Food Analysis Laboratory Manual

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● Chapter 1 Nutrition Labeling Using a Computer Program 3 INTRODUCTION Notes Instructions on how to receive and install the software used Background for this laboratory are located online at On the left hand side of the web page, click on the Food The 1990 Nutrition Labeling and Education Act man- Analysis Students link located under the services heading. dated nutritional labeling of most foods. As a result, a It is possible that the TechWizard™ program has been updated large portion of food analysis is performed for nutri- since the publication of this laboratory manual and any changes tional labeling purposes. A food labeling guide and in the procedures described below will also be found on this links to the complete nutritional labeling regulations web page. are available online at Install the software prior to the laboratory session to ensure flg-toc.html. However, interpretation of these regulations that it works properly with your PC. and the appropriate usage of rounding rules, available nutrient content claims, reference amounts, and serving size can be difficult. METHOD A: PREPARING NUTRITION LABELS Additionally, during the product development FOR SAMPLE YOGURT FORMULAS process, the effect of formulation changes on the nutri- tional label may be important. As an example, a small Procedure change in the amount of an ingredient may determine 1. Start the TechWizard™ program. Enter the if a product can be labeled low fat. As a result, the abil- ity to immediately approximate how a formulation Nutrition Labeling section of the program. (From the Labeling menu, select Labeling Section.) change will impact the nutritional label can be valu- able. In some cases, the opposite situation may occur 2. Enter the ingredients for formula 1 listed in Table 1-1. (Click on the Add Ingredients button, and a concept called reverse engineering is used. In reverse engineering, the information from the nutri- then select each ingredient from the ingredient list window and click on the Add button, click on the X tional label is used to determine a formula for the product. Caution must be used during reverse engi- to close the window after all ingredients have been added.) neering. In most cases, only an approximate formula can be obtained and additional information not pro- 3. Enter the percentage of each ingredient for for- mula 1 in the % (wt/wt) column. Selecting vided by the nutritional label may be necessary. The use of nutrient databases and computer pro- the Sort button above that column will sort the ingredients by the % (wt/wt) in the formula. grams designed for preparing and analyzing nutri- tional labels can be valuable in all of the situations 4. Enter the serving size (common household unit and the equivalent metric quantity) and number described earlier. In this laboratory, you will use a computer program to prepare a nutritional label from of servings. (First, click on the Serving Size button under Common Household unit, enter 8 in the window, a product formula, determine how changes in the for- mula affect the nutritional label, and observe an exam- click on OK, select oz from the units drop down list; ple of reverse engineering. next, click on the Serving Size button under Equiva- lent Metric Quantity, enter 227 in the window, click on Reading Assignment OK, select g from the units drop down list; and finally click on the Number of Servings button, enter 1 in the Metzger, L.E. 2010. Nutrition labeling. Ch. 3, in Food Analysis, window, click on OK.) 4th ed. S.S. Nielsen (Ed.), Springer, New York. Owl Software. 2009. TechWizard™ Version 4 Manual, Columbia, MO. 1-1 table Sample Yogurt Formulas Objective Formula 1 (%) Formula 2 (%) Prepare a nutritional label for a yogurt formula, determine how formulation changes will affect the Milk (3.7% fat) 38.201 48.201 nutritional label, and observe an example of reverse Skim milk no Vit A add 35.706 25.706 engineering. Condensed skim milk 12.888 12.888 (35% total solids) Materials Sweetener, sugar liquid 11.905 11.905 Modified starch 0.800 0.800 TechWizard™ Version 4 – Formulation and Nutrition Stabilizer, gelatin 0.500 0.500 Labeling Software for Office 2007● 4 Chapter 1 Nutrition Labeling Using a Computer Program Note by clicking on the Show Ref. Table METHOD B: ADDING NEW INGREDIENTS TO button, a summary of the CFR 101.12 Table 2 A FORMULA AND DETERMINING HOW THEY Reference Amounts Customarily Consumed INFLUENCE THE NUTRITION LABEL Per Eating Occasion will be displayed. 5. Enter a name and save formula 1. (Click on the Sometimes, it may be necessary to add additional ingredients to a formula. As an example, let us say, Formula Name window, enter “food analysis for- mula 1” in the top Formula Name window, click you decided to add an additional source of calcium to yogurt formula 1. After contacting several suppliers, OK and click on the X to close the window. From the File menu, select Save Formula.) you decided to add Fieldgate Natural Dairy Calcium 1000, a calcium phosphate product produced by First 6. View the nutrition label and select label options. (Click on the View Label button, click on the Label District Association (Litchfield, MN), to the yogurt for- mula. This product is a natural dairy-based whey min- Options button, select the label type you want to dis- play – the standard, tabular, linear or simplified eral concentrate and contains 25% calcium. You want to determine how much Dairy Calcium 1000 you need format can be displayed; select the voluntary nutri- ents you want to declare – you may want to select to add to have 50 and 100% of the Daily Value (DV) of calcium in one serving of your yogurt. The composi- Protein – Show ADV since yogurt is high in pro- tein; the daily value footnote and calories conver- tion of the Dairy Calcium 1000 you will add is shown in Table 1-2. sion chart will be displayed unless Hide Footnote and Hide Calorie Conversion Chart are selected; Procedure when you have finished selecting the label options select Apply and then Close to view the label.) 1. Add and enter the name of the new ingredient 7. Edit the ingredient declarations list. (Click on the to the database. (From the Edit Ingredient tab, View/Edit Declaration button, click Yes when asked select “Edit Ingredient File” from the main toolbar, – Do you wish to generate a formula declaration then Edit Current File, click Add, type the ingredient using individual ingredient declarations? – Each name “ Dairy Calcium 1000” in the enter ingredi- ingredient used in the formula can be selected in the ent name box, click Add. Answer yes to the question, top window and the ingredient declaration can be and click OK.) edited in the middle window.) 