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(HPLC: Reducing Sugar Analysis) When not using and rinsing the system (Size Exclusion - Ligand Exchange Mode)
(HPLC: Reducing Sugar Analysis) When not using and rinsing the system (Size Exclusion - Ligand Exchange Mode)
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Answer
Since there are sugars predisposed to forming certain metal ions and weak complexes, this method performs separation of such sugars using packing that is configured with specific metal ions which are determined in advance. The packing used for this mode is configured with calcium ions or lead ions on a sulfone group base that is combined with resin for aqueous size exclusion chromatography.
• Parts Number:
228-07730-92: Shim-pack SCR-101N (Na-type sulfone group, 300 mm x 7.9 mm I.D.)
228-09619-92: Guard Column SCR(N) (Na-type sulfone group, 50 mm x 4.0 mm I.D.)
228-17889-91: Shim-pack SCR-101C (Ca-type sulfone group, 300 mm x 7.9 mm I.D.)
228-17891-91: Guard Column SCR(C) (Ca-type sulfone group, 50 mm x 4.0 mm I.D.)
228-17890-91: Shim-pack SCR-101P (Pb-type sulfone group, 300 mm x 7.9 mm I.D.)
228-17892-91: Guard Column SCR(P) (Pb-type sulfone group, 50 mm x 4.0 mm I.D.)
228-17889-91: Shim-pack SCR-101C (Ca-type sulfone group, 300 mm x 7.9 mm I.D.)
228-17891-91: Guard Column SCR(C) (Ca-type sulfone group, 50 mm x 4.0 mm I.D.)
228-17890-91: Shim-pack SCR-101P (Pb-type sulfone group, 300 mm x 7.9 mm I.D.)
228-17892-91: Guard Column SCR(P) (Pb-type sulfone group, 50 mm x 4.0 mm I.D.)
• About selecting columns
The "Shim-pack SCR" series features three types of columns, Ca-type, Na-type and Pb-type, and the sugar retention time and separation differs for each type. Select the column appropriate to the type and concentration of the sugars contained in the actual sample. For details on the retention time of sugars in each column, refer to the Shimadzu food data sheet. (C180-E059C)
Note that adjustment of retention time and separation by changing the mobile phase composition is not possible because, in practice, only purified water can be used in these columns.
Note that adjustment of retention time and separation by changing the mobile phase composition is not possible because, in practice, only purified water can be used in these columns.
• Measures to take when performing the same analysis on the following day
Because the mobile phase is purified water in these analysis conditions, the contents of the reaction coil, cooling coil and detection cell can be replaced with purified water by stopping the delivery of reaction reagent while continuing delivery of mobile phase.
• Measures to take when not performing analysis for a number of days and when changing the separation mode
After removing the column from the instrument, purge both pumps for delivering mobile phase and reaction reagent with purified water and then replaceinside of all flow lines with purified water. If the instrument is not to be used for more than one months, replace inside of all flow lines, except the column, with methanol or ethanol.
Lastly, dispose of the waste liquid in the waste container.
Lastly, dispose of the waste liquid in the waste container.
• Flow Line Rinsing
Once installation of the analysis system is finished, rinse any contaminants from the flow lines according to the procedure described below before installing the column. If the analysis system is to be used again after an extended break (more than one month), perform rinsing using this procedure as there is a possibility of flow line contamination.
1. Remove the coupling that connects the 0.13 mm i.d. PEEK tubing to the detector inlet and insert the outlet end of the PEEK tubing into a vessel such as a beaker.
This measure prevents rinsed contaminants from clogging the detection cell.
HINT: The ETFE tube for detector outlet can be connected directly to the PEEK tube outlet.
This measure prevents rinsed contaminants from clogging the detection cell.
HINT: The ETFE tube for detector outlet can be connected directly to the PEEK tube outlet.
2. Connect the column inlet connection pipe directly to the column outlet connection pipe using a coupling.
3. Perform rinsing by turning ON power to the pumps for mobile phase delivery and reaction reagent delivery.
3. Perform rinsing by turning ON power to the pumps for mobile phase delivery and reaction reagent delivery.
4. Pumping 17% phosphoric acid* at a rate of 1.0 mL/min for 10 minutes.
*Phosphoric acid (special grade, 85%) / Purified water = 1 / 4 ( v / v )
HINT: Note that the following rinsing solutions are also delivered at the same rate of 1.0 mL/min.
5. Deliver purified water for 10 minutes.
6. Deliver 2-propanol for 10 minutes.
HINT: This is for flow line rinsing so use a first class reagent grade of 2-propanol.
7. Deliver purified water again for 30 minutes. This time, set the temperature of the CRB to 150 ºC.
8. Open the door on the chemical reaction box while delivering the purified water and wait until it cools to around room temperature before stopping delivery.
*Phosphoric acid (special grade, 85%) / Purified water = 1 / 4 ( v / v )
HINT: Note that the following rinsing solutions are also delivered at the same rate of 1.0 mL/min.
5. Deliver purified water for 10 minutes.
6. Deliver 2-propanol for 10 minutes.
HINT: This is for flow line rinsing so use a first class reagent grade of 2-propanol.
7. Deliver purified water again for 30 minutes. This time, set the temperature of the CRB to 150 ºC.
8. Open the door on the chemical reaction box while delivering the purified water and wait until it cools to around room temperature before stopping delivery.
CAUTION: Do not operate until the temperature inside the system has decreased. The chemical reaction box is very hot. Be careful not to burn yourself.
9. Reconnect the outlet end of the PEEK tubing to the detector inlet using a coupling.
10. Deliver mobile phase for 5 minutes.
HINT: For gradients, deliver either A or B solution.
11. Remove the coupling, detach the column inlet connection pipe and column outlet connection pipe and attach the column.
10. Deliver mobile phase for 5 minutes.
HINT: For gradients, deliver either A or B solution.
11. Remove the coupling, detach the column inlet connection pipe and column outlet connection pipe and attach the column.
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