invertase Powder available separately Vinmetrica Part Number RS 10 causes sucrose which is not active in the assay to be hydrolyzed into its fructose and glucose components which are active Combined value you will add invertase powder 10 mg or one full rounded scoop see Figure 2 above along with the Enzyme powder at step 12 above Proceed with the rest of the assay in the usual way skip step 15 of course Calculation follow steps 16 18 as usual this produces a value that represents sucrose glucose and fructose combined Separate value you will add invertase powder 10 mg or one full rounded scoop see Figure 2 above along with the Enzyme powder at step 15 above Calculation Proceed with the calculation of residual sugar as in steps 16 18 using the initial pH values for the original sample and the final pH value after the second Invertase 60 minute period This will produce a value for combined sugars Subtract the value for residual non sucrose sugar calculated at the first 60 minute time point from the value for combined sugars just done above This represents the separate value for sucrose content Residual Sugar Assay Manual 7 version 3 Appendix B Table of wine sweetness suggested dilution factors and volumes to pipette see step 4 of the Procedures Wine type Dry Medium Dry Medium Sweet Residual Sugar up to 4 g l up to 12 g l up to 45 g l over 45 g l Dilution Factor 50 step
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