2. Enter the new ingredient composition (Table 1-2). Note the rules for ingredient declaration are (Look for the ingredient name in the column named found in the CFR 101.4. “ingredients and properties.” Click Edit Selected 8. Copy and paste the nutritional label and ingredi- under the edit ingredient file tab, the row will turn ent declaration list for formula 1 in a Word file. blue, enter the amount of each component/nutrient in (Click on the Copy button on the labeling tab, select the appropriate column.) standard label, click OK, open a Word document and 3. Edit the ingredient declaration (which will paste the label, click Return on the label window). To appear on the ingredient list) for the new copy and paste the ingredient list for formula 1, ingredient. (Type “Whey mineral concentrate” in click on the View/edit declaration button, click Yes to the column named “default spec text, Ingredient the question, select the Edit formula declaration sec- declaration.”) tion, highlight (Shift + arrow keys) the ingredient decla- ration list from the bottom window, copy the ingredient list and paste it into a Word file, close the View/edit Composition of Fieldgate 1-2 declaration window.) Natural Dairy Calcium 1000 table 9. Return to the Nutrition Info & Labeling (First District Association) section of the program. (Click on the Return Component Amount button.) 10. Enter the percentage of each ingredient for Ash 75% formula 2 in the % (wt/wt) column. Calcium 25,000 mg/100 g 11. Enter a name and save formula 2. (Click on the Calories 40 cal/100 g Formula Name window, enter “food analysis for- Lactose 10% mula 2” in the top Formula Name window, click on Phosphorus 13,000 mg/100 g Protein 4.0% the X to close the window, select Save Formula from Sugars 10 g/100 g the File menu.) Total carbohydrate 10 g/100 g 12. View and print the nutrition label and formula Total solids 92% 2 (follow the procedure described in Step 8 Water 8.0% above).● Chapter 1 Nutrition Labeling Using a Computer Program 5 4. Save the changes to the ingredient file. (Click on 13. View and print the nutritional label for the new the Finish Edit button, answer Yes to the question.) formula for 50% of the calcium DV. Follow the 5. Select close ingredient file. instructions described in section 4.b in this 6. Open food analysis formula 1 in the Formula handout. Development Section of the program. (From 14. Produce a formula and label that has 100% of the the File menu, select Open Formula and select food calcium DV. (Repeat steps 8–13 except using the cal- analysis formula1, click on the Open button, click culated amount of calcium required to meet 100% of on Yes for each question.) the calcium DV. You will have to perform this calcula- 7. Add the new Dairy Calcium 1000 ingredient tion yourself following the example in Step 8.) to “food analysis formula 1”. (Click on the Add Ingredients button, then select Dairy Calcium 1000 METHOD C: AN EXAMPLE OF REVERSE from the ingredient list, click on the Add button, ENGINEERING IN PRODUCT DEVELOPMENT click on the X to close the window.) 8. Calculate the amount of calcium (mg/100 g) Procedure required to meet 50 and 100% of the DV (see In this example, the program will automatically go example below). through the reverse engineering process. Start the example by selecting Cultured Products Automated Calcium required Examples from the Help menu and clicking on example = (DV for calcium/serving size) 4. During this example, you proceed to the next step ´ 100 g ´ % of DV required by clicking on the Next button. Calcium required for 50% of the DV 1. The information from the nutrition label for the product you want to reverse engineer is entered = (1000 mg/227 g) ´ 100 g ´ 0.50 into the program. (Comment: In this example serv- Calcium required for 50% of the DV ing size, calories, calories from fat, total fat, satu- = 220 mg/100 g rated fat, cholesterol, sodium, total carbohydrate, sugars, protein, vitamin A, vitamin C, calcium, and 9. Enter the amount of calcium required in the for- iron are entered.) mula and restrict all ingredients in the formula 2. The minimum and maximum levels of each except skim milk and Dairy Calcium 1000. (Find nutrient are calculated on a 100-g basis. (Comment: calcium in the Properties column and enter 220 in The program uses the rounding rules to determine the the Minimum and Maximum columns for calcium. possible range of each nutrient on a 100-g basis.) This lets the program know that you want to have 3. The information about nutrient minimum and 220 mg of calcium per 100 g. In both the Min and maximums is transferred into the Formula Max columns of the formula ingredients enter 38.201 Development section of the program. (Com- for milk (3.7% fat), 12.888 for condensed skim milk ment: The program has now converted nutrient (35% TS), 11.905 for sweetener, sugar liquid, 0.800 range information into a form it can use during the for modified starch, and 0.500 for stabilizer, gelatin. formulation process.) This lets the program adjust the amount of skim milk 4. Ingredients used in the formula are then selected and Dairy Calcium 1000 (calcium phosphate) and based on the ingredient declaration statement on keeps the amount of all the other ingredients con- the nutrition label. (Comment: Selecting the right stant. Click on the Formulate button, click OK.) ingredients can be difficult and an extensive under- 10. Enter a name and save the modified formula. standing of the ingredient declaration rules is neces- (Click on the Formula Name window, enter “food sary. Additionally, some of the required ingredients analysis formula 1 added calcium 50% DV your may not be in the database and will need to be added.) initials” in the top Formula Name window, click on 5. Restrictions on the amount of each ingredient the X to close the window, select Save Formula from in the formula are imposed whenever possible. the File menu.) (Comment: This is a critical step that requires knowl- 11. Open the new formula on the nutritional label- edge about the typical levels of ingredients used in the ing section. (Click on the Labeling Menu tab, select product. Additionally, based on the order of ingredi- labeling section, click File, Open Formula, and select ents in the ingredient declaration, approximate ranges “food analysis formula 1 added calcium 50% DV,” can be determined. In this example, the amount of click open.) modified starch is limited to 0.80%, the amount of 12. Make sure you have the correct serving size gelatin is limited to 0.50%, and the amount of culture information (see Method A, Step 4). is limited to 0.002%.)