Micro-Fraction Collection Option for the UltiMate 3000 ...tools.thermofisher.com/content/sfs/manuals/Man... · The Micro Fraction Collection option of the UltiMate 3000 Series Nano/Cap

M164208R1

Micro-Fraction Collection Option for the

UltiMate™ 3000 Series Nano/Cap Autosampler

Operating Instructions

Version: 1.2

Date: April 2008

© 2008 DIONEX

Doc.: WPS-3000 NanoCap Micro Fraction Collection_Operating Instructions_V1.2

JEngelhaupt

Thermo Branding Stamp

WPS-3000 Micro Fraction Collection - Operating Instructions

Page II WPS-3000 NanoCap Micro Fraction Collection_Operating Instructions_V1.2

All information in this manual is subject to change without notice and does not represent a commitment on the part of Dionex Corp.

CHROMELEON® and UltiMate™ are (registered) trademarks of Dionex Corp.

Analyst® is a registered trademark of Applied Biosystems.

Cheminert® is a registered trademark of Valco Instruments Company Inc.

HyStar™ is a Trademark of Bruker Daltonics Inc.

Xcalibur™ is a Trademark of Thermo Fisher Scientific, Inc.

Any other mentioned trade or company names are subject to the copyright and the property and trademark rights of the respective companies.

All rights reserved including those for photomechanical reproduction and storage on electronic media. Without the written permission of Dionex, no part of this publication may be reproduced in any form (by means of photocopy, microfilm, or any other process) for any purpose or processed, copied, transmitted, or distributed in any other form, independent from the means, electronic or mechanical, that is used.


WPS-3000 NanoCap Micro Fraction Collection_Operating Instructions_V1.2 Page III

Warnings The Warning sign and the Important sign shown below are included in various locations in this manual or in the manuals provided with the instruments which are to be tested. These signs provide the following information:

Warning: Indicates that failure to take note of the accompanying information may result in personal injury.

Important: Indicates that failure to take note of the accompanying information may result in damage to the instrument.

Tip: Indicates general information intended to optimize the performance of the instrument.

Safety Precautions

Warning: The following precautions should be followed to minimize the possibility of personal injury and/or damage to property.

Tip: Make certain that you are familiar with the contents of this manual and the operating instructions before working on the system.

The operator should follow all safety precautions, warnings, etc. provided with the instruments, in addition, please note the items presented below:

• All components of the system should be plugged into a common power line that is directly connected to a true ground.

• Repair or replace faulty power cords and all communication cables. • If a leak occurs, turn off power to the instrument and remedy the situation immediately. • If the mobile phase includes volatile or flammable solvents, avoid open flames and sparks. • Many organic solvents and buffers are toxic. Make certain that you know the toxicological

properties of all mobile phases that you are using. • The toxicological properties of many samples may not be well known. If you have any doubt

about a sample, treat it as if it contained a potentially harmful substance. • Wear protective eye goggles when handling mobile phases or operating the instrument. An eye

wash facility and a sink should be close to the unit. If any mobile phase splash on the eyes or skin, wash the affected area and seek medical attention.

• Dispose of all waste mobile phase in an environmentally safe manner that is consistent with all local regulations. Do not allow flammable and/or toxic solvents to accumulate. Follow a regulated, approved waste disposal program. Never dispose flammable and/or toxic solvents through the municipal sewage system.

• Wear protective eye goggles when handling fused silica tubing (i.e. installation, cutting etc.). • If a buffer is used as a part of the mobile phase, flush the system with several volumes of an

aqueous solution before it is shut down. This will prevent salt build-up inside the unit. • Do not use the instrument in ways other than those indicated in the instructions given in this

manual.


Page IV WPS-3000 NanoCap Micro Fraction Collection_Operating Instructions_V1.2


WPS-3000 NanoCap Micro Fraction Collection_Operating Instructions_V1.2 - 1 -

Table of Contents

Warnings ……………………………………………………………………….……….. III

Safety Precautions ………………………………………………………………………. III

1 How to use this Manual ...................................................................................... 4

2 Introduction ......................................................................................................... 6

2.1 Basic Requirements .......................................................................................................................6

3 Chromeleon ......................................................................................................... 7

3.1 Server Configuration ......................................................................................................................7 3.2 Programming a 2D-LC Method ................................................................................................... 11

3.2.1 Program for the First Dimension LC Separation ................................................................. 11 3.2.2 Add Fractionation Templates and Parameters to the First Dimension Program File ......... 15 3.2.3 Creation of the Second Dimension Sequence Automatically ............................................. 19 3.2.4 Define Post Acquisition Steps ............................................................................................. 20 3.2.5 Fraction Collection Enabled During the Second Dimension Separation. ........................... 25 3.2.6 Analyzing Multiple Samples in One Sequence ................................................................... 26 3.2.7 Needle Position Before and After Fractionation .................................................................. 27 3.2.8 Programming a Wash .......................................................................................................... 27

3.3 WPS Accessories ........................................................................................................................ 28 3.4 Injection Methods with the Micro Fraction Collection Option ................................................ 28

3.4.1.1 µL-Pickup / Full-Loop 28 3.4.1.2 User Defined Program (UDP) 28

3.5 Reviewing Results ....................................................................................................................... 30 3.6 Numbering of the Fractions ........................................................................................................ 33

4 Applications ....................................................................................................... 35

4.1 2D-LC of Peptides using the MIC SCX/NAN PepMap Configuration ...................................... 35 4.1.1 Fluidic Connections ............................................................................................................. 35 4.1.2 The Workflow ...................................................................................................................... 36 4.1.3 Experimental ........................................................................................................................ 36 4.1.4 Results ................................................................................................................................. 37

4.1.4.1 First Dimension Separation 37 4.1.4.2 Second Dimension Separation 38 4.1.4.3 2D-LC Plot 38

4.1.5 Application Kits .................................................................................................................... 39 4.2 2D-LC of Peptides Using the CAP SCX/CAP Monolith Configuration .................................... 42

Fluidic Connections ........................................................................................................................... 42 4.2.1 Workflow .............................................................................................................................. 43 4.2.2 Experimental ........................................................................................................................ 44 4.2.3 Results ................................................................................................................................. 45



- 2 - WPS-3000 NanoCap Micro Fraction Collection_Operating Instructions_V1.2

4.2.4 Application Kits .................................................................................................................... 47 4.3 2D-LC of Proteins using the MIC WAX / 500 µm I.D Monolith Configuration ........................ 50

4.3.1 Fluidic Connections ............................................................................................................. 51 4.3.2 The Workflow ...................................................................................................................... 51 4.3.3 Experimental ........................................................................................................................ 52 4.3.4 Results ................................................................................................................................. 52


4.3.5 Application Kits .................................................................................................................... 55 4.3.6 Automated in-well digestion for tandem MS detection and protein identification ............... 57

4.3.6.1 Tryptic digest fractions 57 4.4 2D-LC of Proteins using the MIC WAX / 200 µm I.D. Monolith Configuration ....................... 59

4.4.1 Fluidic Connections ............................................................................................................. 60 4.4.2 The Workflow ...................................................................................................................... 60 4.4.3 Experimental ........................................................................................................................ 61 4.4.4 Results ................................................................................................................................. 61


4.4.5 Application Kits .................................................................................................................... 64

5 Appendixes ........................................................................................................ 67

5.1 DCMSlink Version 2.0 .................................................................................................................... 67 5.1.1 Xcalibur Version 2.0 ............................................................................................................ 68 5.1.2 HyStar Version 3.2 .............................................................................................................. 70 5.1.3 Analyst Version 1.4.2 .......................................................................................................... 73

5.2 Maintenance of the 8-Port Switching Valve .............................................................................. 75 5.2.1 Cleaning and Replacing Parts ............................................................................................. 75 5.2.2 Disassembly of the Valve .................................................................................................... 76 5.2.3 Reassembly of the Valve ..................................................................................................... 76

5.3 CM programs for 2D-LC of Peptides using the MIC SCX/NAN PepMap Configuration ........ 77 5.3.1 First Dimension Separation ................................................................................................. 77 5.3.2 Prepare System for Second Dimension Separation (Wash SCX prepare for RP) ............. 81 5.3.3 Second Dimension Separation ............................................................................................ 84 5.3.4 Prepare System for First Dimension Separation (Wash RP prepare for SCX) ................... 87

5.4 CM programs for 2D-LC of Peptides Using the CAP SCX/CAP Monolith Configuration ..... 90 5.4.1 First Dimension Separation ................................................................................................. 90 5.4.2 Prepare System for Second Dimension Separation (Wash SCX prepare for RP) ............. 94 5.4.3 Second Dimension Separation ............................................................................................ 98 5.4.4 Prepare System for First Dimension Separation (Wash RP prepare for SCX) ................. 102

5.5 CM programs for 2D-LC of Proteins using the MIC WAX / 500 µm I.D Monolith Configuration 106 5.5.1 First Dimension Separation ............................................................................................... 106 5.5.2 Prepare System for Second Dimension Separation (Wash IEX prepare for RP) ............. 110 5.5.3 Second Dimension Separation .......................................................................................... 113 5.5.4 Prepare System for First Dimension Separation (Wash RP prepare for IEX) .................. 117 5.5.5 Add Trypsin by WPS to fractions of interest ...................................................................... 120

5.6 CM programs for 2D-LC of Proteins using the MIC WAX / 200 µm I.D. Monolith Configuration 123 5.6.1 First Dimension Separation ............................................................................................... 123



5.6.2 Prepare System for Second Dimension Separation (Wash IEX prepare for RP) ............. 127 5.6.3 Second Dimension Separation .......................................................................................... 130 5.6.4 Prepare System for First Dimension Separation (Wash RP prepare for IEX) .................. 134



1 How to use this Manual The Micro Fraction Collection option of the UltiMate 3000 Series Nano/Cap Autosamplers (WPS-3000 [T][B] with the extended automation features will only be available from Chromeleon 6.80 Service Pack 2 (or later) in combination with the Extended Fraction Collection license.

The material included in this manual is intended as a supplementary guide to the Operating Instructions of the UltiMate 3000 Series Nano/Cap Autosamplers (WPS-3000 [T][B]) and is provided to assist the use of the Micro Fraction Collection option. It is assumed that the individual using this manual has sufficient training in the installation of analytical instrumentation and is aware of the potential hazards including (but not limited to) electrical hazards, chemical solvent hazards, exposure to UV radiation and the exposure to pressurized solvents.

The layout of this manual is designed to provide quick reference to the sections of interest to the user. However, we recommend that you review the manual thoroughly before starting any application in order to obtain full understanding of the procedure.

To perform Off-line 2D-LC using the Micro Fraction Collection option, hardware and software changes are required. Hardware modifications are described in the manual ‘WPS-3000 NanoCap Micro Fraction Collection_Installation Instructions_V1.2’ (part no. 164210) provided with the Micro Fraction Collection Option kit.

This manual describes the required modifications in the Server Configuration, Program Files in Chromeleon and includes four examples for the Off-line 2D-LC separation of peptides and proteins using the Micro Fraction Collection option.

Required actions in the Server Configuration and in Chromeleon are numbered; each section starts with number 1.

Required modifications in a Chromeleon Tab Page are listed alphabetical, starting with a) in each section.

The DGP/LPG-3600M is equipped with two ternary gradient pumps. In this manual, the ‘LoadingPump’ refers to the left pump and the ‘Micro Pump’ refers to the right pump.

This manual describes four applications, ‘MIC SCX/NAN PepMap’ and ‘CAP SCX/CAP Monolith’.

MIC SCX/NAN PepMap refers to Section 4.1, 2D-LC of Peptides using the MIC SCX/NAN PepMap Configuration.

• In this experiment, the first dimension separation was performed split-less on a 1 mm I.D. x 15 cm SCX column. The second dimension separation was performed on a 75 µm I.D. x 15 cm nano column using a split ratio of 1:1000.

CAP SCX/CAP Monolith refers to Section 4.2, 2D-LC of Peptides Using the CAP SCX/CAP Monolith Configuration.

• The first dimension separation was performed on a 300 µm I.D. x 15 cm SCX column, and the second dimension separation was performed on a 200 µm I.D. x 5 cm PepSwift Monolithic column, both using a split ratio of 1:100.

MIC WAX/500 µm I.D. Monolith refers to Section 4.3, 2D-LC of Proteins using the MIC WAX / 500 µm I.D Monolith Configuration

• In this experiment, the first dimension separation was performed split-less on a 1 mm I.D. x 5 cm ProSwift WAX column. The second dimension separation was performed on a 500 µm I.D. x 5 cm PepSwift Monolithic column using a split ratio of 1:15

MIC WAX/200 µm I.D. Monolith refers to Section 4.4, 2D-LC of Proteins using the MIC WAX / 200 µm I.D. Monolith Configuration



• In this experiment, the first dimension separation was performed split-less on a 1 mm I.D. x 5 cm ProSwift WAX column. The second dimension separation was performed on a 200 µm I.D. x 5 cm PepSwift Monolithic column using a split ratio of 1:100.

An UV detector for the second dimension separation is required to generate the 2D retention map. To display the UV data for both dimensions in the 2D retention map, two UV detectors (first dimension and second dimension) are required.

This manual is provided 'as is'. Every effort has been made to supply complete and accurate information and all technical specifications and programs have been developed with the utmost care. However, Dionex assumes no responsibility and cannot be held liable for any errors, omissions, damage, or loss that might result from any use of this manual or the information contained therein. We appreciate your help in eliminating any errors that may appear in this document.



2 Introduction With the new Micro Fraction Collection enabled (patent pending), injection, fractionation, and re-injection can all be performed on a single system. The system uses the three well capacity to allow for fractionating and re-injecting over 1000 samples without the need for any manual handling.

In combination with the Extended Fraction Collection capabilities of the Chromeleon® Chromatography Management Software, set up, system control, reviewing and data reporting are straightforward and easily performed. A new overlay view, the 2D Retention Map, provides a 2D gel-like representation of UV data for easy comparison. Dynamically linked sample and fraction displays make reviewing and finding the fractions of interest easy.

2.1 Basic Requirements The procedures described in this manual apply to the following instruments:

TABLE2-1 List of supported UltiMate 3000 System Components

Instrument Supported Model UltiMate 3000 Series Micro/Loading Pump (a)

LPG-3600M LPG-3600MB (biocompatible version) DGP-3600M DGP-3600MB (biocompatible version)

UltiMate 3000 Series Flow Manager (b)

FLM-3100 FLM-3100B (biocompatible version) FLM-3300 (x2) FLM-3300B (x2) (biocompatible version)

UltiMate 3000 Series UV Detector (c)

UVD-3000 VWD-3100 VWD-3400

UltiMate 3000 Series Microautosampler (d)

WPS-3000 (non-cooled version) WPS-3000B (non-cooled, biocompatible version) WPS-3000T (cooled version) WPS-3000TB (cooled, biocompatible version)

Additional license Extended Fraction Collection license Note: a) In conjunction with the UltiMate 3000 Degasser SRD-3600.

b) Two 10-port switching valves are required. c) A UV detector for the second dimension separation is required to generate the 2D retention map.

To display the UV data for both dimensions in the 2D retention map, two UV detectors (first dimension and second dimension) are required.

d) Nano/Cap Autosampler equipped with the Micro Fraction Collection Option



3 Chromeleon The Micro Fraction Collection option of the UltiMate 3000 Series Nano/Cap Autosamplers (WPS-3000 [T][B] with the extended automation features will only be available from Chromeleon 6.80 Service Pack 2 (or later) in combination with the Extended Fraction Collection license. The standard Fraction Collection license is not sufficient. However, data created with the Extended Fraction Collection license can be reviewed with any Chromeleon 6.80 Service Pack 2 Client.

Tip: DCMSLink version 2.0 in combination with a full Chromeleon licence including the Extended Fraction Collection Licence is compatible with the WPS Micro Fraction Collection Option.

The Micro Fractionation Collection Option is not compatible with DCMS Link Version 1.1

3.1 Server Configuration To perform a successful Off-line 2D-LC experiment using the Fraction Collection option, the Server Configuration has to be configured correctly.

FIGURE 3-1 display the server configuration for a typical installation of an UltiMate 3000 system. FIGURE 3-2 through FIGURE 3-5 displays the required changes made to the Server Configuration.

FIGURE 3-1: Typical Server Configuration for an UltiMate 3000 System

The UV detector which is typically included in the UltiMate 3000 system is used to display data of the second dimension separation and to generate 2D gel view images. If data of the first dimension should be monitored a second UV detector is required.

To add the second UV detector to the server configuration; choose ‘UV_2’ as Device name and ‘UV_VIS_1_2’ as Signal name.

Section 4 Applications, describes two applications using two UV detectors. In these examples, the detector UV_2 with the signal name UV_VIS_1_2 was used as detector for the First Dimension separation.



1. In the WPS-3000 Autosampler box, select the ‘Micro Fraction Collection option installed’.

FIGURE 3-2: Options Tab Page (for the WPS-3000 Autosampler)

Tip: After selecting the ‘Micro Fraction Collection option’ in the WPS-3000 Autosampler Options tab, the modified Server Configuration must be saved and either the ‘Reset’ or ‘SelfTest’ command must be sent to the autosampler (use F8 key or the corresponding button of the Sampler Tab Set panel).



2. Add the Fraction Collection Driver to the timebase.

FIGURE 3-3: Devices Selection Dialog

a) Select Add Device on the context menu b) Select Generic from the Manufacturers box (left) c) Select Fraction Collection from the Devices box (right) d) Press OK to add the Fraction Collection driver to the Timebase. The Fraction Collection

dialog will appear as presented in FIGURE 3-4.



FIGURE 3-4 Fraction Collection Settings Dialog

3. Make sure that the ‘DeviceName’ is ‘FractionCollection' and that the ‘Maximum number of Detection channels’ is set to ‘1’. Press ‘OK’.

4. Save the Server Configuration (FIGURE 3-5).

FIGURE 3-5 Typical Timebase for an UltiMate 3000 system with the Micro Fraction Collection Option



3.2 Programming a 2D-LC Method For a complete 2D-LC experiment with fractionation in the first dimension and injection of the fractions into the second dimension, 4 programming steps are required.

1. Create the program for the first dimension separation using the Program Wizard (Section 3.2.1).

2. After you have created the program for the first dimension separation, insert the fractionation template (Section 3.2.2).

3. Create the program for the second dimension separation using the Program Wizard (Section 3.2.3).

4. Define the ‘Post Acquisition Steps’ in the program made in step 2 (Section 3.2.4).

3.2.1 Program for the First Dimension LC Separation

The program used for the first dimension separation is created via the wizard and is similar to programming a direct injection. (‘File>new>Program File’). In the Program Wizard, the parameters for fraction collection are programmed in the ‘Fraction Collection’ dialog box.

The specified flow rates on a Nan/Cap UltiMate 3000 always refer to the column flow rates. If a split flow is employed, the master flow rate is calculated automatically. However, typically only one pump is connected to one splitter. In case that the loading pump and the micro pump are both connected to one flow splitter through the 10-port switching valve (see Section 4.2 2D-LC of Peptides Using the CAP SCX/CAP Monolith Configuration,) the master flow rate of the loading pump must be calculated manually. Therefore the column flow rate, which is used in the first dimension separation, must be converted to the master flow rate by using the split ratio from the currently installed flow splitter.

As an example, if the desired column flow rate is 6 µL/min and the split ratio on the chip card of the CAP flow splitter is 95, the loading pump flow is:

6 µL/min * 95 = 570 µL/min

In FIGURE 3-6, the desired column flow rate is 6 µL/min using a CAP flow slitter with a split ratio of 1:95.

… LoadingPump.Flow = 570 µL/min LoadingPump.%B = 60.0 [%] LoadingPump.%C = 0.0 [%] …

FIGURE 3-6 Example for Programming the Flow Rate when two Pumps are connected to one Splitter

Tip: When two pumps are connected to one flow splitter as in the CAP/CAP application (Section 4.2), the master pump flow rate on the loading pump (First Dimension) depends on the split ratio and must be calculated manually.

When all programming steps in the wizard are completed, select the desired parameters in the ‘Fraction Collection’ tab page (FIGURE 3-7).



FIGURE 3-7 Fraction Collection Tab Page

a) Select the Collection period (Time per fraction) b) Select the Collection Time Frame




c) Select the pump which delivers the flow for fraction collection.

The ‘Pump device’ must be assigned to calculate the fractionation volume.




d) Enter the Delay Time or the Delay Volume between the detector and the fraction collector.

The delay time is the time that a substance needs to travel from the detector cell of the first detector to the collection tube. The delay volume is the volume between the detector cell and the tube. Typical delay volumes are listed in TABLE3-1.

TABLE3-1 Typical Delay Volume (µL) between the Center of the Flow Cell and the Fraction Collection Vial in a typical Configuration

Configuration 2.4 µL Sample Needle 15 µL Sample Needle 1 mm I.D. SCX – 180 nL Flow cell* 8.3 µL 21.0 µL 300 µm I.D. SCX – 45 nL Flow cell 3.7 µL 16.3 µL * Flow cell connected to valve with 5 cm x 130 µm I.D. tubing

Tip: If you want to use the delay time approach, the flow must be constant. If the flow rate changes, the actual delay time between the detector and the fraction collector will change during the run, which is not supported by the system. If you find it easier to determine the delay volume rather than the delay time, enter the ‘Delay volume’ instead and let the system use the (constant) flow to calculate the appropriate ‘Delay time’.

Tip: If you use several detectors, this entry always refers to the first detector after the column.



3.2.2 Add Fractionation Templates and Parameters to the First Dimension Program File

In comparison with a standard fraction collector, the WPS-3000 [T][B] fractionation needle uses the needle in needle principle (one to puncture and one to withdraw). This makes it possible to fractionate in vials with a cap or well-plate protected by a film. However, when fractionating into capped vials, there is a risk of losing some eluent in the puncturing needle with a potential carryover or contamination risk. Fractionation is possible in capped and uncapped 1.5 mL vials, 250 µL vials, 96 well plates and 384 well plates.

The program file created from the wizard contains the fractionation commands; ‘start’, ‘collection period’ and ‘stop’, but does not contain the commands for needle height during fractionation. These commands, provided in a template format, need to be inserted manually.

The directory which contains the required template is located on the CD delivered with the application kits. “CM templates for installation and applications of micro fraction collection option WPS-3000”.

Fraction_Collection_WPS\ Templates Capped and Un-Capped Vials

Use the template for capped vials when fractionating in capped vials and use the template for uncapped vials when fractionating in uncapped vials.

1. Copy the appropriate template to the program file just before time = 0.000

Template for capped vials is given in FIGURE 3-10

FIGURE 3-10 Template for Capped Vials



Template for uncapped vials is given in FIGURE 3-11

FIGURE 3-11 Template for Uncapped Vials

The vertical position of the fractionation needle during fractionation is defined by ‘NeedleExtension’ and ‘FractionHeight’. The values can be changed in the copied templates. A lower value will result in a lower needle position.

‘NeedleExtension’: Distance (µm) that the fused silica injection / fractionation needle remains outside the metal pre-puncture needle during vial change (only available when using uncapped vials)

‘FractionHeight’: Distance (µm) between the bottom of the vial and the injection / fractionation needle.

FIGURE 3-12 Defining the ‘NeedleExtension’ and ‘Fractionation Height’

When fractionating at low flow rates, e.g. 6 µL/min, the fractionation needle needs to touch the bottom of the plate. If the needle does not touch the bottom of the plate, the droplet will stick on the needle. When fractionating high flow rates, e.g. 250 µL/min, it is not necessary for the needle to touch the bottom of the vial.

