The ICP spectrometry is a relatively new analytical technique used for the analysis of major and trace minerals. It is a type of emission spectroscopy that uses the ICP to produce excited atoms and ions that emit electromagnetic radiation at wave-lengths characteristic of a particular element. The intensity of this emission is indicative of the concentration of the element within the sample. The ICP is based in the production of a plasma or a gaseous mixture containing cations and electrons produced by an argon torch which generates temperatures of 5000 K to 10,000 K resulting in a very effective atomization.
The ICP-AES is composed of three fundamental parts: the ICP, the optical spectrometer, and a computer for data collec-tion and treatment. The heart of the ICP is the plasma torch consisting of two or three quartz glass tubes centered in a cop-per coil. Argon gas is typically used to create the plasma. Wet or dry-ash diluted solutions are almost always introduced as aerosols carried by a stream of argon in the injector tube. The temperature in the zone of the load coil ranges from 1000 to 3000 K. The plasma is heated to a temperature ranging from 1000 K to 5000 K to excite the atoms and produce an emission light that is read by a spectrometer. All minerals can be quantified in the same run because the equipment is equipped with monochromators or polychromators capable of scanning over a wavelength range. When the torch is turned on, an intense electromagnetic field is created within the coil by the high-power radio frequency signal flowing in the coil.
This radio frequency signal is created by the radio frequency generator which is, effectively, a high-power radio transmitter driving the “work coil” the same way a typical radio trans-mitter drives a transmitting antenna. The argon gas flowing through the torch is ignited with a Tesla unit that creates a brief discharge arc through the argon flow to initiate the ion-ization process. Once the plasma is “ignited,” the Tesla unit is turned off. The argon gas is ionized in the intense electromag-netic field and flows in a particular rotationally symmetrical pattern toward the magnetic field of the radio frequency coil.
A stable, high-temperature plasma of approximately 7000 K is then generated as a result of the inelastic collisions created between the neutral argon atoms and the charged particles.
2.4.3.1 analysis of minerals with IcP spectroscopy A. Samples, Ingredients, and Reagents
• Dry or wet ash samples (refer to procedures in Sections 2.4.1.1 or 2.4.1.2)
• Distilled deionized water
• Mineral standards (1000 mg/L) or commer-cially available standard solutions
36 Cereal Grains: Laboratory Reference and Procedures Manual
B. Materials and Equipment
• ICP analyzer
• Diluter or automatic dispensing system
• Test tube shaker or vortex
• Volumetric flasks (50 mL)
• Argon gas tank (high purity grade)
• Test tubes
• Pipettes (5 mL and 10 mL) C. Procedure
1. Obtain the wet or dry ash stock solution (refer to procedures in Sections 2.4.1.1 or 2.4.1.2).
2. Dilute the test sample with distilled deionized water preferably using the automatic diluter.
Then, with the test tube shaker, agitate the contents. Make sure to use test tubes that were previously acid-washed. Prepare at least five different dilutions of the mineral standard.
3. Turn on at least 45 minutes before the test and set up operating conditions of the ICP and initi-ate configuration of instrument computer. Make sure to set up the peristaltic pump tubing on the pump.
4. When the instrument is operating at steady state, nebulize calibration standards to obtain a reliable calibration curve covering the appropri-ate calibration range. The sample is nebulized and the resulting aerosol transported by argon gas into the plasma torch. The ions produced by high temperatures are entrained in the plasma gas and extracted through a differentially pumped vacuum interface and separated on the basis of their mass-to-charge ratio by a mass spectrometer.
5. Nebulize the test samples and determine con-centrations of the different minerals by a chan-nel electron multiplier or Faraday detector and the instrument’s data-handling system.
6. First, adjust values according to the dilution factor and then according to the original sample weight.
2.4.4 PhosPhorus analysis
Phosphorus is the mineral found in highest concentrations in all cereal grains. Most of this essential mineral is bound to phytic acid and its salts. Phytic acid has several relevant physiological functions such as antioxidant protection during dormancy, storage of phosphorus and cations, and precur-sor of cell walls. In addition, phytic acid plays an important and critical role during germination. Approximately 80%
of the total phosphorus is bound to phytates in maize, rice, and wheat. Most phytic acid is found in the aleurone cells, although in the special case of maize, 80% of the phytic acid is located in the germ. The phosphorus bound to phytate has a low bioavailability (40–80%) and binds other minerals, such as calcium, magnesium, zinc, copper, and iron, lowering their availability. The availability of phosphorus and other
minerals improves after germination or mating and fermen-tation due to the production of phytases.