● 6 Chapter 1 Nutrition Labeling Using a Computer Program 1-3 table a,b Recipe for Chocolate Chip Cookies Ingredients Amount Grams Wheat flour, white, all purpose, enriched, unbleached 2.25 cup 281.15 Sugars, granulated 0.75 cup Baking chocolate, unsweetened, squares 100 grams 100 Sugar, brown 0.75 cup Butter (salted) 1 cup 227 Egg, whole, extra large 2 unit 200 Salt 0.75 tsp a Source for Ingredients: TechWizard™, USDA ingredients as source b Conversion Data Source: USDA webpage 3. If Dairy Calcium 1000 costs 2.50/lb and you are going 6. The program calculates an approximate formula. to have 100% of the DV for calcium in your yogurt, how (Comment: The program uses the information on nutrient much extra will you have to charge for a serving of yogurt ranges and composition of the ingredients to calculate to cover the cost of this ingredient? the amount of each ingredient in the formula.) 4. Assume you added enough Dairy Calcium 1000 to 7. The program compares the nutrition informa- claim 100% of the DV of calcium, would you expect tion for the developed formula to the original the added calcium to cause any texture changes in the nutrition label. (Comment: This information is yogurt? viewed in the Nutrition Label to Formula Spec 5. Make a nutrition label using the chocolate chip cookie section of the program accessed by selecting View recipe and other information in Table 1-3. Conversion fac- Reverse Engineering Section then Label to Spec from tors to get the weight of sugars and salt can be found in the Reverse Engineering menu.) the U.S. Department of Agriculture Nutrient Database for Standard Reference website: http://www.nal.ars.usda. gov/ba/bhnrc/ndl (Assume: 25% loss of water during baking; Number of servings = 1, 30 g). QUESTIONS 1. Based on the labels you produced for yogurt formula 1 and 2 in Method A, what nutrient content claims could RESOURCE MATERIALS you make for each formula (a description of nutrient content claims is found in Tables 3-7 and 3-8 in the Nielsen Metzger LE (2010) Nutrition labeling. Ch. 3. In: Nielsen SS (ed) Food Analysis text)? Food analysis, 4th edn. Springer, New York 2. How much Dairy Calcium 1000 did you have to add to the Owl Software (2009) TechWizard™ Version 4 Manual, yogurt formula to have 50 and 100% of the DV of calcium Columbia, MO. in the formula?2 chapter Assessment of Accuracy and Precision S.S. Nielsen, Food Analysis Laboratory Manual, Food Science Texts Series, 9 DOI 10.1007/978-1-4419-1463-7_2, © Springer Science+Business Media, LLC 2010● Chapter 2 Assessment of Accuracy and Precision 11 INTRODUCTION repair is necessary before the pipettor can be reliably used again. Background It is generally required that reported values minimally include the mean, a measure of precision, Volumetric glassware, mechanical pipettes, and balances and the number of replicates. The number of are used in many analytical laboratories. If the basic significant figures used to report the mean reflects skills in the use of this glassware and equipment the inherent uncertainty of the value, and it needs are mastered, laboratory exercises are easier, more to be justified based on the largest uncertainty in enjoyable, and the results obtained are more accurate making the measurements of the relative precision of and precise. Measures of accuracy and precision can the assay. The mean value is often expressed as part be calculated based on the data generated, given the of a confidence interval (CI) to indicate the range glassware and equipment used, to evaluate the skill of within which the true mean is expected to be found. the user as well as the reliability of the instrument Comparison of the mean value or the CI to a standard and glassware. or true value is the first approximation of accuracy. Determining mass using an analytical balance is A procedure or instrument is generally not deemed the most basic measurement made in an analytical inaccurate if the CI overlaps the standard value. laboratory. Determining and comparing mass is fun- Additionally, a CI that is considerably greater than the damental to assays such as moisture and fat determi- readability indicates that the technician’s technique nation. Accurately weighing reagents is the first step needs improvement. In the case of testing the accuracy in preparing solutions for use in various assays. of an analytical balance with a standard weight, if Accuracy and precision of the analytical balance the CI does not include the standard weight value, it are better than for any other instrument commonly would suggest that either the balance needs calibration used to make analytical measurements, provided the or that the standard weight is not as originally issued. balance is properly calibrated, and the laboratory Accuracy is sometimes estimated by the relative error personnel use proper technique. With proper cali- ) between the mean analysis value and the true (%E rel bration and technique, accuracy and precision are value. However, %E only reflects tendencies, and rel limited only by the readability of the balance. in practice is often calculated even when there is no Repeatedly weighing a standard weight can yield statistical justification that the mean and true value valuable information about the calibration of the differ. Also, note that there is no consideration of balance and the technician’s technique. the number of replicates in the calculation of %E , rel Once the performance of the analytical balance suggesting that the number of replicates will not and the technician using it has been proven to be affect this estimation of accuracy to any large extent. acceptable, determination of mass can be used to Absolute precision is reflected by the standard assess the accuracy and precision of other analytical deviation, while relative precision is calculated as the instruments. All analytical laboratories use volumetric coefficient of variation (CV). Calculations of precision glassware and mechanical pipettes. Mastering