FractionHeight

NeedleExtension



TABLE3-2, TABLE3-3, TABLE3-4 and TABLE3-5 present some typical values for fractionation in uncapped and capped 250µL vials, 96 well plates and 384 well plates. The listed values are valid for 250 µL vials (part no. 6820.0027), 96 V-bottom well plate (part no. 164282) and V-bottom 384 well plate (part no. 164275).

Tip: The FractionHeight can be different from system to system and needs to be optimized. Optimization can be done by programming a blank run with fractionation start at time 0.

TABLE3-2 Typical Settings for different Vial/well Types uncapped, Needle above the Vial/well

250 µL vials 96 well plate 384 well plate NeedleExtension 10000 10000 10000 FractionHeight 30000 15000 15000

TABLE3-3 Typical Settings for different Vial/well Types uncapped, Needle at the Bottom of the Vial/well

250 µL vials 96 well plate 384 well plate NeedleExtension 10000 10000 10000 FractionHeight 10000 8000 8000

TABLE3-4 Typical Settings for different Vial/well Types capped, Needle above the Vial/well

250 µL vials 96 well plate 384 well plate FractionHeight 15000 6000 6000

TABLE3-5 Typical Settings for different Vial/well types capped, Needle at the Bottom of the Vial/well

250 µL vials 96 well plate 384 well plate FractionHeight 2500 5000 4000

Warning: With a ‘NeedleExtension’ set above 10000, the fused silica needle will move too far outside the metal puncturer needle and will break when changing fraction position.

The values displayed in TABLE3-2, TABLE3-3, TABLE3-4 and TABLE3-5 are only valid when with a ‘SampleHeightOffset’ of 0. Make certain that the ‘SampleHeightOffset’ is set to 0 (→ FIGURE 3-13).



FIGURE 3-13: Chromeleon F8 Command Dialog for Setting the ‘SampleHeightOffset’ for a 384 Well Plate

The ‘SamleHeightOffset’ settings can only be changed in expert mode. The Expert mode can be selected by right clicking on a property in the left box.

Tip: Set the ‘SampleHeightOffset’ to 0

When entering a ‘FractionHeight’ or ‘NeedleExtension’ position which can not be reached by the WPS, the following error will be displayed:

FIGURE 3-14 Error Message when Destination ‘FractionHeight’

is out of Range

In most instances, the ‘FractionHeight’ is set too high and a lower ‘FractionHeight’ should be entered.

The position where fractionation starts (‘TubePosition’) must be specified in the inserted template (e.g. start position Gc1).

… -0.001 Sampler.TubePosition = Gc1 FractionCollection.TubePosition = Sampler.TubePosition AirDry = Off …

FIGURE 3-15: Insert the Location where the Fractionation starts and specify the State of the Air pump when Fractionation Position Changes (as an Example: Position Gc1 and AirDry = off)



Tube position: Enter the start position of the tube (vial or well) to which the fractionation needle shall be moved.

Tip: The fractionation needle follows the path shown in FIGURE 3-16. This pattern cannot be changed.

1 2 3 4 5 6 7 8

C

A

B

E

D Next

Position ‘Ba1’

Green Segment (40 Vials)

Start Position

‘Gc1’

Sample Rack Positions

Green Blue

Red

FIGURE 3-16: The Path of the Fractionation Needle with Start Position Gc1

For fractionation into two trays, the fractionation will continue in position Ba1 after position Ge8 and will follow the same fractionation pattern as presented in FIGURE 3-16.

During fractionation, it is possible to activate the air pump to blow the droplet from the fused silica needle into the vial. An AirDry command is programmed with the option to activate the pump or deactivate the pump during fractionation.

AirDry: When On, the air pump is activated when the needle changes fractionation position

When Off, the air pump is de-activated when the needle changes fractionation position

The air pump setting (AirDry = On or Off) can be changed during the run.

Tip Dionex recommends that you do not use the AirDry feature while fractionating into a 384 well plate, as the liquid may be blown out when the air pump is activated.

3.2.3 Creation of the Second Dimension Sequence Automatically

Chromeleon can create the second dimension sequence automatically after the first dimension sequence is finished. To do this, it is necessary to create the second dimension program file and incorporate it in to the post acquisition steps as described in Section 3.2.4.



3.2.4 Define Post Acquisition Steps

. A wizard guides you in creating this step.

1. Open the program file used for the first dimension separation.

FIGURE 3-17: Create ‘Post Acquisition Steps’

a) Select ‘Post-acquisition steps’ in the program file commands window. b) Click on ‘Click on this line to add a new step’. c) Select ‘Create Fraction Analysis Samples’.

Select ‘Create Fraction Analyses Samples’

a) Select ‘Post acquisition step’

c)

Click once

b)



FIGURE 3-18: Create Fraction Analysis Samples Tab Page

d) Select ‘Reanalyze all tubes’.

Tip If it is not necessary to reanalyze all tubes, the sequence for the second dimension separation created by Chromeleon needs to be adjusted manually.




e) Enable ‘Add sequence to batch’ to start the second dimension sequence automatically after the first dimension sequence is completed.




f) Select the program for the second dimension separation.

FIGURE 3-21: Post Acquisition Steps are added to the First Dimension Program File



The post acquisition step will be executed when the first dimension sequence is completed. A Sequence to re-inject all fractions in the second dimension is automatically created and added to the running batch. This sequence will start after the first sequence is completed. The post acquisition steps do not have to be in the last program of the first dimension sequence, but should be in all programs used to fractionate a sample.

FIGURE 3-22: Typical Sequence used for Separation and Fractionation of One Sample

In the CAP SCX/CAP Monolith configuration as described in Section 4.2, the tubing from port 1 on the right switching valve to the flow splitter inlet (250 µm I.D. x 15 cm) is shared for both dimensions. Therefore a wash program must be added to the first dimension separation sequence to prevent salt entering the mass spectrometer. FIGURE 3-22 shows a sequence used in the first dimension separation and FIGURE 3-24 shows the sequence automatically made by Chromeleon for the second dimension separation.

2. A second sequence will be created to analyze the fractions when you start the Batch.

FIGURE 3-23: Automatically created Sequence for Analyzing the second Dimension

FIGURE 3-24: Typical Sequence for Injecting all Collected Fractions (Gc1 was the Fractionation Start Position Specified in the First Dimension Program Template)

After completing a 2D experiment and a new set of 2D experiments needs to be started, a wash program after the last sample in the second dimension is required to prepare the system for the first dimension separation and equilibrate the first dimension (SCX) column.

FIGURE 3-25: Typical Sequence for Injecting Collected Fractions with a Wash Program to Prepare the System for a New Set of 2D Experiments.

Sequence created automatically by Chromeleon



3.2.5 Fraction Collection Enabled During the Second Dimension Separation.

Separation of complex protein or peptide mixtures may require a second fractionation step. This second fractionation step is easy to perform using the Extended Fraction Collection capabilities of the Chromeleon® Chromatography Management Software.

To enable fraction collection in the second dimension separation, the fraction collection templates must be added to the second dimension program file as described in section 3.2.2.

The second dimension program file is added to the first dimension program file via the “Post Acquisition Steps” as described in section 3.2.4. Since only one program file can be added to the “Post Acquisition Steps” but the fractionation needs to start at a different position each run, the start position can be programmed using the following formula for the “Sampler.TubePosition command:

Ga1 + 10 * (Sample.number - 1)

Ga1 = Start position for fraction collection in the second dimension

10 = Amount of fractions collected during one second dimension separation

Sample.number -1 = Filled in by Chromeleon

… -0.001 Sampler.TubePosition = GA1 + 10 * (Sample.number - 1) FractionCollection.TubePosition = Sampler.TubePosition AirDry = Off …

FIGURE 3-26: Insert the Location where the Fractionation starts and fill in the amount of fractions collected during one second dimension run, specify the State of the Air pump when Fractionation Position Changes (as an Example: Position Gc1 and AirDry = off)

When performing a ready

Example

The original sample is injected from position Ra1. From the first dimension separation, 15 fractions are collected starting at position Ba1. During the second dimension program, 5 fractions are collected starting at position Ga1.

For the example described above, the second dimension program requires the following formula: Ga1 + 5 * (Sample.number - 1).

The first fraction from the first dimension separation is located at position Ba1. The second dimension program injects the fraction Ba1 onto the second dimension column. The fractionation in the second dimension starts at position Ga1 and ends at position Ga5. => Ga1 + 5*(1-1) = Ga1

The second fraction from the first dimension separation is located at position Ba2. The second dimension program injects the fraction Ba2 onto the second dimension column. The fractionation in the second dimension starts at position Ga6 and ends at position Ga10. => Ga1 + 5*(2-1) = Ga6

The third fraction from the first dimension separation is located at position Ba3. The second dimension program injects the fraction Ba3 onto the second dimension column. The fractionation in the second dimension starts at position Ga11 and ends at position Ga15. => Ga1 + 5*(3-1) = Ga11

The fourth fraction from the first dimension separation is located at position Ba4. The second dimension program injects the fraction Ba4 onto the second dimension column. The fractionation in the second dimension starts at position Ga16 and ends at position Ga20. => Ga1 + 5*(4-1) = Ga16



3.2.6 Analyzing Multiple Samples in One Sequence

If two or more samples are considered for 2D-LC analysis, e.g. by SCX and RP separations, the following sequence needs to be programmed;

-Sequence 1. SCX1-SCX2-…SCXn

Each sample which is fractionated in the first dimension requires a unique program with a unique fractionation start position (tube position). The second sample will start fractionating at the position programmed for the previous sample without adjusting the ‘Tube Position’.

Chromeleon automatically creates one sequence for the second dimension (RP) separation that contains all fractions from all first dimension separations.

Sequence 2 is generated automatically by Chromeleon and added to the Batch-list, and is started after the first dimension sequence (SCX) is completed.

FIGURE 3-27: Typical Sequence used for Separation and Fractionation of Multiple Samples

FIGURE 3-28: Typical Sequence for Injecting all Collected Fractions Generated from Sample 1 and Sample 2



3.2.7 Needle Position Before and After Fractionation

On power up or during any manual self test (e.g. through the F8 key or the Tab Set panel), the 8-port inject/fractionation valve (if selected in the Server Configuration) will switch to the ‘load’ position to prevent droplets falling into vials/wells when the flow is on. In this valve position the flow is directed to waste through ports 7 and 8.

After the self test, the needle is moved to the wash/drain port and the inject/fractionation valve is switched to ‘inject’ to flush the loop with mobile phase.

Tip When operating the autosampler at flow rates higher than 50 µL/min it may happen that the leak sensor is activated because of an overflow of the drip tray. In such a case remove the liquid from the drip tray on a routine basis and reduce the sensitivity of the leak sensor.

Warning: The sampler must be switched to standby before switching off for transport (i.e. shipped to factory). Going to Standby drives the needle back into the puncturer needle.

3.2.8 Programming a Wash

To fractionate the breakthrough, the fractionation must start within one minute after injection, (depending on column size and flow rate). This is only possible with no rinsing after injection. To disable rinsing after injection, program a Wash command after fractionation and set the ‘InjectWash’ to ‘NoWash’ in Expert mode.

FIGURE 3-29: Select ‘Expert’ Mode



FIGURE 3-30: Chromeleon F8 Tab Page (Example)

To move the syringe to the home position after injection, program a wash after one minute or after fractionation. To perform a wash, the inject valve must be in the ‘Inject’ position.

Tip: A Wash cannot be executed during fractionation. When fractionation starts at time=0, program the wash after fractionation.

3.3 WPS Accessories For applications with flow rates higher than 50 µL/min and/or for injection volumes larger than 50 µL it is recommended that you use the large volume injection upgrade kit (part no. 6820.0031 for a standard system and part no. 6821.0031 for a biocompatible system). The standard syringe (25 µL), injection needle (2.4 µL), buffer tubing (50 µL) and injection loop (20 µL) must be replaced then.

When using the MIC SCX/NAN PepMap setup with a 50 µL sample loop and injection volumes larger than 20 µL, the 500 µL buffer tubing (part no. 6820.0020) must be installed and a User Defined Program is required.

3.4 Injection Methods with the Micro Fraction Collection Option One of the consequences of the Micro Fraction Collection option is that a needle pre-flush cannot be executed. The injection method Partial Loop is therefore not executable. Injection of sample can be performed with a Full-loop injection, a µL-pick up injection or preferably with a User Defined Program.

When using a User Defined Program or µL-pickup in both separation dimensions, two transport vials (ReagentAVial, ReagentBVial) are required, one for each dimension. Make sure that the proper eluent is present in the transport vial. E.g. fill the transport vial located at R1 (ReagentAVial) with buffer A used in the first dimension separation, and fill the transport vial located at R2 (ReagentBVial) with loading solvent used in the second dimension separation.

Tip: When using a User Defined Program or µL-pickup in both dimensions, make sure that the proper eluent is present in transport vial R1 and R2 (Sampler.ReagentAvial, Sampler.ReagentBvial).

3.4.1.1 µL-Pickup / Full-Loop

To perform a µL-pickup or Full-Loop injection with the 8-port injection/fraction valve installed, it is necessary to flush the sample loop completely with transport liquid before drawing the sample plug. This prevents the injection of rinsing liquid. The required buffer loop volume prior to drawing sample is:

Sample Loop Volume * LoopOverfill Factor + Needle Volume

It is not possible to flush the needle with Flushvolume (or Flushvolume2 with RinseBetweenInjection=No) with the 8-port valve.

3.4.1.2 User Defined Program (UDP)

Injection of sample is preferably performed with a User Defined Program.

Make sure that the proper eluent is present in the transport vial. Fill the transport vial located at R1 with mobile phase A used in the first dimension separation, and fill the transport vial located at R2 with loading solvent used in the second dimension separation.

When using the MIC SCX/NAN PepMap setup with a 50 µL sample loop and injection volumes larger than 20 µL, the 500 µL buffer tubing (part no. 6820.0020) must be installed and an User Defined Program are required.

An example for a 50 µL injection by a User Defined Program is presented below, when using the MIC SCX/NAN PepMap setup. The 50 µL sample loop, 500 µL Buffer Tubing and 25 µL Syringe are installed.



The example below is valid for an injection of sample in the first dimension.

Sampler.InjectMode = UserProg Sampler.ReagentAVial= R1 Sampler.UdpDispense To = Drain, Volume = 0.000, SyringeSpeed = GlobalSpeed, SampleHeight = GlobalHeight Sampler.UdpSyringeValve Position = Needle Sampler.UdpInjectValve Position = Load Sampler.UdpDraw From = SampleVial, Volume = 25.000, SyringeSpeed = GlobalSpeed, SampleHeight =

GlobalHeight Sampler.UdpMixWait Duration = 5 Sampler.UdpSyringeValve Position = Waste Sampler.UdpDispense To = Waste, Volume = 25.000, SyringeSpeed = GlobalSpeed, SampleHeight = GlobalHeight Sampler.UdpSyringeValve Position = Needle Sampler.UdpDraw From = SampleVial, Volume = 25.000, SyringeSpeed = GlobalSpeed, SampleHeight =

GlobalHeight Sampler.UdpMixWait Duration = 5 Sampler.UdpSyringeValve Position = Waste Sampler.UdpDispense To = Waste, Volume = 25.000, SyringeSpeed = GlobalSpeed, SampleHeight = GlobalHeight Sampler.UdpSyringeValve Position = Needle Sampler.UdpDraw From = ReagentAVial, Volume = 2.500, SyringeSpeed = GlobalSpeed, SampleHeight =

GlobalHeight Sampler.UdpMixWait Duration = 5 Sampler.UdpDispense To = Drain, Volume = 0.000, SyringeSpeed = GlobalSpeed, SampleHeight = GlobalHeight Sampler.UdpInjectValve Position = Inject Sampler.UdpInjectMarker

An example for a 10µL injection by a User Defined Program is presented below, when using the CAP SCX/CAP Monolith setup. The 20µL sample loop, 50µL Buffer Tubing and 25µL Syringe are installed.

Sampler.InjectMode = UserProg Sampler.ReagentAVial= R1 Sampler.UdpDispense To=Drain, Volume=0.000, SyringeSpeed=GlobalSpeed, SampleHeight=GlobalHeight Sampler.UdpSyringeValve Position=Needle Sampler.UdpInjectValve Position=Load Sampler.UdpDraw From=ReagentAVial, Volume=5.000, SyringeSpeed=GlobalSpeed, SampleHeight=GlobalHeight Sampler.UdpMixWait Duration=5 Sampler.UdpDraw From=SampleVial, Volume=10.000, SyringeSpeed=GlobalSpeed, SampleHeight=GlobalHeight Sampler.UdpMixWait Duration=5 Sampler.UdpDraw From=ReagentAVial, Volume=7.500, SyringeSpeed=GlobalSpeed, SampleHeight=GlobalHeight Sampler.UdpMixWait Duration=5 Sampler.UdpDispense To=Drain, Volume=0.000, SyringeSpeed=GlobalSpeed, SampleHeight=GlobalHeight Sampler.UdpInjectValve Position=Inject Sampler.UdpInjectMarker

Tip: No commands can be entered after the ‘UdpInjectMarker’ command. If one is entered, the error message ‘Inject valve must be in load position for this operation’ will be presented.

To move the syringe to the home position after the User Defined Program, program a wash after one minute or after fractionation. To perform a wash, the inject valve must be in the ‘Inject’ position.

Tip: A Wash cannot be executed during fractionation. When fractionation starts at time=0, program the wash after fractionation.



3.5 Reviewing Results The 3-dimensional dataset that is obtained (First dimension separation – Second dimension separation – Intensity) can be visualized in a 2D retention map, see FIGURE 4-8

To display the 2D retention map, open a run from the second dimension separation and open the Chromatogram decoration menu from the toolbar (FIGURE 3-31). Select ‘Extended fractionation’

FIGURE 3-31: Display the Chromatogram decoration menu

FIGURE 3-32: Comparison Tab Page from Chromatogram Decoration Options



FIGURE 3-33: Extended Fractionation Tab Page from Chromatogram Decoration Options

a) Select the channel used for recording the first dimension separation. b) and c) Select the (same) channel used for recording the second dimension separation.

a)

b)

c)



In the ‘2D Retention Map’ the signal scaling and the displayed colour can be changed.

FIGURE 3-34: 2D Retention Map Tab Page from Chromatogram Decoration Options



3.6 Numbering of the Fractions

In the 2D retention map presented in FIGURE 3-35, the tube position located on top, represents the location of the fractions in the WPS.

10,0 12,0 14,0 16,0 18,0 20,0 23,07,50

9,00

10,00

11,00

12,00

13,00

14,00

15,00 2D-OFFLINE_SCX #1 PMD 10 pmol 2D retention mapmin

min

min

min

T89 T90 T91 T92 T93 T94 T95 T96 T97 T98 T99 T100 T101

FIGURE 3-35: Typical 2D Retention Map, the Tube Position located on top, represents the location of the fractions in the WPS

The tube position follows the same path as shown in FIGURE 3-37.

The inject tray and fractionation tray can be visualized in Chromeleon. To display the inject- or fractionation tray, open a run from the second dimension and click on ‘Show Inject Tray’ or ‘Show Fractionation Tray’ from the toolbar as shown in FIGURE 3-36.

FIGURE 3-36: Display the ‘Show Fractionation Tray’ in the Chromeleon Toolbar.



The location of the sample is visualized in the inject tray and the location of the fractions are visualized in the fractionation tray. The fractionation tray presented in FIGURE 3-37, shows the location of the fractions marked in yellow.

FIGURE 3-37: Example of the Fractionation Tray

BA8

BB8

GA1

RA1

RE8

BA1

GP24

RB8

RA8

BE8



4 Applications 4.1 2D-LC of Peptides using the MIC SCX/NAN PepMap Configuration

The fractionation option of the WPS-3000 [T][B] allows the user to perform automated off-line two-dimensional separation of peptides. In this set-up, the first dimension separation was performed ‘Split-less’ on a 1 mm I.D. x 15 cm SCX column. The second dimension separation was performed on a 75 µm I.D. x 15 cm RP column using a split ratio of 1:1000. A schematic drawing for automated off-line 2D-LC of peptides as shown in FIGURE 4-1

FIGURE 4-1: LC Setup for Automated Offline 2D-LC of Peptides with Fractionation.

4.1.1 Fluidic Connections

The SCX column, the C18 PepMap trap column, the C18 PepMap nano column, the connection tubing and waste tubing to the FLM switching valves are connected according TABLE4-1.

TABLE4-1 Valve Connections on a Standard and Biocompatible System

Left Valve Right Valve Port No. Connect to Port No. Connect to 1 - 1 Waste 2 - 2 - 3 - 3 - 4 - 4 - 5 - 5 Left valve, port 8 (75 µm I.D. x 50 cm) 6 - 6 WPS port 4 (100 µm I.D. x 50 cm) 7 SCX column (1mm I.D. x 15cm) 7 C18 trap column (300 µm I.D. x 5 mm) 8 Right valve, port 5

(75 µm I.D. x 50 cm) 8 Nano column (75 µm I.D. x 15 cm)

9 Waste 9 Flow splitter outlet, (20 µm I.D. x 30 cm) 10 - 10 C18 trap column (300 µm I.D. x 5 mm) Loading pump to WPS port 3: PEEK connection tubing, 130 µm I.D. x 75 cm.



4.1.2 The Workflow

Injection of sample is performed by a User Defined Program. The loading pump delivers the salt gradient for the SCX column through channels A and B. The eluent from the column is directed to the injection needle for fractionation. An example for the program used in the first dimension separation is described in Appendix 5.3.1

After the fractionation, a wash program is used to switch from the SCX dimension to the reversed phase dimension. For the wash program, see Appendix 5.3.2.

After the wash program, an aqueous solution (channel C of loading pump) consisting of TFA or formic acid is used to load the SCX fractions onto the reversed phase trap column. Desalting of the fractions occurs on the reversed phase trap column. The micro pump delivers the gradient for elution of the peptides. The reversed phase program is described in Appendix 5.3.3.

After the reversed phase separations are completed, a wash program is used to switch back to the first dimension separation. For the wash program, see Appendix 5.3.4.

The valve positions for various stages in the experiment are listed in TABLE4-2.

TABLE4-2 Valve Positions during Automated Off-line 2D-LC

Injector Left Valve Right Valve SCX 1st dimension Loop fill and effluent to waste 1-2 1-2 1-2 SCX separation and fractionation 8-1 1-2 1-2 Wash SCX 8-1 1-2 1-2 RP 2nd dimension Loop fill 1-2 10-1 10-1 Inject fractions and wash free of salt 8-1 10-1 10-1 Elute and separate fractions 8-1 10-1 1-2 Equilibrate RP trap 8-1 10-1 10-1

4.1.3 Experimental

The experimental conditions listed below are optimized for the application automated off-line 2D-LC of peptides, MIC SCX/NAN PepMap Configuration First dimension separation Column - PolySULFOETHYL, 1 mm I.D. x 15 cm, 5 µm, 300 Å* Mobile Phase A - 95/5 water/ACN, 5 mM NaH2PO4 buffer pH=3 Mobile Phase B - Mobile Phase A + 1M NaCl Flow rate - 50 µL/min (Loading Pump) Gradient - 0-60 %B in 20 min, 100 %B for 5 min, 20 min equilibration UV detection - 214 nm, 180 nL flow cell Sample - Protein Mix Digest (PMD), 0.5 pmol/µL, lyophilized

Tryptic digest of Cytochrome-C, Lysozyme, Alcohol dehydrogenase, Bovine serum albumin, Apo-transferrin, Beta-galactosidase

Loop: - 50 µL Injection volume - 20 µL for PMD (UDP, 10 pmol injected) Fractionation - 16 fractions of 1 minute Temperature - 25 ºC Injection needle - 2.4 µL Fused silica (100 µm I.D. x 30 cm)



* The PolyLC column is not compatible with TFA; avoid contact with TFA under all circumstances. Second dimension separation Column - C18 PepMap, 75 μm I.D. x 15 cm, 3 μm, 100 Å - C18 PepMap, 300 μm I.D. x 0.5 cm, 5 μm, 100 Å Mobile Phase A - Water + 0.05%TFA Mobile Phase B - 20/80 water/ACN +0.04 %TFA Loading Solvent - 98/2 water/ACN +0.05 %TFA Flow rate - 300 nL/min (Micro Pump) - 20 µL/min (Loading Pump) Loading time - 10 minutes Gradient - 4-55 %B in 30 min, 90 %B for 5 min, 25 min equilibration UV detection - 214 nm, 3 nL flow cell Sample - SCX fractions Loop: - 50 µL Injection volume - 50 µL by UDP Oven Temperature - 25 ºC

4.1.4 Results

In this experiment, UV detection was used for both separation dimensions.

