Procedures

Inspection

1. All fresh plant tissue should be carefully examined to determine physical condition, type of material, extent of contamination, and freedom from disease.
2. When whole plants are received, remove the roots, stems and foreign material if included in the sample.
3. Remove soil particles from plant tissue by gentle brushing. Washing under cold water tap is sometimes necessary to remove water soluble contamination. Remove excess water before drying.
4. Plant material that is decayed or moldy should be discarded.
5. Make note of the physical condition of the sample and what preparation steps were taken if other than normal.
6. Assign laboratory number to sample bag, data sheet, and sample submission form. The first digit of the lab number is 1 for plant. The second two digits are the month, the last four digits are the sample number for the year.
7. Check analyses desired on the data sheet.

Drying

Oven Drying

1. Place paper sample bag (do not use waxed or plastic bags) in oven set at 70-80oC. Drying may require 12 to 24 hours depending on the original condition of sample.
2. To facilitate rapid drying, loosen and separate plant parts in sample bag.
3. Samples should be dried to a crisp and brittle state and ground immediately.
4. Assign laboratory number to data sheet and sample submission form.
5. Use sharpie to write lab number on sample bag and oven dried paper bag.
6. The first digit of the lab number is 2 for feed, the next two digits are the month, the last four digits are the sample number for the year.
7. Check analyses desired on the data sheet. Place an "A" next to the sample number if the sample is to be analyzed "as-is".

1. For sample to be determined on an dry basis i.e., non- 'A' sample weigh dried paper bag and record weight to 2 decimal places (keep extra bags in oven set at 60oC). For sample to be determined on an as-sampled basis, i.e., an 'A' sample, go to step 7.
2. Place a representative portion of the sample into the paper bag.
3. Weigh paper bag and sample and record weight to 2 decimal places.
4. Dry sample at 60oC overnight.
5. To prepare for the next morning, label 100 mL beaker. Weigh and record weight to 2 decimal places.
6. After sample has been dried overnight, weigh bag and sample and record weight to 2 decimal places. (NOTE--When samples await grinding, they should be left in the oven with heat to prevent moisture absorption by the dried material.)
7. Grind sample.
8. Mix ground sample thoroughly then fill labeled glass vial with ground sample for analysis.
9. Pour approximately 10 grams ground sample into pre-labeled and pre-weighed 100 mL beaker. Weigh and record weight to 2 decimal places.
10. Dry beaker plus sample at 105oC for 3 hours.
11. After sample has been dried at 105oC, place in desiccator, then weigh beaker and sample and record weight to 2 decimal places.

Calculations

Calculation of non-'A' samples 

• bag weight = a
• bag weight plus sample = b
• bag weight plus sample after drying overnight at 60oC = c
• beaker weight = x
• beaker weight plus sample = y
• beaker weight plus sample after drying overnight at 105oC = z

 % DM = (c-a)/(b-a) * (z-x)/(y-x) * 100

Calculation of 'A' samples 

% DM = (z-x)/(y-x) * 100

Calculation for % Moisture

% MOISTURE = 100 - DM

Grinding

Large Sample

1. Using a large Wiley mill, grind the samples to pass a 1 mm sieve.
2. Grind the entire sample and mix thoroughly before transferring the sample or an aliquot to a labeled glass vial.

Small Sample

1. Grind in the small Wiley mill to pass a 20-mesh sieve.

Feed Sample to be Determined on an As-Sampled Basis - 'A' Sample

1. Pour about 50 grams of sample into coffee grinder. Grind for approximately 30 seconds.
2. Mix sample then transfer to a labeled glass vial.

NOTE -- When samples await grinding, they should be left in the oven with heat to prevent moisture absorption by the dried material.

1. Fill a 150 mL beaker half full with fresh silage and add sufficient water to set sample leaving about 1/2 inch of free water over the top.
2. Stir and let stand 30 minutes.
3. Stir and remove the water from the silage by pouring into another beaker.
4. Read the pH of the solution immediately using a calibrated pH meter and buffers, pH 4.0 and pH 7.0. Record value with one decimal.

Reagents

1. 1 N HYDROCHLORIC ACID - Dilute 83.3 mL conc. HCl to 1 L Deionized H2O (dH2O) or 170 mL to 2 liters d H2O).

Apparatus

1. MUFFLE FURNACE
2. "HIGH FORM" PORCELAIN CRUCIBLES
3. 100 mL VOLUMETRIC FLASKS
4. 13 X 100 mm FLINT GLASS TEST TUBES

Procedure

1. Weigh 1.000 g sample into "high form" porcelain crucible.
2. Place sample into furnace and increase temperature gradually until temperature reaches 500oC.
3. Ash for 1 h after reaching temperature.
4. Remove sample from furnace and let cool.
5. Dissolve ash by adding 10 mL 1 N HCl to crucible.
6. Allow to equilibrate for 15 min.
7. Quantitatively transfer dissolved ash into 100 mL volumetric flasks.
8. Wash down sample with dH2O.
9. Dilute to volume with dH20 and shake.
10. Place aliquot into ICP test tube.
11. Record values directly from ICP sheet with no decimal.

Quality Control

1. Ash NIST peach sample as needed. Run solution along with standards and samples daily. Prior to use, the NIST peach sample should be dried overnight at 80oC and stored in the desiccator.
2. Check ICP standards at midpoints and end of run.

References

1. See page Dry Ash Procedure.

Reagents

1. 1 N HYDROCHLORIC ACID - Dilute 83.3 mL conc. HCl to 1 L Deionized H20.
2. 6 N HYDROCHLORIC ACID - Dilute 50 mL conc. HCl to 100 mL deionized H20.

