Using Dynamic Load and Wash (DLW). Thermo Fisher Scientific Thermo PAL

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Using Dynamic Load and Wash (DLW)

This chapter describes how to use the Dynamic Load and Wash (DLW) option, which includes how to install the Cycle Composer Macros or ICC Cycles, how to operate the DLW, and a step-by-step illustration of the DLW cycles.

Contents

•

Overview

•

Installing Cycle Composer Macros or ICC Cycles

•

Operating Dynamic Load and Wash (DLW)

•

DLW Cycle Step-by-Step

Overview

This section contains an overview of the Dynamic Load and Wash (DLW) option.

Figure 35. Dynamic Load and Wash (DLW)

Thermo Scientific Thermo PAL User Guide

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The DLW option represents a new wash station concept that combines an injection cycle with wash steps. The linked combination of the two steps, which are usually separate, minimizes cycle time and carryover.

There are two characterizing features for the DLW option:

• The sample solution never contacts the syringe itself; it is held between the holding loop.

• Wash solvents are pumped from back to front into the DLW system to intensely flush all critical parts that are in contact with the sample.

The DLW option consists of two self-priming micro pumps (mounted on a dedicated bracket), and the wetted parts are Ryton PPS and Kalrez (FFPM). The pump IN ports are connected to the wash solvent bottles and the OUT ports are connected to the DLW manifold, which is part of the assembly of the dedicated DLW syringe holder. A holding loop separates the syringe and the DLW actuator to avoid sample from contacting these parts.

The syringe and holding loop are preloaded with wash solvent #1 at the start. The sample is picked up and remains separated from wash solvent #1 by an air gap. After loading the loop and injection, wash solvent #1 is pushed into the system, followed directly by wash solvent #2 to flush the critical valve paths.

The DLW syringe assembly is moved to the wash station for further cleaning steps and for preparing the Syringe and holding loop for the next cycle.

For further details, refer to

“DLW Cycle Step-by-Step” on page 73

Installing Cycle Composer Macros or ICC Cycles

You can only operate the autosampler DLW option in combination with PAL control software, such as Thermo PAL driver for Xcalibur or Cycle Editor, for ICC interpretation in another data handling system software.

For software control, three macros or three cycles are provided.

The first of three macros covers the initial and daily priming of the solvent lines and covers a complete injection cycle, a second macro is used for Standard speed with optimized washing possibilities, and the last for a Fast cycle for optimized throughput and less focus on carryover.

See

Table 14

.

For detailed descriptions of all macros (or cycles), see Table 14

, Table 15 , and

Table 16 .

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Table 14. The DLW macro definitions

Macro name

Standard Injection

Accela Open

Macro description

Priming Accela Open For initial and daily routine priming of the solvent lines and DLW manifold. The Clean Time for both wash solvents is a variable for the user to define the intensity of washing.

Standard injection cycle using all possibilities of the DLW option.

The injection valve inlet port and the needle are washed with both wash solvents (inside and out). You can add an extra Stator Wash for intensive washing of the injection valve (valve toggle).

Fast Injection Accela

Open

Tuned for speed and high-throughput application. It differs from the Standard DLW macro in that some steps are left out to shorten the cycle time.

Installing the Cycle Composer Macros or ICC Cycles

The autosampler is shipped with a CD-ROM containing various cycles for the DLW option.

Macros for Xcalibur are installed by the LC Devices 2.4 installer.

Copy these macros to your Thermo PAL driver method folder or corresponding folder for application within the integrated system.

Y

To copy cycles to your Cycle Composer

1. Navigate in Windows Explorer to the Thermo PAL driver folder. The usual folder location is as follows:

C:\Thermo\Instruments\LC Devices\ThermoPAL\PAL

2. If you wish to add the DLW option macros to an existing method folder, copy the macros

(*.pma) and the methods (*.pme) files from the DLW Option folder on the CD-ROM to the method folder.

3. If the PAL system is integrated in a data system software that controls the PAL using the

Cycle Editor for PAL ICC interpretation (for example, Analyst, ChemStation, Empower,

EZChrom, MassLynx, Xcalibur), an ICC Cycle is used and not the Cycle Composer

Macro, the cycle extension is *.cyx.

