Standard Operating Procedure
Title: Analysis of maize kernel carbohydrates by HPAE-PAD
Department: Agronomy
Created by: Jason Haegele
Laboratory: Crop Production & Physiology
Supervisor: Dr. Mark Westgate
Lab Supervisor: Maria Hartt me
Date approved: 26 September 2007
Procedure Overview: Sucrose, fructose, and glucose from immature/mature maize kernels
are separated by high performance anion exchange chromatography (HPAE) followed by
pulsed amperometric detection (PAD).
Equipment and reagents necessary:
50% w/w sodium hydroxide solution (Fisher SS254-1)
Stir plate
Magnetic stir bars (2)
Glucose (Fisher D16-500)
Fructose (Fisher L95-500)
Sucrose (Fisher S5-3)
Maize kernel carbohydrate samples (obtained during Starch Quantification procedure)
Wood applicator sticks
Deionized water
50 ml volumetric flasks (4)
50 ml glass bottles (4)
5 ml volumetric pipettes (3)
Sonicating bath
Vacuum tubing (2 pieces)
1 L vacuum flasks (2)
Micropipettor & tips: 1 ml, 5 ml
Electronic scale
Weighing paper
Spatulas
5 ml plastic syringes with Luer-Lok tip (BD Syringe 309603)
25 mm nylon syringe filters, 0.45 m (Fisher 09-730-17)
HPLC vials (Dionex 055058)
CarboPac PA20 analytical chromatography column, 3 x 150 mm (Dionex 060142)
CarboPac PA20 guard column, 3 x 30 mm (Dionex 060144)
Electrochemical detector (Dionex ED40)
Gradient pump (Dionex GP40)
Autosampler (Dionex AS50)
Chromatography enclosure (Dionex LC20)
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Solution preparation:
1. 200 mM sodium hydroxide eluent solution: In a properly labeled 1 L vacuum flask
add 1 L of deionized water along with a magnetic stir bar. Attach vacuum tubing to flask
and attach other end of tubing to laboratory vacuum valve. Place flask in sonicating bath
and sonicate for 20 minutes. Remove vacuum tube from flask and place flask on stir
plate. While stirring at medium speed, gently add 10.4 ml of 50% w/w sodium hydroxide
solution. When pipetting from the sodium hydroxide bottle, make sure to transfer liquid
from the center of the bottle avoiding the bottom and sides. This avoids any carbonate
contamination in the sodium hydroxide. After addition of sodium hydroxide, transfer to
the plastic eluent bottle provided with the HPLC system. Be careful to pour slowly along
the side of the plastic bottle to avoid introducing any air bubbles into the solution.
2. 16 mM sodium hydroxide eluent solution: Proceed as in step 1 except add 0.832 ml
of 50% w/w sodium hydroxide solution instead of 10.4 ml.
3. Carbohydrate standard solutions: Prepare a standard stock solution containing 2.0
mg/ml each glucose, fructose, and sucrose. Use weighing paper and a scale to weigh
out 100 mg of each and add to a small volume of deionized water in a 50 ml volumetric
flask. Once dissolved bring to volume with additional deionized water. Use a funnel to
transfer the stock solution to a properly labeled 50 ml glass bottle. Prepare standard
solutions of 0.2, 0.02, and 0.002 mg/ml by serial dilution. Three 50 ml volumetric flasks
and 5 ml volumetric pipettes will be needed. Transfer to properly labeled 50 ml glass
bottles using a funnel. Use these standard solutions for HPLC analysis.
Method set-up:
1. Click on the MenuDx icon located on the desktop.
2. Click on Method > File > New. Add the modules (Gradient pump, detector, and
autosampler), click Exit.
3. Double-click on GP40 Gradient Pump. Turn pump on; set the high and low pressure
limits to 3600 and 200 psi, respectively; select standard piston size; select psi for
pressure units; and label eluents (200 mM NaOH, 16 mM NaOH, DI water, etc.). Click
OK.
4. Develop the sequence of pump events. Begin by typing “Initial” in the time drop-down
menu. Enter 0.50 in the flow drop-down menu and select 100% Eluent A (if eluent A is
the 200 mM NaOH). Click Enter. Repeat this routine for the following pump events:
Time (Min):
Flow (ml/min):
Eluent:
Comments:
Initial
0.50
100% A
-
0.00
0.50
100% A
-
14.95
0.50
100% A
Regeneration
15.00
0.50
100% B
Equilibration
25.00
0.50
100% B
Inject
40.00
0.50
100% B
End
After all events are entered, click Exit.