Apparatus

1. MUFFLE FURNACE
2. "HIGH FORM" PORCELAIN CRUCIBLES
3. 100 mL VOLUMETRIC FLASKS
4. 13 X 100 mm FLINT GLASS TEST TUBES

Procedure

1. Weigh 1.000 g sample into "high form" porcelain crucible.
2. Place sample into furnace and increase temperature gradually until temperature reaches 500oC.
3. Ash for 3 h after reaching temperature.
4. Wet sample with a small amount of dH20, then add 5-10 mL of 6 N HCl and bring to near dryness on hot plate.
5. Dissolve ash by adding 10 mL 1 N HCl to crucible.
6. Quantitatively transfer dissolved ash into 100 mL volumetric flasks.
7. Wash down sample with dH20.
8. Dilute to volume with dH20 and shake.
9. Place aliquot into ICP test tube.

References

1. Ellis, R. J., Jr., J. J. Hanway, G. Holmgren, D. R. Keeney, and O. W. Bidwell. Sampling and Analysis of Soils, Plants, Waste Waters, and Sludge-Suggested Standardization and Methodology. Agricultural Exp. Station, Kansas State University, Manhattan, Res. Pub. 170. North Central Regional Pub. 230.
2. Haynes, R. J. 1980. A Comparison of Two Modified Kjeldahl Digestion Techniques for Multi-Element Plant Analysis with Conventional Wet and Dry Ashing Methods. Comm. in Soil Sci. and Plant Anal. 11(5): 459-467.

Reagents

1. CONCENTRATED NITRIC ACID - HN03
2. 30% HYDROGEN PEROXIDE - H202 (stored in refrigerator)

Apparatus

1. DIGESTION BLOCK
2. 100 mL STRAIGHT DIGESTION TUBES - CALIBRATED AT 50 mL.
3. SILICONE-RUBBER STOPPERS
4. PIPET DISPENSERS
5. TEST TUBES FOR ICP AUTOSAMPLER

Procedure

1. Weigh 0.5000 + 0.0005 g into labeled 100 mL digestion tubes.
2. Under hood, add 5 mL conc. HNO3 rinsing down sides during the addition. Predigest samples for 30 min. by leaving under hood with no heat.
3. Place digestion tubes on digestion block and heat at 125oC for 1 and 1/2 h. Make sure front and back shields are on rack. Close sash. Record ending heat time on board.
4. Remove samples from block and let cool for a few minutes.
5. Add 3 mL 30% H202 slowly to each sample rinsing down sides during the addition. Allow frothing to settle. Place tubes back on block. Close sash.
6. Heat for 1 h. Record ending heat time on board.
7. Repeat steps 4-6.
8. Increase block temperature to 200oC. Heat for 1 h. Record ending heat time on board. This should bring the samples to dryness. If sample is still wet, heat a little longer.
9. Remove dry samples one by one from the block adding 10 mL of 1:10 HN03. Set samples in second rack under hood.
10. Let samples cool for 15 minutes.
11. Dilute to 50 mL with deionized water.
12. Stopper and shake vigorously.
13. Transfer aliquot to labeled ICP tubes.
14. For P, K, Ca, Mg, and S move decimal on ICP sheet two places to the left and record 2 decimals. For Zn, Cu, Mn, and Fe record values directly from ICP sheet with no decimals.

Quality Control

1. Ash an NIST peach sample on Monday along with samples. Prior to use, the NIST peach sample is dried at 80oC and stored in the desiccator.
2. Save solution and run daily on ICP with standards and samples.
3. Check ICP standards at midpoints and end of run.

References

1.  Jones & Case. Analyzing plant tissue samples.
2. Jones, J. B., Wolf, B., and Mills, H. A. Plant Analysis Handbook. Micro-Macro Publishing, Inc. p.197.
3. Plank, C. O. Plant Analysis Reference Procedures for the Southern Region of the United States. Southern Cooperative Series Bulletin 368, May 1992. p.9.

(This procedure is not recommended for ICP analysis.)

Reagents

1. CONCENTRATED NITRIC ACID - HNO3
2. 70% PERCHLORIC ACID - HCl04

Apparatus

1. PERCHLORIC ACID FUME HOOD
2. HOT PLATE*
3. 150 mL BEAKERS
4. WATCH GLASS COVERS
5. 100 mL VOLUMETRIC FLASKS

Procedure

1. Weigh 1.000 g sample into 150 mL beakers.
2. Add 10 mL conc. HNO3.
3. Place beakers on hot plate and cover with watch glass covers and heat at 4.5 until all organic matter is digested - clear yellow. Remove watch glasses and continue to heat to reduce volume to near dryness (do not bring to dryness).
4. Remove beakers from hot plate and add 5 mL of 70% HCl04.
5. Return to hot plate and digest at 5.5 until white fumes appear and solution is clear (silicates will be in bottom). If S is to be determined, allow to fume a minimum of 1 1/2 h.
6. Remove beakers from hot plate. While still under the hood add about 10 mL of dH20 to beakers to cool samples and stop fuming.
7. Quantitatively transfer sample into 100 mL volumetric flask with dH2O - cool to room temperature.
8. Dilute to volume, stopper and shake flasks.
9. Allow silicates to settle and transfer aliquot to test tubes. 
   *The setting 4.5 on the hot plate corresponds to about 100oC, the setting 5.5 corresponds to about 200-250oC.

References

1. Ellis, R. J., Jr., J. J. Hanway, G. Holmgren, D. R. Kenney, and O. W. Bidwell. Sampling and Analysis of Soils, Plants, Waste Waters, and Sludge-Suggested Standardization and Methodology. Agricultural Exp. Station, Kansas State University, Manhattan, Res. Pub. 170. North Central Regional Pub. 230.
2. Haynes, R. J. 1980. A Comparison of Two Modified Kjeldahl Digestion Techniques for Multi-Element Plant Analysis with Conventional Wet and Dry Ashing Methods. Comm. in Soil Sci. and Plant Anal. 11(5): 459-467. 

Blanks

EDTA 

Samples 

Print Report 

Clean Up 

If the power is out for an extended period of time and the furnace cools down, it must be powered up for at least 1 h before using.  Combustion furnace temp can be checked by clicking on Diagnostics - Ambients.  Temp should be 825 - 875 C.