their are largely independent of the number of replicates, use is necessary to obtain reliable analytical results. except that more replicates may give a better estimate To report analytical results from the laboratory in of the population variance. a scientifically justifiable manner, it is necessary to Validation of a procedure or measurement tech- understand accuracy and precision. nique can be performed, at the most basic level, as a A procedure or measurement technique is vali- single trial validation, as is described in this laboratory dated by generating numbers that estimate their that includes estimating the accuracy and precision accuracy and precision. This laboratory includes of commonly used laboratory equipment. However, assessment of the accuracy and precision of automatic for more general acceptance of procedures, they are pipettors. An example application is determining the validated by collaborative studies involving several accuracy of automatic pipettors in a research or qual- laboratories. Collaborative evaluations are sanctioned ity assurance laboratory, to help assess their reliability by groups such as AOAC International, AACC Inter- and determine if repair of the pipettors is necessary. national, and the American Oil Chemists’ Society Laboratory personnel should periodically check the (AOCS). Such collaborative studies are prerequisite to pipettors to determine if they accurately dispense procedures appearing as approved methods in manu- the intended volume of water. To do this, water dis- als published by these organizations. pensed by the pipettor is weighed, and the weight is converted to a volume measurement using the appro- Reading Assignment priate density of water based on the temperature of the water. If replicated volume data indicate a prob- Literature on how to properly use balances, volumetric glass- ware, and mechanical pipettes. lem with the accuracy and/or precision of the pipettor, ● 12 Chapter 2 Assessment of Accuracy and Precision Nielsen, S.S. 2010. Introduction to food analysis. Ch. 1, in Food (a) Tare a 100-ml beaker, deliver 10 ml of water Analysis, 4th ed. S.S, Nielsen (Ed.), Springer, New York. from a volumetric pipette into the beaker, Smith, J.S. 2010. Evaluation of analytical data. Ch. 4, in Food and record the weight. Repeat this proce- Analysis, 4th ed. S.S. Nielsen (Ed.), Springer, New York. dure of taring the beaker, adding 10 ml, and recording the weight, to get six determina- Objective tions on the same pipette. (Note that the total volume will be 60 ml.) (It is not necessary to Familiarize, or refamiliarize, oneself with the use empty the beaker after each pipetting.) of balances, mechanical pipettes, and volumetric (b) Repeat the procedure as outlined in Step 2a glassware, and assess accuracy and precision of data but use a 20- or 30-ml beaker and a 1.0-ml generated. volumetric pipette. Do six determinations. 3. Analytical balance and buret. Principle of Method (a) Repeat the procedure as outlined in Step 2a, but use a 100-ml beaker, a 50-ml (or 25-ml) Proper use of equipment and glassware in analytical buret filled with water, and dispense 10 ml tests helps ensure more accurate and precise results. of water (i.e., tare a 100 ml beaker, deliver 10 ml of water from the buret into the bea- Supplies ker, and record the weight). (Handle the ●● 1 Beaker, 100 ml beaker wearing gloves, to keep oils from ●● 1 Beaker, 20 or 30 ml your hands off the beaker.) Repeat this pro- ●● 1 Beaker, 250 ml cedure of taring the beaker, adding 10 ml, ●● Buret, 25 or 50 ml and recording the weight, to get six deter- ●● Erlenmeyer flask, 500 ml minations on the buret. (Note that the total ●● Funnel, approximately 2 cm diameter (to fill volume will be 60 ml.) (It is not necessary to buret) empty the beaker after each addition.) ●● Mechanical pipettor, 1000 ml, with plastic tips (b) Repeat the procedure as outlined in Step 3a ●● Plastic gloves but use a 20- or 30-ml beaker and a 1.0-ml ●● Ring stand and clamps (to hold buret) volume from the buret. Do six determinations. ●● Rubber bulb or pipette pull-up 4. Analytical balance and mechanical pipette. ●● Standard weight, 50 or 100 g Repeat the procedure as outlined in Step 2a but ●● Thermometer, to read near room temperature use a 20- or 30-ml beaker and a 1.0-ml mechanical ●● Volumetric flask, 100 ml pipette (i.e., tare a 20- or 30-ml beaker, deliver ●● 2 Volumetric pipettes, one each of 1 and 10 ml 1 ml of water from a mechanical pipettor into the beaker, and record the weight). Repeat this procedure of taring the beaker, adding 1 ml, and Equipment recording the weight to get six determinations ●● Analytical balance on the same pipettor. (Note that the total vol- ●● Top loading balance ume will be 6 ml.) (It is not necessary to empty the beaker after each pipetting.) Notes 5. Total content (TC) versus total delivery (TD). Tare a 100-ml volumetric flask on a top loading Before or during the laboratory exercise, the instructor is encouraged to discuss the following: (1) Difference between balance. Fill the flask to the mark with water. dispensing from a volumetric pipette and a graduated pipette, Weigh the water in the flask. Now tare a 250-ml (2) difference between markings on a 10-ml versus a 25- or beaker and pour the water from the volumetric 50-ml buret. flask into the beaker. Weigh the water delivered from the volumetric flask. 6. Readability versus accuracy. Zero a top loading PROCEDURES balance and weigh a 100-g (or 50-g) standard weight. Record the observed weight. Use gloves (Record data in tables that follow.) or finger cots as you handle the standard weight 1. Obtain 400 ml deionized distilled (dd) H O to keep oils from your hands off the weight. 2 in a 500-ml Erlenmeyer flask for use during this Repeat with the same standard weight on at laboratory session. Check the temperature of least two other top loading balances, recording the water with a thermometer. the observed weight and the type and model 2. Analytical balance and volumetric pipettes. (e.g., Mettler, Sartorius) of balance used.