North Carolina State University is a landGrant university and a constituent institution
of The University of North Carolina
Department of Wood and Paper Science
NC STATE UNIVERSITY
STANDARD OPERATING PROCEDURE (SOP)
Dionex HPLC System for Sugar Analysis
Location:
3218B, Pulp and Paper Labs
Contact:
Dr. Sunkyu Park (sunkyu_park@ncsu.edu), 919-515-0473
Junyeong Park (jpark7@ncsu.edu), 919-515-0321
Standard Operating Procedure
A. Start-up Process
1) Prepare deionized water (HPLC grade, 0.2 µm filtered) in a solvent bottle with GL45 thread.
2) Sonicate the solvent for minimum 30 min for degassing.
3) Connect the bottle to solvent line ‘D’ of HPLC system. The pump should be stopped before
connecting the bottle.
4) Set the maximum pressure as less than 3.0 MPa. The maximum pressure for the SP0810
column is 3.0 MPa.
5) Remove air-bubble in the solvent line before running.
a. Open purge valve by turning the valve about one turn counterclockwise.
b. Press the pump selector D on the pump control panel.
c. Press ‘Purge’, and let the purge line flow for minimum 3 min. Vacuum the purge line with
a syringe, if applicable.
d. After minimum 3 min, press ‘Stop’ and finger-tighten the purge valve.
6) Power on the whole system (auto-sampler, column oven, and RI detector).
7) Set flow-rate for pump ‘D’ as 0.1 mL/min, and run the pump ‘D’.
8) Wait for the column temperature to reach 75 °C.
9) Increase the flow-rate gradually to 0.5 mL/min, and wait until the pump-pressure being stabilized.
10) Press the ‘Purge’ menu on RI detector every 10 seconds to turn on and off the purge valve for a
few minute, and let ‘Purge’ on for about 30 min.
11) Press the ‘Purge’ menu to turn off the valve, and wait until the baseline stabilizes.
B. Standards and Sample Preparation
12) Prepare high purity standard sugars and make standard mixture solution with different
concentration of each sugar. Suggested concentration range is 1.2 - 24 mg/mL.
13) Make standard curves for each sugar using a four-point calibration.
14) For samples with pH <5, neutralization is necessary.
a. Aliquot sample (5 - 10 mL) in a small flask or beaker.
b. Neutralize to pH 5 - 6 using calcium carbonate (ACS grade).
c. After reaching pH 5 - 6, allow sample to settle and decant off the clear liquid. The pH of
the liquid after settling will be approximately 7.
d. Prepare the sample vial by passing the decanted liquid through 0.2 µm filter.
- Caution When calcium carbonate is used excessively, solid precipitation might be
occurred even after filtering, and could damage the column. Use calcium
carbonate as little as possible, and wait sufficient time for settlement after
neutralization.
C. Shut-down Process
15) Decrease the flow-rate gradually to 0.2 mL/min.
16) Turn off the auto-sampler and the RI detector.
17) Turn off the column oven and open the cover-lid.
- Caution Interior of the column oven is hot. Pay attention to avoid contacting with
the oven interior before cooling down.
18) Let the pump flow at 0.2 mL/min for minimum 30 min.
19) Stop the flow and turn of f the pump.