Note You can convert a Cycle Composer macro to a cycle (extension *.cyx) by using the

Cycle Editor software. Conversion is available starting with Cycle Editor version 1.4.0.4.

Note The provided macros are written for standard injection valve drives, which are controlled and activated through the AUX interface.


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General Considerations

You must establish the duration of the wash steps for each configuration and application.

Consider factors such as the viscosity and surface tension of the individual wash solvent composition and the backpressure of the system.

Be aware that a higher backpressure builds up if the valve bore size (standard valve bore

0.25 mm) or the installed loop internal diameter is lower. Standard loop internal diameter

(ID) for Thermo-defined loops with a volume of 5, 10, and 20 μL is 0.25 mm. The loop with

2 μL content volume has an ID of 0.125 mm.

Keep the tubing internal diameters of the tubing in line with the valve dimensions, loop ID, and flow rate. For details, see “Tips & Hints for HPLC Technique.”

Priming the Solvent Lines, Wash1 and Wash2

This macro is used at installation to prime the entire system. Set the wash time to approximately 120 seconds for each solvent.

After installation, for best results prime the system before activating the first run. For daily preparation of the system, the wash time can be much shorter: approximately. 20 seconds.

The goal is an entire liquid system free of any air bubbles.

Macro Name: Priming Accela Open AS_Rev01

Table 15. Macro Priming Accela Open AS (Sheet 1 of 2)


The injection unit moves to the DLW Wash

Station, position Wash1.


Station, position Wash2.

The DLW system is rinsed with Wash Solvent 1 in position Waste.

Macro variable

Clean Time solvent 1

Eject Speed DLW Syringe

Clean Time solvent 1

Eject Speed DLW Syringe

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Table 15. Macro Priming Accela Open AS (Sheet 2 of 2)

Macro description Macro variable

The injection valve is cleaned first with the content of DLW syringe (Wash Solvent 1), followed by Wash Solvent 2 and finally, the last wash to prepare the system for injection cycle, rinsed with Wash Solvent 1 again.

Needle Gap is a parameter from Rinse Inj

Atom. The variable in this macro is:

Needle Gap Valve Clean.

The function of this parameter is to raise the needle in the injection port very little to allow rinsing around the needle tip. Remark: The Atom Rinse Inj is new, available starting with FW 4.1.x. The DLW actuator/solenoid is activated; the Wash Solvent

(pump), the Needle Gap and the Rinse Time are selectable.

The pressure of the spring-loaded balls in the DLW Syringe Holder assembly is released by moving approx 3 mm up

(default). This leaves a gap, between the needle tip and the valve bottom, of approximately. 1 mm to enable a flush at this contact point.

A Repeat-End loop enables adding an extra rinsing step, valve toggle.

Stator Wash:

Counter 0 = disable valve toggle steps

Counter 1 = enable valve toggle steps.

If the counter is set to 1, follow the described steps below. If the counter is set to 0, the macro finishes at this point.

The injection moves to the injection valve.

The valve is switched to the Active position.

The valve is rinsed with Wash Solvent 2, followed by Wash Solvent 1

Stator Wash Time Solvent 2


Remark: The loop is filled with the last rinse with Wash Solvent 1. Verify the composition of Wash Solvent1. The solvent should have a lower elution power than the solvent gradient starting conditions or sample solvent composition. This is important for partial loop filling.

The injection valve is switched back to the

Standby position.

End of macro DLW Priming.

Thermo PAL User Guide

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Standard DLW Injection Cycle

Macro Name: Standard Injection Accela Open_Rev03

Table 16. Macro Standard Injection Accela Open (Sheet 1 of 3)


The PAL system waits first for the Sync Signal

Ready before the injection cycle is started.

The injection valve is brought in a defined

Standby position.

The Rear air segment is pulled into the Holding

Loop.

The sum of Rear-, Sample List-, and

Front-Volume is aspirated into the

Holding Loop.

Macro variable

Remark:

Sync Signal setting Start

Inject to

Standby

Airgap Volume

Filling Speed

Front Volume

Rear Volume

(SL.volume)

The Front air segment is aspirated.

Airgap Volume

Filling Speed

Pullup Delay


Station, Wash1 position.