● Chapter 2 Assessment of Accuracy and Precision 13 Part 6 data: DATA AND CALCULATIONS Type/Model Wt. of standard Calculate the exact volume delivered in Parts 2–5, using Balance of balance weight each weight measurement and the known density of water (see Table 2-1). Using volume data, calculate the 1 following indicators of accuracy and precision: mean, 2 standard deviation, coefficient of variation, percent 3 relative error, 95% confidence interval. Use your first three measurements for n = 3 values requested, and all six measurements for n = 6 values. QUESTIONS 2-1 Viscosity and Density of Water 1. Theor etically, how are standard deviation, coefficient of table at Various Temperatures variation, mean, percent relative error, and 95% confidence interval affected by: (1) more replicates, and (2) a larger Tempera- Density Viscosity Tempera- Density Viscosity size of the measurement? Was this evident in looking at ture (°C) (g/ml) (cps) ture (°C) (g/ml) (cps) the actual results obtained using the volumetric pipettes and the buret, with n = 3 versus n = 6, and with 1 ml versus 20 0.99823 1.002 24 0.99733 0.9111 10 ml? (see table below) 21 0.99802 0.9779 25 0.99707 0.8904 22 0.99780 0.9548 26 0.99681 0.8705 23 0.99757 0.9325 27 0.99654 0.8513 Actual, with Theoretical results obtained Data for Parts 2, 3, and 4: More Larger More Larger Volumetric Mechanical replicates measurement replicates measurement pipette Buret pipettor Standard 1 ml 10 ml 1 ml 10 ml 1 ml deviation Coefficient Rep Wt. Vol. Wt. Vol. Wt. Vol. Wt. Vol. Wt. Vol. of variation Mean 1 Percent 2 relative 3 error 4 95% 5 Confidence 6 interval n = 3 – – – – – Mean S – – – – – 2. Why are percent relative error and coefficient of variation CV – – – – – used to compare the accuracy and precision, respectively, – – – – – %E rel of the volumes from pipetting/dispensing 1 and 10 ml – – – – – CI 95% with the volumetric pipettes and buret in Parts 2 and n = 6 3, rather than simply the mean and standard deviation, – – – – – Mean respectively? – – – – – S 3. Compare and discuss the accuracy and the precision of – – – – – CV the volumes from the 1 ml pipetted/dispensed using a – – – – – %E volumetric pipette, buret, and mechanical pipettor (Parts rel – – – – – CI 2, 3, and 4). Are these results consistent with what would 95% be expected? 4. If accuracy and/or precision using the mechanical pipettor Part 5 data: are less than should be expected, what could you do to improve its accuracy and precision? Wt. Vol. 5. In a titration experiment using a buret, would you expect to use much less than a 10-ml volume in each titration? Water in flask= Would you expect your accuracy and precision to be better Water in beaker= using a 10-ml buret or a 50-ml buret? Why?● 14 Chapter 2 Assessment of Accuracy and Precision 6. How do your results from Part 5 of this lab dif ferentiate ACk NOw LEDg MENT “to contain” from “to deliver”? Is a volumetric flask “to contain” or “to deliver”? Which is a volumetric pipette? This laboratory was developed with inputs from Dr 7. From your results from Part 6 of this lab, would you now Charles E. Carpenter, Department of Nutrition and assume that since a balance reads to 0.01 g, it is accurate to Food Sciences, Utah State University, Logan, UT. 0.01 g? 8. What sources of error (human and instrumental) were evident or possible in Parts 2–4, and how could these be reduced or eliminated? Explain. RESOURCE MATERIALS 9. You are considering adopting a new analytical method in your lab to measure the moisture content of cereal products. Nielsen SS (2010) Introduction to food analysis, Ch. 1. In: How would you determine the precision of the new method Nielsen SS (ed) Food analysis, 4th edn. Springer, New York and compare it to the old method? How would you determine Smith JS (2010) Evaluation of analytical data, Ch. 4. In: (or estimate) the accuracy of the new method? Nielsen SS (ed) Food Analysis, 4th edn. Springer, New York● Chapter 2 Assessment of Accuracy and Precision 15 NOTES 3 chapter Determination of Moisture Content S.S. Nielsen, Food Analysis Laboratory Manual, Food Science Texts Series, 17 DOI 10.1007/978-1-4419-1463-7_3, © Springer Science+Business Media, LLC 2010 ● Chapter 3 Determination of Moisture Content 19 INTRODUCTION METHOD A: FORCED DRAFT OVEN Background Objective The moisture (or total solids) content of foods is Determine the moisture content of corn syrup and important to food manufacturers for a variety of corn flour using a forced draft oven method. reasons. Moisture is an important factor in food Principle of Method quality, preservation, and resistance to deterioration. Determination of moisture content also is necessary The sample is heated under specified conditions and to calculate the content of other food constituents on the loss of weight is used to calculate the moisture a uniform basis (i.e., dry weight basis). The dry matter content of the sample. that remains after moisture analysis is commonly referred to as total solids. Supplies While moisture content is not given on a nutrition ●● Basil (fresh), 15 g (ground) label, it must be determined to calculate total carbohy- ●● drate content. Moisture content of foods can be deter- Beaker, 25–50 ml (to pour corn syrup into pans) ●● Corn flour, 10 g mined by a variety of methods, but obtaining accurate ●● and precise data is commonly a challenge. The vari- Corn syrup, 15 g ●● 3 Crucibles (preheated at 550°C for 24 h) ous methods of analysis have different applications, ●● advantages, and disadvantages (see Reading Assign- 2 Desiccators (with dried desiccant) ●● Liquid milk, 20 ml ment). If the ash content also is to be determined, it ●● is often convenient to combine the moisture and ash Nonfat dry milk (NFDM), 10 g ●● Plastic gloves (or tongs) determinations. In this experiment, several methods to ●● determine the moisture content of foods will be used 2 Spatulas ●● 5 Trays (to hold/transfer samples) and the results compared. Summarized below are the ●● food samples proposed for analysis and the methods 2 Volumetric pipettes, 5 ml ●● 6 Weighing pans – disposable aluminum open used. However, note that other types of food sam- ples could be analyzed and groups of students could pans (for use with corn syrup) (predried at 100°C for 24 h) analyze different types of food samples. It is recom- ●● mended that all analyses be performed in triplicate, as 6 Weighing pans – metal pans with lids (for use with corn flour and NFDM) (predried at 100°C time permits. for 24 h) Equipment Corn Corn Milk Nonfat syrup flour (liquid) dry milk Basil ●● Forced draft oven ●● Analytical balance, 0.1 mg sensitivity Forced draft oven X X X X X Vacuum oven X Note Microwave drying X X Rapid moisture X X Glass microfiber filters (e.g., GF/A, Whatman, Newton, MA), analyzer predried for 1 h at 100°C, can be used to cover