Waters 2690/5 Separations Module
Based on the Quick Start Guide
http://www.forumsci.co.il/HPLC/Waters_2690.html
Put all inlets in the appropriate solvents according to the rules of HPLC work (see guide for HPLC
work)
Power on the Separations Module. Wait until STARTUP DIAGNOSTICS is over – do not open any
door!
1 1. "Dry prime"in the “Direct function” (To be used if the instrument is dry or filled with unkown
solvents or after wet prime fails).
To perform a dry prime:
Attach an empty syringe to the prime/vent valve, then open the valve by turning it counterclockwise
1-2-turn.
1. Note: The syringe does not lock onto the prime/vent valve; hold it in place while you pull on the
plunger.
2. Press the Direct Function screen key in the Status screen. Select Dry Prime, then press Enter. The
Dry Prime dialog box appears
1. Press the screen key corresponding to the solvent line you want to prime.
2. Withdraw the syringe plunger to pull solvent through the tubing. You may need to exert force to
pull the air and solvent through the system. Continue until you pull all the air through the solvent
line into the syringe.
3. Repeat steps 1 and 2 for each solvent line you want to prime, then close the prime/vent valve.
Optional:
In the Enter a Duration field, enter the length of time (in minutes) to prime the solvent management
system. Start with a value of 5 minutes. Press Continue.
When the system is dry, Waters recommends that you prime using the solvent with the lowest
viscosity to help purge air from the lines, especially if the in-line vacuum degasser is installed.
2. Wet Prime the Solvents using the “Direct function” and “wet priming” for all the solvents to be
used. Check that the reservoirs are full and that liquid comes out at the outlet
Perform a wet prime when you want to change reservoirs or solvents, or if the Separations Module
has been idle for some time. Use an intermediate solvent such as water when you change from
buffers to high-organic-content solvents.
Wet prime:
1. In the Status screen Composition field, enter 100% for the each solvent you want to use for wet
priming..
2. Press the Direct Function screen key in the Status screen. The Direct Functions menu appears.
3. Select the Wet Prime option, then press Enter. The Wet Prime dialog box
appears.
3. Degas Eluents– Note: For proper operation of the in-line vacuum degasser, you must fill each
vacuum chamber with solvent
To control the operation of the in-line vacuum degasser from the Status screen:
1. Press the Menu/Status key.
2. Select the Degasser mode field, then press Enter to display a list of degasser modes. The modes
are:
On - The degasser is always on
Off- The degasser is off
4. Select the desired operating mode, then press Enter.
5. Wet prime again after vacuum pressure was established.
4. Prime the Seal Wash Pump
For reversed-phase HPLC applications, use an aqueous plunger seal-wash solution with enough
organic content to inhibit bacterial growth. For example, use an 90% water:10% methanol solution
or an 90% water:10% acetonitrile solution, depending on your application. For normal phase
separations, use a 50% methanol:50% IPA seal wash solution. If required, use an intermediate
solution to prevent immiscibility or precipitate problems when you switch between normal phase
and reversed-phase analytical solvents.
To prime the plunger seal wash pump:
1. Press the Diag screen key in the Main screen. The Diagnostics screen appears.
2. Press the Prime SealWsh screen key, then press the Start screen key. The seal wash procedure
begins. Push on a syringe plunger to force seal wash solvent through the system if needed.
3. Press Close to return to the Diagnostics screen.
5. Priming the Needle-Wash Pump
The needle-wash pump flushes the needle in the sample management system, preventing carryover
of sample between injections. The needle wash also extends the life of the injector seals by
removing buffered mobile phase and sample from the needle..
To prime the needle-wash pump:
1. Press the Diag screen key in the Main screen. The Diagnostics screen appears.
2. Press the Prime NdlWsh screen key. The 30-second needle-wash procedure begins. If solvent does
not flow out of the waste tube within 30 seconds, press the Start Again screen key.
Selecting a Needle-Wash Solvent
Use a needle-wash solvent based on the sample and mobile phase chemistries, making sure all
solutions/buffers are miscible and soluble. Remember: the composition of the needle-wash solvent
should be the most solubilizing compatible solvent (your strongest diluent), selecting it is part of the
method development.
Generic Chromatographic Condition Needle Wash Solvent
Buffered aqueous reversed-phase mobile phases: 50% up to 100% organic solvent in water,
whatever dissolves the sample best..
Non-aqueous, reversed-phase: 100% MeOH
Normal phase Mobile phase or GPC Mobile phase: IPA or EtOH
Ion exchange: H 2 O.•
6. Purge the Injector (autosampler)
Purge the sample management system with water (in reversed phase) whenever you:
• Dry-Prime the solvent management system
• Change solvents
• See bubbles in the syringe
• Start using the Separations Module at the beginning of each day
Attention: To avoid precipitating salts in the Separations Module, use an intermediate solvent such
as water when you change from buffers to high-organic-content solvents and vice versa. It is
recommended to purge the injector with the intermediate solvent after working with high
concentration of buffers and/or before running them. After using the Purge Injector function with
water, repeat it with your mobile phase.
To purge the injection system:
1. In the Main screen, press the Menu/Status key to display the Status screen.
2. Enter the appropriate solvent composition in the Composition fields.
3. Press Direct Functions to display the Direct Functions menu.
4. Select Purge Injector, then press Enter. The Purge Injector dialog box appears.
5. Enter the number of sample loop volumes in the Sample Loop Volumes field (use the default value
of 6.0 volumes), then press Enter.
6. Leave the “Compression Check” check box blank ordinarily.
7. Press OK. The purge cycle begins.Do not interfere while the process is going on!
7. Check for stability of the pressure and enter the Run Samples (Quick set) in Millennium to monitor
the baseline.
Summary: Steps in the Preparation of Alliance 2690/5
Remark Instrument's Function
Screen
A, B, C, D are mobile phase reservoirs and Check for solvents in:
wash solvents
Solvents in A, B, C, D;
For "Needle wash" solvent: use the most
dissolving solvent Needle Wash
For seal wash solvent: use aqueous solvent. Seal Wash
Check for pressure stability Status: Dry Prime:
Direct Function - 2
Wet Prime:
A, B, C, D.
Build up the vacuum in the degasser Stop Flow:
Degas ("Continuous" in
2690).
Verify full & degassed vacuum chambers Status: Wet Prime again
shortly.
Direct Function - 2
Check for liquids in yellow tubing outlet Main: Prime Needle Wash
Diag
Check for liquids out Main: Prime Seal Wash
Diag
1. Wash remains from previous work Status: Purge Injector
(with water in reversed phase)
Direct Function - 3
2. Purge again with mobile phase.
Set the flow. It will pass through the Status Set Flow
column.