The needle is inserted (dipped) for 1 second to wash the outer needle surface. No plunger movement at this step.

The injection unit moves to the specified injection valve.

The Front- and Airgap-Volume is ejected.

The PAL system waits for the data system.

The injection valve is switched to Active position.

The time Pre Inject Delay is awaited.

The loop is filled with the sample volume as specified in the sample List.

Inject to

Front Volume

Airgap Volume

Injection Speed

Wait for DS

Inject to

Pre Inject Delay

(SL.volume)

Injection Speed

The injection valve is switched to Standby position, the loop content is injected.

Timer 1 Delay Stator Wash is started and a Start signal to the HPLC system is sent.

Post Inject Delay

Timer 1

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The plunger of the DLW Syringe is pushed down to dispense the Rear Sample and Air Segment to

Waste. The Holding Loop is still filled with

Wash Solvent 1.

(Syr. Eject Speed)

The DLW Actuator/Solenoid is activated to deliver Wash Solvent 2 into the Holding Loop to clean the injection valve from Port 1 to Port 2.

Wash2

Inject to

Needle Gap Valve Clean

Valve Clean Time Solvent 2

For this step the needle tip is lifted, releasing the sealing pressure to enable rinsing around the tip sealing point.

Remark: For a detailed explanation of the Needle Gap Valve Clean, see DLW

Priming.

The injection unit is moved to the DLW Wash

Station, Wash2 position.

Wash2

(Syr.Eject Speed)

The needle is rinsed inside and out with Wash

Solvent 2.

The injection unit is moved back to the injection valve. The Inlet Port and engraving to waste Port are flushed with Wash Solvent 1 to prepare the valve for the next injection.

The injection unit is moved back to the DLW

Wash Station, Wash1 position to flush the syringe needle inside and out with Wash

Solvent 1.

Post Clean Time Solvent2

Wash1

Inject to



Wash1

Post Clean Time Solvent 1

This is a preparation step for next injection, and especially important for biofluid samples.

Cycle end for LC-Inj DLW Standard macro.

An optional cleaning step is attached to the DLW

Standard injection cycle: Stator Wash or valve toggle.

Stator Wash

Stator Wash count: 1 = Cleaning step active

Stator Wash count: 0 = Cleaning step disabled

A Repeat-End loop can be activated with the

Count.

If Stator Wash is activated, the following steps will be executed.


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The injection unit is moved to the injection valve. From the last step above, the Holding

Loop is filled with Wash Solvent 1.

Macro variable

Inject to

Delay Stator Wash

(Active)

Timer 1 is awaited to switch the valve (Toggle) into Active position (fill loop).

The DLW Actuator/Solenoid is activated to deliver Wash Solvent 2 to the Holding Loop and into the valve system.

Inject to

Wash2


The first solvent flush arriving at the valve is

Wash Solvent 1 parked in the Holding Loop at the beginning, followed by Wash Solvent 2.

Wash Solvent is changed to Wash Solvent 1.

The injection valve is switched back to the

Standby position.

Inject to

Wash1


Inject to

(Standby)

Fast DLW Injection Cycle

The Fast injection cycle differs from the Standard cycle as follows:

• The needle is not dipped in the Wash station Wash1 after sample pickup and before it moves to the injection valve.

• The wash steps after injection are reduced to Valve Clean with Wash Solvent 1 and Wash

Solvent 2. The DLW needle is flushed in the DLW Wash Station with Wash Solvent 1 only.

• Stator Wash (valve toggle) is not available.

Macro Name: Fast Injection Accela Open_Rev03

Table 17. Macro Fast Injection Accela Open (Sheet 1 of 3)


The PAL system waits first for the Sync Signal

Ready before the injection cycle is started.

The injection valve is brought to a defined position: Standby.

The Rear air segment is pulled into the Holding

Loop.

Macro variable

Remark:

Sync Signal setting Start

Inject to

Standby

Airgap Volume

Filling Speed

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The sum of Rear-, Sample List-, and

Front-Volume is aspirated into the

Holding Loop.

The Front air segment is aspirated.

The injection unit moves to the specified injection valve.

The Front- and Airgap-Volume is ejected to

Waste.

The PAL system waits for the data system

The injection valve is switched to Active position.

The Pre Inject Delay time is awaited.

