PERFORMING A SYSTEM ACID CLEANSING FLUSH

System contamination can cause the flow cell to become contaminated. If the system
becomes contaminated, perform a system acid cleansing flush. This procedure cleans
the binary solvent manager, solvent manager, and flow cell.

Cleaning the system

IMPORTANT: This cleaning procedure must be performed when installing the ACQUITY
UPLC H-Class and H-Class Bio Amino Acid Analysis System.

CAUTION: TO AVOID FLOW PATH CONTAMINATION, YOU MUST REMOVE THE BOTTLE FILTERS
BEFORE CLEANING THE SYSTEM. AFTER THE CLEANING IS FINISHED, REPLACE THE FILTERS.

Prepare the cleaning solvents

1. Prepare a mixture of 50:50 (v/v) methanol:water as follows:

a. Measure 500 mL of water in a graduated cylinder.

b. In another graduated cylinder, measure 500 mL of methanol.
c. Add the methanol to the water and mix for 5 minutes.
d. Fill a 1L mobile phase reservoir with the 50:50 (v/v) methanol:water.

2. Prepare a mixture of 30:70 (v/v) 85% phosphoric acid:water as follows:

a. Measure 700 mL of water in a graduated cylinder.

b. In another graduated cylinder, measure 300 mL of 85% phosphoric acid.
c. Add the 85% phosphoric acid to the water and mix for 5 minutes.
d. Fill a 1L mobile phase reservoir with the 30:70 (v/v) phosphoric
acid:water.

3. Fill a 1L mobile phase reservoir with 100% water.

4. Fill a 1L mobile phase reservoir with 100% isopropanol.

Prepare the sample vials for cleaning

1. Add 1 mL of the 50:50 (v/v) methanol:water to a sample vial.

2. Using a new vial, repeat step 1, using the 30:70 (v/v) phosphoric acid:water.
3. Using a new vial, repeat step 1, using the 100% water.
4. Using a new vial, repeat step 1, using the 100% isopropanol.

STARTING CLEAN THE SYSTEM

CAUTION: THIS CLEANING PROCEDURE USES A CONFIGURATION THAT ROUTES EFFLUENT TO

WASTE. DO NOT ROUTE EFFLUENT THROUGH THE COLUMN OR OPTICAL DETECTOR.

NOTE: The cleaning procedure takes approximately 3.5 hours to run.

1. Place solvent lines A, B, C, D, seal wash, purge solvent, and wash solvent into
 the 50:50 methanol:water reservoir.
2. Prime the solvent lines for 5 minutes each.
3. Prime the seal wash for one minute.
4. Prime the wash solvent for 60 seconds.
5. Prime the purge for 50 cycles.
6. Carefully connect a flow restrictor (capable of creating at least 2000 psi of
system back pressure) to the outlet of the APH assembly in the CH-A.
7. Connect a waste line from the outlet of the flow restrictor to waste container.
8. Transfer the sample vial with the 50:50 (v/v) methanol:water, and place it in
tray position 1:A,1.
9. Create an instrument method with the following parameters:
Flow rate = 0.5 mL/min
Gradient composition = 25%A/25%B/25%C/25%D
10. Set the Run Time to 0.5 minutes, then make 10 injections from the vial. This
step takes approximately 10 minutes.
11. Remove solvent lines A, B, C, D, seal wash, purge solvent, and wash solvent
from the existing reservoir, then place them into the 100 % Isopropanol
reservoir. See below
NOTE: Do not pass effluent through optical detector for this wash step. Restrictor
should be routed to waste.
12. Repeat step 2 through step 10 for the isopropanol, making sure you use the
sample vial that contains isopropanol.
13. Remove solvent lines A, B, C, D, seal wash, purge solvent, and wash solvent
from the existing reservoir, then place them into the 100 % water reservoir.
14. Repeat step 2 through step 10 for the water, making sure you use the sample
vial that contains water.
15. Remove solvent lines A, B, C, D, purge solvent, and wash solvent from the 100
% water reservoir, then place the six lines into the 30:70 phosphoric acid:water
reservoir.

NOTE: Leave the seal wash line in the 100% water reservoir.

16. Reinsert the waste line to separate waste container for the phosphoric acid
waste.
17. Repeat step 2 through step 10, making sure you use the sample vial that
contains the 30:70 phosphoric acid:water. After repeat step 17, do below!

NOTE: To completely remove all flow cell contaminants, continue pumping the phosphoric
acid mixture for an additional 3 hours at a low flow of 0.05 to 0.1 ml/min. Do not
exceed 1000 psi (69 bar,6895 kPa).
18. Remove solvent lines A, B, C, D, purge solvent, and wash solvent from the 30:70
phosphoric acid:water reservoir, then place the lines into the 100% water
reservoir.
19. Reinsert the waste line to the original waste container.
20. Repeat step 2 through step 10 for the water, making sure you use the sample
vial that contains water.
21. Remove solvent lines A, B, C, D, seal wash, purge solvent, and wash solvent
from the existing reservoir, then place them into the 50:50 (v/v) methanol:water
reservoir.
22. Repeat step 2 through step 10 for the 50:50 (v/v) methanol:water, making
sure you use the sample vial that contains 50:50 (v/v) methanol:water.
23. Carefully remove the flow constrictor from the APH assembly.

24. Replace the sample and solvent manager bottle filters.