Residual Sugar Assay
Contents
1. 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 Buffer Solution Keep in Refrigerator Reaction Buffer Keep in Freezer IN NaOH 2 is etn Standard Solution Keep in Refrigerator Diluent Solution Keep in Refrigerator Duk wd 24 Enzymatic Powder Vials Keep in Freezer ef Things you will need 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 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 8 mL plastic bottles 2 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 2 Distilled water which usually can be found at your local grocery store It s handy to have a wash bottle for rinsing Rinse bottle available from Vinmetrica Part number SC 100 17 3 Optional Invertase Powder Via2. 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 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
3. 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 Viametrica 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 2010 14 Sportsman Consulting LLC DBA Vinmetrica All rights reserved Residual Sugar Assay Manual 6 version 1 0 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 sugars 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 a
4. alues are taken from EU regulation 753 2002 Residual Sugar Assay Manual 9 version 1 0
5. es The kit provides 24 microtubes with Enzymatic Powder This allows you to run up to 22 tests with 2 extra microtubes for the Residual Sugar Standard and the Blank Note that you must al ways run a blank and a standard each time you assay one or more samples With the 12 bottles provided 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 need to be purchased Ad ditional bottles are available Part Number RS 8 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 Num ber RS 10 and follow one of the two possible sucrose methods in the Procedure see steps 12 15 and Appendix A Plastic bottles may be cleaned with DI water after completion of assay and reused Residual Sugar Assay Manual 2 version 1 0 Setup l 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 Reaction buffer may be placed in room temperature water bath Procedures l With the 5 mL serological pipette measure exactly 3 0 mL of wine that is to be tested and place in the 100 mL plastic beaker Rinse the i
6. in a stable pH value This value should be between 8 00 and 8 30 Record this measurement for each sample 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 Take one microtube that contains the Enzymatic Powder and add its entire contents to one of the 8 mL bottles then gen tly swirl bottle with contents Repeat this step for each sample including Standard and Blank If you are measuring sucrose as a combined value add invertase powder now See Appendix A Allow samples to sit at room temperature for 60 minutes agitate gently at least once over this time At 60 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 for the non sucrose sugars At the end of this second 60 minute
7. ls for measuring sucrose levels Available from Vinmetrica Part Number RS 10 4 Optional Additional 8 mL plastic bottles Available from Vinmetrica Part Number RS 8 Residual Sugar Assay Manual 1 version 1 0 Why Test for Residual Sugar Residual sugar refers to any significant concentration of sugar that is contained in wine Winemakers are typically most interested in knowing the concentration of the fermentable hexoses glucose and fructose The enzymes present in Vinmetrica s Enzymatic Powder convert residual sugar in wine into glucose 6 phosphate 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 and comparing that value to a known sugar standard you can reliably calculate residual sugar of your wine sample If you want to measure sucrose you can add Invertase Powder available separately Part Number RS 10 that will enable this measurement Assay Notes Vinmetrica s residual sugar assay has an incubation period of 60 minutes with approximately 10 20 minutes of sample preparation time 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 It is necessary to run one Residual Sugar Assay Standard and one Blank each time you use the kit whether for one sample or multiple sampl
8. nd glucose components which are active Combined value you will add invertase powder 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 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 1 0 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 4 of assay none 5 25 100 if over 100 g L Volume of pH d 0 20 mL wine sample to 1 0 mL 0 40 mL take 0 10 mL Volume of diluent 9 8 mL to add 4 0 mL 9 6 mL 9 9 mL Appendix B Table of wine sweetness suggested dilution factors and volumes to pipette see step 4 of the Procedures Residual Sugar Assay Manual 8 version 1 0 Wine type designations and corresponding residual sugar v
9. nside and outside of the 5 mL pipette with dH20 into a waste container and drain thoroughly Then use the pipette to measure 3 0 mL of Buffer solu tion 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 2 0 1 i e 8 10 to 8 30 As you approach pH 7 5 it is important to only add one drop at a time as you may over shoot past 8 3 If you do go past 8 3 you may use the acid solution that is provided as part of the SO kit to drop pH back to 8 2 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 using the 1 mL serological pipette 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 You may have to use the 5 mL pipette in the kit twice e g to add 9 6 mL you can add exactly 5 0 mL Diluent then an additional 4 6 mL Mix well Reminder This s
10. 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 Asto standard and As blank Plug these values into this equation residual sugar g L 2 1 25 Aws As Astp Ap Residual Sugar Assay Manual 4 version 1 0 This gives your residual sugar concentration in g L For example if Aws 0 11 Astp 0 39 Ag 0 07 then residual sugar 2 1 25 0 11 0 07 0 39 0 07 0 3 g L 18 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 1 1 Turn off your pH meter and store the electrode properly 1 2 If you have unused solutions or Enzymatic Powder vials store back immediately as directed on bottle 1 3 Rinse all plastic ware with DI water Technical assistance info vinmetrica com tel 760 494 0597 Residual Sugar Assay Manual 5 version 1 0 WARRANTIES AND LIABILITIES l The materials provided in the kit as
11. tep is only required if as saying a medium dry medium or sweet wine With the wine solution at pH 8 2 and further diluted if necessary use the 1 mL serological pipette to add 0 5 mL of diluted wine sample to a clean 8 mL bottle Rinse 1 mL serological pipette with dH2O into a waste container drain thoroughly then mea sure exactly 1 0 mL of Reaction Mix and add to the 8 mL bottle 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 and add this to a clean 8 mL bottle Rinse and drain the pipette thor Residual Sugar Assay Manual 3 version 1 0 10 11 12 13 14 15 16 17 oughly then add exactly 1 0 mL of Reaction Mix to this bottle Label this bottle with Stan dard For the blank measure exactly 0 50 mL of Diluent and add this to a clean 8 mL bottle Rinse and drain the pipette thoroughly then add exactly 1 0 mL of Reaction Mix to this bottle Label this bottle Blank You should now have at least three 3 8 mL bottles a blank a standard and at least one sam ple You may have more if you are running more wine samples With all bottles lined up and labeled measure and record the initial pH Note Electrode will not be completely submerged in solution By gently swirling solution with electrode contacting it you should be able to obta
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