If the sliding head is not sliding to drop the samples - Click Diagnostics - Solenoids.  Click 4th box down (Block Seal).  Click 1st box (Block open).  You should hear and see the block open.  Unclick both boxes.  Close.

Reagents

1. DIGESTION MIXTURE - 3 kg K2S04 and 600 g CuS04.5H20 (or 384 g CuS04 anhydrous) (use CuS04.5H20 if available). (This mixture can be purchased as Kjeltab catalyst tablets.)
2. CONC H2S04.
3. NITROGEN STANDARD - Accurately weigh 4.7166 g (NH4)2S04 (dried and stored in the desiccator), dissolve in deionized water, then dilute to 100 mL. This will give you a 1% N solution. To check distillation procedure, pipet 1 mL into digestion tube, dilute with about 80 mL deionized water and follow the distillation procedure. For calculations, 1 mL = 1 g.
4. Glycine - Weigh 0.1000 g (stored in desiccator) and digest along with samples. Value should be 18.66 + 0.9% N.

Apparatus

1. DIGESTION BLOCK
2. FUME MANIFOLD
3. 250 mL DIGESTION TUBES

Procedure

1. Weigh 0.500 g plant tissue or 0.500 g feed sample into a 250 mL digestion tube.
2. Add 2 Kjeltab catalyst tablets.
3. Add 12 mL of conc. H2S04.
4. Place on digestion block with front and back shields.
5. Heat at 400oC for 45 min. (should be 30 min. after samples have cleared).
6. Remove from block and allow to cool 15 - 20 min. with shields off.
7. Add approximately 75 mL dH20.
8. Sample is ready for distillation.

Quality Control

1. Ash 0.1 g glycine along with samples on first set. Ensure value of N is 18.66 + 0.9%.
2. Prior to distilling samples, pipet 1 mL of (NH4)2SO4 solution into a digestion tube for each distillation unit, dilute each with 80 mL deionized water, and distill to ensure each sample is 1.00 + 0.05% N.

References

1. Brenner, J. M. 1965. Total Nitrogen. In C. A. Black (ed) Methods of Soil Analysis, Part II, SSSA., Madison, Wis. p.1149-1178.
2. Horwitz, W. (ed). 1970. Official Methods of Analysis of the AOAC, 11th Edition, Washington, D. C. p.16-17.
3. Tecator Manual. Kjeltec System 2300 Distilling Unit. Tecator, Hoganas, Sweden.

Reagents

1. 40% NaOH solution (w/w) (16.67 N NaOH)
2. BORIC ACID INDICATOR SOLUTION - Dissolve 400 g H3B03 in about 2 L of hot deionized water (use a 4 L beaker, heat and stir on a hot plate). When H3BO3 is dissolved, transfer to 20 L carboy. Dilute to approximately 18 L with dH20. Add 200 mL of mixed indicator solution and dilute to 20 L. Adjust pH to 5.0 using 2 mL of 1.67 N NaOH.
3. STOPPER TIGHTLY!
4. STANDARDIZED H2S04 SOLUTION - Dilute 64 mL conc. H2S04 to 20 L with deionized H20. Be sure to standardize solution by following the standardization of acids and bases procedure. (Final concentration should be close to 0.1142 N.)
5. MIXED INDICATOR SOLUTION - Weigh 0.33 g Bromocresol green and 0.165 g Methyl red. Transfer weighings to a 500 mL volumetric flask using an ethanol squirt bottle. Dilute to volume with ethanol. Carefully slide a stir bar into flask and stir until the bromocresol green and methyl red are dissolved. Remove stir bar and store solution in plastic bottle.
6. NITROGEN STANDARD - Accurately weigh 4.6445 g (NH4)2S04 (dried and stored in desiccator), dissolve in deionized water, then dilute to 100 mL. This will give you a 1% N solution. To check distillation procedure, pipet 1 mL into digestion tube, dilute with about 80 mL deionized water and follow the distillation procedure. For calculation, 1 mL = 1 g.

Apparatus

1. KJELTEC 2300 DISTILLATION UNIT
2. 250 mL DIGESTION TUBES

Operation

1. Turn power on.
2. Turn printer on.
3. Turn water on.
4. Go to manual mode.
5. Flick titrating tube to get bubbles out. Dispense titrant several times to get bubbles out.
6. Go to steam on. Press arrow key to turn steam on. Allow to warm up for 5 minutes.
7. Press arrow key to turn steam off.
8. Go to analysis mode.
9. Select program.
10. Go to result. Select blank.
11. Have tube containing water in place.
12. Close door.
13. Run at least two blanks. Make sure the blank value is around 0.10 or less.
14. Go to result and change to % Nitrogen. (The blank number above should read the last blank value you ran.)
15. Put a tube with 1 mL of the 1% N solution standard and some water in place. Input 1.0 for the weight.
16. Close door and let run. Readout should be close to 1% (+ 0.05%).
17. Proceed with samples. Run the glycine check first. Input the correct weight for each sample. (The result for the glycine check should be close to 18.66 % N.)
18. If you have blanks within the set run a tube with just water first to flush out the system, then run the blank. (Change result to blank for running the blank then back to % N for the samples. Input the weight for the samples as above.)
19. Record numbers from printout into nitrogen book and onto lab sheets with 2 decimals.

Calculations

1. %N = 14.01 x N H2SO4 x (mL acid)/(sample wt(g) x 10)
2. % Crude Protein = %N X 6.25

Shut Down and Maintenance

Daily

• Place tube with water in unit. Select manual. Go to steam on. Press arrow key and allow generator to run for about 5 minutes. Press arrow key to turn steam off. Remove tube and dispose of contents.
• Turn power off.
• Turn water off.
• Turn printer off.
• Clean drip tray. Clean the shield. Rinse and inspect the rubber adapter. Wipe any spillage with a damp cloth. Squirt water into titration vessel and onto pins to clean.
• Place empty tube in unit and close shield.