4.1.4.1 First Dimension Separation

The UV chromatogram of the peptide separation from the first dimension separation is shown in FIGURE 4-2.

0,1 5,0 10,0 15,0 20,0 25,0 30,0 35,2-12,3

0,0

12,5

25,0

37,5

50,0

60,2 MIC_NAN CONFIGURATION PEPTIDES #21 [modif ied by BDolman] UV_VIS_1_2mAU

min

WVL:214 nm

FIGURE 4-2 First Dimension Separation of Peptides on a 1 mm I.D. SCX Column.



4.1.4.2 Second Dimension Separation

The UV chromatogram of the peptide separation from the second dimension is shown in FIGURE 4-3.

7,5 15,0 20,0 25,0 30,0 35,0 40,0 45,0 50,0-20

0

25

50

75

100

140 mAU

min

10

9

8

7

6

5

4

3

2

1

WVL:214 nm

FIGURE 4-3 Second Dimension Separation of Peptide Fractions from 10 Minutes to 20 Minutes on a 75

µm I.D. Nano Column

4.1.4.3 2D-LC Plot

This 3-dimensional dataset that is obtained (SCX time – RP time – Intensity) can be visualized following the procedure described in Section 3.5. A typical 2D-LC plot that is obtained is shown in FIGURE 4-4.

5,0 7,5 10,0 12,5 15,0 17,5 20,0 22,5 25,0 27,5 30,0-10,0

60,0 1mAU

min1

WVL:214 nm

0,0 5,0 10,0 15,0 20,0 25,0 30,0 35,020,0

45,0 2 PMD 10pmol polyLC #2 2D retention mapmin

min

min

min

15,0 20,0 25,0 30,0 35,0 40,0 45,0 50,0-5,0

40,0 3mAU

min3

WVL:214 nm

FIGURE 4-4 2D-LC Plot of SCX fractions from 10 to 26 Minutes



4.1.5 Application Kits

All required materials are provided in the UltiMate 3000 Application Kit for automated off-line 2D-LC of peptides, including the 1.0 mm I.D. SCX column and 75 µm I.D. Nano PepMap column. For the standard system, use part no. 6720.0101 and for the biocompatible system, use part no. 6721.0101.

TABLE4-3 and TABLE4-4 list all accessories and standards which are provided with the application kits.

TABLE4-3 UltiMate 3000, Application Kit for automated off-line 2D-LC of peptides, MIC SCX/NAN PepMap Configuration, standard system, Part No. 6720.0101

Part Number Description Qty 164208 (a) Operating Instructions for automated Off-line 2D-LC of peptides and proteins,

Version V1.2 1

164281 (a) CD with CM templates for installation and applications of micro fraction collection option WPS-3000

1

160321 75 µm I.D. x 15 cm, packed with C18 PepMap™, 3 µm, 100 Å 1 160454 300 µm I.D. x 5 mm, packed with C18 PepMap™, 5 µm, 100 Å (set of 5

cartridges) 1

6720.0012 µ-Precolumn™ holder, 5 mm, with connecting tubing (30 µm I.D.) 1 164262 1.0 mm I.D. x 15 cm, packed with PolySULFOETHYL ASP, 5 µm, 300 Å 1 161088 Protein mixture digest, 100 pmol, Lyophilized 1 161089 Cytochrome C digest, 1.6 nmol, Lyophilized 1 6820.4113 96 Well plate PP, V-Bottom 1 6035.2556 Connection LPG to FLM (w/ UV) for 2D-LC Comprehensive 1 6720.0105 100 µm I.D. x 50 cm, connecting tubing, PEEKsil, FLM to WPS-3000 [T] [B], MIC 1 6720.0104 Tubing set consisting of 130 µm I.D. x 50 cm PEEK tubing 2 6720.0033 20 µm I.D. x 30 cm, connecting tubing, PEEKsil, NANO Splitter outlet to FLM-3100 1 6720.0038 75 µm I.D. x 50 cm, connecting tubing, PEEKsil, FLM to WPS-3000 [T][B], MIC 2 6720.0032 130 µm I.D. x 75 cm, connecting tubing, PEEK, loading pump outlet to WPS-3000

[T][B] injector (w/ detector) with SR7 and SS7 nut and ferrule 1

6720.0074 Micro tight Union inclusive 2 fittings and 1 gauge plug 1 6720.0075 PEEK sleeves, precision cut and polished for connections with Micro tight Union

(280 µm O.D.), 10 pieces 1

6720.0076 PEEK sleeves, precision cut and polished for connections with Micro tight Union (380 µm O.D.), 10 pieces

1

6720.0064 PEEK sleeves, precision cut and polished for connections with fused silica tubing (280 µm O.D.), 5 each

1


1

6720.0015 1/16’ Universal Finger tight Fitting, one-piece design, extra long thread, 4 pc. 2 6720.0077 Teflon® tubing, 500 µm I.D. 100 cm, used as waste tubing 2 6820.0055 50 µL sample loop, Stainless Steel tubing, specially designed for minimal

dispersion 1

6820.0020 Buffer Tubing 500 µl for WPS 1 6820.0027 Polypropylene vials for WPS with glass insert, 250 µL, 25 pcs 1

Notes: a) Is only available as part of the kit.



TABLE4-4 UltiMate 3000, Application Kit for automated off-line 2D-LC of peptides, MIC SCX/NAN PepMap Configuration, Inert, Part No. 6721.0101

Part Number Description Qty 164208 (a) Operating Instructions for automated Off-line 2D-LC of peptides and proteins,

Version V1.2 1


1

160321 75 µm I.D. x 15 cm, packed with C18 PepMap™, 3 µm, 100 Å 1 160454 300 µm I.D. x 5 mm, packed with C18 PepMap™, 5 µm, 100 Å (set of 5

cartridges) 1

6720.0012 µ-Precolumn™ holder, 5 mm, with connecting tubing (30 µm I.D.) 1 164262 1.0 mm I.D. x 15 cm, packed with PolySULFOETHYL ASP, 5 µm, 300 Å 1 161088 Protein mixture digest, 100 pmol, Lyophilized 1 161089 Cytochrome C digest, 1.6 nmol, Lyophilized 1 6820.4113 96 Well plate PP, V-Bottom 1 6037.2556 Connection LPG to FLM (w/ UV) for 2D-LC Comprehensive, INERT 1 6721.0105 100 µm I.D. x 50 cm, connecting tubing, PEEKsil, FLM to WPS-3000 [T] [B], MIC,

INERT 1

6720.0104 Tubing set consisting of 130 µm I.D. x 50 cm PEEK tubing 2 6721.0033 20 µm I.D. x 30 cm, connecting tubing, PEEKsil, NANO Splitter outlet to FLM-3100,

INERT 1

6721.0029 75 µm I.D. x 50 cm, connecting tubing, PEEKsil, FLM to WPS-3000 [T][B], MIC, INERT

2

6721.0032 Capillary LP-WPSinj, long, IDxL 130µmx75cm, PEEK fittings, INERT 1 6720.0074 Micro tight Union inclusive 2 fittings and 1 gauge plug 1 6720.0075 PEEK sleeves, precision cut and polished for connections with Micro tight Union

(280 µm O.D.), 10 pieces 1


1


1


1

6720.0015 1/16’ Universal Finger tight Fitting, one-piece design, extra long thread, 4 pc. 2 6720.0077 Teflon® tubing, 500 µm I.D. 100 cm, used as waste tubing 2 6821.0055 50 µL loop for FAMOS/WPS-3000 INERT, PEEK tubing, specially designed for

minimal dispersion 1

6821.0020 Buffer Tubing 500µl, biocomp.,WPS-3000 PL 1 6820.0027 Polypropylene vials for WPS with glass insert, 250 µL, 25 pcs 1




TABLE4-5 Required materials which are NOT provided with Application Kit for automated off-line 2D-LC of peptides, MIC SCX/NAN PepMap Configuration P/N 6720.0101 and P/N 6721.0101

Part Number Description Qty 6720.3150A Splitter Cartridge 1:1000 (NAN) 1 6721.3150A Splitter Cartridge 1:1000, biocompatible (NAN) 1 VWD-3x00 - Flow Cells 6074.0290(b) UZ-View™ flow cell for VWD 3x00, 10 mm path length, volume 180 nL 1 6074.0270 UZ-View™ flow cell for VWD 3x00, 10 mm path length, volume 3 nL 1 UVD-3000 - Flow Cells 6073.0004(b) UZ-View™ flow cell for UVD-3000, 10 mm path length, volume 180 nL, 1 6073.0002 UZ-View™ flow cell for UVD-3000, 10 mm path length, volume 3 nL 1 Notes: b) Required when two UV detectors are used



4.2 2D-LC of Peptides Using the CAP SCX/CAP Monolith Configuration The fractionation option of the WPS-3000 [T][B] allows the user to perform automated off-line two-dimensional separation of peptides. In this set-up, both dimensions were operated in capillary mode. This means that only one FLM and one dual gradient pump (DGP) was required for the 2D separation. The first dimension separation (SCX) was performed on a 300 µm I.D. x 15 cm SCX column, and the second dimension separation (RP) was performed on a 200 µm I.D. x 5 cm PepSwift Monolithic column, both using a split ratio of 1:100. A schematic for automated off-line 2D-LC of peptides is shown in FIGURE 4-5.

FIGURE 4-5 LC Setup for Automated Offline 2D-LC of Peptides with Fractionation.

Fluidic Connections

The SCX column, the Monolithic trap column, the Monolithic separation column, the connection tubing and waste tubing to the FLM switching valves are connected according TABLE4-6.


Left Valve Right Valve Port No. Connect to Port No. Connect to 1 Right valve, port 9 (50 µm I.D. x 50

cm) 1 Flow splitter inlet (250 µm I.D. x 15 cm)

2 Waste 2 Loading pump ( 600 µm I.D. x 70 cm) 3 - 3 WPS port 3 (50 µm I.D. x 50 cm) 4 - 4 Flow splitter outlet (50 µm I.D. x 30 cm) 5 - 5 Left valve, port 8 (50 µm I.D. x 50 cm) 6 Waste 6 - 7 Monolithic trap column

(200 µm I.D. x 5 mm) 7 SCX column (50 µm I.D. x 30 cm)

8 Right valve, port 5 (50 µm I.D. x 50 cm)

8 WPS port 4 (50 µm I.D. x 50 cm)

9 Monolithic column (200 µm I.D. x 5 cm)

9 Left valve, port 1 (50 µm I.D. x 50 cm)

10 Monolithic trap column 10 Micro pump ( 600 µm I.D. x 70 cm)



(200 µm I.D. x 5 mm) The monolithic trap column and the monolithic separation column are located in the column compartment. For the separation on the monolithic column, the oven is heated to 60 °C.

Warning: The SCX separation column cannot withstand temperatures above 40 ºC, therefore the SCX column is located outside the column oven.

4.2.1 Workflow

Both dimensions, SCX as well as RP are using the same splitter cartridge so there is no need fore a second FLM or an additional pump for desalting the peptides on the trap column.

In the server configuration, only one pump can be assigned to the splitter. In this experiment, the micro pump is assigned to the splitter and the master flow rate on the loading pump must be calculated manually. This is done as described in Section 3.2.1.

In this example, the split ratio of the flow splitter used was 1:100. For the first dimension separation, a flow rate of 600 µL/min was programmed for the loading pump to generate a flow rate of 6 µL/min after the flow splitter.

Warning: The master flow rate on the loading pump depends on the split ratio of the splitter cartridge (→ Section 3.2.1).

Valve B controls in which dimension the separation takes place. In position 1_2, the loading pump is in-line with the splitter and the peptides are separated on the first dimension SCX column. When valve B is in position 10_1, the micro pump is in-line with the splitter and the fractionated samples are separated on the second dimension RP column.

Injection of sample is performed by a User Defined Program. The loading pump delivers the salt gradient for the SCX column through channels A and B. The eluent from the column is directed to the injection needle for fractionation. An example for the program used in the first dimension separation is described in Appendix 5.4.1.

After the fractionation, a wash program is used to switch from the SCX dimension to the reversed phase dimension. An aqueous solution (channel C of loading pump) consisting of 0.05% HFBA is used to flush out all the salts to prepare the reversed phase analysis. For the wash program, see Appendix 5.4.2.

After the wash, the loading solvent, which consists of water + 0.05% HFBA, is delivered by channel C of the loading pump to load the SCX fractions onto the RP trap column. Desalting of the fractions occurs on the trap column located on the left valve which is in position 10_1.

After 5 minutes, the peptides are eluted onto the monolithic separation column by switching the left valve into position 1_2. The micro pump delivers the gradient for elution of the peptides. 5 minutes before the end of the run, the left valve is switched back to prepare the trap column for the next injection. The reversed phase program is described in Appendix 5.4.3.

After the reversed phase separations are completed, a wash program is used to switch back to the first dimension separation. A blank is programmed inside the wash program to prepare for the next injection on the first dimension. For the wash program, see Appendix 5.4.4.


TABLE4-7 Valve Positions During Automated Off-line 2D-LC

Injector Left valve Right valve SCX 1st dimension Loop fill and effluent to waste 1-2 1-2 1-2 SCX separation and fractionation 8-1 1-2 1-2



Wash SCX 8-1 1-2 1-2 RP 2nd dimension Loop fill 1-2 10-1 10-1 Inject fractions and wash free of salt 8-1 10-1 10-1 Elute and separate fractions 8-1 1-2 10-1 Equilibrate RP trap 8-1 10-1 10-1

4.2.2 Experimental

The experimental conditions listed below are optimized for the application automated off-line 2D-LC of peptides, 300 µm i.d. SCX column, capillary PS-DVB monolithic column First dimension separation Column - PolySULFOETHYL, 300 µm I.D. x 15 cm, 5 µm, 300 Ǻ, placed outside the column oven* Mobile Phase A - 95/5 water/ACN, 5 mM NaH2PO4 buffer pH=3 Mobile Phase B - Mobile Phase A + 1 M NaCl Flow rate - 6 µL/min (600 µL/min programmed on the Loading Pump) Gradient - 0-60 %B in 20 min, 100 %B for 5 min, 20 min equilibration UV detection - 214 nm, 45 nL flow cell Sample - Protein Mix Digest (PMD), 1 pmol/µL, lyophilized

Tryptic digest of Cytochrome-C, Lysozyme, Alcohol dehydrogenase, Bovine serum albumin, Apo-transferrin, Beta-galactosidase

Loop: - 20 µL Injection volume - 10 µL for PMD (UDP, 10 pmol injected) Fractionation - 20 fractions of 1 minute Oven Temperature - Ambient Injection needle - 2.4 µL Fused silica (100 µm I.D. x 30 cm) * The PolyLC column is not compatible with TFA and HFBA; avoid contact with TFA and HFBA under all

circumstances. The maximum operating temperature is 40ºC, therefore the PolyLC column must be placed outside the column oven

Second dimension separation Column - PepSwift PS-DVB Monolith, 200 µm I.D. x 5 cm - PepSwift PS-DVB Monolithic trap, 200 µm I.D. x 0.5 cm Mobile Phase A - water + 0.05 %TFA Mobile Phase B - 20/80 water/ACN + 0.04 %TFA Loading Solvent - water + 0.05 % HFBA Flow rate - 2.5 µL/min (Micro Pump) - 20 µL/min (Loading Pump) Loading time - 5 minutes Gradient - 0-4 5%B in 10 min, 90 %B for 2 min, 6 min equilibration UV detection - 214 nm, 3 nL flow cell Sample - SCX fractions Loop: - 20 µL Injection volume - 6 µL by UDP Oven Temperature - 60 ºC



4.2.3 Results

In this experiment UV detection was applied for both dimensions.


The UV chromatogram of the peptide separation from the first dimension is shown in FIGURE 4-6.

FIGURE 4-6: First Dimension Separation of Peptides on a 300µm I.D. SCX Column.



FIGURE 4-7 Second Dimension Separation of Peptide Fractions from 10 minutes to 25 minutes on a

200 µm I.D. PS-DVB Monolithic Column.

4,3 6,3 7,5 8,8 10,0 11,3 12,5 13,8 15,0 16,3 17,5 18,8 20,2-63,7

-50,0

-37,5

-25,0

-12,5

0,0

19,9 mAU

min

1514131211109 8 7 6 5 4 3 2 1



4.2.3.3 2D-LC Plot

This 3-dimensional dataset that is obtained (SCX time – RP time – Intensity) can be visualized following the procedure described in Section 3.5. A typical 2D-LC plot that is obtained is shown in FIGURE 4-8.

FIGURE 4-8 2D-LC plot of SCX fractions from 10 to 25 Minutes

Tip: To display the UV chromatograms for both dimensions in the 2D retention map, two UV detectors are required

0,0 5,0 10,0 15,0 20,0 25,0 30,0 35,0 40,0-50

125

250 mAU

min

1

WVL:214 nmT1 T3 T4 T5 T6 T7 T8 T9T10T11T12T13T14T15

0,0 5,0 10,0 15,0 20,0 25,0 30,0 35,0 40,07,0

15,0

20,0 2D retention mapmin

min

min

0,0 2,0 4,0 6,0 8,0 10,0 12,0 14,0 16,0 18,0 20,0 22,0 25,0-4,0

16,0 mAU

min

3

WVL:214 nm




All required materials are provided in the UltiMate 3000 Application Kit for automated off-line 2D-LC of peptides, including the 300 µm I.D. SCX column, 200 µm I.D. PS-DVB monolithic column and monolithic trap column. For the standard system, use part no. 6720.0100 and for the biocompatible system, use part no. 6721.0100

TABLE4-8 and TABLE4-9 list all accessories and standards which are provided with the application kits.

TABLE4-8 UltiMate 3000, Application Kit for automated off-line 2D-LC of peptides, CAP SCX/CAP monolith, standard system, Part No. 6720.0100

Part Number Description Qty164208 (a) Operating Instructions for automated Off-line 2D-LC of peptides and proteins,

Version V1.2 1


1

161409 Monolithic Capillary Column, 200 µm I.D. x 5 cm (PS-DVB) 1 163972 Monolithic Trap Column, 200 µm I.D. x 5 mm (PS-DVB), set of 2 1 164263 300 µm I.D. x 15 cm, packed with PolySULFOETHYL ASP, 5 µm, 300Å 1 161088 Protein mixture digest, 100 pmol, Lyophilized 1 161089 Cytochrome C digest, 1.6 nmol, Lyophilized 1 6820.4114 384 Well Micro plate, PP, V-Bottom 1 6035.2556 Connection LPG to FLM (w/ UV) for 2D-LC Comprehensive 2 6720.0107 Connection tubing from switching valve FLM to Splitter inlet, 250 µm I.D. x 15 cm,

stainless steel 1

6720.0034 50 µm I.D. x 30 cm, connecting tubing, PEEKsil, CAP Splitter outlet to FLM-3100 2 6720.0037 50 µm I.D. x 50 cm, connecting tubing, PEEKsil, WPS-3000 [T][B] to valve FLM-

3100 4

6720.0039 1/16" zero-dead-volume union 1 6720.0074 Micro tight Union inclusive 2 fittings and 1 gauge plug 2 6720.0075 PEEK sleeves, precision cut and polished for connections with Micro tight Union

(280 µm O.D.), 10 pieces 1


1

160477 Fused silica tubing I.D. 50µm±3µm/O.D. 280µm ±10µm, 5 meters 1 6720.0064 PEEK sleeves, precision cut and polished for connections with fused silica tubing

(280 µm O.D.), 5 each 1


1

6000.0069 1/16’ Valco Ferrule and Nut (long) stainless steel (for 10-port valve) set of 10 1 6720.0015 1/16’ Universal Finger tight Fitting, one-piece design, extra long thread, 4 pc. 2 6720.0077 Teflon® tubing, 500 µm I.D. 100 cm, used as waste tubing 3 6820.0018 20 µL sample loop, PEEK tubing, specially designed for minimal dispersion 1 6820.0027 Polypropylene vials for WPS with glass insert, 250 µL, 25 pcs 1 Notes: a) Part is available as part of the kit only.



TABLE4-9 UltiMate 3000, Application Kit for automated off-line 2D-LC of peptides, CAP SCX/CAP monolith, Inert, Part No. 6721.0100


Version V1.2 1


1

161409 Monolithic Capillary Column, 200 µm I.D. x 5 cm (PS-DVB) 1 163972 Monolithic Trap Column, 200 µm I.D. x 5 mm (PS-DVB), set of 2 1 164263 300 µm I.D. x 15 cm, packed with PolySULFOETHYL ASP, 5 µm, 300Å 1 161088 Protein mixture digest, 100 pmol, Lyophilized 1 161089 Cytochrome C digest, 1.6 nmol, Lyophilized 1 6721.0108 Union Body-NanoTight .005in 1 6820.4114 384 Well Micro plate, PP, V-Bottom 1 6721.0106 250µm x 60 cm, connection tubing, PEEK, pump outlet to switching valve FLM (w/

detector), INERT 2

6721.0107 Connection tubing from switching valve FLM to Splitter inlet, 250 µm i.d. x 15 cm, PEEK

1

6721.0034 Capillary Cap Splitter-FLM, IDxL 50µmx30cm, PEEKsil, inert 2 6720.0037 50 µm I.D. x 50 cm, connecting tubing, PEEKsil, WPS-3000 [T][B] to valve FLM-

3100, INERT 4


(280 µm O.D.), 10 pieces 1


1

160477 Fused silica tubing I.D. 50µm±3µm/O.D. 280µm ±10µm, 5 meters 1 6720.0064 PEEK sleeves, precision cut and polished for connections with fused silica tubing

(280 µm O.D.), 5 each 1


1

6721.0017 PEEK 1/16’ Universal Fitting, INERT, long hex nut and ferrule with groove, set of 10 pcs.

1

6720.0015 1/16’ Universal Finger tight Fitting, one-piece design, extra long thread, 4 pc. 2 6720.0077 Teflon® tubing, 500 µm I.D. 100 cm, used as waste tubing 3 6821.0018 20 µL Sample loop , biocomp.,PEEK, WPS-3000 PL 1 6820.0027 Polypropylene vials for WPS with glass insert, 250 µL, 25 pcs 1 Notes: a) Part is available as part of the kit only.



