Waters’ Support Documents
Written by: Dr. Shulamit Levin , Waters (TC) Israel
(For printing choose: 2.5 cm margins-top, bottom, left, right in A4 page)
Waters 2690/5 Separations Module
Based on the Quick Start Guide
34 Maple Street
Milford, MA 01757
WAT553-03TP, Revision 4


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!


When the initial part of the startup diagnostics routine is successful, the Main screen appears in the front panel display. The startup diagnostics routine continues by initializing the:

• Needle, syringe, and valves

• Carousel carrier

• Solvent management system

The status of these mechanical diagnostics tests appear in the banner area of the Main screen, while the diagnostics are running. When the startup diagnostics routine is complete, the Separations Module enters the Idle mode.



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.

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




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


A, B, C, D are mobile phase reservoirs and wash solventsFor “Needle wash” solvent: use the most dissolving solvent

For seal wash solvent: use aqueous solvent.

Check for solvents in:Solvents in A, B, C, D; 

Needle Wash 

Seal Wash

Check for pressure stability Status:Direct Function – 2 Dry Prime:
Wet Prime: A, B, C, D.
Build up the vacuum in the degasser Stop Flow:
Degas (“Continuous” in 2690).
Verify full & degassed vacuum chambers StatusDirect Function – 2 Wet Prime again shortly.
Check for liquids in yellow tubing outlet Main:
Prime Needle Wash
Check for liquids out Main:
Prime Seal Wash
1.  Wash remains from previous work (with water in reversed phase)2.   Purge again with mobile phase. Status:Direct Function – 3 Purge Injector 
Set the flow.  It will pass through the column. Status Set Flow 

Copyright (C)2003 Dr. Shulamit Levin