Most phosphorus is analyzed by the molybdate tests.
The original acid molybdate method was developed by Osmond in 1887 and became more widely used after the modifications suggested by Murphy and Riley (1962). The principle of this test is that phosphate reacts with ammo-nium molybdate to form the compound (NH4)3PO4·12MoO4
which, after it is reduced with aminonaptholsulfonic acid, forms a blue-colored complex that is measured in a spec-trophotometer. Free (unbound) molybdates will not reduce under these conditions so only the molybdate that is bound with phosphate will form the blue compound. This blue color has a maximum absorption of light at a wavelength of 690 nm or 710 nm. The other test is known as vanadate–
molybdate. In this similar assay, the vanadate–molybdate yields an orange complex that is measured in the colorim-eter at 420 nm (James 1995).
2.4.4.1 analysis of Phosphorus with the Blue molybdate colorimetric analysis
Phosphorus has been traditionally analyzed with the blue molybdate colorimetric assay, in which the phosphorus pres-ent as orthophosphate reacts with ammonium molybdate yielding a purple complex commonly known as molybdenum blue. The absorbance at 710 nm of the solution is directly related to the phosphorus concentration.
2.4.4.1.1 Molybdenum Blue Colorimetric Assay A. Samples, Ingredients, and Reagents
• Dry or wet ashed samples (refer to procedures in Sections 2.4.1.1 or 2.4.1.2)
• Distilled deionized water
• Ammonia
• Sulfuric acid
• Phenolphthalein indicator
• Phosphorus standard (0.1 mg/mL)
• Ammonium molybdate
• Tin chloride
B. Materials and Equipment
• Spectrophotometer
• Spectrophotometer cuvettes
• Test tube shaker or vortex
• Volumetric flasks (100 mL)
• Graduated cylinders (100 mL and 500 mL)
• Dropper bottle
• Diluter or automatic dispensing system
• Test tubes (10 mL capacity)
• Pipettes (1 mL, 5 mL, and 10 mL)
• Thermometer
• Volumetric flask (100 mL) C. Procedure
1. Prepare a 1:4 ammonia solution, a 4% ammo-nium molybdate in sulfuric acid solution, and a 2% tin chloride solution. Use distilled deion-ized water for the preparation of the various solutions.
37 Determination of Chemical and Nutritional Properties of Cereal Grains and Their Products
2. Prepare the standard phosphorus solution by adding 0, 0.25, 0.5, 1.0, and 2 mL of the stan-dard phosphorus solution containing 0.1 mg/mL to 100 mL volumetric flasks. Add one drop of phenolphthalein, neutralize with 1:4 ammonia and make up to approximately 85 mL with dis-tilled deionized water. Add 4 mL of ammonium molybdate reagent and shake well. Then, add 0.7 mL of the 2% tin chloride solution and add water to the 100 mL mark.
3. Dilute the wet ash test sample with distilled deionized water, preferably using the automatic diluter. The dilution factor will vary according to the phosphorus concentration and calibration standard curve. For instance, pipette 5 mL of the wet ash solution into a 100 mL volumetric flask. Next, add distilled deionized water to the 100 mL mark and shake contents. Then, obtain exactly 10 mL of the diluted test sample and place it in a 100 mL volumetric flask. Add one drop of phenolphthalein, neutralize with 1:4 ammonia and make up to approximately 85 mL with distilled deionized water. Add 4 mL of ammonium molybdate reagent and shake well.
Then, add 0.7 mL of the 2% tin chloride solution and add distilled water to the 100 mL mark.
4. Turn on the spectrophotometer at least 15 min-utes before the test and set up the wavelength to 710 nm.
5. Read the absorbance of the standard solutions to construct the calibration curve. The standard solutions contain 0, 0.25, 0.5, 0.75, and 1 mg of phosphorus/100 mL. Determine the slope of the curve and the r2 factor (should be >.99).
Then, read the absorbance of the test solution.