Weekly

• Check and clean around reagent tank screw caps to remove crystals. Every one to three months:
• Clean alkali pump. Put 2 L of warm distilled water in a beaker. Put alkali tube into beaker. Select manual mode. Select add alkali several times. Empty distillation tube as needed. (A rinse using a half a liter of warm water is sufficient.) Replace hose into alkali tank. Flush out residual water and remove entrapped air.
• Clean receiver solution dispensing system as above only if contamination has occurred.
• Check volume of alkali. Go to manual mode. Select the pump you want. Measure dispensed liquid. Adjust as necessary.
• Check volume of receiver solution. Put drain tube into measuring cylinder. Go to manual mode. Select add receiver. Adjust as necessary.
• Clean splash head. Put 25 mL distilled water and 25 mL acetic acid into distillation tube. Select manual. Go to steam on for 5 to 10 minutes. Turn off steam. Replace tube with tube containing 50 mL distilled water. Distill for 5 minutes. Repeat the distillation three times with fresh water.

Yearly

• Inspect all tubing and connectors.

References

1. Brenner, J. M. 1965. Total Nitrogen. In C. A. Black (ed) Methods of Soil Analysis, Part II, SSSA., Madison, Wis. p.1149-1178.
2. Horwitz, W. (ed). 1970. Official Methods of Analysis of the AOAC, 11th Edition, Washington, D. C. p.16-17.
3. Tecator Manual. Kjeltec System 2300 Distilling Unit. Tecator, Hoganas, Sweden.

PHENOLPHTHALEIN SOLUTION - Dissolve 0.5 g phenolphthalein in 85 mL 95% ethanol and dilute to 100 mL with deionized H20.

Standardization of Na0H

1. Accurately weigh out three 0.2300 to 0.2500 g portions of KHP (Potassium Hydrogen Phthalate) on weighing paper and quantitatively transfer to three 250 mL Erlenmeyer flasks. (KHP must be dry; dry in oven before weighing and cool in desiccator before using.)
2. Add 50 mL deionized water and dissolve by stirring.
3. Add 3 drops of phenolphthalein solution.
4. Titrate with unknown NaOH solution (approximately 0.05 M) to endpoint (very faint persistent pink).

Calculations

1. # equivalents KHP = wt. KHP - 204.23 g/eq.
2. N NaOH = (# equivalents KHP)/(mL NaOH)(0.001)

Standardization of H2SO4

1. Pipet 20 mL standardized Na0H into four 150 mL beakers.
2. Add 3 drops of phenolphthalein solution.
3. Titrate with unknown H2SO4 solution to endpoint.

Calculations

1. N H2SO4 = (N NaOH x mL NaOH)/mL H2SO4
   (Take out the high and low numbers and average middle two. If there is too much discrepancy, try again.)

References

1. Peters, D. G., J. M. Hayes, and G. M. Hieftje. 1976. A Brief Introduction to Modern Chemical Analysis, p.87-118. W. B. Sanders Company, Philadelphia.

Reagents

1. ADS - ACID DETERGENT SOLUTION - Weigh 400 g Hexadecyltrimethyl-ammonium bromide in a 2 L beaker under the hood. (This material is harmful if inhaled or absorbed through the skin so a respirator and gloves should be worn while making this solution.) Add deionized water to this beaker and stir until mixture can be poured. Transfer this solution to a 4 L beaker.  Bring the volume up to about 3 L with water  and stir thoroughly with a long stirring rod. Very slowly add 556 mL conc. H2S04 to the solution and continue to stir vigorously with the stirring rod. (If the acid is poured in too quickly without stirring, the solution may turn brown. If this happens add a little more water and continue to stir.) At this point there may still be some undissolved material, so place the beaker on a heater/stirrer and stir with heat until the material is dissolved. Transfer this solution to a 20 L carboy and dilute to 20 L with deionized H20. This may be purchased as a concentrate or straight solution.
2. ACETONE

Apparatus

1. ANKOM FIBER ANALYZER #F200
2. 3 3000 mL BEAKERS 
3. HOT PLATE
4. FILTER BAGS (ANKOM CO. #F57)  
5. HEAT SEALER #1915
6. BAG HOLDER

Procedure

1. Prepare Filter Bags/Samples.
   1. Weigh labeled filter bag (ANKOM Co. #F57). Record weight.  
   2. Tare bag holder containing sample bag.  Weigh 0.5 g (+0.01 g) of sample  into bag. Record weight of sample.
   3. Seal the bag closed within 1 cm from the open edge using the heat sealer.
   4. Spread sample uniformly inside the filter bag. This should be done by shaking and tapping the bag to eliminate clumping. A uniform distribution of the sample inside the bag is very important to achieving good precision and accuracy in the analysis.
   5. Place 24 bags in the bag suspender. The bag suspender is composed of 9 individual baskets and a center post. The 9th basket acts as a top of the 8th. Place three bags in each basket (separated 120 degrees apart). Stack baskets on center post ensuring bags on different levels are not positioned directly above each other in order to maximize the flow of solution around each bag. (Run a check with each batch.)
2. Fill pressure vessel with 2000 mL of ambient temperature AD or ND solution. Less bags and solution can be used per study but a minimum of 1500 mL of solution has to be used in the digestion vessel. (See note below*.)
3. Place bag suspender with samples into the solution in vessel. Place weight on top of samples.
4. Start timer (60 min. for ADF and 75 min. for NDF), screw down vessel top and turn agitation and heat on.
5. While samples are being digested heat 9 L of rinse water to 90-100oC.
6. Turn heat and agitation off when indicated by timer, slowly open the valve and exhaust hot solution before opening lid. NOTE: THE SOLUTION IN THE DIGESTION VESSEL IS UNDER PRESSURE. THEREFORE, THE VALVE NEEDS TO BE OPENED FIRST IN ORDER TO RELEASE THE PRESSURE BEFORE THE VESSEL LID CAN BE OPENED.
7. After the solution has been exhausted close valve and open the lid. Add approximately 2 L of hot rinse water. Lower lid and turn agitate on. Repeat hot water  rinse three more times. For NDF assays add 4 mL of heat stable alpha-amylase  (ANKOM Co. #FAA) to each of the first two rinses (8 mL total). Add amylase to  beaker to mix before adding solution to vat.
8. 8. Remove filter bags from bag suspender and gently press excess water out. Place bags in a small beaker and pour in enough acetone (240 mL) to cover bags. Allow bags to soak 3 min. Remove and lightly press excess acetone out.
9. 9. Spread bags out and let air dry. Complete drying in oven (105oC) overnight.
10. Transfer samples to plastic bags. Take one fiber bag out at a time and close plastic bag with remaining samples to exclude moisture. Weigh. Record weight.
11. Calculate percent fiber: % Fiber = 100(C-(A*D))/B
    Where:
    