Macro variable

Front Volume

Rear Volume

(SL.volume)

Airgap Volume

Filling Speed

Pullup Delay

Inject to

Front Volume

Airgap Volume

Injection Speed

Wait for DS

Inject to

Pre Injection Delay

The loop is filled with the sample volume as specified in the sample list.

(SL.volume)

The injection valve is switched to Standby position, the loop content is injected.

The plunger of the DLW Syringe is pushed down to dispense the Rear Sample and Air Segment to

Waste. The Holding Loop is still filled with

Wash Solvent 1.

Injection Speed

Post Inject Delay

Injection Speed

The DLW Actuator/Solenoid is activated to deliver Wash Solvent 2 into the Holding Loop to clean the injection valve from Port 1 to Port 2.

Inject to

Wash2



For this step the needle tip is lifted, releasing the sealing pressure to enable rinsing around the tip sealing point.

Remark: For complete details on the

Needle Gap Valve Clean, see DLW

Priming.

Wash Solvent 1 follows to prepare the valve for the next injection.

Wash1

Inject to




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The injection unit is moved back to the DLW

Wash station, Wash1 position to flush the syringe needle inside and out with Wash Solvent 1.

Wash1

Post Clean Time Solvent 1

This is a preparation step for the next injection, and is especially important for biofluid samples.

Cycle end for LC-Inj DLW Fast macro.

Operating Dynamic Load and Wash (DLW)

This section describes how to operate the Dynamic Load and Wash (DLW) option.

•

Priming the Solvent Lines

•

Location of Solvent and Waste Bottles

•

DLW Pumps

Priming the Solvent Lines

Note For trouble-free DLW operation, make sure the two solvent lines are free of air bubbles at all times. If the solvent lines are being connected for the first time or during a solvent change, you must prime the solvent lines properly until air bubbles are no longer visible. Solvent degassing is recommended.

In order to make the initial and daily priming efficient and controllable, the Open

Autosampler comes with a Cycle Composer macro, or ICC cycle.

Y

To prime the solvent lines

1. Load the macros and methods into the folder.

2. Start the corresponding macro for initial or daily priming.

3. Check the solvent lines and prime until air bubbles are no longer visible.

4. Press F4 forHome.

Location of Solvent and Waste Bottles

The DLW option contains self-priming membrane pumps. The solvent bottles can be placed either in the fast wash station holder or on the lab bench.

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You must place the Waste bottle must be placed > 30 cm ( in.) below the injection valve.

Make sure that the waste liquid can flow into the waste bottle without restriction. Place the waste tubing above the level of the liquid. Ideally, the tube is fixed at the neck of the waste bottle.

Note Use good lab practice to avoid contaminating the wash solvents and the wash bottles. Avoid biological growth in pure water by either replacing it regularly or adding a small percentage of organic solvents, such as methanol or acetonitrile. Certain buffer solutions can decompose at room temperature when exposed to light. Filtering the wash solvents before filling the bottle, especially if salt buffers are used, is mandatory to avoid any clogging of the solvent paths.

DLW Pumps

From the control point of view, the DLW pumps respond in the same manner as the fast wash station. Power-out signals activate the pumps. Because the electric current setting for the

DLW is different, the corresponding PAL Firmware Objects must be loaded for the DLW wash station type.

The wetted parts in the pump are made from the following materials:

• Membrane: Kalrez (FFPM)

• Body, valves: Ryton PPS

The pumps are self priming with a suction lift of up to 3 m water column.

DLW Actuator/Solenoid

The DLW Actuator/Solenoid has the function of separating and completely shutting-off the lines in the direction of the syringe (sample loading) or the wash solvent lines.

After opening the DLW Actuator/Solenoid for the wash solvent lines, you can pump the desired wash solvent into the system by activating the corresponding DLW pump.

Figure 67 illustrates this functionality.

The wetted parts in the DLW Actuator Solenoid are made from the following materials:

• Solenoid body: PEEK

• Seal material: FFKM (Simriz)

Note PEEK exhibits excellent chemical resistance to most of the chemicals used.

However, the following solvents are not recommended for use with PEEK: DMSO, THF, methylene chloride (dichloromethane), nitric acid, or sulfuric acid. For more details refer to the compatibility tables provided by the manufacturer of PEEK material or components.


