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(HPLC: Reducing Sugar Analysis) When not using and rinsing the system (Borate Complex - Anion Exchange Mode)
(HPLC: Reducing Sugar Analysis) When not using and rinsing the system (Borate Complex - Anion Exchange Mode)
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Answer
• Parts Number:
228-59512-41: Shim-pack ISA-09 (250 mm x 4.0 mm I.D.)
228-59512-42: Shim-pack ISA-09(G) (50 mm x 4.0 mm I.D.)
• Precautions when using new columns
When using a "Shim-pack ISA-07/S2504"or "Shim-pack ISA-09" separation column for the first time after purchase, the separation column and guard column must be flushed with about 720 mL of mobile phase B (column temperature: 65 ºC, flow rate: 0.6 mL/min, duration: about 20 hours) and the column interiors evenly equilibrated with borate ions.
Reaction reagent does not need to be delivered for this equilibration process.
Reaction reagent does not need to be delivered for this equilibration process.
• Measures to take when performing the same analysis on the following day
Purge the pump for reaction reagent delivery with purified water and substitute the contents of the reaction coil, cooling coil and detection cell with purified water. Failing to perform this task will allow salt to deposit inside the tubing and cause clogging.
When performing the same analysis on the following day, the mobile phases and separation column can be left set on the instrument. However, note that because the mobile phase is an alkaline, leaving mobile phases unsealed over a number of days will lead to absorption of carbon dioxide and a change in pH.
When performing the same analysis on the following day, the mobile phases and separation column can be left set on the instrument. However, note that because the mobile phase is an alkaline, leaving mobile phases unsealed over a number of days will lead to absorption of carbon dioxide and a change in pH.
• 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 replace inside of all flow lines with purified water. If the instrument is not to be used for more than one month, 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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