    • A = Bag tare weight
    • B = Sample weight
    • C = Final bag weight after digestion and drying
    • D = Blank bag correction (final oven dried weight/original blank bag weight)
    •      (use value of D=0.997)
    *NOTE ADF
    
    • < 6 samples - hold until next day
    • 7-15 samples - use 1500 mL ADF
    • 16 samples - use 1600 mL ADF
    • 17 samples - use 1700 mL ADF
    • 18 samples - use 1800 mL ADF
    • 19 samples - use 1900 mL ADF
    • 20-24 samples - use 2000 mL ADF
12. Record values with one decimal.

Quality Control

1. Run an NFTA check sample with each batch.

References

1. Goering, H. K., and P. J. Van Soest. 1970. Forage Fiber Analysis (Apparatus, Reagents, Procedures, and Some Applications). Agri. Handbook, No. 379, ARS, USDA.

Reagents

1. ADS - see page 20 for ADF determination.

Apparatus - see page 20.

Procedure

1. Weigh out 0.500 g sample into a filter bag.
2. Use procedure for ADF determination (page 20).
3. Dry in oven at 100oC.
4. Place filter bag with sample into digestion tube. Run a blank (an empty filter bag) before the sample. Determine % N by following Kjeldahl procedure (page 12) with this exception: add 3 Kjeltab tablets and 18 mL H2S04.
5. Distill sample using procedure (page 14). (Press Blank Value on control unit before running blank. This will automatically subtract the blank value from the samples.)
6. Record values with two decimals.

Calculation

1. %N = 14.01 X N H2S04 X (mL acid used - mL acid for blank)/(sample wt (g) x 10)

Reagents

1. NEUTRAL DETERGENT SOLUTION - NDS - In a 4 L beaker combine 600 g sodium lauryl sulfate, 200 mL ethylene glycol monoethyl ether, and 2 L deionized water. Mix this solution thoroughly and pour it into a 20 L carboy. Combine 372.2 g disodium ethylenediaminetetra-acetate (EDTA) dihydrate crystals and 136.2 g of sodium borate decahydrate (Na2B407.10H20) with 3 L of deionized water in 4 L beaker. Heat and stir this solution until the crystals are dissolved, then add it to the 20 L carboy. Add 91.2 g of anhydrous sodium phosphate dibasic (Na2HP04) to 1 L of deionized water, heat and stir to dissolve, then add it to the 20 L carboy. Dilute this mixture to 20 L with deionized water and mix thoroughly. pH should be between 6.9-7.1. This solution may be purchased ready made.
2. ANHYDROUS SODIUM SULFITE
3. ACETONE
4. ALPHA-AMYLASE (ANKOM CO. #FAA) (stored in refrigerator)

Apparatus - see ADF determination.

Procedure - see ADF Procedure.

*NOTE ADF

• < 6 samples - hold until next day
• 7-15 samples - use 1500 mL NDS
• 16 samples - use 1600 mL NDS
• 17 samples - use 1700 mL NDS
• 18 samples - use 1800 mL NDS
• 19 samples - use 1900 mL NDS
• 20-24 samples - use 2000 mL NDS

1. Use 1 g/(100 mL NDS) Na sulfite
2. Put solution in beaker, then sodium sulfite - stir w/stirring bar on stir plate. Add 4 mL alpha amylase. Mix solution then pour into vat. Place basket with samples into vat and weight on top.
3. See ADF Procedure.

Calculations - see ADF Procedure.

Reagents

1. DEIONIZED WATER
2. SODIUM PHOSPHATE, MONOBASIC, MONOHYDRATE (NaH2PO4.H20)
3. SODIUM TETRA BORATE, DECAHYDRATE (Na2B407.10 H20)
4. SODIUM BICARBONATE

Solution Preparation

BUFFER SOLUTION - Carefully weigh exactly 89.10 g of Sodium Tetra Borate, Decahydrate into a 250 mL beaker. Next, carefully weigh exactly 122.0 g Sodium Phosphate mono-basic, mono-hydrate into a 250 mL beaker. To a 4 L beaker, add approximately 3500 mLs deionized water and a stir bar (Large > 1"). With stir bar rotating slowly, carefully add Sodium Tetra Borate, Decahydrate. Use a squirt bottle containing deionized water to thoroughly rinse the weigh boat. Next, add the Sodium Phosphate monobasic, monohydrate. Again, use squirt bottle to thoroughly rinse weigh boat. Allow to stir until all crystals are dissolved.

IMPORTANT: DO NOT TURN STIRRER TOO HIGH AND CAUSE EXCESSIVE SPLASHING! After all crystals are dissolved, with a clean, dry stir bar retriever, remove stir bar from beaker. Rinse retriever and stir bar into beaker as bar is removed. DO NOT EXCEED THE 4000 mL VOLUME LINE DURING THIS RINSE. Bring beaker to 4000 mL mark. Transfer beaker contents into 20 L buffer carboy. Continue by adding 6000 mLs of deionized water to the 20 L carboy. This addition of deionized water is used to thoroughly rinse the beaker and bring the solution to the proper volume of 10 L (concentration). Mix the contents of the carboy. Before using, measure the pH of the solution. The normal range is 6.70-6.75.