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Note Current applied from the actuator control PCB to the actuator/solenoid activates a green LED. This activation does not indicate that the solenoid opens or closes.

Figure 36. DLW Manifold and Actuator/Solenoid

Figure 37. Inserting DLW Flow Diverter

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DLW Cycle Step-by-Step

This section provides illustrations to demonstrate a step-by-step DLW cycle.

•

Cycle for Standard Injection

•

Additional Valve Toggle Step to DLW Standard Cycle

•

Cycle for Fast Injection

Cycle for Standard Injection

Figure 38 to

Figure 51 illustrates a step-by-step cycle for the standard injection.

Figure 38. Standard: Cycle start


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Figure 39. Standard: Step 1 – A spirate rear air segment

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Figure 40. Standard: Step 2 – Get sample, aspirate rear, inject, and front volume


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Figure 41. Standard: Steps 3 - 4 – A spirate front air segment

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Figure 42. Standard: Steps 5 - 6 – Passive needle clean outside (dip) in wash position 1


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Figure 43. Standard: Steps 7 - 8 – Dispense front air segment and front sample volume to waste

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Figure 44. Standard: Steps 9 - 10 – Valve is switched to LOAD position, loop is filled with “Inject

Volume”


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Figure 45. Standard: Step 11– Valve is switched to INJECT position, start chromatographic process

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Figure 46. Standard: Step 12 – Rear sample volume and air segment are dispensed to waste


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Figure 47. Standard: Steps 13 - 14 – Valve clean with Wash Solvent 2

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Figure 48. Standard: Steps 15 - 16 – Active syringe needle wash with Wash Solvent 2

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Figure 49. Standard: Steps 17 - 18 – Valve clean with Wash Solvent 1

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Figure 50. Standard: Steps 19 - 20 – Active syringe needle wash with Wash Solvent 1


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Figure 51. Standard: Cycle end

Additional Valve Toggle Step to DLW Standard Cycle

This section contains information about additional steps that are necessary for a DLW

Standard Cycle.

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Considerations for Additional Stator Wash Cleaning Step

The DLW Standard Cycle has the built-in option for the user to toggle the injection valve at the end of the chromatographic run before equilibration of the column to the start conditions.

If the method variable “Stator Wash” is set to “1”, the extra cleaning process for the valve, with

“Valve Toggle”, is part of the standard cycle.

If the method variable is deactivated (setting “0”), the DLW Standard cycle ends as shown in

Figure 51 .

The macro (cycle) is written so that the optional valve toggle steps can be executed before re-equilibration of the column. You must synchronize the time to switch the valve with the chromatographic method using the method variable Delay Stator Wash. The two wash solvents are timed by the method variables Stator Wash Time Solvent 1 and Stator Wash Time

Solvent 2. After these wash times have elapsed, the valve is switched back to the start position.

Figure 52 illustrates the recommended retention time for Stator Wash or Valve Toggle times.

Figure 52. Timing for Stator Wash Step

Thermo Scientific

From the chromatographic point of view, the optional cleaning step is important to understand. Assuming that the valve stator between ports 1 and 6 (example standard

Cheminert valve) is contaminated and cannot be cleaned during the injection process, the valve toggle brings the engraving back between the two ports. Flushing the valve with both wash solvents eliminates remaining sample material located between stator ports 1 and 6.

What points must you consider when you use the Stator Wash or Valve Toggle option?

Observe the rules if biofluid samples are injected. First sample contact should always be with an aqueous solution to avoid protein precipitation. After washing with organic solvent (higher elution power) the system must be flushed again with wash solvent 1.

The first toggle near the end of the chromatographic cycle provides the advantage that the sample loop is already flushed out first with the mobile phase with a solvent of high elution power (assuming gradient application).


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The second valve toggle time follows immediately after finishing the second solvent flush. A second switching time cannot be programmed. The waiting time for the second valve toggle should be long enough so that the entire system is flushed out by both wash solvents.

Consider the entire delay volume to determine the second valve switch. The DLW internal volumes are:

Manifold, 90 μL

Holding Loop,108 μL

Syringe Needle Gauge 22, 6.7 μL

Installed Injection Loop

Total delay volume: 205 μL + Loop content volume

Do the second valve toggle (back to starting condition) before the system equilibration time has started. The Loop content is ideally a solvent of a low elution power when switched back.