If the color of the obtained solution is greater or inferior compared with the highest and small-est standard, repeat the procedure adjusting the dilution factor.
6. Calculate the % phosphorus = (absorbance × 10)/sample weight (g) × volume of ash solution diluted to 100 mL.
2.4.4.1.2 Analysis of Phosphorus with the Molybdate–
Vanadate Colorimetric Method
Phosphorus is, in most cases, analyzed by the vanadium phosphomolybdate colorimetric method in which the phos-phorus present as the orthophosphate reacts with a vanadate–
molybdate reagent to produce a yellow–orange complex. The absorbance at 420 nm of the solution is directly related to the phosphorus concentration (James 1995).
A. Samples, Ingredients, and Reagents
• Dry or wet ash samples (refer to procedure in Section 2.4.1)
• Phosphorus standard (1000 mg/L)
• Nitric acid
• Test tube shaker or vortex
• Volumetric flasks (100 mL)
• Graduated cylinders (100 mL and 500 mL)
• Diluter or automatic dispensing system
• Test tubes (10 mL capacity)
• Pipettes (1 mL, 5 mL, and 10 mL)
• Thermometer
• Volumetric flask (1 L) C. Procedure
1. Prepare the vanadate–molybdate reagent by dissolving 20 g of ammonium molybdate in 400 mL of water at approximately 50°C.
Dissolve 1 g of ammonium vanadate in 300 mL of boiling distilled water, cool and gradu-ally add 140 mL of concentrated nitric acid.
Obtain the wet or dry ash stock solution (refer to procedures in Sections 2.4.1.1 or 2.4.1.2).
Add the vanadate– molybdate solution to the acid vanadate solution and dilute to exactly 1 L with distilled water.
2. Prepare the standard phosphorus solution by dissolving 4.39 g of potassium dihydrogen phosphate (KH2PO4) in 1 L of distilled water.
Dilute 1 to 10 with distilled water to give a solu-tion containing 0.1 mg phosphorus/mL.
3. Prepare the series of phosphorus standards by adding 0, 2.5, 5, 7.5, and 10 mL of the stand ard phosphorus solution of step 2 to 100 mL volu-metric flasks and dilute with 30 mL of distilled deionized water. Then, add 25 mL of the vana-date–molybdate reagent and mix contents and then add distilled deionized water to the 100 mL mark. Label each of the standard solutions.
4. Dilute the wet ash test sample with distilled deion-ized water, preferably using the automatic diluter.
The dilution factor will vary according to the phosphorus concentration and calibration stan-dard curve. For instance, pipette 2 mL of the wet ash solution into a 100 mL volumetric flask. Next, add 25 mL of the vanadate– molybdate reagent and mix contents and then add distilled deionized water to the 100 mL mark. Allow the solution to stand 10 minutes before measuring absorbance.
5. Turn on the spectrophotometer at least 15 min-utes before the test and set up the wavelength at 420 nm.
6. Read the absorbance of the standard solution to construct the calibration curve. The standard solutions contain 0, 0.25, 0.5, 0.75, and 1 mg of phosphorus/100 mL. Determine the slope of the curve and the r2 factor (should be >.99).
Then, read the absorbance of the test solution.
38 Cereal Grains: Laboratory Reference and Procedures Manual If the color of the solution obtained is greater or
inferior compared with the highest and small-est standard, repeat the procedure adjusting the dilution factor.
7. Calculate the % phosphorus = (absorbance × 10)/sample weight (g) × volume of ash solution diluted to 100 mL.
2.4.5 sodiuM Chloride analysis
Sodium chloride is widely used to prepare a wide range of cereal-based food items. Almost all wheat-based foods (breads, cookies, pasta, and tortillas), breakfast cereals, and snack foods contain significant amounts of sodium chlo-ride that is added to enhance gluten formation, as a preser-vative, and as a seasoning agent. In addition, the sodium of the sodium chloride molecule should be declared in the nutritional label because it has negative health implications (i.e., hypertension). There are many analytical procedures to determine the sodium and sodium chloride present in foods.
The most popular methods are the Mohr and Volhard titra-tion tests, the Dicromat analyzer, and the Quantab chloride strip tests. The Dicromat analyzer is a fast method in which soluble salt is read directly from a digital readout whereas the Quantab chloride titrator is ideal for quickly and quanti-tatively determining salt.