To adjust pH up- use Sodium Bicarbonate.

To adjust pH down - use Hydrochloric Acid.

Procedure

1. Weigh 0.5 g of sample into a clean, dry 250 mL Erlenmeyer Flask. Label flask with sample number.
2. Add 50 mL of Borate-Phosphate buffer to each flask.
3. Place caps on flasks, swirl gently and let stand 1 h.
   1. IMPORTANT NOTE 3: Soy Products (461,462,463,703,704,764) must be incubated in a water bath at 39oC for 1 h.
4. After 1 h, samples are ready to filter. Filter samples through filter paper and air dry overnight.
5. Analyze filter paper and residue for crude protein according to ADFN procedure (page 23). This is insoluble protein.
6. Record values with 2 decimals.

Calculation

1. Soluble Protein % = (Sample CP% - Residue CP%) x 100/Sample CP%

References

1. NYDHIA
   Cornell Nutrition Conference proceedings (10/23/92).

Reagent

1. HEXANE

Apparatus

1. SOXHLET EXTRACTION APPARATUS
2. THIMBLES (should be kept in oven at 100^oC)
3. KIMWIPES

Procedure

1. Weigh approximately 2 g of sample. Wrap sample up in kimwipe and place kimwipe inside thimble.
2. Record weight to 4 decimal places.
3. Place thimble in labeled beaker. Dry overnight at 100^oC, reweigh immediately from oven and record weight to 4 decimals.
4. Place thimble inside extraction unit.
5. Fill boiling flask « to 2/3 full with hexane. (After cleaning flasks add a small amount of zinc metal to serve as boiling chips.)
6. Turn on cooling water.
7. Turn on heating units.
8. Allow to extract for 6 h.
9. Remove thimbles and place in beakers. Pour excess hexane into reserve jug.
10. Place thimbles in oven at 100^oC to dry overnight.
11. Weigh immediately from oven. Record weight to 4 decimal places.
12. Calculate fat % and record with one decimal.

Calculations

1. %FAT = ((wt. after drying) - (wt. after extraction)) x 100/(thimble and sample wt.) - (thimble wt.))

Quality Control

1. Analyze AAFCO check sample along with each set.

References

1. Horwitz, W. (ed). 1970. Official Methods of Analysis of the Association of Official Analytical Chemists, 11th Edition, Washington, D. C., p.128.

Reagent

1. Pet Ether (BP 35 - 65 degrees C)

Apparatus

1. ANKOM XT15
2. Analytical Balance
3. Oven
4. Ankom Fat filter bags
5. Heat sealer
6. Marking pen
7. Desiccant Pouch

Procedure

1. If the sample is > 20 % fat, refer to the manual, else continue.
2. Label filter bag and weigh into spreadsheet.
3. Weigh ~ 1 g of sample into the bag and enter sample weight into spreadsheet (W1).
4. Heat seal the bag closed within 4 mm of the top.
5. Place samples in the drying oven for 3 hrs at 105 degrees C.
6. Cool the dried samples in the Desiccant Pouch, weigh and enter the weight into the spreadsheet (W2).
7. Place filter bags with sample into the bag holder and place in the extractor.
8. Proceed according to the extraction instrument instructions.
9. When the extraction is complete, place the samples in the oven for 30 minutes.
10. Cool in the Pouch and Weigh into spreadsheet (W3).

Calculations

1. %FAT = 100 x (W2 - W3)/W1

Quality Control

1. Analyze AAFCO check sample along with each set.

Apparatus

1. 150 mL BEAKERS
2. 18 X 150 mL TEST TUBES
3. METAL FILTER

Procedure

1. Weigh 1.000 g sample into a 150 mL beaker.
2. Add 100 mL H20 and place on stirrer for 30 min.
3. Filter mixture with metal filter and pour filtrate into a large test tube.
4. Before analyzing, pour filtrate into ICP test tube.
5. Take reading directly from ICP printout with no decimals.

Calculation

1. Na (2.54) ppm = NaCl ppm

Reagents

1. EXTRACTION SOLUTION - In a 4 L beaker, add 173.2 g Al2(S04)3, 12.8 g H 3B0 3, and 0.7218 g KNO3 and 25.2 g sulfamicacid (NH2S03H) to 3 L deionized water. Stir to dissolve. Pour solution in carboy and dilute to 10 L with deionized water. Adjust to pH 3.0 adding at least 15 mL conc. NaOH (may need more).
2. N03-N STANDARDS - To make 1000 ppm N stock solution, dissolve 0.7218 g KNO3 (dry and stored in desiccator) in deionized water in a 100 mL volumetric flask. Add 1 mL preservation solution then dilute to 100 mL. (Make up blank, 10 ppm, and 100 ppm N03-N standards using stock solution and diluting with extracting solution.)
3. OUTER FILLING SOLUTION - 0.04 M ammonium sulfate - Add 0.52 g (NH4)2S04 to 100 mL vol. flask. Dilute with H20.
4. INNER FILLING SOLUTION - Orion cat. 900002
5. PRESERVATION SOLUTION - Dissolve 6.2 g Boric Acid in 100 mL hot deionized water. Allow to cool.

Apparatus

1. ORION ION ANALYZER
2. NITRATE ION ELECTRODE (ORION 93-07)
3. REFERENCE ELECTRODE (ORION 90-02)
4. MAGNETIC STIRRER

Procedure

1. Weigh 2 g plant or feed sample into 100 mL beaker. (When testing cotton petioles, weigh 0.25 g sample, follow steps 2-11, then multiply final answer by 8).
2. Add 50 mL extraction solution.
3. Let stand for 30 minutes.
4. Check levels of inner and outer filling solutions in reference electrode (see reference electrode manual for specifications).
5. To set up instrument, press 2nd function - 2 to select appropriate electrode.
6. Press speed-0 to calibrate N03-N.
7. Follow prompts - select 2 standards - Do blank correction. (Rinse electrodes and blot dry before switching solutions.)
8. Set 10 ppm standard to read 25.
9. Set 100 ppm standard to read 250.
10. Measure N03-N in sample and multiply reading by 10. Record as ppm with no decimal.
11. When finished place meter in standby mode by pressing speed-8. Submerge electrodes in proper solutions.