TABLE4-10 Required materials which are NOT provided in the Automated Off-line Application Kit CAP SCX/CAP Monolith P/N 6720.0100 and P/N 6721.0100

Part Number Description Qty 6720.3150A Splitter Cartridge 1:100 (CAP) 1 6721.3150A Splitter Cartridge 1:100, biocompatible (CAP) 1 VWD-3x00 - Flow Cells 6074.0280(b) UZ-View™ flow cell for VWD 3x00, 10 mm path length, volume 45 nL 1 6074.0270 UZ-View™ flow cell for VWD 3x00, 10 mm path length, volume 3 nL 1 UVD-3000 - Flow Cells 6073.0003(b) UZ-View™ flow cell for UVD-3000, 10 mm path length, volume 45 nL 1 6073.0002 UZ-View™ flow cell for UVD-3000, 10 mm path length, volume 3 nL 1 Notes: b) Required when two UV detectors are used



4.3 2D-LC of Proteins using the MIC WAX / 500 µm I.D Monolith Configuration The fractionation option of the WPS-3000 [T][B] allows the user to perform automated off-line two-dimensional separation of proteins. In this set-up, the first dimension separation was performed ‘Split-less’ on a 1 mm I.D. x 5 cm ProSwift Weak Anion Exchange (WAX) column. The second dimension separation was performed on a 500 µm I.D. x 5 cm PepSwift Reversed Phase Monolithic column, using a split ratio of 1:15. Both column outlets are connected to the right switching valve and share the UV detector and fractionation needle. This enables “dual fractionation”, where the first dimension separation as well as the second dimension separation is fractionated. The schematic for automated off-line 2D-LC of proteins is shown in FIGURE 4-9.

FIGURE 4-9: LC Setup for Automated Offline 2D-LC of Proteins with First and Second Dimension Fractionation.




The ProSwift WAX column, the PepSwift monolithic column, the connection tubing and waste tubing to the FLM switching valves are connected according TABLE4-11.


Left Valve Right Valve Port No. Connect to Port No. Connect to 1 WPS port 4 (75 µm I.D. x 50 cm) 1 Right valve, port 1 (75 µm I.D. x 50 cm) 2 Right valve, port 1 (75 µm I.D. x 50 cm) 2 ProSwift WAX column (tubing 75µm I.D.

x 30 cm INERT) (inlet) 3 - 3 - 4 - 4 Monolithic column (500 µm I.D. x 5 cm)

(outlet) 5 - 5 UV (inlet) 6 Waste 6 ProSwift WAX column (tubing 75µm I.D.

x 30 cm INERT) (outlet) 7 Monolithic trap column

(200 µm I.D. x 5 mm) 7 -

8 Slitter Outlet (50 µm I.D. x 30 cm) 8 - 9 Monolithic column (500 µm I.D. x 5 cm)

(inlet) 9 -

10 Monolithic trap column (200 µm I.D. x 5 mm)

10 Waste

4.3.2 The Workflow

Injection of sample is performed by a User Defined Program. The loading pump delivers the salt gradient for the ProSwift WAX column through channels A and B. The eluent from the WAX column is directed to the injection needle for fractionation. An example for the program used in the first dimension separation is described in Appendix 5.5.1

After the fractionation, a wash program is used to switch from the WAX dimension to the reversed phase dimension. For the wash program, see Appendix 5.5.2.

After the wash program, an aqueous solution (channel C of loading pump) consisting of 0.05% HFBA is used to load the WAX fractions onto the monolithic reversed phase trap column. Desalting of the fractions occurs on the monolithic reversed phase trap column. The micro pump delivers the gradient for elution of the proteins. The eluent from the PepSwift RP column is directed to the injection needle for fractionation. The reversed phase program is described in Appendix 5.5.3.




Injector Left Valve Right Valve WAX 1st dimension Loop fill and effluent to waste 1-2 1-2 1-2 WAX separation and fractionation 8-1 1-2 1-2 Wash WAX 8-1 1-2 1-2 RP 2nd dimension



Loop fill 1-2 10-1 10-1 Inject fractions and wash free of salt 8-1 10-1 10-1 Elute and separate fractions 8-1 1-2 10-1 Equilibrate RP trap 8-1 10-1 10-1

4.3.3 Experimental

The experimental conditions listed below are optimized for the application automated off-line 2D-LC of proteins, MIC WAX/MON CAP500 First dimension separation Column - ProSwift WAX, 1 mm I.D. x 5 cm Mobile Phase A - 20 mM Tris-HCl buffer pH=7.6 Mobile Phase B - Mobile Phase A + 1 M NaCl Flow rate - 50 µL/min (Loading Pump) Gradient - 0-70 %B in 15 min, 100 %B for 2 min, 18 min equilibration UV detection - 220 nm, 45 nL flow cell Sample - E-coli lysate (2 µg/µL) Loop: - 50 µL Injection volume - 20 µL (UDP, 40 µg injected) Fractionation - 10 fractions of 1 minute Oven Temperature - 50 ºC Injection needle - 2.4 µL Fused silica (100 µm I.D. x 30 cm) Second dimension separation Column - PepSwift PS-DVB Monolith, 500 µm I.D. x 5 cm - PepSwift PS-DVB Monolithic trap, 200 µm I.D. x 0.5 cm Mobile Phase A - Water + 0.05 %TFA Mobile Phase B - 20/80 water/ACN + 0.04 % TFA Loading Solvent - Water + 0.05% HFBA Flow rate - 15 µL/min (Micro Pump) - 20 µL/min (Loading Pump) Loading time - 5 minutes Gradient - 15-70 %B in 11 min, 90 %B for 2 min, 13 min equilibration UV detection - 220 nm, 45 nL flow cell Sample - WAX fractions Loop: - 50 µL Injection volume - 25 µL by UDP Fractionation - 10 fractions of 1 minute Oven Temperature - 60 ºC

4.3.4 Results

The flow rate used in the first dimension separation and second dimension separation are both compatible with a 45 nL flow cell. The UV traces from both dimension separations are recorded using one UV detector equipped with a 45 nL flow cell.




The UV chromatogram of the E-coli protein separation from the first dimension separation is shown in FIGURE 4-10

0,0 5,0 10,0 15,0 20,0 25,0 30,0 35,0-10

100

200

250 MIC_CAP500 CONFIGURATION PROTEINS #1 UV_VIS_1mAU

min

WVL:220 nm

FIGURE 4-10 First Dimension Separation of E-coli Proteins on a 1 mm I.D. ProSwift WAX Column.


The UV chromatogram of the protein separation from the second dimension is shown in FIGURE 4-11.

5,0 8,0 10,0 12,0 14,0 16,0 18,0 20,0-65

0

50

87 mAU

min

141312111098765432

WVL:220 nm

FIGURE 4-11 Second Dimension Separation of E-coli Protein Fractions from 8 Minutes to 18 Minutes on a 500 µm I.D. Monolithic Column

Fraction collection of the second dimension ProSwift RP separation is described in section 3.2.5 Fraction Collection Enabled During the Second Dimension Separation.



4.3.4.3 2D-LC Plot

This 3-dimensional dataset that is obtained (WAX time – RP time – Intensity) can be visualized following the procedure described in Section 3.5. A typical 2D-LC plot that is obtained is shown in FIGURE 4-12.

0,0 5,0 10,0 15,0 20,0 25,0 30,0 35,0-10

250 1mAU

min1

WVL:220 nm

0,0 5,0 10,0 15,0 20,0 25,0 30,0 35,08,00

17,00 2 E-coli Protein 2D retention mapmin

min

min

min

0,0 5,0 10,0 15,0 20,0 25,0 30,0-5,0

50,0 3mAU

min3

WVL:220 nm

FIGURE 4-12 2D-LC Plot of WAX fractions from 0 to 10 Minutes




All required materials are provided in the UltiMate 3000 Application Kit for automated off-line 2D-LC of proteins, including the 1 mm I.D. ProSwift WAX column, 500 µm I.D. PS-DVB monolithic column. For the standard system, use part no. 6720.0103 and for the biocompatible system, use part no. 6721.0103. TABLE4-13 and TABLE4-14 list all accessories and standards which are provided with the application kits.

TABLE4-13 UltiMate 3000, Application Kit for automated off-line 2D-LC of proteins, MIC WAX/MON CAP500, Standard System, Part No. 6720.0103


Version V1.2 1


1

164087 Monolithic Capillary Column, 500 µm I.D. x 5 cm (PS-DVB) 1

163972 Monolithic Trap Column, 200µm I.D. x 5mm (PS-DVB), set of 2 1

066642 ProSwift WAX-1S, 1 x 50 mm 1 164405 E. coli Protein Sample, lyophilized, 2.7 mg 1 6820.4114 384 Well Micro plate, PP, V-Bottom 2 6035.2556 Connection LPG to FLM (w/ UV) for 2D-LC Comprehensive 1 6720.0035 Capillary MicSplitter-FLM, IDxL 75 µmx30 cm, PEEKsil 1 6720.0038 Capillary Mic, IDxL 75 µmx50 cm, PEEKsil 2 6721.0059 PEEKsil Bridge, ID 75 µmx30 cm, inert 2 6720.0026 75 µm I.D. x 50 cm, connecting tubing, PEEKsil, WPS-3000 to MIC Column in FLM,

incl. 1/16" zero-dead-volume union 1

6720.0032 130 µm I.D. x 75 cm, connecting tubing, PEEK, loading pump outlet to WPS-3000 [T][B] injector (w/ detector) with SR7 and SS7 nut and ferrule

1

6720.0081 Fused silica tubing I.D. 75 µm±3 µm/O.D. 280 µm ±10 µm, 5 meters 1 6720.0074 Micro tight Union inclusive 2 fittings and 1 gauge plug 1


1


1


1


1

6000.0069 1/16’ Valco Ferrule and Nut (long), stainless steel (for 10-port valve) set of 10 1

6720.0077 Teflon® tubing, 500 µm I.D. 100 cm, used as waste tubing 3 6720.0015 1/16’ Universal Finger tight Fitting, one-piece design, extra long thread, 4 pc. 1 6820.0055 50 µL sample loop, Stainless Steel tubing, specially designed for minimal dispersion 1

6820.0020 Buffer Tubing 500 µl for WPS 1

6820.0027 Polypropylene vials for WPS with glass insert, 250 µL, 25 pcs 1




TABLE4-14 UltiMate 3000, Application Kit for automated off-line 2D-LC of proteins, MIC WAX/MON CAP500 Inert, Part No. 6721.0103


Version V1.2 1


1

164087 Monolithic Capillary Column, 500 µm I.D. x 5 cm (PS-DVB) 1 163972 Monolithic Trap Column, 200µm I.D. x 5mm (PS-DVB), set of 2 1 066642 ProSwift WAX-1S, 1 x 50 mm 1 164405 E. coli Protein Sample, lyophilized, 2.7 mg 1 6820.4114 384 Well Micro plate, PP, V-Bottom 2 6037.2556 Connection LPG to FLM (w/ UV) for 2D-LC Comprehensive, INERT 1 6721.0035 Capillary MicSplitter-FLM, IDxL 75 µmx30 cm, PEEKsil, INERT 1 6721.0060 Capillary Mic, IDxL 75 µmx50 cm, PEEKsil, INERT 2 6721.0059 PEEKsil Bridge, ID 75 µmx30 cm, inert 2 6721.0026 Conn. tubing, IDxL 75µmx50cm, PEEKsil, WPS-FLM + Union, Mic, inert 1 6721.0032 Capillary LP-WPSinj, long, IDxL 130µmx75cm, PEEK fittings, inert 1 6720.0081 Fused silica tubing I.D. 75 µm±3 µm/O.D. 280 µm ±10 µm, 5 meters 1 6720.0074 Micro tight Union inclusive 2 fittings and 1 gauge plug 1 6720.0075 PEEK sleeves, precision cut and polished for connections with Micro tight Union

(280 µm O.D.), 10 pieces 1


1


1


1

6721.0017 PEEK 1/16’ Universal Fitting, INERT, long hex nut and ferrule with groove, set of 10 pcs.

1

6720.0077 Teflon® tubing, 500 µm I.D. 100 cm, used as waste tubing 3 6720.0015 1/16’ Universal Finger tight Fitting, one-piece design, extra long thread, 4 pc. 1 6821.0055 50 µl loop, PEEK tubing, specially designed for minimal dispersion 1 6821.0020 Buffer Tubing 500 µl, INERT 1 6820.0027 Polypropylene vials for WPS with glass insert, 250 µL, 25 pcs 1




TABLE4-15 Required materials which are NOT provided in the Automated Off-line Application Kit MIC WAX/CAP 500 µm I.D. Monolith P/N 6720.0103 and P/N 6721.0103

Part Number Description Qty 6720.3165A Splitter Cartridge 1:15 (CAP) 1 6721.3165A Splitter Cartridge 1:15, biocompatible (CAP) 1 VWD-3x00 - Flow Cells 6074.0280 UZ-View™ flow cell for VWD 3x00, 10 mm path length, volume 45 nL 1 UVD-3000 - Flow Cells 6073.0003 UZ-View™ flow cell for UVD-3000, 10 mm path length, volume 45 nL 1

4.3.6 Automated in-well digestion for tandem MS detection and protein identification

With the set-up described in section 4.3 it is possible to fractionate the first WAX separation as well as the second dimension RP separation in well plates. The separations and the resulting chromatograms are presented in a 2D retention map. As a follow up analysis, from the 2D retention map selected protein fractions can be in-well digested automatically. The in-well digestion is performed by adding trypsin solution by the WPS to fractions of interest.

The system set-up described in section 4.3 can be reconfigured for peptide analysis or the peptides can be separated on a second system connected to a mass spectrometer

4.3.6.1 Tryptic digest fractions

The complete 2D-LC separation of intact proteins described in section 4.3 was achieved in 4 hours. Based on the generated 2D retention map, nine fractions were digested with trypsin in 3 hours. For the addition of trypsin, ReagentCVial was filled with trypsin solution and added to the fractions by a User Defined Program (UDP). For the program, see Appendix 5.5.5.

Separation of the Tryptic peptides Column - PepSwift PS-DVB Monolith, 200 µm I.D. x 5 cm (not part of the kit) - PepSwift PS-DVB Monolithic trap, 200 µm I.D. x 0.5 cm Mobile Phase A - Water + 0.05 %TFA Mobile Phase B - 20/80 water/ACN + 0.04 %TFA Loading Solvent - Water + 0.05 % HFBA Flow rate - 2.5 µL/min (Micro Pump) - 20 µL/min (Loading Pump) Loading time - 5 minutes Gradient - 0-45 %B in 7.5 min, 90 %B for 1.5 min, 5 min equilibration UV detection - 214 nm, 3 nL flow cell Sample - Tryptic peptides from second dimension fractionation Loop: - 50 µL Injection volume - 25 µL by UDP Oven Temperature - 60 ºC



0,0 2,0 4,0 6,0 8,0 10,08,50

10,00

12,00

14,00

16,00

18,00 1 MIC_CAP500 CONFIGURATION PROTEINS #1 2D retention mapmin

min

min

min

0,0 10,0 25,0-0,50

1,00

2,00

3,00 MON-500 fraction digestmAU

min

WVL:214 nm

0,0 10,0 25,0-0,50

1,00

2,00

3,00 MON-500 f raction digestmAU

min

WVL:214 nm

FIGURE 4-13 2D-LC Plot of WAX fractions from 0 to 10 Minutes (upper), two reversed phase separations of tryptic peptides.

The tryptic peptides were then analyzed by capillary LC-MS/MS for protein identification. FIGURE 4-13 shows the 2D retention map, with two tryptic digests separated on a 200 µm I.D. PepSwift monolithic column.

The directory which contains templates and DEMO data is located CD ROM provided with the kit.

Fraction_Collection_WPS\ DEMO\ Proteins\ MIC_CAP500 including in well digestion



4.4 2D-LC of Proteins using the MIC WAX / 200 µm I.D. Monolith Configuration The fractionation option of the WPS-3000 [T][B] allows the user to perform automated off-line two-dimensional separation of proteins. In this set-up, the first dimension separation was performed ‘Split-less’ on a 1 mm I.D. x 5 cm ProSwift Weak Anion Exchange (WAX) column. The second dimension separation (RP) was performed on a 200 µm I.D. x 5 cm PepSwift Monolithic column, using a split ratio of 1:100. A schematic for automated off-line 2D-LC of proteins is shown in FIGURE 4-14.

FIGURE 4-14: LC Setup for Automated Offline 2D-LC of Proteins with Fractionation.




The WAX column, the monolithic trap column, the PepSwift monolithic column, the connection tubing and waste tubing to the FLM switching valves are connected according TABLE4-16


Left Valve Right Valve Port No. Connect to Port No. Connect to 1 WPS port 4 (75 µm I.D. x 50 cm) 1 Left valve, port 5 (Waste tubing) 2 Right valve, port 2

(75 µm I.D. x 50 cm) 2 Left valve, port 2 (75 µm I.D. x 50 cm)

3 - 3 ProSwift WAX column (tubing 75µm I.D. x 30 cm INERT)

4 - 4 - 5 Right valve, port 1

(Waste tubing) 5 -

6 Waste 6 - 7 Monolithic trap column

(200 µm I.D. x 5 mm) 7 -

8 Slitter Outlet (50 µm I.D. x 30 cm)

8 -

9 Monolithic column (200 µm I.D. x 5 cm) 9 - 10 Monolithic trap column

(200 µm I.D. x 5 mm) 10 -

Loading pump to WPS port 3: PEEK connection tubing, 130 µm I.D. x 75 cm.

4.4.2 The Workflow

Injection of sample is performed by a User Defined Program. The loading pump delivers the salt gradient for the ProSwift WAX column through channels A and B. The eluent from the column is directed to the injection needle for fractionation. An example for the program used in the first dimension separation is described in Appendix 5.6.1

After the fractionation, a wash program is used to switch from the WAX dimension to the reversed phase dimension. For the wash program, see Appendix 5.6.2.

After the wash program, an aqueous solution (channel C of loading pump) consisting of 0.05% HFBA is used to load the WAX fractions onto the reversed phase trap column. Desalting of the fractions occurs on the reversed phase trap column. The micro pump delivers the gradient for elution of the proteins. The reversed phase program is described in Appendix 5.6.3.




Injector Left Valve Right Valve WAX 1st dimension Loop fill and effluent to waste 1-2 1-2 10-1 WAX separation and fractionation 8-1 1-2 10-1 Wash WAX 8-1 1-2 10-1 RP 2nd dimension



Loop fill 1-2 10-1 1-2 Inject fractions and wash free of salt 8-1 10-1 1-2 Elute and separate fractions 8-1 1-2 1-2 Equilibrate RP trap 8-1 10-1 1-2

4.4.3 Experimental

The experimental conditions listed below are optimized for the application automated off-line 2D-LC of proteins, MIC WAX/MON CAP200 First dimension separation Column - ProSwift WAX, 1 mm I.D. x 5 cm Mobile Phase A - 20 mM Tris-HCl buffer pH=7.6 Mobile Phase B - Mobile Phase A + 1M NaCl Flow rate - 50 µL/min (Loading Pump) Gradient - 0-70 %B in 15 min, 100 %B for 2 min, 18 min equilibration UV detection - 220 and 280 nm, 45 nL flow cell Sample - E-coli lysate Loop: - 50 µL Injection volume - 10 µL (UDP, 40 µg injected) Fractionation - 14 fractions of 1 minute Temperature - 50 ºC Injection needle - 2.4 µL Fused silica (100 µm I.D. x 30 cm) Second dimension separation Column - PepSwift PS-DVB Monolith, 200 µm I.D. x 5 cm - PepSwift PS-DVB Monolithic trap, 200 µm I.D. x 0.5 cm Mobile Phase A - Water + 0.05 %TFA Mobile Phase B - 20/80 water/ACN + 0.04 %TFA Loading Solvent - Water + 0.05 % HFBA Flow rate - 2.5 µL/min (Micro Pump) - 20 µL/min (Loading Pump) Loading time - 5 minutes Gradient - 10-70 %B in 15 min, 80 %B for 3 min, 10 min equilibration UV detection - 220 nm, 3 nL flow cell Sample - WAX fractions Loop: - 50 µL Injection volume - 25 µL by UDP Oven Temperature - 60 ºC

4.4.4 Results

In this experiment, UV detection was used for both separation dimensions.




The UV chromatogram of the E-coli protein separation from the first dimension separation is shown in FIGURE 4-15

0,0 5,0 10,0 15,0 20,0 25,0 30,0 35,0-10

100

200

250 MIC_CAP200 CONFIGURATION PROTEINS #1 UV_VIS_1_2mAU

min

WVL:220 nm

FIGURE 4-15 First Dimension Separation of E-coli Proteins on a 1 mm I.D. ProSwift WAX Column.



5,0 8,0 10,0 12,0 14,0 16,0 18,0 20,0 22,0 25,0-40

-20

0

20

40

60 mAU

min

14

13

12

11

10

9

8

7

6

5

4

3

2

1

WVL:220 nm

FIGURE 4-16 Second Dimension Separation of E-coli protein Fractions from 6 Minutes to 20 Minutes on

a 200 µm I.D. Monolithic Column



4.4.4.3 2D-LC Plot

This 3-dimensional dataset that is obtained (WAX time – RP time – Intensity) can be visualized following the procedure described in Section 3.5. A typical 2D-LC plot that is obtained is shown in FIGURE 4-17.

0,0 5,0 10,0 15,0 20,0 25,0 30,0 35,0-10

100

250 1mAU

min1

WVL:220 nm

0,0 5,0 10,0 15,0 20,0 25,0 30,0 35,08,0

15,0

20,0 2 E-coli proteins 2D retention mapmin

min

min

min

0,0 5,0 10,0 15,0 20,0 25,0 30,0-2,0

5,0

10,0 3mAU

min

3

WVL:220 nm

FIGURE 4-17 2D-LC Plot of WAX fractions from 6 to 20 Minutes




All required materials are provided in the UltiMate 3000 Application Kit for automated off-line 2D-LC of proteins, including the 1 mm I.D. ProSwift WAX column, 200 µm I.D. PS-DVB monolithic column and monolithic trap column. For the standard system, use part no. 6720.0102 and for the biocompatible system, use part no. 6721.0102

TABLE4-18and TABLE4-19 list all accessories and standards which are provided with the application kits.