2.4.5.1 mohr titration method
The Mohr method uses chromate ions as an indicator in the titration of chloride ions with 0.1 M silver nitrate standard solution. After the precipitation of the whole amount of chlo-ride (usually as white silver chlochlo-ride), the first excess of titrant results in the production of a silver chromate precipitate, which indicates the endpoint. When the stoichiometry and moles consumed at the endpoint are identified, the amount of chloride in the experimental sample can be determined.
A. Samples, Ingredients, and Reagents
• Samples of cereal-based products
• 5% Silver nitrate solution
• Distilled and deionized water
• 5% Potassium chromate solution
• Silver nitrate (0.1 N) B. Materials and Equipment
• Analytical scale
• Volumetric flasks (0.1 L, 0.5 L, and 1 L)
• Graduated cylinders (25 mL)
• 250 mL and 500 mL Erlenmeyer flasks
• 125 mL Erlenmeyer flask
• Filter paper
• Chronometer
• Laboratory or coffee mill
• 10 mL pipette
• Stirring rod
• Burette with 0.1 mL divisions
• 125 and 250 mL funnels
• 500 mL graduated cylinder
C. Procedure
1. Prepare the following reagents:
a. 5% K2CrO4 indicator solution by dissolving 5 g K2CrO4 in 100 mL of distilled deionized water.
b. Standard AgNO3 solution. Dissolve 8.49 g of AgNO3 in a 500 mL volumetric flask and make up to volume with distilled water. The resulting solution is approximately 0.1 M.
2. Mill the test sample using a laboratory or coffee mill. For fat-rich products, the material can be crushed by hand.
3. Weigh 25 g of the test sample. Transfer sam-ple to 500 mL beaker or Erlenmeyer flask and add 250 mL of distilled deionized water from a graduated cylinder.
4. Stir and let stand at least 5 minutes (make sure the entire sample is covered by water).
5. Stir again and filter into a beaker.
6. Pipette 10 mL of the clear filtrate into a clean 250 mL Erlenmeyer flask.
7. Add 25 mL of distilled water and stir.
8. Add six to seven drops of potassium chromate indicator and stir.
9. Fill burette with 0.1 M of silver nitrate and titrate the salt solution with indicator until the solution changes to a faint brick-red color.
10. Register the volume (in mL) of 0.1 N silver nitrate solution dispensed by the burette.
11. Determine the percentage of chloride and sodium chloride in the sample using the follow-ing equations:
2.4.5.2 Volhard titration method
Chloride ions can also be determined by the Volhard pro-cedure. Briefly, the protocol starts when the food sample is boiled in diluted nitric acid, and then the addition of excess silver nitrate and back-titration with potassium thiocyanate.
The addition of excess silver nitrate to a solution containing chloride ions results in the precipitation of silver chloride.
The concentration of chloride can then be analyzed by back- titrating of the excess silver ions with a thiocyanate solution to create a silver thiocyanate precipitate. Ferric ion (Fe3+) is usu-ally used as an indicator for the titration because as soon as all the silver ions have reacted, the minimum excess of thiocyanate will react with the indicator to yield a bright-red complex.
A. Samples, Ingredients, and Reagents
• Samples of cereal-based products
• HNO3
• KMnO4
• Ferric ammonium sulfate [Fe2NH4(SO4)2]
39 Determination of Chemical and Nutritional Properties of Cereal Grains and Their Products
• Distilled and deionized water
• AgNO3
• Erlenmeyer flasks (300 mL)
• Magnetic stirrer
• Volumetric flasks (1 L)
• Graduated cylinders (25 mL)
• Laboratory or coffee mill
• Hot stirring plate
• Burette
• 5 mL and 20 mL pipettes
• Brown glass bottles (1 L) C. Procedure
1. Prepare the following reagents:
a. Standard silver nitrate solution (0.5 M). In a 1-L volumetric flask, dissolve 84.94 g of AgNO3 in distilled deionized water. Bring volume to 1 L and store in a brown bottle.
b. In a 100 mL volumetric flask, dissolve 7.42 g of KMnO4 in 100 mL of distilled deionized water and store in a brown bottle.
c. In a 100 mL flask, dissolve 22.4 g of ferric ammonium sulfate [FeNH4(SO4)2] in 100 mL of distilled deionized water and store in brown bottle.
d. Dissolve 29.2 g of reagent grade NaCl in distilled deionized water and bring volume to exactly 1 L (0.5 M solution).
e. Dissolve 38.06 g of NH4SCN in distilled deionized water, bring volume to exactly 1 L and store in a brown glass bottle (0.5 M).