References

1. Baker, A. S., and R. Smith. 1969. Extracting Solution for Potentiometric Determination of Nitrate in Plant Tissue. J. Agr. Food Chem. 17:1284-1287.
2. Barker, A. V. 1974. Nitrate Determination in Soil, Water, and Plants. Massachusetts Agricultural Experiment Station Bulletin 611, p.35.
3. Cantliffe, D. J., G. E. MacDonald, and N. H. Peck, 1970. The Potentiometric Determinations of Nitrate and Chloride in Plant Tissue. New York's Food and Life Sciences Bulletin 3, p.7.

Apparatus

1. MUFFLE FURNACE
2. PORCELAIN CRUCIBLES

Procedure

1. Weigh crucible and record weight to 4 decimal places.
2. Weigh approximately 2 g into crucible.
3. Record weight to 4 decimal places.
4. Ash sample at 600^oC for 2 h.  (Bring temperature rapidly to 600)
5. Cool in desiccator and weigh within 1 h after reaching room temperature.
6. Weigh ashed sample and record weight to 4 decimal places.
7. Calculate % Ash and record value with one decimal.

Calculation

1. % ASH = ((ashed wt.) - (crucible wt.)) x 100/((crucible and sample wt.) - (crucible wt.))

Reference

1. St. John (1939), JOACI,XXII, 630.

Reagents

1. ACID MOLYBDATE STOCK SOLUTION - In a 2 L volumetric flask, dissolve 125 g ammonium molybdate in 400 mL deionized H20 by heating to 60^oC until solution is clear. Allow to cool, then dissolve 2.9 g antimony potassium tartrate in the molybdate solution. Place the flask in an ice bath in the sink and very slowly add 1500 mL con. H2S04. Cool in the ice bath and very slowly dilute to 2 L with H20. (It is very important to not let this solution get too hot. It may cause severe burns if mishandled.) Store in brown bottle under refrigeration.
2. ASCORBIC ACID STOCK SOLUTION - Dissolve 211.2 g ascorbic acid in 1500 mL deionized water and dilute to 2 L. Store in brown bottle under refrigeration.
3. WORKING SOLUTION (Prepare Fresh Daily) - Add 20 mL acid molybdate stock to about 800 mL water. Mix, then add 10 mL ascorbic acid stock and dilute to 1 L.
4. STANDARDS - 10, 80 ppm phosphorous. To make 1000 ppm P stock solution, dissolve 4.3937 g of dried KH2P04 in deionized H20 then dilute to 1 L. (10 ppm: 1 mL of 1000 ppm stock diluted to 100 mL dH20. 80 ppm: 8 mL of 1000 ppm stock diluted to 100 mL dH20.)

Apparatus

1. SPECTROPHOTOMETER
2. TEST TUBES

Procedure for Preparing Samples

1. Wet or dry ash sample as previously described.
2. Set diluter to 1:100.
3. Place hose from diluter into flask containing the P working solution.
4. Run diluter through several cycles to draw the working solution into diluter.
5. See diluter manual for instructions on diluting samples.
6. Use Vortex stirrer to insure proper mixing.
7. Allow color to develop for 30 minutes.
8. Set up spectrophotometer as follows.

Set Up of Spectrophometer

1. Turn power on.
2. Let instrument run through self test.
3. Enter wavelength at 660 nm.
4. Press <Second Function> <Go To >.
5. Allow for a 30 min. warm up.
6. Press <T% / A/C> until you get to concentration mode.
7. Asp Blk by pressing <Sample> (hold under tubing until set up).
8. Press <Second Function> <100% T / Zero A>
9. After set up, remove Blk from tubing.
10. Asp high standard by pressing <Sample> (hold under tubing until set).
11. Enter value for high standard - Press <%T / A/C> (80 ppm corresponds to 80% in sample).
12. Let set up then remove tube.
13. Read cks and samples by pressing <Sample>, record value, remove tube (can read accurately to 140 ppm or 1.40% P in sample).
14. Clean cell, leaving water in cell.
15. Disconnect pump.
16. Turn power off.
17. Cover instrument.

References

1. Murphy, J. and J. P. Riley. 1962. A Modified Single Solution Method for the Determination of Phosphate in Natural Waters. Anal Chim. Acta. 27:31-36.
2. Nelson, W. L., A. Mehlich and E. Winters. 1953. The Development, Evaluation and Use of Soil Tests for Phosphorus Availability. In W. H. Pierre and A. G. Norman (ed.). Soil and Fertilizer Phosphorus, Vol. IV Agronomy, A Series of Monographs, Academic Press, Inc., NY pp.153-188.
3. Watanbe, F. S. and S. R. Olsen. 1965. Test of an Ascorbic Acid Method for Determining Phosphorus in Water and NaHC03 Extracts from Soil. Soil Sci. Soc. Amer. Proceedings, 29:677-78.

Reagents

1. CONDITIONING SOLUTION - Dissolve 150 g NaCl in 600 mL deionized water. Add 60 mL conc. HCl, 100 mL glycerine, and 200 mL 96% isopropyl alcohol. Add glycerine first and mix well before adding isopropanol.
2. 2 N NH4OAc - Dissolve 150 g ammonium acetate (NH4OAc) in 1 L deionized water.
3. BaSO4 SEED SUSPENSION - (Prepare fresh daily.) - Dissolve 10 g BaCl2.2H20 in 50 mL hot demineralized water in a 250 mL Erlenmeyer flask. Add 1 mL of the 1000 ppm S standard. Swirl and heat to boiling. Cool rapidly under flowing water. Add 5 mL of conditioning solution.
4. STANDARDS - Prepare a 1000 ppm Sulfur standard by dissolving 0.5435 g of oven-dried K2S04 in 100 mL 0.1 N HCl. Prepare working standards of 10, 20, 30, 40 ppm S. Prepare S stock solution fresh weekly and prepare working standards fresh daily.
5. EDTA REAGENT - (Used for cleaning cell in spectrophotometer.) Dissolve 20.2 g ammonium chloride in 500 mL H20. Add 171 mL ammonium hydroxide and mix, then add 40 g of sodium (di)ethylene-diaminetetraacetate (EDTA) and dissolve. Dilute to 1 L with H20.

