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Waters' Support Documents

Written by: Dr. Shulamit Levin
Medtechnica, Analytical Department
Efal St. 5 Petach Tikva Tel: 03-9254040 Fax: 03-9249977 Email

(For printing choose: 2.5 cm margins-top, bottom, left, right in A4 page)

Waters Alliance HT 2790/5 Separations Module

Based on the Quick Start Guide
34 Maple Street
Milford, MA 01757
WAT553-03TP, Revision 4

2795

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 STARTUP DIAGNOSTICS. The Main Screen

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.

Note: The syringe does not lock onto the prime/vent valve; hold it in place while you pull on the plunger.
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:

2790:
•Normal– The degasser cycles on and off on a pressure-regulated cycle

•Continuous– The degasser is always on

•Disable– The degasser is always off

2795:

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: 90% H 2 O, 10% MeOH

Non-aqueous, reversed-phase: 100% MeOH

Normal phase Mobile phase/or GPC: Mobile phase IPA or EtOH

Ion exchange: H 2 O

6. Refresh Syringe (autosampler)

Refresh the syringe whenever you:

• Prime the solvent management system

• Change solvents

• See bubbles in the syringe

• Start using the 2795 Separations Module at the beginning of each day

<>Attention: To prevent degradation to the syringe and valves, avoid the use of salt buffers in the purge solvent. Use a low concentration (0% to 50%) organic-aqueous solvent that is <>similar to the mobile phase, initial gradient conditions, or sample diluent.

Note: When you refresh (purge) the syringe for the first time or when you change the purge solvent, set the number of syringe strokes to 10 and the replacement volume to 600 <>This volume fills the entire degasser chamber and the associated inlet tubing (with the standard 500-.L syringe installed).

<>To refresh the syringe: <>

<>

<>
1. In the Status screen, activate the degasser by selecting the On mode in the
Degasser field. <>
2. Press the Direct Function screen key in the Status screen. The Direct Functions Menu. <>
3. Select the Refresh Syringe option, then press Enter. The Refresh Syringe dialog box appears. <>
4. Enter the number of syringe strokes and the replacement volume of degassed solvent in the appropriate field. Start with 3 syringe strokes and 600 uL replacement volume.
<>5. Press OK. The Refresh Syringe cycle begins.

<>7. Washing the needle

<> <>The Wash Needle function:

• Washes the inject port and the interior and exterior of the needle

<>• Flushes the wash solvent out of the needle and inject port and replaces it with

degassed purge solvent

Use the Wash Needle function to:

• Ensure that the needle and inject port are clean before the start of a new method

•Verify that the needle-wash system is primed and operating properly by checking for

flow through the waste tube

Note: If the 2795 Separations Module does not detect that a needle wash was performed

since the last time the 2795 Separations Module was powered on, it performs an

automatic needle wash before beginning any injections, either those specified in a sample

set or by using the Inject Samples direct function.

Note: Waters recommends using a high percentage organic wash solvent that is

compatible with your mobile phase and sample diluent, such as methanol or acetonitrile.

Attention: Make sure the needle-wash solvent line is primed before you perform a needle

wash.

Wash Needle:

Menu/Status
Direct Functions screen key.
Select Wash Needle-> Enter
Enter the number of wash cycles in the Wash cycles field.
Enter the amount of time (in seconds) to wash the inject port and the needle exterior in the appropriate fields. Enter the replacement volume of degassed solvent in the appropriate field. Start with 600 uL replacement volume.
Press OK.

8. Check for stability of the pressure and enter the Run Samples (Quick set) in Millennium/Empower to monitor the baseline.

Summary: Steps in the Preparation of Alliance 2790/5

Remark Instrument's
Screen Function

A, B, C, D are mobile phase reservoirs and wash solvents

For "Needle wash" solvent: use the most dissolving solvent

For seal wash solvent: use aqueous solvent.

For Purge Solvent use either diluent's or mobile phase's solvents composition

Check for solvents in:

Solvents in A, B, C, D;

Purge solvent

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 2790).

Verify full & degassed vacuum chambers

Status:

Direct Function - 2

Wet Prime again shortly.

Check for liquids in yellow tubing outlet

Main:

Diag

Prime Needle Wash

Check for liquids out

Main:

Diag

Prime Seal Wash

1. Refresh Syringe: wash the injector interior tubings with the diluent/mobile phase solvents.

2. Wash Needle : wash the injector interior with the strong solvent

Status:

Direct Function - 3

Refresh syringe

Set the flow. It will pass through the column.

Status

Set Flow

Remark	Instrument's Screen	Function
A, B, C, D are mobile phase reservoirs and wash solvents For "Needle wash" solvent: use the most dissolving solvent For seal wash solvent: use aqueous solvent. For Purge Solvent use either diluent's or mobile phase's solvents composition		Check for solvents in: Solvents in A, B, C, D; Purge solvent 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 2790).
Verify full & degassed vacuum chambers	Status: Direct Function - 2	Wet Prime again shortly.
Check for liquids in yellow tubing outlet	Main: Diag	Prime Needle Wash
Check for liquids out	Main: Diag	Prime Seal Wash
1. Refresh Syringe: wash the injector interior tubings with the diluent/mobile phase solvents. 2. Wash Needle : wash the injector interior with the strong solvent	Status: Direct Function - 3	Refresh syringe
Set the flow. It will pass through the column.	Status	Set Flow