2. Mill the sample using a laboratory or coffee mill.
3. In an analytical scale, weigh 2.5 g of the sample and carefully place it in an Erlenmeyer flask (250 mL).
4. Add 29 mL of the 0.5 M AgNO3 and mix. Then, carefully add 15 mL of HNO3 and place on a hot plate inside a laboratory hood. Boil the result-ing acid solution for 10 minutes. Add 2 mL of the KMnO4 and continue boiling until the blue–
purple color disappears or until the sample has a slight yellowish coloration.
5. Add 25 mL of distilled deionized water and keep boiling for 5 minutes. Remove the Erlenmeyer flask from the hot plate and allow contents to cool down. After cooling, add distilled deion-ized water to a volume of 150 mL.
6. Add 25 mL of diethyl ether and mix contents.
7. Add 5 mL of the ferric ammonium sulfate indi-cator right before titration. Place a magnetic stirrer in the Erlenmeyer flask and titrate with 0.5 M of NH4SCN until the solution turns light
brown–orange in color. Register the volume (in mL) of NH4SCN dispensed by the burette.
8. Calculate the percentage of NaCl with the fol-lowing formula:
{[(Mol AgNO3) (mL AgNO3)/100] − [(Mol NH4SCN/100)
× 58.5 × 100]}/sample weight.
2.4.5.3 analysis of salt content with the dicromat analyzer
This method was previously described by Rooney (2007).
A. Samples, Ingredients, and Reagents
• Samples of cereal-based products
• Distilled and deionized water B. Materials and Equipment
• Digital scale
• Dicromat salt analyzer (Noramar Co., Chagrin Falls, OH)
• Beaker (250 mL)
• Coffee filters
• Laboratory or coffee mill
• Electric blender
3. Add exactly 150 mL of distilled deionized water.
4. Grind the sample in the electric blender to obtain a slurry.
5. After 10 minutes of resting, filter the slurry through a coffee filter. Obtain 100 to 120 mL of the filtrate.
6. Determine salt content of the filtrate by pouring the filtrate through the dicromat cylinder which has been calibrated following instructions from the manufacturer.
7. Salt content is read directly from the digital readout.
2.4.5.4 analysis of chloride (salt) with the Quantab strip test
The Quantab chloride titrator is ideal for quickly and quantita-tively determining salt. The test simply consists of dipping the chloride strip in the sample. When test strips are placed on a solution, the fluid reacts with the strip. The height of the col-umn is proportional to the total chloride or salt concentration. A calibration table converts the strip value to parts per million of chloride ion. The approximate titration range is 0.005% to 1%
NaCl equivalent to 30 ppm to 6000 ppm, respectively.
A. Samples, Ingredients, and Reagents
• Samples of cereal-based products
• Distilled and deionized water
40 Cereal Grains: Laboratory Reference and Procedures Manual
• Quantab chloride titrator strips
• 2.5% Salt solution B. Materials and Equipment
• Analytical scale
• Erlenmeyer flasks (300 mL)
• Volumetric flasks (1 L)
• Graduated cylinders (100 mL)
• Laboratory or coffee mill
• Conical funnel
• Whatman no. 1 filter paper
• Chronometer C. Procedure
1. Obtain a representative sample of the food and grind it in a coffee or laboratory mill. If the sample is rich in fat (i.e., snacks), crush sample by hand.
2. Weigh 10 g of finely blended sample and add 90 mL of boiling distilled water and blend for 30 seconds. Allow to sit for 1 minute then blend again. Cool to room temperature. Filter at least 5 mL through a Whatman no. 1 filter paper folded into a cone as specified and supported in a conical funnel.
3. Place lower end of Quantab chloride titrator in the solution. Allow test solution to saturate the
3. Place lower end of Quantab chloride titrator in the solution. Allow test solution to saturate the