Equipment & Supplies

1. SPECTROPHOTOMETER
2. TEST TUBES
3. VORTEX MIXER

Procedure

1. Wet ash sample as previously described.
2. Pipet 2 mL working standards and samples into clean test tubes.
3. Add 10 mL of 2 N NH4OAc.
4. Add 2 mL of conditioning solution.
5. Mix on vortex mixer.
6. Add 1.0 mL of seed suspension. (Swirl seed suspension frequently to insure uniform distribution of BaS04 crystals.)
7. Mix each tube uniformly on the Vortex mixer. This step is important to insure uniform precipitation of BaS04.
8. Wait 30 minutes and estimate turbidity using a visible spectrophotometer. See page 32 for set up of spectrophotometer and make the following changes. Set wavelength on 440 nm. 40 ppm S corresponds to 0.40% S in sample. Can read accurately to 60 ppm or 0.60% S in sample.) Mix each sample on Vortex mixer before reading.

References

1. Butters, B., and E. M. Chenery. 1959. A Rapid Method for the Determination of Total Sulfur in Soils and Plants. Analyst 84:239-245.
2. Massoumi, A., and A. H. Cornfield. 1963. A Rapid Method for Determining Sulfate in Water Extracts of Soils. Analyst 88:321-322.
3. Standard methods for the examination of water and wastewater. 1960. 11th edition. p.241-243. Am. Public Health Assoc., Inc., New York.

Reagents

1. CALCIUM OXIDE
2. ACETIC ACID (25%) - Dilute concentrated acid with distilled water, 1:4.
3. POTASSIUM CHROMATE INDICATOR - Dissolve 5 g K2Cr04 in 100 mL deionized water.
4. STANDARD SILVER NITRATE TITRANT, 0.03 N - Dissolve 5.0964 g AgN03 in deionized water and carefully dilute to 1 L. This solution can be checked against a standard sodium chloride solution if desired. Store silver nitrate solution in a brown bottle and keep in the dark.

Apparatus

1. 50 mL BURET, FUNNEL, AND STAND
2. 1 L VOLUMETRIC FLASK
3. 250 mL ERLENMEYER FLASKS
4. MAGNETIC STIRRER
5. FUNNEL, FUNNEL STAND, AND FILTER PAPER
6. HOT PLATE AND HOT WATER

Procedure

1. Accurately weigh 1 g of ground sample into a porcelain crucible.
2. Add 0.25 g of calcium oxide and mix.
3. Weigh out 0.25 g of calcium oxide into a separate crucible to serve as a blank. To each crucible add sufficient water to give a thin paste.
4. Place sample & blk into furnace and increase temperature gradually until temperature reaches 550oC.
5. Ash at this temperature for at least 90 min.
6. Remove the sample & blk from the muffle furnace and cool.
7. Add about 15 mL of hot water to the crucibles and break the ash into a fine powder.
8. Pour the mixtures through filter paper into 250 mL Erlenmeyer flasks and rinse the crucibles with water into filter.
9. Wash the residue in the filters with five 10 mL portions of hot water. Bring up the final volume to the 100 mL mark on the Erlenmeyer with dH20.
10. Cool the filtrate and add dilute acetic acid dropwise until the solution is about pH 6.0 to 7.0. (Check pH with pH paper.)
11. Cool and add 5 drops of the potassium chromate indicator to the sample and to the blank.
12. Titrate the blank, then the sample, with standard AgN03 to a persistent light orange-red. (Be consistent in end-point recognition.)
13. Calculate value and report with 2 decimals.
14. Pour the waste into a carboy for pick up by the hazardous waste manager.

Calculation

1. %Cl = ((mL AgN03 for sample - mL AgN03 for blank) X NAgN03 X 3.545)/g sample
2. %NaCl = %Cl (1.65)

1. Weigh 1 gram sample.
2. Add 100 mL deionized water
3. Stir or shake for 30 minutes.
4. Filter.
5. Analyze solution on ICP.

Test Tube Washing

Pour acetone into bucket under hood. Swirl basket with test tubes in acetone to remove numbers. Soak test tubes overnight in detergent solution. Fill clean rinsing bucket with deionized water. Rinse test tubes in basket three times in rinsing bucket using new water for each rinse. Dry test tubes in oven.

Washing Pipets

Put all pipets in one holder. Move pipet washer to sink. Place one alcotab tablet (under sink)in bottom of washer. Insert holder with pipets. Fill with water. Let stand 1 h. Rinse with water until no suds remain (make sure rinser is refilling). Rinse with deionized water 2 or 3 times. Remove clean pipets and drain tips up in racks. Change acid water in pipet holders periodically. (2N HCL)

Washing Glassware

Run deionized water in dishpan adding some citranox detergent. All glassware, plastic bottles, stirbars, etc. are washed with soap with the exception of the volumetric flasks and their stoppers. First wash glassware with soapy water, rinse three times thoroughly with deionized water. Soak wet ash digestion tubes in a second dishpan of citranox for 30 minutes. If yellow deposits remain on tubes, soak overnight. Rinse three times with deionized water. If yellow deposits still remain, scrub with brush. Drain on cart. Next rinse stoppers or volumetric flasks three times thoroughly with deionized water. Drain on cart.

Fresh feed samples containing greater than 15% moisture are stored under refrigeration for at least one month. Dried feed and plant samples are stored in bins for at least one month. Dried and ground feed and plant samples are stored in vials for at least two months. Samples are held for rechecks if necessary. After the holding period, samples are disposed of by a laboratory technician.