TABLE4-18 UltiMate 3000, Application Kit for automated off-line 2D-LC of proteins, MIC WAX/MON CAP200, standard system, Part No. 6720.0102


Version V1.2 1


1

161409 Monolithic Capillary Column, 200 µm I.D. x 5 cm (PS-DVB) 1 163972 Monolithic Trap Column, 200 µm I.D. x 5 mm (PS-DVB), set of 2 1 066642 ProSwift WAX-1S, 1 x 50mm 1 164405 E. coli Protein Sample, lyophilized, 2.7 mg 1 6820.4114 384 Well Micro plate, PP, V-Bottom 2 6035.2556 Connection LPG to FLM (w/ UV) for 2D-LC Comprehensive 1 6720.0034 50 µm I.D. x 30 cm, connecting tubing, PEEKsil, CAP Splitter outlet to FLM-3100 1 6720.0038 Capillary Mic, WPS-FLM, IDxL 75µmx50cm, PEEKsil 4 6721.0038 Capillary Mic, WPS-FLM, IDxL 75µmx50cm, PEEKsil, INERT 1 6720.0059 PEEKsil Bridge, ID 75µmx30cm(FLM to FLM) 1 6721.0059 PEEKsil Bridge, ID 75µmx30cm(FLM to FLM), inert 2 6720.0032 130 µm I.D. x 75 cm, connecting tubing, PEEK, loading pump outlet to WPS-3000

[T][B] injector (w/ detector) with SR7 and SS7 nut and ferrule 1

6720.0081 Fused silica tubing I.D. 75 µm±3 µm/O.D. 280 µm ±10 µm, 5 meters 1 6720.0039 1/16" Zero-Dead-Volume, 0.15 µm I.D. union 2 6000.0069 1/16’ Valco Ferrule and Nut (long), stainless steel (for 10-port valve) set of 10 1 6720.0064 PEEK sleeves, precision cut and polished for connections with fused silica tubing

(280 µm O.D.), 5 each 1


(280 µm O.D.), 10 pieces 1


1

6720.0077 Teflon® tubing, 500 µm I.D. 100 cm, used as waste tubing 3 6720.0015 1/16’ Universal Finger tight Fitting, one-piece design, extra long thread, 4 pc. 2 6820.0055 50 µL sample loop, Stainless Steel tubing, specially designed for minimal dispersion 1 6820.0020 Buffer Tubing 500 µl for WPS 1 6820.0027 Polypropylene vials for WPS with glass insert, 250 µL, 25 pcs 1




TABLE4-19 UltiMate 3000, Application Kit for automated off-line 2D-LC of proteins, MIC WAX/MON CAP200 Inert, Part No. 6721.0102


Version V1.2 1


1

161409 Monolithic Capillary Column, 200 µm I.D. x 5 cm (PS-DVB) 1 163972 Monolithic Trap Column, 200 µm I.D. x 5 mm (PS-DVB), set of 2 1 066642 ProSwift WAX-1S, 1 x 50mm 1 164405 E. coli Protein Sample, lyophilized, 2.7 mg 1 6820.4114 384 Well Micro plate, PP, V-Bottom 2 6037.2556 Connection LPG to FLM (w/ UV) for 2D-LC Comprehensive, INERT 1 6721.0034 50 µm I.D. x 30 cm, connecting tubing, PEEKsil, CAP Splitter outlet to FLM-3100,

INERT 1

6721.0060 PEEKsil Bridge, ID 75µmx50cm, INERT 4 6721.0059 PEEKsil Bridge, ID 75µmx30cm(FLM to FLM), INERT 2 6721.0032 130 µm i.d. x 75 cm, connecting tubing, PEEK, loading pump outlet to WPS-3000

injector (w/ detector) INERT 1

6720.0081 Fused silica tubing I.D. 75 µm±3 µm/O.D. 280 µm ±10 µm, 5 meters 1 6721.0108 Union Body-NanoTight .005in 2 6720.0064 PEEK sleeves, precision cut and polished for connections with fused silica tubing

(280 µm O.D.), 5 each 1


(280 µm O.D.), 10 pieces 1


1

6720.0077 Teflon® tubing, 500 µm I.D. 100 cm, used as waste tubing 3 6721.0017 PEEK 1/16’ Universal Fitting, INERT, long hex nut and ferrule with groove, set of

10 pcs. 1

6720.0015 1/16’ Universal Finger tight Fitting, one-piece design, extra long thread, 4 pc. 2 6821.0055 50 µl loop, PEEK tubing, specially designed for minimal dispersion 1 6821.0020 Buffer Tubing 500 µl, INERT 1 6820.0027 Polypropylene vials for WPS with glass insert, 250 µL, 25 pcs 1




TABLE4-20 Required materials which are NOT provided in the Automated Off-line Application Kit MIC WAX/200 µm I.D. Monolith P/N 6720.0102 and P/N 6721.0102 Part Number Description Qty 6720.3150A Splitter Cartridge 1:100 (CAP) 1 6721.3150A Splitter Cartridge 1:100, biocompatible (CAP) 1 VWD-3x00 - Flow Cells 6074.0280(b) UZ-View™ flow cell for VWD 3x00, 10 mm path length, volume 45 nL 1 6074.0270 UZ-View™ flow cell for VWD 3x00, 10 mm path length, volume 3 nL 1 UVD-3000 - Flow Cells 6073.0003(b) UZ-View™ flow cell for UVD-3000, 10 mm path length, volume 45 nL 1 6073.0002 UZ-View™ flow cell for UVD-3000, 10 mm path length, volume 3 nL 1 Notes: b) Required when two UV detectors are used



5 Appendixes 5.1 DCMSlink Version 2.0

Dionex Chromatography Mass Spectrometry Link (DCMSLink) provides an interface for controlling a wide range of Dionex chromatography instruments with Xcalibur, HyStar and Analyst software.

DCMSLink version 2.0 in combination with a full Chromeleon licence including the Extended Fraction Collection Licence is compatible with the WPS Micro Fraction Collection Option. Fractionation capabilities of the WPS are supported, however DCMSLink works through the MS software interface, so the following automation and data review features are not available;

- Automatic generation of the second sequence, (Sample and Fraction injections need to be programmed manually)

- Dynamically linked sample and fractions

- The 2D Retention Map, which provides a 2D gel-like representation of UV data.

DCMSLink version 2.0 is based on Chromeleon technology, i.e. it installs and uses certain portions of the Dionex Chromeleon Chromatography Management System such as the Chromeleon Server, the Chromeleon Server Configuration, Control panels and the Program Editor. Adding the Fraction Collection Driver to the Timebase and adding the Fraction Collection Templates to the program file with DCMSLink are similar and are described in this manual.

Refer to Section 3.1 for information about how to add the Fractionation Driver to the Timebase and which modifications are required in the Server Configuration.

Refer to Sections 3.2.1, 3.2.2, 3.2.7 and 3.2.8 for information about how to create a program file and how to add the Fraction Collection Template to the Program File.

This section provides some basic information about how to use the Micro Fraction Collection Option with the DCMSLink software version V2.0 for Xcalibur, Hystar and Analyst. Dionex assumes that the user is familiar with the operation of Xcalibur, Hystar or Analyst.



5.1.1 Xcalibur Version 2.0

This section shows an example of a Timebase, Program File and Sequence for the DCMSLink version 2.0 for Xcalibur version 2.0. To be able to perform Off-line 2D-LC experiments using the Fraction Collection Option by Xcalibur, the Fraction Collection Driver must be added to the Sever Configuration as shown in FIGURE 5-1, and as described in section 3.1.

FIGURE 5-1 Typical Timebase in Xcalibur for an UltiMate 3000 system with the Micro Fraction Collection

Option installed. FIGURE 5-1 displays the server configuration for a typical installation of an UltiMate 3000 system with the Micro Fraction Collection Option installed. Note that both the DCMSLink and Fraction Collection Driver are added to the Server Configuration.

The program file created with the wizard contains the fractionation commands; ‘start’, ‘collection period’ and ‘stop’, but does not contain the commands for needle height during fractionation. These commands, provided in a template format on the Chromeleon CD, need to be inserted manually. Copy the appropriate template to the program file just before time = 0.000 as described in section 3.2.2 Add Fractionation Templates and Parameters .



FIGURE 5-2 Fraction Collection Template Copied in a Typical Xcalibur Program File for an UltiMate 3000

System with the Micro Fraction Collection Option installed. Since it is not possible to generate the second sequence automatically with DCMSLink, the injection of sample and re-injections of the fractions must be programmed manually. For Xcalibur, typically a sequence is used as displayed in FIGURE 5-3.

FIGURE 5-3 Typical Sequence in Xcalibur for an UltiMate 3000 system with the Micro Fraction Collection

Option installed. For more information on how to use DCMSLink in general, please see the DCMSLink for Xcalibur Installation Guide and Quick Start Guide Version 2.0.



5.1.2 HyStar Version 3.2

This section shows an example of a Time Base, Program File and Sequence for the DCMSLink version 2.0 for HyStar version 3.2. To be able to perform Off-line 2D-LC experiments using the Fraction Collection Option by HyStar, the Fraction Collection Driver must be added to the Sever Configuration as shown in FIGURE 5-4, and as described in section 3.1.

FIGURE 5-4 Typical Timebase in HyStar for an UltiMate 3000 System with the Micro Fraction Collection

Option Installed. FIGURE 5-4 displays the server configuration for a typical installation of an UltiMate 3000 system with the Micro Fraction Collection Option installed. Note that both the DCMSLink and Fraction Collection Driver are added to the Server Configuration.




FIGURE 5-5 Fraction Collection Template copied in a typical HyStar Program File for an UltiMate 3000

System with the Micro Fraction Collection Option Installed. Since it is not possible to generate the second sequence automatically with DCMSLink, the injection of sample and re-injections of the fractions must be programmed manually. For HyStar, typically a sequence is used as displayed in FIGURE 5-6.



FIGURE 5-6 Typical Sequence in HyStar for an UltiMate 3000 System with the Micro Fraction Collection

Option Installed. For more information on how to use DCMSLink in general, please see the DCMSLink for HyStar Installation Guide and Quick Start Guide Version 2.0.



5.1.3 Analyst Version 1.4.2

This section shows an example of a Time Base and Program File for the DCMSLink version 2.0 for Analyst version 1.4.2. To be able to perform Off-line 2D-LC experiments using the Fraction Collection Option by Analyst, the Fraction Collection Driver must be added to the Sever Configuration as shown in FIGURE 5-7, and as described in Section 3.1.

FIGURE 5-7 Typical Timebase in Analyst for an UltiMate 3000 system with the Micro Fraction Collection

Option Installed. FIGURE 5-7 displays the server configuration for a typical installation of an UltiMate 3000 system with the Micro Fraction Collection Option installed. Note that both the DCMSLink and Fraction Collection Driver are added to the Server Configuration.




FIGURE 5-8 Typical Program File in Analyst for an UltiMate 3000 system with the Micro Fraction Collection

Option Installed Since it is not possible to generate the second sequence automatically as described in section 3.2.3, the injection of sample and re-injections of the fractions must be programmed manually. For more information on how to use DCMSLink in general, please see the DCMSLink for Analyst Installation Guide and Quick Start Guide Version 2.0.



5.2 Maintenance of the 8-Port Switching Valve The WPS-3000 Micro-autosampler is standard equipped with one Cheminert® Model C2 6-port Injection Valve manufactured by VICI™ Valco Instruments Co. Inc. When upgrading the instrument with Micro Fraction Collection Option; the 6-port injection valve is replaced by a Cheminert® Model C2 8-port switching valve. Both valves are designed to offer optimal results with the switching system as they are made from materials that are inert to materials used in liquid chromatography and are designed to provide exceedingly low carryover when switched.

5.2.1 Cleaning and Replacing Parts

Description Part No. Rotor for 2 position/8 port valve, standard version 6820.0054 Rotor for 2 position/8 port valve, biocompatible version 6821.0053 Stator for 2 position/8 port valve, standard version 6820.0053 Stator for 2 position/8 port valve, biocompatible version 6821.0053

In most instances, the only maintenance that is required is cleaning of the valve. Cleaning can often be accomplished by flushing all the lines with (an) appropriate solvent(s). The selection of the solvent is dependent on the nature of the sample and the mobile phases that are used. Typically solvents such as methanol, acetonitrile, methanol/water (80/20) or acetonitrile/water (80/20) should be used.

Warning: Follow the safety precautions when handling solvents (→ page iii).

Important: To avoid any contamination of the surfaces, use disposable gloves when reassembling the valve.

Tip: Do not disassemble the valve unless system malfunction is definitely isolated to the valve.

Tip: A detailed discussion on the Installation, Use and Maintenance of the valve is presented in Technical Note 801 from VICI Valco Instruments, Co. Inc. and can be obtained at the Valco website (www.Valco.com).

http://www.valco.com/�



5.2.2 Disassembly of the Valve

To disassemble the valve:

1. Use a 9/64’ hex driver to remove the socket head screws which secure the stator to the valve (→ Fig. 1). Driver

RotorStator

Fig. 1: Exploded View of the Valco Model C2 Valve (6-port Version)

2. To insure that the sealing surface of the stator is not damaged, rest it on its outer face. If the tubing is

still attached, leave it suspended by the tubing.

3. Gently pry the rotor away from the driver with your fingers or a small screwdriver.

4. Examine the rotor sealing surface for scratches.

- If scratches are visible to the naked eye, the rotor must be replaced. - If no scratches are visible, clean all parts thoroughly with an appropriate solvent. Take care that no

surfaces get scratched while you are cleaning the components.

Tip: The most common problem in the use of the valve with HPLC is the formation of buffer crystals, which are usually water-soluble. After cleaning, it is not necessary to dry the rotor.

5.2.3 Reassembly of the Valve

Important: To avoid any contamination of the surfaces, clean all parts (→ section 5.2.1) and use disposable gloves when reassembling the valve.

To reassemble the valve:

1. Replace the rotor in the driver, making sure that the rotor sealing surface with its engraved flow passages is facing out. The pattern is asymmetrical to prevent improper placement.

2. Replace the stator. Insert the two socket head screws and tighten them gently until both are snug.

Important: Do not over tighten the screws, they simply hold the assembly together and do not affect the sealing force, which is automatically set as the screws close the stator against the valve body.

Test the valve by pressurizing the system. If the valve does not hold pressure it should be replaced.

Tip: When re-installing the valve, make certain that the proper tubing is attached using the appropriate fitting.

Tip: After you have replaced the seals, perform the RotorSealChanged command in Chromeleon to update the related service information.



5.3 CM programs for 2D-LC of Peptides using the MIC SCX/NAN PepMap Configuration

5.3.1 First Dimension Separation

;============================================================================ ; First Dimension SCX Program ; --------------------------------------------------------------------------- ; PGM-Version June 2007 ; Sample: Protein Mix Digest (PMD) ;============================================================================ AcquireExclusiveAccess ; Settings for Pump LoadingPump.Pressure.LowerLimit = 0 [bar] LoadingPump.Pressure.UpperLimit = 350[bar] LoadingPump.MaximumFlowRampDown = 50 [µl/min²] LoadingPump.MaximumFlowRampUp = 50 [µl/min²] LoadingPump.%A.Equate = ‘95/5 W/ACN, 5mmol NaH2PO4 pH3’ LoadingPump.%B.Equate = ‘as A + 1M NaCl’ LoadingPump.%C.Equate = ‘98/2 W/ACN + 0.05% TFA’ MicroPump.Pressure.LowerLimit = 0 [bar] MicroPump.Pressure.UpperLimit = 350 [bar] MasterPressure.LowerLimit = 0 [bar] MasterPressure.UpperLimit = 350 [bar] MicroPump.MaximumFlowRampDown = 0.300 [µl/min²] MicroPump.MaximumFlowRampUp = 0.300 [µl/min²] MicroPump.%A.Equate = ‘%A’ MicroPump.%B.Equate = ‘%B’ MicroPump.%C.Equate = ‘%C’ LoadingPump_Pressure.Step = Auto LoadingPump_Pressure.Average = On MicroPump.Flow = 0.000 [µl/min] MicroPump.%B = 0.0 [%] MicroPump.%C = 0.0 [%] MicroPump.Curve = 5 ; Settings for UV Detector ; In the Server Configuration the first UV Detector was named UV with Signal name UV_VIS_1 ; In the Server Configuration the second UV Detector was named UV_2 with Signal name

UV_VIS_1_2 UV.Data_Collection_Rate = 2.5 [Hz] UV.TimeConstant = 0.60 [s] UV_VIS_1.Wavelength = 214 [nm] UV_VIS_1.Average = On UV_2.Data_Collection_Rate = 2.5 [Hz] UV_2.TimeConstant = 0.60 [s] UV_VIS_1_2.Wavelength = 214 [nm] UV_VIS_1_2.Average = On ; Settings for ColumnOven



ColumnOven.TempCtrl = On ColumnOven.Temperature.Nominal = 25.0 [°C] ColumnOven.Temperature.LowerLimit = 5.0 [°C] ColumnOven.Temperature.UpperLimit = 70.0 [°C] ColumnOven.ReadyTempDelta = 1.0 [°C] ColumnOven_Temp.Step = Auto ColumnOven_Temp.Average = On ColumnOven.ValveLeft = 1_2 ColumnOven.ValveRight = 1_2 ; Settings for Autosampler ;Sampler.TempCtrl = On ; Valid for AutoSampler with cooling ;Sampler.Temperature.Nominal = 5[°C] ;Sampler.Temperature.UpperLimit = 45 [°C] ;Sampler.Temperature.LowerLimit = 4 [°C] Sampler.DrawSpeed = 200 [nl/s] Sampler.DrawDelay = 5000 [ms] Sampler.DispSpeed = 2000 [nl/s] Sampler.DispenseDelay = 2000 [ms] Sampler.WasteSpeed = 4000 [nl/s] Sampler.WashSpeed = 4000 [nl/s] Sampler.SampleHeight = 4.000 [mm] Sampler.PunctureDepth = 8.000 [mm] Sampler.WashVolume = 25.000 [µl] Sampler.SyncWithPump = On Sampler.PumpDevice = ‘LoadingPump’ Sampler.LowDispersionMode = Off Sampler.InjectMode = UserProg Sampler.ReagentAVial= R1 ; Mobile phase A must be present in the

ReagentAVial located at position R1 Sampler.ReagentBVial= R1 Sampler.ReagentCVial= R1 Sampler.ReagentDVial= R1 Sampler.PrepVial= R1 Sampler.UdpDispense To=Drain, Volume=0.000,

SyringeSpeed=GlobalSpeed, SampleHeight=GlobalHeight Sampler.UdpSyringeValve Position=Needle Sampler.UdpInjectValve Position=Load Sampler.UdpDraw From=SampleVial, Volume=20.000,

SyringeSpeed=GlobalSpeed, SampleHeight=GlobalHeight Sampler.UdpMixWait Duration=5 Sampler.UdpDraw From=ReagentAVial, Volume=3.500,

SyringeSpeed=GlobalSpeed, SampleHeight=GlobalHeight Sampler.UdpMixWait Duration=5 Sampler.UdpDispense To=Drain, Volume=0.000,

SyringeSpeed=GlobalSpeed, SampleHeight=GlobalHeight Sampler.UdpInjectValve Position=Inject Sampler.UdpInjectMarker ; Settings for Fraction Collection FractionCollection.PumpDevice = ‘LoadingPump’ TubeMaxVolume = Unlimited MaxTubesPerFraction = Unlimited TubeWrapping = No TubeChangeDuration = 2.0 [s] DelayTime = 5.0 [s]



;============================================================================ ; Definition of triggers for fraction collection starts here. ; ; Vials have to be uncapped (Commands: StartFraction/ChangeTube: VialType= Uncapped) ; Needle remains outside the puncture (e.g. 10 mm -> NeedleExtension) ; during fractionation / vial change ;============================================================================ Trigger FracStart FracStartDetected Sampler.TubePosition = FractionCollection.TubePosition StartFractionUncapped NeedleExtension=10000, fractionheight=8000 EndTrigger Trigger TubeChange FracTubeChange Sampler.TubePosition = FractionCollection.TubePosition ChangeTubeUncapped NeedleExtension=10000, Fractionheight=8000 EndTrigger Trigger FracEnd FracEndDetected EndFraction EndTrigger ;============================================================================ ; Definition of triggers for fraction collection ends here. ;============================================================================ -0.001 Sampler.TubePosition = Ba1 ;Fill in start position for fractionation FractionCollection.TubePosition = Sampler.TubePosition Airdry = off ; Select Airdry = on or Airdry = off 0.000 UV.Autozero UV_2.Autozero LoadingPump.Flow = 50 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 0.0 [%] Wait UV.Ready and ColumnOven.Ready and

Sampler.Ready Sampler.Inject ColumnOven_Temp.AcqOn LoadingPump_Pressure.AcqOn CollectFractions = By_Time CollectPeriod = 60 [s] UV_VIS_1.AcqOn ; Select the correct UV detector UV_VIS_1_2.AcqOn ; Select the correct UV detector LoadingPump.Flow = 50 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 0.0 [%] 20.000 LoadingPump.Flow = 50 [µl/min] LoadingPump.%B = 60.0 [%] LoadingPump.%C = 0.0 [%] 20.100 LoadingPump.Flow = 50 [µl/min] LoadingPump.%B = 100.0 [%] LoadingPump.%C = 0.0 [%] 25.000 LoadingPump.Flow = 50 [µl/min]



LoadingPump.%B = 100.0 [%] LoadingPump.%C = 0.0 [%] 25.100 LoadingPump.Flow = 50 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 0.0 [%] 30.000 CollectFractions = No 40.000 Sampler.Wash ; This command needs to be programmed after

the fractionation ; to empty the syringe and prepare for next injection

45.000 ColumnOven_Temp.AcqOff LoadingPump_Pressure.AcqOff UV_VIS_1.AcqOff ; Select the correct UV detector UV_VIS_1_2.AcqOff ; Select the correct UV detector LoadingPump.Flow = 50 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 0.0 [%] ReleaseExclusiveAccess End



5.3.2 Prepare System for Second Dimension Separation (Wash SCX prepare for RP)

;============================================================================ ; Wash Program for Switching from SCX to RP ; --------------------------------------------------------------------------- ; PGM-Version June 2007 ;============================================================================ AcquireExclusiveAccess ; Settings for Pump LoadingPump.Pressure.LowerLimit = 0 [bar] LoadingPump.Pressure.UpperLimit = 350 [bar] LoadingPump.MaximumFlowRampDown = 50 [µl/min²] LoadingPump.MaximumFlowRampUp = 50 [µl/min²] LoadingPump.%A.Equate = ‘%A’ LoadingPump.%B.Equate = ‘%B’ LoadingPump.%C.Equate = ‘%C’ MicroPump.Pressure.LowerLimit = 0 [bar] MicroPump.Pressure.UpperLimit = 300 [bar] MasterPressure.LowerLimit = 0 [bar] MasterPressure.UpperLimit = 300 [bar] MicroPump.MaximumFlowRampDown = 0.6 [µl/min²] MicroPump.MaximumFlowRampUp = 0.6 [µl/min²] MicroPump.%A.Equate = ‘%A’ MicroPump.%B.Equate = ‘%B’ MicroPump.%C.Equate = ‘%C’ LoadingPump_Pressure.Step = Auto LoadingPump_Pressure.Average = On ; Settings for UV Detector ; no UV detection ; Settings for ColumnOven FlowSplitter_1.FSControlMode = Off ColumnOven.TempCtrl = on ColumnOven.temperature = 25 [°C] ColumnOven_Temp.Average = On ColumnPressure.Average = On ColumnOven.ValveLeft = 1_2 ColumnOven.ValveRight = 1_2 ; Settings for Autosampler Sampler.DrawSpeed = 200 [nl/s] Sampler.DrawDelay = 7000 [ms] Sampler.DispSpeed = 2000 [nl/s] Sampler.DispenseDelay = 2000 [ms] Sampler.WasteSpeed = 4000 [nl/s] Sampler.WashSpeed = 4000 [nl/s] Sampler.PunctureDepth = 8.000 [mm] Sampler.WashVolume = 0.000 [µl] Sampler.SyncWithPump = On Sampler.LowDispersionMode = Off



Sampler.InjectMode = UserProg Sampler.SampleHeight = 4.000 [mm] Sampler.ReagentAVial= R1 Sampler.PrepVial= RA1 Sampler.UdpDispense To=Drain, Volume=0.000,

SyringeSpeed=GlobalSpeed, SampleHeight=GlobalHeight Sampler.UdpSyringeValve Position=Needle Sampler.UdpInjectValve Position=Load Sampler.UdpDraw From=ReagentAVial, Volume=5.000,

SyringeSpeed=GlobalSpeed, SampleHeight=GlobalHeight Sampler.UdpMixWait Duration=5 Sampler.UdpDraw From=SampleVial, Volume=10.000,



SyringeSpeed=GlobalSpeed, SampleHeight=GlobalHeight Sampler.UdpInjectValve Position=Inject Sampler.UdpInjectMarker 0.000 LoadingPump.Flow = 50 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 0.0 [%] MicroPump.Flow = 0.000 [µl/min] MicroPump.%B = 4.0 [%] MicroPump.%C = 0.0 [%] Wait Sampler.Ready Sampler.Inject LoadingPump_Pressure.AcqOn ColumnOven_Temp.AcqOn ColumnPressure.AcqOn LoadingPump.Flow = 50 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 0.0 [%] 2.000 LoadingPump.Flow = 0 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 0.0 [%] 2.100 Columnoven.ValveLeft = 10_1 2.500 LoadingPump.Flow = 0 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 100.0 [%] MicroPump.Flow = 0.000 [µl/min] MicroPump.%B = 4.0 [%] MicroPump.%C = 0.0 [%] 2.6 FlowSplitter_1.FSControlMode = Auto 4.000 LoadingPump.Flow = 20 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 100.0 [%]