samples to Toluene distillation X X prevent splattering in the forced draft oven and the vacuum Karl Fischer X X X oven. Instructors may want to have students compare results Near infrared X with and without these fiberglass covers. Cautions and Hazards Reading Assignment Be sure to label all containers used with complete Bradley, R.L., Jr. 2010. Moisture and total solids analysis, information, or record container information linker to Ch. 6, in Food Analysis, 4th ed. S.S. Nielsen (Ed.), Springer, each sample. Use gloves or tongs when handling sam- New York. ple plans and crucibles. These pans and crucibles have been dried and stored in desiccators prior to weighing. Overall Objective They will pick up moisture by sitting on the counter, The objective of this experiment is to determine and so remove them from the desiccator only just before compare the moisture contents of foods by various use. Open desiccators slowly to avoid damage and methods of analysis. danger from broken glass.● 20 Chapter 3 Determination of Moisture Content Note: Ash content of this milk sample could be Procedure determined by placing the milk sample, dried at Instructions are given for analysis in triplicate. 100°C for 3 h, in a muffle furnace at 550°C for 18–24 h. After cooling in a desiccator, the crucibles containing I. Moisture in Corn Syrup ashed milk would be weighed and the ash content 1. Label dried pans (disposable aluminum calculated. open pans) and weigh accurately. 2. Place 5 g of sample in the pan and weigh IV. Moisture of Nonfat Dry Milk accurately. (Because corn syrup is very 1. Weigh accurately the dried pan with lid. hygroscopic, work quickly, using a plastic (Note identifier number on pan and lid.) transfer pipette, as you weigh the corn 2. Place 3 g of sample in the pan and weigh syrup.) accurately. 3. Place in a forced draft oven at 98–100°C 3. Place pan in a forced draft oven at 100°C for 24 h. for 24 h. 4. Store in a desiccator until samples are 4. Store in a desiccator until samples are weighed. weighed. 5. Calculate percentage moisture (wt/wt) as 5. Calculate percentage moisture (wt/wt) as described below. described below. II. Moisture in Corn Flour (Method 44-15A of V. Moisture in Fresh Basil AACC International, one-stage procedure) 1. Label dried pans (disposable aluminum 1. Weigh accurately dried pan with lid. open pans) and weigh accurately. (Note identifier number on pan and lid.) 2. Place 3 g of ground sample in the pan and 2. Place 2–3 g of sample in the pan and weigh weigh accurately. accurately. 3. Place in a forced draft oven at 98–100°C 3. Place in a forced draft oven at 130°C for for 24 h. 1 h. Be sure metal covers are ajar, to allow 4. Store in a desiccator until samples are water loss. weighed. 4. Remove from oven, realign covers to 5. Calculate percentage moisture (wt/wt) as close, cool, and store in desiccator until described below. samples are weighed. 5. Calculate percentage moisture (wt/wt) as described below. Data and Calculations Calculate percentage moisture (wt/wt): III. Moisture in Liquid Milk (AOAC Method 990.19, 990.20) wt of H O in sample 2 1. Label and weigh accurately predried cruci- % moisture= ´ 100 wt of wet sample bles (550°C for 24 h). (Note identified num- ber on crucible.) % moisture 2. Place 5 g of sample in the crucible and æ wt of wet sampleöæ wt of dried sampleö weigh accurately. - ç ÷ç ÷ ++ pan pan è øè ø 3. Evaporate a majority of water on a hot =´ 100 plate; do not dry the sample completely. (wt of wet sample+- pan)(wt of pan) (Gently heat the milk in the crucibles. % ash, wet weight basis (wwb) Wear gloves as you handle the crucibles, swirling the milk to coat the sides of the wt of ash =´ 100 crucible. Try to avoid development of a wt of wet sample film on the surface, until most of the water % ash, wwb has been evaporated.) 4. Place in a forced draft oven at 100°C for 3 h. wt of ashed sample æö - wt of crucible () 5. Store in a desiccator until samples are ç÷ + crucible èø =´ 100 weighed. wt of wet sample æö 6. Calculate percentage moisture (wt/wt) as -(wt of crucible) ç÷ + crucible èø described below.● Chapter 3 Determination of Moisture Content 21 Equipment Pan Pan + Wet Pan + Dried ●● Sample Rep (g) sample (g) sample (g) % Moisture Vacuum oven (capable of pulling vacuum to 100 mm of mercury) Corn 1 ●● Analytical balance, 0.1 mg sensitivity syrup 2 3 Cautions and Hazards – X = See same information in Method A. SD = Corn flour 1 Procedure 2 3 I. Moisture of Corn Syrup, Without Use of Drying Sand – X = 1. Label weighing pans (i.e., etch identifier SD = into tab of disposable aluminum pan) and Liquid milk 1 2 weigh accurately. 3 2. Place 5 g of sample in the weighing pan and – X = weigh accurately. SD = 3. Dry at 70°C and a vacuum of at least 26 in. for Nonfat 1 24 h, but pull and release the vacuum slowly. dry milk 2 (Note that samples without drying sand will 3 bubble up and mix with adjoining samples if – pans are too close together.) Bleed dried air X = into the oven as vacuum is released. SD = 4. Store in a desiccator until samples are cooled Fresh basil 1 2 to ambient temperature. Weigh. 3 – X = II. Moisture of Corn Syrup, with Use of Drying Sand SD = 1. Label weighing pan, add 10 g dried sand and stirring rod, then weigh accurately. 2. Add 5 g of sample and weigh accurately. Add 5 ml of deionized distilled (dd) water. Mix with METHOD B: VACUUM OVEN stirring rod being careful not to spill any of the Objective sample. Leave the stirring bar in the pan. 3. Dry at 70°C and a vacuum of 100 mm Determine the moisture content of corn syrup by the mercury for 24 h. Bleed dried air into the vacuum oven method, with and without the addition oven as vacuum is released. of sand to the sample. 