5. Click on ED40 Detector. Enter 15.00 into the time drop-down menu.
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6. Double-click on ED40 Detector. In the time drop-down menu enter “Initial” and in the
range drop-down menu enter 10000. Click Enter. Repeat this step for times 0.00 and
25.00. For time 25.00 make sure that the Begin Collection box is checked.
7. Click on Waveform. Enter the following waveform one step at a time, clicking on Enter
after each addition:
Step:
Time (Sec):
Potential (V):
Integrate
1
0.00
0.10
-
2
0.20
0.10
Begin
3
0.40
0.10
End
4
0.41
-2.00
-
5
0.42
-2.00
-
6
0.43
0.60
-
7
0.44
-0.10
-
8
0.50
-0.10
-
After all waveform events are entered, click on Exit. Click on Exit again to finish the
ED40 Editor.
8. Double-click on AS50 Autosampler. Enter the following events one at a time, clicking
on Enter after each addition. Click Exit once finished.
Time (min):
Valve:
Init
-
0.00
-
23.00
Load
25.00
Inject
9. The method is now complete and ready for use. Save the method with a descriptive file
name and exit the Method Editor.
Schedule set-up:
1. Click on the MenuDx icon located on the desktop. Click on Schedule.
2. Click on File > New > AS50 Schedule > OK.
3. In column 1 enter the vial position (number) within the autosampler.
4. In column 2 enter 1.
5. In column 3 enter 25.
6. In column 4 select full as the injection type.
7. In column 5 enter 10.
8. In column 6 enter 3.
9. In column 7 enter the sample name or number.
10. In column 10 select sample type. Calibration std. for standards and sample for samples
to be analyzed.
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11. In column 11 enter the level of standard solution if Calibration std. was selected in step
10.
12. Double click in column 12 and select the method file previously created.
13. Double click in column 14 and create a file name to which the results should be saved.
14. Insert 1 in columns 15-17. Columns 13 and 18 can be left blank.
15. Complete steps 3-14 for all samples/standards. Save the schedule with a descriptive
name/date and exit the Schedule editor. The schedule is now ready for use.
Sample preparation:
1. Remove kernel carbohydrate samples from the 1522 freezer. Using a pipette, add 4 ml
of deionized water to the glass culture tube. Use a wood applicator stick to break apart
the protein/carbohydrate pellet at the bottom of the tube. Allow samples to sit for
approximately 30 minutes to allow for complete solubilization of sugars.
2. Transfer the contents from one of the glass tubes to a 5 ml plastic syringe fitted with a
syringe filter. Carefully filter the contents into a new, appropriately labeled glass culture
tube. Discard the syringe and filter. Repeat step 2 for all samples.
3. Use a pipette to transfer approximately 1 ml of sample to a labeled HPLC vial.
Procedure:
1. Follow the instructions available for start up of the HPLC system (see HPLC Operating
Procedure).
2. Place the labelled HPLC vials into the autosampler. Press any key on the autosampler
to bypass the warning message.
3. Click on the MenuDx icon located on the desktop. Click on Run.
4. Click on File > Load Schedule. Select and open the schedule previously created. Click
OK.
5. Click Start (arrow icon located at top of screen).
6. After analysis is complete follow the instructions available for shut down of the HPLC
system.
Personal Protective Equipment / Engineering Controls:
Eye protection (goggles & shield)
Skin protection (proper shoes, gloves, lab coat, etc.)
Ventilation system
Safety shower
Eye wash station
Hazard Controls & Storage Precautions:
Sodium hydroxide: Keep container closed when not in use. Store in a cool, dry, well-ventilated area
away from incompatible substances. Keep away from strong acids, metals, flammable liquids and
organic halogens.
Glucose: Store in a cool, dry, well-ventilated area away from incompatible substances.
Fructose: Store in a cool, dry, well-ventilated area away from incompatible substances.
Sucrose: Store in a cool, dry, well-ventilated area away from incompatible substances.
Waste Disposal & Decontamination Procedures:
Sodium hydroxide: Place excess solution in appropriately labeled hazardous waste container for
EH&S disposal.
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Glucose: Rinse glucose solution down the drain with excess water.
Fructose: Rinse fructose solution down the drain with excess water.
Sucrose: Rinse sucrose solution down the drain with excess water.
Health & Safety Info for Required Reagents:
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Chemical name
Sodium hydroxide
Glucose
Fructose
Sucrose
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Eyes, skin, mucous
membranes.
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Eyes, skin.
Eyes.
Eyes, skin, lungs.
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Target Organ
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Incompatibilities
Metals, acids, aluminum,
nitro compounds, zinc, tin,
halogenated hydrocarbons,
nitromethane, flammable
liquids.
Oxidizing agents.
Strong oxidizing agents.
Strong oxidizing agents.
 The above summary consists of guidelines for proper handling & disposal of
chemicals used in this procedure. You must read complete MSDS(s) for more
specific information before beginning this procedure.
Reference:
Dionex Corporation. 2000. Analysis of carbohydrates by high performance anion exchange
chromatography with pulsed amperometric detection (HPAE-PAD). Technical Note 20.
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