MicroPump.Flow = 0.300 [µl/min] MicroPump.%B = 4.0 [%] MicroPump.%C = 0.0 [%] 7.500 Columnoven.ValveRight = 10_1 ; Switch from the first dimension to

the second dimension 15.000 LoadingPump.Flow = 20 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 100.0 [%] LoadingPump_Pressure.AcqOff ColumnOven_Temp.AcqOff ColumnPressure.AcqOff ReleaseExclusiveAccess End



5.3.3 Second Dimension Separation

;============================================================================ ; Second Dimension RP Program ; --------------------------------------------------------------------------- ; PGM-Version June 2007 ; Sample: First Dimension Fractions ;============================================================================ AcquireExclusiveAccess ; Settings for Pump LoadingPump.Pressure.LowerLimit = 0 [bar] LoadingPump.Pressure.UpperLimit = 350 [bar] LoadingPump.MaximumFlowRampDown = 20 [µl/min²] LoadingPump.MaximumFlowRampUp = 20 [µl/min²] LoadingPump.%A.Equate = ‘%A’ LoadingPump.%B.Equate = ‘%B’ LoadingPump.%C.Equate = ‘%C’ MicroPump.Pressure.LowerLimit = 0 [bar] MicroPump.Pressure.UpperLimit = 350 [bar] MasterPressure.LowerLimit = 0 [bar] MasterPressure.UpperLimit = 350 [bar] MicroPump.MaximumFlowRampDown = 0.300 [µl/min²] MicroPump.MaximumFlowRampUp = 0.300 [µl/min²] MicroPump.%A.Equate = ‘100% water + 0.05% TFA’ MicroPump.%B.Equate = ‘20/80 water/ACN + 0.04% TFA’ MicroPump.%C.Equate = ‘%C’ LoadingPump_Pressure.Step = Auto LoadingPump_Pressure.Average = On MicroPump_MasterPressure.Step = Auto MicroPump_MasterPressure.Average = On LoadingPump.Flow = 20 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 100.0 [%] LoadingPump.Curve = 5 ; Settings for UV Detector ; In the Server Configuration the first UV Detector was named UV with Signal name UV_VIS_1 ; In the Server Configuration the second UV Detector was named UV_2 with Signal name

UV_VIS_1_2 UV.Data_Collection_Rate = 2.5 [Hz] UV.TimeConstant = 0.60 [s] UV_VIS_1.Wavelength = 214 [nm] UV_VIS_1.Average = On UV_2.Data_Collection_Rate = 2.5 [Hz] UV_2.TimeConstant = 0.60 [s] UV_VIS_1_2.Wavelength = 214 [nm] UV_VIS_1_2.Average = On ; Settings for ColumnOven ColumnOven.TempCtrl = On ColumnOven.Temperature.Nominal = 25.0 [°C] ColumnOven.Temperature.LowerLimit = 5.0 [°C]



ColumnOven.Temperature.UpperLimit = 70.0 [°C] ColumnOven.ReadyTempDelta = 1.0 [°C] ColumnOven_Temp.Step = Auto ColumnOven_Temp.Average = On ColumnPressure.Step = Auto ColumnPressure.Average = On ColumnOven_FC_BridgeFlow.Step = Auto ColumnOven_FC_BridgeFlow.Average = On ColumnOven_FC_Stepper.Step = Auto ColumnOven_FC_Stepper.Average = On ColumnOven.ValveLeft = 10_1 ColumnOven.ValveRight = 10_1 ; Settings for Autosampler ;Sampler.TempCtrl = On ; Valid for AutoSampler with cooling ;Sampler.Temperature.Nominal = 5[°C] ;Sampler.Temperature.UpperLimit = 45 [°C] ;Sampler.Temperature.LowerLimit = 4 [°C] Sampler.DrawSpeed = 200 [nl/s] Sampler.DrawDelay = 5000 [ms] Sampler.DispSpeed = 2000 [nl/s] Sampler.DispenseDelay = 2000 [ms] Sampler.WasteSpeed = 4000 [nl/s] Sampler.WashSpeed = 4000 [nl/s] Sampler.SampleHeight = 1.000 [mm] Sampler.PunctureDepth = 8.000 [mm] Sampler.WashVolume = 25.000 [µl] Sampler.SyncWithPump = On Sampler.PumpDevice = ‘MicroPump’ Sampler.LowDispersionMode = Off Sampler.InjectMode = UserProg Sampler.ReagentAVial= R2 ; loading solvent must be present in the

ReagentAVial loacated at position R2 Sampler.ReagentBVial= R2 Sampler.ReagentCVial= R2 Sampler.ReagentDVial= R2 Sampler.PrepVial= R2 Sampler.UdpDispense To=Drain, Volume=0.000,

SyringeSpeed=GlobalSpeed, SampleHeight=GlobalHeight Sampler.UdpSyringeValve Position=Needle Sampler.UdpInjectValve Position=Load Sampler.UdpDraw From=SampleVial, Volume=10.000,



SyringeSpeed=GlobalSpeed, SampleHeight=GlobalHeight Sampler.UdpInjectValve Position=Inject Sampler.UdpInjectMarker 0.000 UV.Autozero UV_2.Autozero MicroPump.Flow = 0.300 [µl/min] MicroPump.%B = 4.0 [%] MicroPump.%C = 0.0 [%] Wait UV.Ready and ColumnOven.Ready and

Sampler.Ready



Sampler.Inject ColumnOven_Temp.AcqOn ColumnPressure.AcqOn ColumnOven_FC_BridgeFlow.AcqOn ColumnOven_FC_Stepper.AcqOn LoadingPump_Pressure.AcqOn MicroPump_MasterPressure.AcqOn CollectFractions = No UV_VIS_1.AcqOn ; Select the correct UV detector UV_VIS_1_2.AcqOn ; Select the correct UV detector MicroPump.Flow = 0.300 [µl/min] MicroPump.%B = 4.0 [%] MicroPump.%C = 0.0 [%] 1.000 Sampler.Wash ; This command needs to be programed after the

fractionation ; to empty the syringe and prepare for next injection

5.000 MicroPump.Flow = 0.300 [µl/min] MicroPump.%B = 4.0 [%] MicroPump.%C = 0.0 [%] 7.000 Columnoven.ValveRight = 1_2 35.000 MicroPump.Flow = 0.300 [µl/min] MicroPump.%B = 55.0 [%] MicroPump.%C = 0.0 [%] 35.100 MicroPump.Flow = 0.300 [µl/min] MicroPump.%B = 90.0 [%] MicroPump.%C = 0.0 [%] 40.000 MicroPump.Flow = 0.300 [µl/min] MicroPump.%B = 90.0 [%] MicroPump.%C = 0.0 [%] 40.100 MicroPump.Flow = 0.300 [µl/min] MicroPump.%B = 4.0 [%] MicroPump.%C = 0.0 [%] 55.000 Columnoven.ValveRight = 10_1 60.000 ColumnOven_Temp.AcqOff ColumnPressure.AcqOff ColumnOven_FC_BridgeFlow.AcqOff ColumnOven_FC_Stepper.AcqOff LoadingPump_Pressure.AcqOff MicroPump_MasterPressure.AcqOff UV_VIS_1.AcqOff ; Select the correct UV detector UV_VIS_1_2.AcqOff ; Select the correct UV detector MicroPump.Flow = 0.300 [µl/min] MicroPump.%B = 4.0 [%] MicroPump.%C = 0.0 [%] ReleaseExclusiveAccess End



5.3.4 Prepare System for First Dimension Separation (Wash RP prepare for SCX)

;============================================================================ ; Wash Program for Switching from RP to SCX ; --------------------------------------------------------------------------- ; PGM-Version June 2007 ;============================================================================ AcquireExclusiveAccess ; Settings for Pump LoadingPump.Pressure.LowerLimit = 0 [bar] LoadingPump.Pressure.UpperLimit = 350 [bar] LoadingPump.MaximumFlowRampDown = 50 [µl/min²] LoadingPump.MaximumFlowRampUp = 50 [µl/min²] LoadingPump.%A.Equate = ‘%A’ LoadingPump.%B.Equate = ‘%B’ LoadingPump.%C.Equate = ‘%C’ MicroPump.Pressure.LowerLimit = 0 [bar] MicroPump.Pressure.UpperLimit = 300 [bar] MasterPressure.LowerLimit = 0 [bar] MasterPressure.UpperLimit = 300 [bar] MicroPump.MaximumFlowRampDown = 0.6 [µl/min²] MicroPump.MaximumFlowRampUp = 0.6 [µl/min²] MicroPump.%A.Equate = ‘%A’ MicroPump.%B.Equate = ‘%B’ MicroPump.%C.Equate = ‘%C’ LoadingPump_Pressure.Step = Auto LoadingPump_Pressure.Average = On ; Settings for UV Detector ; no UV detection ; Settings for ColumnOven FlowSplitter_1.FSControlMode = auto ColumnOven.TempCtrl = on ColumnOven.temperature = 25 [°C] ColumnOven_Temp.Average = On ColumnPressure.Average = On ColumnOven.ValveLeft = 10_1 ColumnOven.ValveRight = 10_1 ; Settings for Autosampler Sampler.DrawSpeed = 200 [nl/s] Sampler.DrawDelay = 7000 [ms] Sampler.DispSpeed = 2000 [nl/s] Sampler.DispenseDelay = 2000 [ms] Sampler.WasteSpeed = 4000 [nl/s] Sampler.WashSpeed = 4000 [nl/s] Sampler.PunctureDepth = 8.000 [mm] Sampler.WashVolume = 0.000 [µl] Sampler.SyncWithPump = On Sampler.LowDispersionMode = Off



Sampler.InjectMode = UserProg Sampler.SampleHeight = 4.000 [mm] Sampler.ReagentAVial= R1 Sampler.PrepVial= RA1 Sampler.UdpDispense To=Drain, Volume=0.000,





SyringeSpeed=GlobalSpeed, SampleHeight=GlobalHeight Sampler.UdpInjectValve Position=Inject Sampler.UdpInjectMarker 0.000 LoadingPump.Flow = 20 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 100.0 [%] MicroPump.Flow = 0.300 [µl/min] MicroPump.%B = 4.0 [%] MicroPump.%C = 0.0 [%] Wait Sampler.Ready Sampler.Inject LoadingPump_Pressure.AcqOn ColumnOven_Temp.AcqOn ColumnPressure.AcqOn LoadingPump.Flow = 20 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 100.0 [%] MicroPump.Flow = 0.300 [µl/min] MicroPump.%B = 4.0 [%] MicroPump.%C = 0.0 [%] 2.000 LoadingPump.Flow = 0 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 100.0 [%] MicroPump.Flow = 0.000 [µl/min] MicroPump.%B = 4.0 [%] MicroPump.%C = 0.0 [%] 2.100 FlowSplitter_1.FSControlMode = Off 2.500 columnoven.ValveRight = 1_2 ; Switch from the second dimension to the

first dimension 2.600 LoadingPump.Flow = 0 [µl/min] LoadingPump.%B = 0.0 [%]



LoadingPump.%C = 0.0 [%] MicroPump.Flow = 0.000 [µl/min] MicroPump.%B = 4.0 [%] MicroPump.%C = 0.0 [%] 4.000 LoadingPump.Flow = 50 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 0.0 [%] MicroPump.Flow = 0.000 [µl/min] MicroPump.%B = 4.0 [%] MicroPump.%C = 0.0 [%] 7.500 Columnoven.ValveLeft = 1_2 15.000 LoadingPump.Flow = 50 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 0.0 [%] LoadingPump_Pressure.AcqOff ColumnOven_Temp.AcqOff ColumnPressure.AcqOff ReleaseExclusiveAccess End



5.4 CM programs for 2D-LC of Peptides Using the CAP SCX/CAP Monolith Configuration


;============================================================================ ; First Dimension SCX Program ; --------------------------------------------------------------------------- ; PGM-Version June 2007 ; Sample: Protein Mix Digest (PMD) ;============================================================================ AcquireExclusiveAccess ; Settings for Pump LoadingPump.Pressure.LowerLimit = 0 [bar] LoadingPump.Pressure.UpperLimit = 400 [bar] LoadingPump.MaximumFlowRampDown = 600 [µl/min²] LoadingPump.MaximumFlowRampUp = 600 [µl/min²] LoadingPump.%A.Equate = ‘%A’ LoadingPump.%B.Equate = ‘%B’ LoadingPump.%C.Equate = ‘%C’ MicroPump.Pressure.LowerLimit = 0 [bar] MicroPump.Pressure.UpperLimit = 350 [bar] MasterPressure.LowerLimit = 0 [bar] MasterPressure.UpperLimit = 500 [bar] MicroPump.MaximumFlowRampDown = 2.505 [µl/min²] MicroPump.MaximumFlowRampUp = 2.505 [µl/min²] MicroPump.%A.Equate = ‘%A’ MicroPump.%B.Equate = ‘%B’ MicroPump.%C.Equate = ‘%C’ MicroPump.Flow = 0.000 [µl/min] MicroPump.%B = 0.0 [%] MicroPump.%C = 0.0 [%] MicroPump.Curve = 5 LoadingPump_Pressure.Step = Auto LoadingPump_Pressure.Average = On MicroPump_MasterPressure.Step = Auto MicroPump_MasterPressure.Average = On ; Settings for UV Detectors ; In the Server Configuration the first UV Detector was named UV with Signal name UV_VIS_1 ; In the Server Configuration the second UV Detector was named UV_2 with Signal name

UV_VIS_1_2 UV_2.Data_Collection_Rate = 2.5 [Hz] UV_2.TimeConstant = 0.60 [s] UV.Data_Collection_Rate = 2.5 [Hz] UV.TimeConstant = 0.60 [s] UV_VIS_1.Wavelength = 214 [nm] UV_VIS_1.Step = Auto UV_VIS_1.Average = On UV_VIS_1_2.Wavelength = 214 [nm] UV_VIS_1_2.Step = Auto



UV_VIS_1_2.Average = On ; Settings for Columnoven ColumnOven.TempCtrl = On ColumnOven.Temperature.Nominal = 25.0 [°C] ColumnOven.Temperature.LowerLimit = 5.0 [°C] ColumnOven.Temperature.UpperLimit = 85.0 [°C] ColumnOven.ReadyTempDelta = 1.0 [°C] ColumnOven_Temp.Step = Auto ColumnOven_Temp.Average = On ColumnPressure.Step = 0.20 [s] ColumnPressure.Average = Off ColumnOven_FC_BridgeFlow.Step = Auto ColumnOven_FC_BridgeFlow.Average = On ColumnOven_FC_Stepper.Step = Auto ColumnOven_FC_Stepper.Average = On ColumnOven_FC_Filter.Step = Auto ColumnOven_FC_Filter.Average = On ColumnOven_FC_Deviation.Step = Auto ColumnOven_FC_Deviation.Average = On ValveLeft = 1_2 ValveRight = 1_2 ; Settings for Autosampler ;Sampler.TempCtrl = On ; Valid for AutoSampler with cooling ;Sampler.Temperature.Nominal = 5[°C] ;Sampler.Temperature.UpperLimit = 45 [°C] ;Sampler.Temperature.LowerLimit = 4 [°C] Sampler.DrawSpeed = 250 [nl/s] Sampler.DrawDelay = 5000 [ms] Sampler.DispSpeed = 2000 [nl/s] Sampler.DispenseDelay = 2000 [ms] Sampler.WasteSpeed = 4000 [nl/s] Sampler.WashSpeed = 6000 [nl/s] Sampler.SampleHeight = 4.000 [mm] Sampler.PunctureDepth = 8.000 [mm] Sampler.WashVolume = 25.000 [µl] Sampler.SyncWithPump = On Sampler.PumpDevice = ‘LoadingPump’ Sampler.LowDispersionMode = Off Sampler.InjectMode = UserProg Sampler.ReagentAVial= R1 Sampler.UdpDispense To=Drain, Volume=0.000,





SyringeSpeed=GlobalSpeed, SampleHeight=GlobalHeight Sampler.UdpInjectValve Position=Inject



Sampler.UdpInjectMarker ; Settings for Fraction Collection FractionCollection.PumpDevice = ‘LoadingPump’ TubeWrapping = Yes DelayTime = 5.0 [s] ;============================================================================ ; Definition of triggers for fraction collection starts here. ; ; Vials have to be uncapped (Commands: StartFraction/ChangeTube: VialType= Uncapped) ; Needle remains outside the puncture (e.g. 10 mm -> NeedleExtension) ; during fractionation / vial change ;============================================================================ Trigger FracStart FracStartDetected Sampler.TubePosition = FractionCollection.TubePosition StartFractionUncapped NeedleExtension=10000, fractionheight=8000 EndTrigger Trigger TubeChange FracTubeChange Sampler.TubePosition = FractionCollection.TubePosition ChangeTubeUncapped NeedleExtension=10000, Fractionheight=8000 EndTrigger Trigger FracEnd FracEndDetected EndFraction EndTrigger ;============================================================================ ; Definition of triggers for fraction collection ends here. ;============================================================================ -0.010 Sampler.TubePosition = Ba1 FractionCollection.TubePosition = Sampler.TubePosition Airdry = off ; Select Airdry = on or Airdry = off 0.000 UV_2.Autozero UV.Autozero LoadingPump.Flow = 600 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 0.0 [%] Wait UV.Ready and ColumnOven.Ready and

UV_2.Ready and Sampler.Ready Sampler.Inject ColumnOven_Temp.AcqOn ColumnPressure.AcqOn ColumnOven_FC_BridgeFlow.AcqOn ColumnOven_FC_Stepper.AcqOn ColumnOven_FC_Filter.AcqOn ColumnOven_FC_Deviation.AcqOn LoadingPump_Pressure.AcqOn MicroPump_MasterPressure.AcqOn UV_VIS_1.AcqOn ; Select the correct UV detector UV_VIS_1_2.AcqOn ; Select the correct UV detector LoadingPump.Flow = 600 [µl/min]



LoadingPump.%B = 0.0 [%] LoadingPump.%C = 0.0 [%] 1.000 Sampler.Wash ; When fractionation starts at time = 0, the wash must be programmed after the

fractionation 10.000 CollectFractions = By_Time CollectPeriod = 60 [s] 20.000 LoadingPump.Flow = 600 [µl/min] LoadingPump.%B = 60.0 [%] LoadingPump.%C = 0.0 [%] 20.100 LoadingPump.Flow = 600 [µl/min] LoadingPump.%B = 100.0 [%] LoadingPump.%C = 0.0 [%] 25.000 LoadingPump.Flow = 600 [µl/min] LoadingPump.%B = 100.0 [%] LoadingPump.%C = 0.0 [%] 25.100 LoadingPump.Flow = 600 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 0.0 [%] 30.000 CollectFractions = No 45.000 LoadingPump.Flow = 600 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 0.0 [%] ColumnOven_Temp.AcqOff ColumnPressure.AcqOff ColumnOven_FC_BridgeFlow.AcqOff ColumnOven_FC_Stepper.AcqOff ColumnOven_FC_Filter.AcqOff ColumnOven_FC_Deviation.AcqOff LoadingPump_Pressure.AcqOff MicroPump_MasterPressure.AcqOff UV_VIS_1.AcqOff ; Select the correct UV detector UV_VIS_1_2.AcqOff ; Select the correct UV detector ReleaseExclusiveAccess End



5.4.2 Prepare System for Second Dimension Separation (Wash SCX prepare for RP)

;============================================================================ ; Wash Program for Switching from SCX to RP ; --------------------------------------------------------------------------- ; PGM-Version August 2007 ;============================================================================ ; Settings for Pump LoadingPump.Pressure.LowerLimit = 0 [bar] LoadingPump.Pressure.UpperLimit = 400 [bar] LoadingPump.MaximumFlowRampDown = 600 [µl/min²] LoadingPump.MaximumFlowRampUp = 600 [µl/min²] LoadingPump.%A.Equate = ‘%A’ LoadingPump.%B.Equate = ‘%B’ LoadingPump.%C.Equate = ‘%C’ MicroPump.Pressure.LowerLimit = 0 [bar] MicroPump.Pressure.UpperLimit = 300 [bar] MasterPressure.LowerLimit = 0 [bar] MasterPressure.UpperLimit = 300 [bar] MicroPump.MaximumFlowRampDown = 2.5 [µl/min²] MicroPump.MaximumFlowRampUp = 2.5 [µl/min²] MicroPump.%A.Equate = ‘%A’ MicroPump.%B.Equate = ‘%B’ MicroPump.%C.Equate = ‘%C’ LoadingPump_Pressure.Step = Auto LoadingPump_Pressure.Average = On ; Settings for UV Detector ; no UV detection ; Settings for ColumnOven FlowSplitter_1.FSControlMode = auto ColumnOven.TempCtrl = on ColumnOven.temperature = 25 [°C] ColumnOven_Temp.Average = On ColumnPressure.Average = On ColumnOven.ValveLeft = 1_2 ColumnOven.ValveRight = 1_2 ; Settings for Autosampler Sampler.DrawSpeed = 200 [nl/s] Sampler.DrawDelay = 7000 [ms] Sampler.DispSpeed = 2000 [nl/s] Sampler.DispenseDelay = 2000 [ms] Sampler.WasteSpeed = 4000 [nl/s] Sampler.WashSpeed = 4000 [nl/s] Sampler.PunctureDepth = 8.000 [mm] Sampler.WashVolume = 0.000 [µl] Sampler.SyncWithPump = On Sampler.LowDispersionMode = Off Sampler.InjectMode = UserProg Sampler.SampleHeight = 4.000 [mm]



Sampler.ReagentAVial= R1 Sampler.PrepVial= RA1 Sampler.UdpDispense To=Drain, Volume=0.000,





SyringeSpeed=GlobalSpeed, SampleHeight=GlobalHeight Sampler.UdpInjectValve Position=Inject Sampler.UdpInjectMarker 0.000 LoadingPump.Flow = 600 [µl/min] ; programmed flowrate depends on

the split ratio LoadingPump.%B = 0.0 [%] LoadingPump.%C = 0.0 [%] MicroPump.Flow = 0.000 [µl/min] MicroPump.%B = 0.0 [%] MicroPump.%C = 0.0 [%] Wait Sampler.Ready Sampler.Inject LoadingPump_Pressure.AcqOn ColumnOven_Temp.AcqOn ColumnPressure.AcqOn LoadingPump.Flow = 600 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 0.0 [%] 0.200 Sampler.InjectValveToInject 1.00 LoadingPump.Flow = 600 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 0.0 [%] 3.00 LoadingPump.Flow = 0 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 0.0 [%] 3.1 FlowSplitter_1.FSControlMode = off 3.20 LoadingPump.Flow = 0 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 0.0 [%] 3.5 LoadingPump.Flow = 0 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 100.0 [%] ; Change eluent from buffer A to

loading solvent



5.00 LoadingPump.Flow = 600 [µl/min] ; Flush SCX flowpath free of buffer pre split

LoadingPump.%B = 0.0 [%] LoadingPump.%C = 100.0 [%] 10.00 LoadingPump.Flow = 600 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 100.0 [%] 12.00 LoadingPump.Flow = 0 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 100.0 [%] 12.1 FlowSplitter_1.FSControlMode = auto 13.5 LoadingPump.Flow = 300 [µl/min] ; Flush SCX flowpath free of buffer

post split LoadingPump.%B = 0.0 [%] ; without flushing the SCX column LoadingPump.%C = 100.0 [%] 14.0 LoadingPump.Flow = 300 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 100.0 [%] 15.5 LoadingPump.Flow = 0 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 100.0 [%] 15.6 FlowSplitter_1.FSControlMode = off 16.00 Columnoven.ValveRight = 10_1 ; Change from SCX to RP split LoadingPump.Flow = 0 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 100.0 [%] MicroPump.Flow = 0.0 [µl/min] MicroPump.%B = 0.0 [%] MicroPump.%C = 0.0 [%] Columnoven.TempCtrl = on Columnoven.temperature = 25 [°C] 16.1 Columnoven.TempCtrl = on Columnoven.temperature = 60 [°C] ; Set temperature for separation on

monolithic column ; Make sure the PolyLC column is NOT located in the ColumnOven

16.5 LoadingPump.Flow = 50 [µl/min] ; Pre flush tubing with loading solvent LoadingPump.%B = 0.0 [%] LoadingPump.%C = 100.0 [%] MicroPump.Flow = 0 [µl/min] MicroPump.%B = 0.0 [%] MicroPump.%C = 0.0 [%] 22.00 LoadingPump.Flow = 50 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 100.0 [%]