4. Store in a desiccator until samples are cooled to ambient temperature. Weigh. Principle The sample is heated under conditions of reduced Data and Calculations pressure to remove water and the loss of weight Calculate percentage moisture (wt/wt) as in Method A. is used to calculate the moisture content of the sample. Pan + Wet Pan + Dried % Sample Sample Rep pan (g) sample (g) (g) Moisture Supplies ●● Corn syrup 1 Corn syrup, 30 g without 2 ●● Desiccator (with dried desiccant) sand 3 ●● 3 Glass stirring rods (ca. 2–3 cm long, predried at 100°C for 3 h) – X = ●● Plastic gloves (or tongs) SD = ●● Pipette bulb or pump Corn syrup 1 ●● Sand, 30 g (predried at 100°C for 24 h) with sand 2 ●● 2 Spatulas 3 ●● Volumetric pipette, 5 ml – ●● 6 Weighing pans – disposable aluminum open X = SD = pans (pre-dried at 100°C for 3 h)● 22 Chapter 3 Determination of Moisture Content METHOD C: MICROWAVE DRYING OVEN Principle The sample placed on a digital balance is heated under Objective controlled high heat conditions, and the instrument Determine the moisture content of corn syrup and automatically measures the loss of weight to calculate milk (liquid) using a microwave drying oven. the percentage moisture or solids. Principle Supplies ●● The sample is heated using microwave energy, and the Corn flour, 10 g ●● loss of weight is used to calculate the moisture content Milk, 10 ml ●● of the sample. Plastic gloves ●● Spatula Supplies Equipment ●● Corn syrup, 4 g ●●●● Glass stirring rod (to spread corn syrup) Rapid Moisture Analyzer (e.g., from ●● Milk (liquid), 4 g Computrac®, Arizona Instrument LLC., ●● 6 Paper pads (for use in microwave oven) Chandler, AZ) ●● Pasteur pipette and bulb (to spread milk sample) ●● Plastic gloves Procedure Follow instructions from manufacturer for use of the Equipment rapid moisture analyzer, regarding the following: ●● Microwave drying oven (e.g., from CEM Cor- ●● Turning on instrument and warming up poration, Matthew, NC) ●● Select test material ●● Taring instrument Procedure ●● Testing sample Follow instructions from manufacturer for use of the ●● Obtaining results microwave drying oven, regarding the following: Data and Calculations ●● Turning on instrument and warming up ●● Loading method for specific application (i.e., sets time, power, etc.) % Moisture ●● Taring instrument ●● Testing sample Sample 1 2 3 Mean ●● Obtaining results Corn flour Milk Data and Calculations Sample Rep % Moisture g Water/g Dry matter METHOD E: TOLUENE DISTILLATION Corn syrup 1 Objective 2 3 Determine the moisture content of basil by the toluene – – X = X = distillation method. SD = SD = Milk (liquid) 1 Principle 2 The moisture in the sample is codistilled with toluene, 3 – – which is immiscible in water. The mixture that distills X = X = SD = SD = off is collected, and the volume of water removed is measured. Chemicals METHOD D: RAPID MOISTURE ANALYZER Objective CAS No. Hazards Determine the moisture content of corn flour using a Toluene 108-88-3 Harmful, highly flammable rapid moisture analyzer.● Chapter 3 Determination of Moisture Content 23 6. Continue refluxing until two consecutive Hazards, Cautions, and Waste Disposal readings 15 min apart show no change. Toluene is highly flammable and is harmful if Dislodge any water held up in the condenser inhaled. Use adequate ventilation. Wear safety glasses with a brush or wire loop. Rinse the condenser and gloves at all times. For disposal of toluene carefully with ca. 5 ml toluene. Dislodge any waste, follow good laboratory practices outlined by moisture droplets adhering to the Bidwell– environmental health and safety protocols at your Sterling trap or toluene trapped under the institution. collected moisture. For this, use the wire. Rinse wire with a small amount (10 ml) of toluene Supplies before removing from apparatus. ●● Fresh basil, 40–50 g 7. Continue refluxing for 3–5 min, remove the ●● NFDM, 40–50 g heat, and cool the trap to 20°C in a suitable ●● Toluene, A.C.S. grade water bath. 8. Calculate the moisture content of the sample: Equipment % Moisture ●● Analytical balance, 0.1 mg sensitivity. ●● Glass distillation apparatus with ground glass =´ éù vol. of water ml /wt of sample g 100 ()() ëû joints: (1) Boiling flask, 250 ml or 300 ml, round- bottom, shortneck flask with a T.S. 24/40 joint; Notes (2) West condenser with drip tip, 400 mm in 1. Flask, condenser, and receiver must be scrupulously length with a T.S. 24/40 joint; (3) Bidwell–Ster- clean and dry. For example, the apparatus, including ling trap, T.S. 24/40 joint, 3- ml capacity gradu- the condenser, could be cleaned with potassium ated in 0.1 ml intervals. dichromate-sulfuric acid cleaning solution, rinsed ●● Heat source, capable of refluxing toluene in with water, rinsed with 0.05 N potassium hydroxide the apparatus above (e.g., heating mantle con- solution, rinsed with alcohol, then allowed to drain for nected to voltage controller). No open flame 10 min. This procedure will minimize the adherence ●● of water droplets to the surfaces of the condenser and Nylon bristle buret brush, ½ in. in diameter, and the Bidwell–Sterling trap. a wire loop. (It should be long enough to extend 2. A correction blank for toluene must be conducted through the condenser, ca. 450 mm. Flatten the periodically by adding 2–3 ml of distilled water loop on the buret brush and use this brush, to 100 ml of toluene in the distillation flask, then inverted, as a wire to dislodge moisture drops following the procedure in Steps 2–6 above. in the moisture trap.) Data and Calculations Procedure 1. Grind the fresh basil with a small table-top Wt. sample (g) Vol. water removed (ml) % Moisture food grinder. Pulse grind the sample in 5–10 s intervals. Avoid long pulses and excessive grinding to prevent frictional heat. 2. Weigh approximately 40 g of sample (basil or NFDM) accurately (amount chosen to yield 2–5 ml water). METHOD F: KARL FISCHER 3. Transfer sample quantitatively to distilling flask. Add sufficient toluene to cover the sam- Objective ple completely (not less than 75 ml). Determine the moisture content of NFDM and corn 4 . Assemble the apparatus as shown in Chap. 6 flour by the Karl Fischer (KF) method. of Nielsen’s textbook. Fill the trap with toluene by pouring it through the condenser until Principle it just fills the trap and begins to flow into the flask. Insert a loose nonabsorbing cotton When the sample is titrated with the KF reagent, plug into the top of the condenser to prevent which contains iodine and sulfur dioxide, the iodine condensation of atmospheric moisture in the is reduced by sulfur dioxide in the presence of water condenser. from the sample. The water reacts stoichiometri- 5. Bring to boil and reflux at about two drops per cally with the KF reagent. The volume of KF reagent second until most of the water has been col- required to reach the endpoint of the titration (visual, lected in the trap, then increase the reflux rate conductometric, or coulometric) is directly related to to ca. four drops per second. the amount of water in the sample.