If isocratic chromatography is applied, the remaining contaminants might be washed into the system and can build up higher background noise for the column, the detector, or both over a longer period of time.

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Additional Cleaning Step Stator Wash or Valve Toggle Step-by-Step

Figure 53 to

Figure 58 illustrate additional cleaning steps for “Stator Wash”.

Stator Wash: End of Standard Injection Cycle

Figure 53. Start for additional cleaning step “Valve Toggle”


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Figure 54. Stator Wash: Step 1 - 2 – Valve switched to Load Position (toggle), valve clean with

Wash Solvent 1

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Figure 55. Stator Wash: Step 3 – Valve clean with Wash Solvent 2


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Figure 56. Stator Wash: Step 4 – Wash Solvent 2 dispensed by Wash Solvent 1

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Figure 57. Stator Wash: Step 5 – Second valve clean with Wash Solvent 1


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Figure 58. Stator Wash: Step 6 – Valve switched back to Inject Position (toggle)

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Cycle for Fast Injection

Figure 59 to

Figure 70 illustrate a step-by-step cycle for the Fast Injection.

Figure 59. Fast: Cycle start


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Figure 60. Fast: Step 1 – Aspirate rear air segment

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Figure 61. Fast: Step 2 – Get sample, aspirate rear, inject, and front volume


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Figure 62. Fast: Step 3 - 4 – Aspirate front air segment

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Figure 63. Fast: Steps 5 - 6 – Dispense front air segment and front sample volume to waste


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Figure 64. Fast: Steps 7 - 8 – Valve is switched to LOAD position, loop is filled with “Inject

Volume”

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Figure 65. Fast: Step 9 – Valve is switched to INJECT position, start chromatographic process


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Figure 66. Fast: Step 10 – Rear sample volume and air segment are dispensed to waste

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Figure 67. Fast: Steps 11 - 12 – Valve clean with Wash Solvent 2


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Figure 68. Fast: Steps 13 - 14 – Valve clean with Wash Solvent 1

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Figure 69. Fast: Step 15 – Active syringe needle wash with Wash Solvent 1


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Figure 70. Fast: Cycle end

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Using Dynamic Load and Wash (DLW). Thermo Fisher Scientific Thermo PAL

Using Dynamic Load and Wash (DLW)

Overview

Installing Cycle Composer Macros or ICC Cycles

Installing the Cycle Composer Macros or ICC Cycles

General Considerations

Priming the Solvent Lines, Wash1 and Wash2

Standard DLW Injection Cycle

Fast DLW Injection Cycle

Operating Dynamic Load and Wash (DLW)

Priming the Solvent Lines

Location of Solvent and Waste Bottles

DLW Pumps

DLW Actuator/Solenoid

DLW Cycle Step-by-Step

Cycle for Standard Injection

Additional Valve Toggle Step to DLW Standard Cycle

Considerations for Additional Stator Wash Cleaning Step

Additional Cleaning Step Stator Wash or Valve Toggle Step-by-Step

Stator Wash: End of Standard Injection Cycle

Cycle for Fast Injection

Related manuals

Table of contents

Using Dynamic Load and Wash (DLW). Thermo Fisher Scientific Thermo PAL

Using Dynamic Load and Wash (DLW)

Overview

Installing Cycle Composer Macros or ICC Cycles

Installing the Cycle Composer Macros or ICC Cycles

General Considerations

Priming the Solvent Lines, Wash1 and Wash2

Standard DLW Injection Cycle

Fast DLW Injection Cycle

Operating Dynamic Load and Wash (DLW)

Priming the Solvent Lines

Location of Solvent and Waste Bottles

DLW Pumps

DLW Actuator/Solenoid

DLW Cycle Step-by-Step

Cycle for Standard Injection

Additional Valve Toggle Step to DLW Standard Cycle

Considerations for Additional Stator Wash Cleaning Step

Additional Cleaning Step Stator Wash or Valve Toggle Step-by-Step

Stator Wash: End of Standard Injection Cycle

Cycle for Fast Injection

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Table of contents