MicroPump.Flow = 0 [µl/min] MicroPump.%B = 0.0 [%] MicroPump.%C = 0.0 [%] 22.50 LoadingPump.Flow = 0 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 100.0 [%] MicroPump.Flow = 0 [µl/min] MicroPump.%B = 0.0 [%] MicroPump.%C = 0.0 [%] Columnoven.ValveLeft = 10_1 ; switch valve for pre-concentration

set-up 22.6 Wait columnoven.ready 23.00 LoadingPump.Flow = 0 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 100.0 [%] MicroPump.Flow = 0 [µl/min] MicroPump.%B = 0.0 [%] MicroPump.%C = 0.0 [%] FlowSplitter_1.FSControlMode = Auto 25.00 LoadingPump.Flow = 20 [µl/min] ; Set loading flow for

preconcentration LoadingPump.%B = 0.0 [%] LoadingPump.%C = 100.0 [%] MicroPump.Flow = 2.5 [µl/min] ; Set analytical flowrate MicroPump.%B = 0.0 [%] MicroPump.%C = 0.0 [%] 30.00 LoadingPump.Flow = 20 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 100.0 [%] MicroPump.Flow = 2.5 [µl/min] MicroPump.%B = 0.0 [%] MicroPump.%C = 0.0 [%] ColumnOven_Temp.AcqOff ColumnPressure.AcqOff LoadingPump_Pressure.AcqOff End




;============================================================================ ; Second Dimension RP Program ; --------------------------------------------------------------------------- ; PGM-Version June 2007 ; Sample: First Dimension Fractions ;============================================================================ AcquireExclusiveAccess ; Settings for Pump LoadingPump.Pressure.LowerLimit = 0 [bar] LoadingPump.Pressure.UpperLimit = 400 [bar] LoadingPump.MaximumFlowRampDown = 50 [µl/min²] LoadingPump.MaximumFlowRampUp = 1000 [µl/min²] LoadingPump.%A.Equate = ‘%A’ LoadingPump.%B.Equate = ‘%B’ LoadingPump.%C.Equate = ‘100% water + 0.05% HFBA’ MicroPump.Pressure.UpperLimit = 350 [bar] MicroPump.Pressure.LowerLimit = 0 [bar] MasterPressure.LowerLimit = 0 [bar] MasterPressure.UpperLimit = 400 [bar] MicroPump.MaximumFlowRampDown = 2.500 [µl/min²] MicroPump.MaximumFlowRampUp = 2.500 [µl/min²] MicroPump.%A.Equate = ‘100% water + 0.05% TFA’ MicroPump.%B.Equate = ‘20/80 water/ACN + 0.04% TFA’ MicroPump.%C.Equate = ‘%C’ LoadingPump_Pressure.Step = Auto LoadingPump_Pressure.Average = On MicroPump_MasterPressure.Step = Auto MicroPump_MasterPressure.Average = On LoadingPump.Flow = 20 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 100.0 [%] ; Settings for UV Detector ; In the Server Configuration the first UV Detector was named UV with Signal name UV_VIS_1 ; In the Server Configuration the second UV Detector was named UV_2 with Signal name

UV_VIS_1_2 UV.Data_Collection_Rate = 10.0 [Hz] UV.TimeConstant = 0.10 [s] UV_2.Data_Collection_Rate = 10.0 [Hz] UV_2.TimeConstant = 0.10 [s] UV_VIS_1.Wavelength = 214 [nm] UV_VIS_1.Step = Auto UV_VIS_1.Average = On UV_VIS_1_2.Wavelength = 214 [nm] UV_VIS_1_2.Step = Auto UV_VIS_1_2.Average = On



; Settings for ColumnOven ColumnOven.TempCtrl = On ColumnOven.Temperature.Nominal = 60.0 [°C] ColumnOven.Temperature.LowerLimit = 5.0 [°C] ColumnOven.Temperature.UpperLimit = 85.0 [°C] ColumnOven.ReadyTempDelta = 1.0 [°C] ColumnOven.ValveLeft = 10_1 ColumnOven.ValveRight = 10_1 ColumnOven_Temp.Step = Auto ColumnOven_Temp.Average = On ColumnPressure.Step = Auto ColumnPressure.Average = On ColumnOven_FC_BridgeFlow.Step = Auto ColumnOven_FC_BridgeFlow.Average = On ColumnOven_FC_Stepper.Step = Auto ColumnOven_FC_Stepper.Average = On ; Settings for Autosampler Sampler.DrawSpeed = 200 [nl/s] Sampler.DrawDelay = 5000 [ms] Sampler.DispSpeed = 2000 [nl/s] Sampler.DispenseDelay = 2000 [ms] Sampler.WasteSpeed = 4000 [nl/s] Sampler.WashSpeed = 4000 [nl/s] Sampler.SampleHeight = 0.000 [mm] Sampler.PunctureDepth = 2.000 [mm] Sampler.WashVolume = 100.000 [µl] Sampler.SyncWithPump = On Sampler.LowDispersionMode = Off Sampler.InjectMode = UserProg Sampler.ReagentAVial= R2 Sampler.UdpDispense To=Drain, Volume=0.000,



SyringeSpeed=GlobalSpeed, SampleHeight=GlobalHeight Sampler.UdpDispense To=SampleVial, Volume=10.000,

SyringeSpeed=500, SampleHeight=GlobalHeight Sampler.UdpMixWait Duration=5 Sampler.UdpDispense To=SampleVial, Volume=0.000,

SyringeSpeed=GlobalSpeed, SampleHeight=GlobalHeight Sampler.UdpDraw From=SampleVial, Volume=10.000,

SyringeSpeed=500, SampleHeight=GlobalHeight Sampler.UdpMixWait Duration=5 Sampler.UdpDraw From=SampleVial, Volume=0.000,



SyringeSpeed=GlobalSpeed, SampleHeight=GlobalHeight



Sampler.UdpDraw From=SampleVial, Volume=16.000, SyringeSpeed=GlobalSpeed, SampleHeight=GlobalHeight

Sampler.UdpMixWait Duration=5 Sampler.UdpDraw From=SampleVial, Volume=0.000,

SyringeSpeed=GlobalSpeed, SampleHeight=GlobalHeight Sampler.UdpDraw From=ReagentAVial, Volume=3.400,


SyringeSpeed=GlobalSpeed, SampleHeight=GlobalHeight Sampler.UdpDispense To=Drain, Volume=0.000,

SyringeSpeed=GlobalSpeed, SampleHeight=GlobalHeight Sampler.UdpInjectValve Position=Inject Sampler.UdpInjectMarker 0.000 MicroPump.Flow = 2.500 [µl/min] UV.Autozero UV_2.Autozero MicroPump.%B = 0.0 [%] MicroPump.%C = 0.0 [%] Wait UV.Ready and ColumnOven.Ready and

UV_2.Ready and Sampler.Ready Sampler.Inject LoadingPump_Pressure.AcqOn MicroPump_MasterPressure.AcqOn ColumnOven_Temp.AcqOn ColumnPressure.AcqOn ColumnOven_FC_BridgeFlow.AcqOn ColumnOven_FC_Stepper.AcqOn CollectFractions = No UV_VIS_1.AcqOn ; Select the correct UV detector UV_VIS_1_2.AcqOn ; Select the correct UV detector MicroPump.Flow = 2.500 [µl/min] MicroPump.%B = 0.0 [%] MicroPump.%C = 0.0 [%] 1.000 Sampler.Wash 2.000 MicroPump.Flow = 2.500 [µl/min] MicroPump.%B = 0.0 [%] MicroPump.%C = 0.0 [%] 5.000 ValveLeft = 1_2 12.000 MicroPump.Flow = 2.500 [µl/min] MicroPump.%B = 45.0 [%] MicroPump.%C = 0.0 [%] 12.100 MicroPump.Flow = 2.500 [µl/min] MicroPump.%B = 90.0 [%] MicroPump.%C = 0.0 [%] 15.000 MicroPump.Flow = 2.500 [µl/min] MicroPump.%B = 90.0 [%] MicroPump.%C = 0.0 [%] 15.100 MicroPump.Flow = 2.500 [µl/min] MicroPump.%B = 0.0 [%]



MicroPump.%C = 0.0 [%] 20.000 ValveLeft = 10_1 25.000 LoadingPump_Pressure.AcqOff MicroPump_MasterPressure.AcqOff ColumnOven_Temp.AcqOff ColumnPressure.AcqOff ColumnOven_FC_BridgeFlow.AcqOff ColumnOven_FC_Stepper.AcqOff UV_VIS_1.AcqOff ; Select the correct UV detector UV_VIS_1_2.AcqOff ; Select the correct UV detector MicroPump.Flow = 2.500 [µl/min] MicroPump.%B = 0.0 [%] MicroPump.%C = 0.0 [%] ReleaseExclusiveAccess End



5.4.4 Prepare System for First Dimension Separation (Wash RP prepare for SCX)

;============================================================================ ; Wash Program for Switching from RP to SCX ; --------------------------------------------------------------------------- ; PGM-Version August 2007 ;============================================================================ AcquireExclusiveAccess ; Settings for Pump LoadingPump.Pressure.LowerLimit = 0 [bar] LoadingPump.Pressure.UpperLimit = 400 [bar] LoadingPump.MaximumFlowRampDown = 600 [µl/min²] LoadingPump.MaximumFlowRampUp = 1000 [µl/min²] LoadingPump.%A.Equate = ‘%A’ LoadingPump.%B.Equate = ‘%B’ LoadingPump.%C.Equate = ‘%C’ MicroPump.Pressure.LowerLimit = 0 [bar] MicroPump.Pressure.UpperLimit = 300 [bar] MasterPressure.LowerLimit = 0 [bar] MasterPressure.UpperLimit = 300 [bar] MicroPump.MaximumFlowRampDown = 3.00 [µl/min²] MicroPump.MaximumFlowRampUp = 3.00 [µl/min²] MicroPump.%A.Equate = ‘%A’ MicroPump.%B.Equate = ‘%B’ MicroPump.%C.Equate = ‘%C’ LoadingPump_Pressure.Step = Auto LoadingPump_Pressure.Average = On ; Settings for UV Detectors ; No UV detection ; Settings for ColumnOven FlowSplitter_1.FSControlMode = auto ColumnOven.TempCtrl = on ColumnOven.temperature = 25 [°C] ColumnOven_Temp.Average = On ColumnPressure.Average = On ColumnOven.ValveLeft = 10_1 ColumnOven.ValveRight = 10_1 ; Settings for Autosampler Sampler.DrawSpeed = 200 [nl/s] Sampler.DrawDelay = 7000 [ms] Sampler.DispSpeed = 2000 [nl/s] Sampler.DispenseDelay = 2000 [ms] Sampler.WasteSpeed = 4000 [nl/s] Sampler.WashSpeed = 4000 [nl/s] Sampler.PunctureDepth = 8.000 [mm] Sampler.WashVolume = 0.000 [µl] Sampler.SyncWithPump = On Sampler.LowDispersionMode = Off Sampler.InjectMode = UserProg Sampler.SampleHeight = 3.000 [mm]



Sampler.ReagentAVial= R1 Sampler.PrepVial= RA1 Sampler.UdpDispense To=Drain, Volume=0.000,





SyringeSpeed=GlobalSpeed, SampleHeight=GlobalHeight Sampler.UdpInjectValve Position=Inject Sampler.UdpInjectMarker 0.000 LoadingPump.Flow = 20 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 100.0 [%] MicroPump.Flow = 2.500 [µl/min] MicroPump.%B = 0.0 [%] MicroPump.%C = 0.0 [%] Wait Sampler.Ready Sampler.Inject LoadingPump_Pressure.AcqOn ColumnOven_Temp.AcqOn ColumnPressure.AcqOn 0.200 Sampler.InjectValveToInject 1.00 LoadingPump.Flow = 20 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 100.0 [%] MicroPump.Flow = 2.500 [µl/min] MicroPump.%B = 0.0 [%] MicroPump.%C = 0.0 [%] 2.10 LoadingPump.Flow = 0 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 100.0 [%] MicroPump.Flow = 0 [µl/min] MicroPump.%B = 0.0 [%] MicroPump.%C = 0.0 [%] 2.20 FlowSplitter_1.FSControlMode = Off 2.30 Columnoven.ValveLeft = 1_2 3.100 LoadingPump.Flow = 0 [µl/min] ; Change eluent from loading solvent to

buffer A LoadingPump.%B = 0.0 [%] LoadingPump.%C = 0.0 [%]



MicroPump.Flow = 0 [µl/min] MicroPump.%B = 0.0 [%] MicroPump.%C = 0.0 [%] 3.200 LoadingPump.Flow = 50 [µl/min] ; Pre flush SCX line with buffer A LoadingPump.%B = 0.0 [%] LoadingPump.%C = 0.0 [%] MicroPump.Flow = 0 [µl/min] MicroPump.%B = 0.0 [%] MicroPump.%C = 0.0 [%] 13.0 LoadingPump.Flow = 50 [µl/min] ; Pre flush SCX line with buffer A LoadingPump.%B = 0.0 [%] LoadingPump.%C = 0.0 [%] MicroPump.Flow = 0 [µl/min] MicroPump.%B = 0.0 [%] MicroPump.%C = 0.0 [%] 14.0 LoadingPump.Flow = 0 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 0.0 [%] MicroPump.Flow = 0 [µl/min] MicroPump.%B = 0.0 [%] MicroPump.%C = 0.0 [%] 14.3 Columnoven.ValveRight = 1_2 ; Change from RP to SCX split wait columnoven.ready 15.000 LoadingPump.Flow = 0 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 0.0 [%] MicroPump.Flow = 0.0 [µl/min] MicroPump.%B = 0.0 [%] MicroPump.%C = 0.0 [%] 16.10 LoadingPump.Flow = 600 [µl/min] ; wash tubing post split, LoadingPump.%B = 0.0 [%] ; programmed flowrate depends on

the split ratio LoadingPump.%C = 0.0 [%] MicroPump.Flow = 0 [µl/min] MicroPump.%B = 0.0 [%] MicroPump.%C = 0.0 [%] 20.00 LoadingPump.Flow = 600 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 0.0 [%] MicroPump.Flow = 0 [µl/min] MicroPump.%B = 0.0 [%] MicroPump.%C = 0.0 [%] 22.00 LoadingPump.Flow = 0 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 0.0 [%]



MicroPump.Flow = 0.0 [µl/min] MicroPump.%B = 0.0 [%] MicroPump.%C = 0.0 [%] 22.10 FlowSplitter_1.FSControlMode = Auto 25.000 LoadingPump.Flow = 600 [µl/min] ; start SCX flow LoadingPump.%B = 0.0 [%] LoadingPump.%C = 0.0 [%] 30.000 LoadingPump.Flow = 600 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 0.0 [%] ColumnOven_Temp.AcqOff ColumnPressure.AcqOff LoadingPump_Pressure.AcqOff ReleaseExclusiveAccess End



5.5 CM programs for 2D-LC of Proteins using the MIC WAX / 500 µm I.D Monolith Configuration


;============================================================================ ; First Dimension WAX Program ; --------------------------------------------------------------------------- ; PGM-Version March 2008 ; Sample: E-coli lysate ;============================================================================ AcquireExclusiveAccess ; Settings for Pump LoadingPump.Pressure.LowerLimit = 0 [bar] LoadingPump.Pressure.UpperLimit = 350 [bar] LoadingPump.MaximumFlowRampDown = 100 [µl/min²] LoadingPump.MaximumFlowRampUp = 100 [µl/min²] LoadingPump.%A.Equate = "20 mM Tris-HCl buffer pH 7.6" LoadingPump.%B.Equate = "as A + 1M NaCl" LoadingPump.%C.Equate = "100% water + 0.05% HFBA" MicroPump.Pressure.LowerLimit = 0 [bar] MicroPump.Pressure.UpperLimit = 350 [bar] MasterPressure.LowerLimit = 0 [bar] MasterPressure.UpperLimit = 350 [bar] MicroPump.MaximumFlowRampDown = 5.000 [µl/min²] MicroPump.MaximumFlowRampUp = 5.000 [µl/min²] MicroPump.%A.Equate = "100% water + 0.05% TFA" MicroPump.%B.Equate = "20/80 water/ACN + 0.04% TFA" MicroPump.%C.Equate = "100% water + 0.05% HFBA" LoadingPump_Pressure.Step = Auto LoadingPump_Pressure.Average = On MicroPump.Flow = 0.000 [µl/min] MicroPump.%B = 0.0 [%] MicroPump.%C = 0.0 [%] MicroPump.Curve = 5 ; Settings for UV Detector ; In the Server Configuration the UV Detector was named UV with Signal name UV_VIS_1 UV.Data_Collection_Rate = 4.0 [Hz] UV.TimeConstant = 1.00 [s] UV_VIS_1.Wavelength = 220 [nm] UV_VIS_1.Average = On UV_VIS_2.Wavelength = 280 [nm] UV_VIS_2.Average = On ; Settings for ColumnOven ColumnOven.TempCtrl = On ColumnOven.Temperature.Nominal = 50.0 [°C]



ColumnOven.Temperature.LowerLimit = 5.0 [°C] ColumnOven.Temperature.UpperLimit = 70.0 [°C] ColumnOven.ReadyTempDelta = 1.0 [°C] ColumnOven_Temp.Step = Auto ColumnOven_Temp.Average = On ColumnOven.ValveLeft = 1_2 ColumnOven.ValveRight = 1_2 FlowSplitter_1.FSControlMode = Off ; Settings for Autosampler ;Sampler.TempCtrl = On ; Valid for AutoSampler with cooling ;Sampler.Temperature.Nominal = 5[°C] ;Sampler.Temperature.UpperLimit = 45 [°C] ;Sampler.Temperature.LowerLimit = 4 [°C] Sampler.DrawSpeed = 200 [nl/s] Sampler.DrawDelay = 5000 [ms] Sampler.DispSpeed = 6000 [nl/s] Sampler.DispenseDelay = 2000 [ms] Sampler.WasteSpeed = 8000 [nl/s] Sampler.WashSpeed = 8000 [nl/s] Sampler.SampleHeight = 5.000 [mm] Sampler.PunctureDepth = 8.000 [mm] Sampler.WashVolume = 25.000 [µl] Sampler.SyncWithPump = On Sampler.PumpDevice = "LoadingPump" Sampler.LowDispersionMode = Off Sampler.InjectMode = UserProg Sampler.ReagentAVial= R1 ; Buffer A must be present in the

ReagentAVial loacated at position R1 Sampler.UdpDispense To=Drain, Volume=0.000,


SyringeSpeed=GlobalSpeed, SampleHeight=6 Sampler.UdpMixWait Duration=5 Sampler.UdpDraw From=SampleVial, Volume=20.000,


SyringeSpeed=GlobalSpeed, SampleHeight=6 Sampler.UdpMixWait Duration=5 Sampler.UdpDispense To=Drain, Volume=0.000,

SyringeSpeed=GlobalSpeed, SampleHeight=GlobalHeight Sampler.UdpInjectValve Position=Inject Sampler.UdpInjectMarker ; Settings for Fraction Collection FractionCollection.PumpDevice = "LoadingPump" TubeMaxVolume = Unlimited MaxTubesPerFraction = Unlimited TubeWrapping = No TubeChangeDuration = 2.0 [s] DelayTime = 5.0 [s]



;============================================================================ ; Definition of triggers for fraction collection starts here. ; ; Vials have to be uncapped (Commands: StartFraction/ChangeTube: VialType= Uncapped) ; Needle remains outside the puncture (e.g. 10 mm -> NeedleExtension) ; during fractionation / vial change ;============================================================================ Trigger FracStart FracStartDetected Sampler.TubePosition = FractionCollection.TubePosition StartFractionUncapped NeedleExtension=10000, fractionheight=8000 EndTrigger Trigger TubeChange FracTubeChange Sampler.TubePosition = FractionCollection.TubePosition ChangeTubeUncapped NeedleExtension=10000, Fractionheight=8000 EndTrigger Trigger FracEnd FracEndDetected EndFraction EndTrigger ;============================================================================ ; Definition of triggers for fraction collection ends here. ;============================================================================ -0.001 Sampler.TubePosition = Ge1 ;Fill in start position for fractionation FractionCollection.TubePosition = Sampler.TubePosition Airdry = off ; Select Airdry = on or Airdry = off 0.000 UV.Autozero LoadingPump.Flow = 50 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 0.0 [%] Wait UV.Ready and ColumnOven.Ready and

Sampler.Ready Inject ColumnOven_Temp.AcqOn LoadingPump_Pressure.AcqOn UV_VIS_1.AcqOn UV_VIS_2.AcqOn LoadingPump.Flow = 50 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 0.0 [%] 6.000 CollectFractions = By_Time CollectPeriod = 60 [s] 15.000 LoadingPump.Flow = 50 [µl/min] LoadingPump.%B = 70.0 [%] LoadingPump.%C = 0.0 [%] 15.100 LoadingPump.Flow = 50 [µl/min] LoadingPump.%B = 100.0 [%] LoadingPump.%C = 0.0 [%]



17.000 LoadingPump.Flow = 50 [µl/min] LoadingPump.%B = 100.0 [%] LoadingPump.%C = 0.0 [%] 17.100 LoadingPump.Flow = 50 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 0.0 [%] 20.000 CollectFractions = No 21.000 Sampler.Wash ; This command needs to be programed after the fractionation ; to empty the syringe and prepare for next injection 35.000 ColumnOven_Temp.AcqOff LoadingPump_Pressure.AcqOff UV_VIS_1.AcqOff UV_VIS_2.AcqOff LoadingPump.Flow = 50 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 0.0 [%] ReleaseExclusiveAccess End