● 24 Chapter 3 Determination of Moisture Content Note that the reaction/titration vessel of the KF Chemicals apparatus (and the anhydrous methanol within the vessel) must be changed after analyzing CAS No. Hazards several samples (exact number depends on type of sample). Remember that this entire appara- Karl Fischer reagent Toxic tus is very fragile. To prevent contamination 2-Methoxyethanol 109-86-4 from atmospheric moisture, all openings must Pyridine 110-86-1 be closed and protected with drying tubes. Sulfur dioxide 7446-09-5 Iodine 7553-56-2 Harmful, dangerous to the environment II. Standardizing Karl Fischer Reagent Methanol, anhydrous 67-56-1 Extremely flammable The KF reagent is standardized to determine its Sodium tartrate dihydrate 868-18-8 water equivalence. Normally, this needs to be (Na C H O ⋅• 2H O) 2 4 4 6 2 done only once a day, or when changing the KF reagent supply. Reagents 1. Add approximately 50 ml of anhydrous methanol to reaction vessel through the ●● KF reagent ●● sample port. Methanol, anhydrous 2. Put the magnetic stir bar in the vessel and ●● Sodium tartrate dihydrate, 1 g, dried at 150°C turn on the magnetic stirrer. for 2 h 3. Remove the caps (if any) from drying tube. Turn the buret stopcock to the filling posi- Hazards, Cautions, and Waste Disposal tion. Hold one finger on the air-release hold Use the anhydrous methanol in an operating hood in the rubber bulb and pump the bulb to fill since the vapors are harmful and it is toxic. Otherwise, the buret. Close the stopcock when the KF adhere to normal laboratory safety procedures. Use reagent reaches the desired level (at posi- appropriate eye and skin protection. The KF reagent tion 0.00 ml) in the buret. and anhydrous methanol should be disposed of as 4. Titrate the water in the solvent (anhydrous hazardous wastes. methanol) by adding enough KF reagent to just change the color of the solution from clear Supplies or yellow to dark brown. This is known as the KF endpoint. Note and record the conductance ●● Corn flour meter reading. (You may titrate to any point ●● Graduated cylinder, 50 ml in the brown KF zone on the meter, but make ●● NFDM sure that you always titrate to that same ●● 2 Spatulas endpoint for all subsequent samples in the ●● Weighing paper series.) Allow the solution to stabilize at the endpoint on the meter for at least 1 min before Equipment proceeding to the next step. ●● Analytical balance, 0.1 g sensitivity 5. Weigh, to the nearest milligram, approxi- ●● Drying oven mately 0.3 g of sodium tartrate dihydrate, ●● KF titration unit (e.g., from Barnsted Themaline, previously dried at 150°C for 2 h. Berkeley, CA, Aquametry Apparatus) 6. Fill the buret with the KF reagent, then titrate the water in the sodium tartrate Procedure dihydrate sample as in Step II.4. Record the volume (ml) of KF reagent used. Instructions are given as for a nonautomated unit, and 7. Calculate the KF reagent water (moisture) for analysis in triplicate. If using an automated unit, equivalence (KFR ) in mg H O/ml: follow instructions of the manufacturer. eq 2 I. Apparatus Set Up 36g/mol´´ S1000 KFR= eq 230.08g/mol´ A Assemble titration apparatus and follow instructions of manufacturer. The titration where: apparatus includes the following: buret; res- S = eight of sodium tartrate dihydrate (g) ervoir for reagent; magnetic stirring device; A = ml of KF reagent required for titration of reaction/titration vessel; electrodes; and cir- sodium tartrate dihydrate cuitry for dead stop endpoint determination. ● Chapter 3 Determination of Moisture Content 25 III. Titration of Sample KFR´K eq S % H O=´ 100 2 1. Prepare samples for analysis and place in S reaction vessel as described below. where: KFR = water equivalence of KF reagent (mg If samples are in powder form: eq H O/ml) 2 – Use an analytical balance to weigh out K = ml of KF reagent required for titration of approximately 0.3 g of sample, and s sample record the exact sample weight (S) to the S = weight of sample (mg) nearest milligram. – Remove the conductance meter from Karl Fischer reagent water equivalence (KFReq): the reaction vessel, then transfer your sample to the reaction vessel through the Buret sample port immediately. (Use an extra Volume piece of weighing paper to form a cone- Wt. Sodium tartrate Start End titrant Calculated shaped funnel in the sample port, then Rep dihydrate (g) (ml) (ml) (ml) KFR eq pour your sample through the funnel ●●● 1 into the reaction vessel.) 2 – P ut the conductance meter and stopper – back in the reaction vessel. The color of 3 X = the solution in the vessel should change Calculation for KFR : to light yellow and the meter will regis- eq ter below the KF zone on the meter. Moisture content of samples by Karl Fischer method: If any samples analyzed are in liquid form: Buret – Use a 1-ml syringe to draw up about 0.1 ml Volume of sample. Weigh the syringe with sample Wt. Sample Start End titrant on an analytical balance and record the Sample Rep (g) (ml) (ml) (ml) % Moisture exact weight (S ) to the nearest milligram. 1 – Inject 1–2 drops of sample into the reac- tion vessel through the sample port, then weigh the syringe again (S ), to the 0 nearest milligram. METHOD G: NEAR INFRARED ANALYZER – S ample weight (S) is the difference of S 1 and S . 0 Objective S = S – S 1 0 Determine the moisture content of corn flour using a near infrared analyzer. – Put the stopper back in the sample port of the reaction vessel. The color of the Principle solution in the vessel should change to light yellow and the meter will register Specific frequencies of infrared radiation are absorbed below the KF zone on the meter. by the functional groups characteristic of water (i.e., 2. Fill the buret, then titrate the water in the the –OH stretch of the water molecule). The concen- sample as in Step II.4 above. Record the tration of moisture in the sample is determined by volume (ml) of KF reagent used. measuring the energy that is reflected or transmitted 3. To titrate another sample, repeat Steps II.5–7 by the sample, which is inversely proportional to the above with the new sample. After titrating energy absorbed. several samples (exact number depends on the nature of the sample), it is necessary to Supplies start with fresh methanol in a clean reac- ●● tion vessel. Record the volume (ml) of KF Corn flour ●● reagent used for each titration. Pans and sample preparation tools for near infrared analyzer Data and Calculations Equipment Calculate the moisture content of the sample as follows: ●● Near infrared analyzer

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