5.5.2 Prepare System for Second Dimension Separation (Wash IEX prepare for RP)

;============================================================================ ; Wash Program for Switching from WAX to RP ; --------------------------------------------------------------------------- ; PGM-Version March 2008 ;============================================================================ AcquireExclusiveAccess ; Settings for Pump LoadingPump.Pressure.LowerLimit = 0 [bar] LoadingPump.Pressure.UpperLimit = 400 [bar] LoadingPump.MaximumFlowRampDown = 100 [µl/min²] LoadingPump.MaximumFlowRampUp = 100 [µl/min²] LoadingPump.%A.Equate = "20 mM Tris-HCl buffer pH 7.6" LoadingPump.%B.Equate = "as A + 1M NaCl" LoadingPump.%C.Equate = "100% water + 0.05% HFBA" MicroPump.Pressure.LowerLimit = 0 [bar] MicroPump.Pressure.UpperLimit = 350 [bar] MasterPressure.LowerLimit = 0 [bar] MasterPressure.UpperLimit = 350 [bar] MicroPump.MaximumFlowRampDown = 5.000 [µl/min²] MicroPump.MaximumFlowRampUp = 5.000 [µl/min²] MicroPump.%A.Equate = "100% water + 0.05% TFA" MicroPump.%B.Equate = "20/80 water/ACN + 0.04% TFA" MicroPump.%C.Equate = "100% water + 0.05% HFBA" LoadingPump_Pressure.Step = Auto LoadingPump_Pressure.Average = On MicroPump_MasterPressure.Step = Auto MicroPump_MasterPressure.Average = On ; Settings for UV Detector ; no UV detection ; Settings for ColumnOven ColumnOven.TempCtrl = On ColumnOven.Temperature.Nominal = 60.0 [°C] ColumnOven.Temperature.LowerLimit = 50.0 [°C] ColumnOven.Temperature.UpperLimit = 65.0 [°C] ColumnOven.ReadyTempDelta = 1.0 [°C] ColumnOven.ValveLeft = 1_2 ColumnOven.ValveRight = 1_2 FlowSplitter_1.FSControlMode = Off ColumnOven_Temp.Step = Auto ColumnOven_Temp.Average = On ColumnPressure.Step = Auto ColumnPressure.Average = On ColumnOven_FC_BridgeFlow.Step = Auto ColumnOven_FC_BridgeFlow.Average = On ColumnOven_FC_Stepper.Step = Auto ColumnOven_FC_Stepper.Average = On ; Settings for Autosampler



Sampler.DrawSpeed = 200 [nl/s] Sampler.DrawDelay = 7000 [ms] Sampler.DispSpeed = 2000 [nl/s] Sampler.DispenseDelay = 2000 [ms] Sampler.WasteSpeed = 4000 [nl/s] Sampler.WashSpeed = 4000 [nl/s] Sampler.PunctureDepth = 8.000 [mm] Sampler.WashVolume = 0.000 [µl] Sampler.LowDispersionMode = Off Sampler.InjectMode = UserProg Sampler.SampleHeight = 4.000 [mm] Sampler.ReagentAVial= R1 Sampler.PrepVial= RA1 Sampler.UdpDispense To=Drain, Volume=0.000,





SyringeSpeed=GlobalSpeed, SampleHeight=GlobalHeight Sampler.UdpInjectValve Position=Inject Sampler.UdpInjectMarker 0.000 MicroPump.Flow = 0.000 [µl/min] MicroPump.%B = 10.0 [%] MicroPump.%C = 0.0 [%] MicroPump.Curve = 5 LoadingPump.Flow = 50 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 0.0 [%] LoadingPump.Curve = 5 Wait ColumnOven.Ready and Sampler.Ready Sampler.Inject LoadingPump_Pressure.AcqOn MicroPump_MasterPressure.AcqOn ColumnOven_Temp.AcqOn ColumnPressure.AcqOn ColumnOven_FC_BridgeFlow.AcqOn ColumnOven_FC_Stepper.AcqOn CollectFractions = No LoadingPump.Flow = 50 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 0.0 [%] LoadingPump.Curve = 5 1.000 LoadingPump.Flow = 0 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 0.0 [%] LoadingPump.Curve = 5 1.100 ColumnOven.ValveRight = 10_1



2.000 LoadingPump.Flow = 0 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 100.0 [%] LoadingPump.Curve = 5 3.000 LoadingPump.Flow = 50 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 100.0 [%] LoadingPump.Curve = 5 5.000 LoadingPump.Flow = 50 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 100.0 [%] LoadingPump.Curve = 5 6.000 LoadingPump.Flow = 20 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 100.0 [%] LoadingPump.Curve = 5 7.000 ColumnOven.ValveLeft = 10_1 7.100 MicroPump.Flow = 0.000 [µl/min] MicroPump.%B = 10.0 [%] MicroPump.%C = 0.0 [%] MicroPump.Curve = 5 9.000 MicroPump.Flow = 15.000 [µl/min] MicroPump.%B = 10.0 [%] MicroPump.%C = 0.0 [%] MicroPump.Curve = 5 9.500 FlowSplitter_1.FSControlMode = Auto ; Start column flow 15.000 LoadingPump_Pressure.AcqOff MicroPump_MasterPressure.AcqOff ColumnOven_Temp.AcqOff ColumnPressure.AcqOff ColumnOven_FC_BridgeFlow.AcqOff ColumnOven_FC_Stepper.AcqOff ReleaseExclusiveAccess End




;============================================================================ ; Second Dimension RP Program ; --------------------------------------------------------------------------- ; PGM-Version March 2008 ; Sample: First Dimension Fractions ;============================================================================ AcquireExclusiveAccess ; Settings for Pump LoadingPump.Pressure.LowerLimit = 0 [bar] LoadingPump.Pressure.UpperLimit = 400 [bar] LoadingPump.MaximumFlowRampDown = 100 [µl/min²] LoadingPump.MaximumFlowRampUp = 100 [µl/min²] LoadingPump.%A.Equate = "20 mM Tris-HCl buffer pH 7.6" LoadingPump.%B.Equate = "as A + 1M NaCl" LoadingPump.%C.Equate = "100% water + 0.05% HFBA" MicroPump.Pressure.LowerLimit = 0 [bar] MicroPump.Pressure.UpperLimit = 350 [bar] MasterPressure.LowerLimit = 0 [bar] MasterPressure.UpperLimit = 350 [bar] MicroPump.MaximumFlowRampDown = 5.000 [µl/min²] MicroPump.MaximumFlowRampUp = 5.000 [µl/min²] MicroPump.%A.Equate = "100% water + 0.05% TFA" MicroPump.%B.Equate = "20/80 water/ACN + 0.04% TFA" MicroPump.%C.Equate = "100% water + 0.05% HFBA" LoadingPump_Pressure.Step = Auto LoadingPump_Pressure.Average = On MicroPump_MasterPressure.Step = Auto MicroPump_MasterPressure.Average = On LoadingPump.Flow = 20 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 100.0 [%] ; Settings for UV Detector ; In the Server Configuration the UV Detector was named UV with Signal name UV_VIS_1 UV.Data_Collection_Rate = 10 [Hz] UV.TimeConstant = 0.10 [s] UV_VIS_1.Wavelength = 220 [nm] UV_VIS_1.Step = Auto UV_VIS_1.Average = On ; Settings for ColumnOven ColumnOven.TempCtrl = On ColumnOven.Temperature.Nominal = 60.0 [°C] ColumnOven.Temperature.LowerLimit = 5.0 [°C] ColumnOven.Temperature.UpperLimit = 85.0 [°C] ColumnOven.ReadyTempDelta = 1.0 [°C]



ColumnOven.ValveLeft = 10_1 ColumnOven.ValveRight = 10_1 ColumnOven_Temp.Step = Auto ColumnOven_Temp.Average = On ColumnPressure.Step = Auto ColumnPressure.Average = On ColumnOven_FC_BridgeFlow.Step = Auto ColumnOven_FC_BridgeFlow.Average = On ColumnOven_FC_Stepper.Step = Auto ColumnOven_FC_Stepper.Average = On FlowSplitter_1.FSControlMode = Auto ; Settings for Autosampler Sampler.DrawSpeed = 200 [nl/s] Sampler.DrawDelay = 5000 [ms] Sampler.DispSpeed = 6000 [nl/s] Sampler.DispenseDelay = 2000 [ms] Sampler.WasteSpeed = 8000 [nl/s] Sampler.WashSpeed = 8000 [nl/s] Sampler.SampleHeight = 1.000 [mm] Sampler.PunctureDepth = 1.000 [mm] Sampler.WashVolume = 100.000 [µl] Sampler.LowDispersionMode = Off Sampler.SyncWithPump = On Sampler.PumpDevice = "MicroPump" Sampler.InjectMode = UserProg Sampler.ReagentAVial= R1 Sampler.ReagentBVial= R2 ; loading solvent must be present in the ReagentBVial loacated at position

R2 PrepVial= R2 Sampler.UdpDispense To=Drain, Volume=0.000,

SyringeSpeed=GlobalSpeed, SampleHeight=GlobalHeight Sampler.UdpSyringeValve Position=Needle Sampler.UdpInjectValve Position=Load Sampler.UdpDraw From=ReagentBVial, Volume=20.000,

SyringeSpeed=GlobalSpeed, SampleHeight=6 Sampler.UdpMixWait Duration=5 Sampler.UdpDispense To=SampleVial, Volume=20.000,

SyringeSpeed=GlobalSpeed, SampleHeight=GlobalHeight Sampler.UdpMixWait Duration=5 Sampler.UdpDraw From=ReagentBVial, Volume=20.000,







SyringeSpeed=GlobalSpeed, SampleHeight=GlobalHeight Sampler.UdpMixWait Duration=5



Sampler.UdpSyringeValve Position=Waste Sampler.UdpDispense To=Waste, Volume=25.000,

SyringeSpeed=GlobalSpeed, SampleHeight=GlobalHeight Sampler.UdpSyringeValve Position=Needle Sampler.UdpDraw From=ReagentBVial, Volume=5.000,


SyringeSpeed=GlobalSpeed, SampleHeight=GlobalHeight Sampler.UdpInjectValve Position=Inject Sampler.UdpInjectMarker ; Settings for Fraction Collection ;============================================================================ ; Definition of triggers for fraction collection starts here. ; ; Vials have to be uncapped (Commands: StartFraction/ChangeTube: VialType= Uncapped) ; Needle remains outside the puncture (e.g. 10 mm -> NeedleExtension) ; during fractionation / vial change ;============================================================================ Trigger FracStart FracStartDetected Sampler.TubePosition = FractionCollection.TubePosition StartFractionUncapped NeedleExtension=10000, fractionheight=8000 EndTrigger Trigger TubeChange FracTubeChange Sampler.TubePosition = FractionCollection.TubePosition ChangeTubeUncapped NeedleExtension=10000, Fractionheight=8000 EndTrigger Trigger FracEnd FracEndDetected EndFraction EndTrigger ;============================================================================ ; Definition of triggers for fraction collection ends here. ;============================================================================ -0.001 Sampler.TubePosition = Gi1 + 11 * (Sample.number – 1) ;Please see manual for explanation equation FractionCollection.TubePosition = Sampler.TubePosition Airdry = off 0.000 MicroPump.Flow = 15.000 [µl/min] UV.Autozero MicroPump.%B = 15.0 [%] MicroPump.%C = 0.0 [%] Wait UV.Ready and ColumnOven.Ready and Sampler.Ready Inject LoadingPump_Pressure.AcqOn MicroPump_MasterPressure.AcqOn



ColumnOven_Temp.AcqOn ColumnPressure.AcqOn ColumnOven_FC_BridgeFlow.AcqOn ColumnOven_FC_Stepper.AcqOn CollectFractions = No UV_VIS_1.AcqOn MicroPump.Flow = 15.000 [µl/min] MicroPump.%B = 15.0 [%] MicroPump.%C = 0.0 [%] 0.500 FlowSplitter_1.FSControlMode = Auto 4.000 MicroPump.Flow = 15.000 [µl/min] MicroPump.%B = 15.0 [%] MicroPump.%C = 0.0 [%] 5.000 ColumnOven.ValveLeft = 1_2 15.000 MicroPump.Flow = 15.000 [µl/min] MicroPump.%B = 70.0 [%] MicroPump.%C = 0.0 [%] 15.100 MicroPump.Flow = 15.000 [µl/min] MicroPump.%B = 90.0 [%] MicroPump.%C = 0.0 [%] 17.000 MicroPump.Flow = 15.000 [µl/min] MicroPump.%B = 90.0 [%] MicroPump.%C = 0.0 [%] 17.100 MicroPump.Flow = 15.000 [µl/min] MicroPump.%B = 15.0 [%] MicroPump.%C = 0.0 [%] 20.500 sampler.wash 25.000 ColumnOven.ValveLeft = 10_1 30.000 ColumnOven_Temp.AcqOff ColumnPressure.AcqOff ColumnOven_FC_BridgeFlow.AcqOff ColumnOven_FC_Stepper.AcqOff LoadingPump_Pressure.AcqOff MicroPump_MasterPressure.AcqOff UV_VIS_1.AcqOff MicroPump.Flow = 15.000 [µl/min] MicroPump.%B = 15.0 [%] MicroPump.%C = 0.0 [%] ReleaseExclusiveAccess End



5.5.4 Prepare System for First Dimension Separation (Wash RP prepare for IEX)

;============================================================================ ; Wash Program for Switching from WAX to RP ; --------------------------------------------------------------------------- ; PGM-Version March 2008 ;============================================================================ AcquireExclusiveAccess ; Settings for Pump LoadingPump.Pressure.LowerLimit = 0 [bar] LoadingPump.Pressure.UpperLimit = 400 [bar] LoadingPump.MaximumFlowRampDown = 100 [µl/min²] LoadingPump.MaximumFlowRampUp = 100 [µl/min²] LoadingPump.%A.Equate = "20 mM Tris-HCl buffer pH 7.6" LoadingPump.%B.Equate = "as A + 1M NaCl" LoadingPump.%C.Equate = "100% water + 0.05% HFBA" MicroPump.Pressure.LowerLimit = 0 [bar] MicroPump.Pressure.UpperLimit = 350 [bar] MasterPressure.LowerLimit = 0 [bar] MasterPressure.UpperLimit = 350 [bar] MicroPump.MaximumFlowRampDown = 5.000 [µl/min²] MicroPump.MaximumFlowRampUp = 5.000 [µl/min²] MicroPump.%A.Equate = "100% water + 0.05% TFA" MicroPump.%B.Equate = "20/80 water/ACN + 0.04% TFA" MicroPump.%C.Equate = "100% water + 0.05% HFBA" LoadingPump_Pressure.Step = Auto LoadingPump_Pressure.Average = On MicroPump_MasterPressure.Step = Auto MicroPump_MasterPressure.Average = On ; Settings for UV Detector ; no UV detection ; Settings for ColumnOven ColumnOven.TempCtrl = On ColumnOven.Temperature.Nominal = 50.0 [°C] ColumnOven.Temperature.LowerLimit = 50.0 [°C] ColumnOven.Temperature.UpperLimit = 65.0 [°C] ColumnOven.ReadyTempDelta = 1.0 [°C] ColumnOven.ValveLeft = 10_1 ColumnOven.ValveRight = 10_1 FlowSplitter_1.FSControlMode = Auto ColumnOven_Temp.Step = Auto ColumnOven_Temp.Average = On ColumnPressure.Step = Auto ColumnPressure.Average = On ColumnOven_FC_BridgeFlow.Step = Auto ColumnOven_FC_BridgeFlow.Average = On ColumnOven_FC_Stepper.Step = Auto ColumnOven_FC_Stepper.Average = On ; Settings for Autosampler Sampler.DrawSpeed = 200 [nl/s] Sampler.DrawDelay = 7000 [ms]



Sampler.DispSpeed = 2000 [nl/s] Sampler.DispenseDelay = 2000 [ms] Sampler.WasteSpeed = 4000 [nl/s] Sampler.WashSpeed = 4000 [nl/s] Sampler.PunctureDepth = 8.000 [mm] Sampler.WashVolume = 0.000 [µl] Sampler.LowDispersionMode = Off Sampler.InjectMode = UserProg Sampler.SampleHeight = 4.000 [mm] Sampler.ReagentAVial= R1 Sampler.PrepVial= RA1 Sampler.UdpDispense To=Drain, Volume=0.000,





SyringeSpeed=GlobalSpeed, SampleHeight=GlobalHeight Sampler.UdpInjectValve Position=Inject Sampler.UdpInjectMarker 0.000 MicroPump.Flow = 15.000 [µl/min] MicroPump.%B = 10.0 [%] MicroPump.%C = 0.0 [%] MicroPump.Curve = 5 LoadingPump.Flow = 20 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 100.0 [%] LoadingPump.Curve = 5 Wait ColumnOven.Ready and Sampler.Ready Sampler.Inject LoadingPump_Pressure.AcqOn MicroPump_MasterPressure.AcqOn ColumnOven_Temp.AcqOn ColumnPressure.AcqOn ColumnOven_FC_BridgeFlow.AcqOn ColumnOven_FC_Stepper.AcqOn CollectFractions = No MicroPump.Flow = 15.000 [µl/min] MicroPump.%B = 10.0 [%] MicroPump.%C = 0.0 [%] MicroPump.Curve = 5 LoadingPump.Flow = 20 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 100.0 [%] LoadingPump.Curve = 5 1.000 MicroPump.Flow = 0.000 [µl/min] MicroPump.%B = 10.0 [%] MicroPump.%C = 0.0 [%] MicroPump.Curve = 5



LoadingPump.Flow = 0 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 100.0 [%] LoadingPump.Curve = 5 1.500 FlowSplitter_1.FSControlMode = Off 2.000 ColumnOven.ValveLeft = 1_2 2.100 LoadingPump.Flow = 0 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 0.0 [%] LoadingPump.Curve = 5 4.000 LoadingPump.Flow = 50 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 0.0 [%] LoadingPump.Curve = 5 10.000 ColumnOven.ValveRight = 1_2 15.000 LoadingPump_Pressure.AcqOff MicroPump_MasterPressure.AcqOff ColumnOven_Temp.AcqOff ColumnPressure.AcqOff ColumnOven_FC_BridgeFlow.AcqOff ColumnOven_FC_Stepper.AcqOff ReleaseExclusiveAccess End



5.5.5 Add Trypsin by WPS to fractions of interest

;============================================================================ ; Add Trypsin Solution by WPS ; --------------------------------------------------------------------------- ; PGM-Version March 2008 ; Sample: First Dimension Fractions ;============================================================================ AcquireExclusiveAccess ; Settings for Pump LoadingPump.Pressure.LowerLimit = 0 [bar] LoadingPump.Pressure.UpperLimit = 400 [bar] LoadingPump.MaximumFlowRampDown = 100 [µl/min²] LoadingPump.MaximumFlowRampUp = 100 [µl/min²] LoadingPump.%A.Equate = "10 mM Tris-HCl buffer pH 7.6" LoadingPump.%B.Equate = "as A + 1M NaCl" LoadingPump.%C.Equate = "95% water + 5% ACN" MicroPump.Pressure.UpperLimit = 350 [bar] MicroPump.Pressure.LowerLimit = 0 [bar] MasterPressure.LowerLimit = 0 [bar] MasterPressure.UpperLimit = 400 [bar] MicroPump.MaximumFlowRampDown = 50.000 [µl/min²] MicroPump.MaximumFlowRampUp = 50.000 [µl/min²] MicroPump.%A.Equate = "100% water + 0.05% TFA" MicroPump.%B.Equate = "20/80 water/ACN + 0.04% TFA" MicroPump.%C.Equate = "95% water + 5% ACN" LoadingPump_Pressure.Step = Auto LoadingPump_Pressure.Average = On MicroPump_MasterPressure.Step = Auto MicroPump_MasterPressure.Average = On LoadingPump.Flow = 0 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 100.0 [%] ; Settings for UV Detector ; In the Server Configuration the UV Detector was named UV with Signal name UV_VIS_1 UV.TimeConstant = 0.10 [s] UV_VIS_1.Wavelength = 220 [nm] UV_VIS_1.Step = Auto UV_VIS_1.Average = On ; Settings for ColumnOven ColumnOven.TempCtrl = Off ColumnOven.ValveLeft = 10_1 ColumnOven.ValveRight = 10_1



ColumnOven_Temp.Step = Auto ColumnOven_Temp.Average = On ColumnPressure.Step = Auto ColumnPressure.Average = On ColumnOven_FC_BridgeFlow.Step = Auto ColumnOven_FC_BridgeFlow.Average = On ColumnOven_FC_Stepper.Step = Auto ColumnOven_FC_Stepper.Average = On FlowSplitter_1.FSControlMode = Off ; Settings for Autosampler ;Sampler.TempCtrl = On ;Sampler.Temperature.Nominal = 30.0 [°C] Sampler.DrawSpeed = 2500 [nl/s] Sampler.DrawDelay = 5000 [ms] Sampler.DispSpeed = 6000 [nl/s] Sampler.DispenseDelay = 2000 [ms] Sampler.WasteSpeed = 8000 [nl/s] Sampler.WashSpeed = 8000 [nl/s] Sampler.SampleHeight = 4.000 [mm] Sampler.PunctureDepth = 1.000 [mm] Sampler.WashVolume = 100.000 [µl] Sampler.LowDispersionMode = Off Sampler.InjectMode = UserProg Sampler.ReagentCVial= R3 ; Trypsin solution must be present in the ReagentCVial loacated at position R3 Sampler.PrepVial= R3 Sampler.UdpDispense To=Drain, Volume=0.000,

SyringeSpeed=GlobalSpeed, SampleHeight=GlobalHeight Sampler.UdpSyringeValve Position=Needle Sampler.UdpInjectValve Position=Load Sampler.UdpDraw From=ReagentCVial, Volume=25.000,


SyringeSpeed=GlobalSpeed, SampleHeight=4.000 Sampler.UdpMixWait Duration=5 Sampler.UdpDraw From=SampleVial, Volume=20.000,

SyringeSpeed=GlobalSpeed, SampleHeight=4.000 Sampler.UdpDispense To=SampleVial, Volume=20.000,

SyringeSpeed=GlobalSpeed, SampleHeight=4.000 Sampler.UdpMixWait Duration=5 Sampler.UdpDispense To=Drain, Volume=0.000,

SyringeSpeed=GlobalSpeed, SampleHeight=GlobalHeight Sampler.UdpInjectMarker 0.000 MicroPump.Flow = 0.000 [µl/min] MicroPump.%B = 15.0 [%] MicroPump.%C = 0.0 [%] Wait Sampler.Ready Inject CollectFractions = No MicroPump.Flow = 0.000 [µl/min] MicroPump.%B = 15.0 [%] MicroPump.%C = 0.0 [%]



0.010 MicroPump.Flow = 0.000 [µl/min] MicroPump.%B = 15.0 [%] MicroPump.%C = 0.0 [%] ReleaseExclusiveAccess End



5.6 CM programs for 2D-LC of Proteins using the MIC WAX / 200 µm I.D. Monolith Configuration


;============================================================================ ; First Dimension WAX Program ; --------------------------------------------------------------------------- ; PGM-Version March 2008 ; Sample: E-coli lysate ;============================================================================ AcquireExclusiveAccess ; Settings for Pump LoadingPump.Pressure.LowerLimit = 0 [bar] LoadingPump.Pressure.UpperLimit = 350 [bar] LoadingPump.MaximumFlowRampDown = 100 [µl/min²] LoadingPump.MaximumFlowRampUp = 100[µl/min²] LoadingPump.%A.Equate = "20 mM Tris-HCl buffer pH 7.6" LoadingPump.%B.Equate = "as A + 1M NaCl" LoadingPump.%C.Equate = "100% water + 0.05% HFBA" MicroPump.Pressure.LowerLimit = 0 [bar] MicroPump.Pressure.UpperLimit = 350 [bar] MasterPressure.LowerLimit = 0 [bar] MasterPressure.UpperLimit = 350 [bar] MicroPump.MaximumFlowRampDown = 5 [µl/min²] MicroPump.MaximumFlowRampUp = 5 [µl/min²] MicroPump.%A.Equate = "100% water + 0.05% TFA" MicroPump.%B.Equate = "20/80 water/ACN + 0.04% TFA" MicroPump.%C.Equate = "%C" LoadingPump_Pressure.Step = Auto LoadingPump_Pressure.Average = On MicroPump.Flow = 0.000 [µl/min] MicroPump.%B = 0.0 [%] MicroPump.%C = 0.0 [%] MicroPump.Curve = 5 ; Settings for UV Detector ; In the Server Configuration the first UV Detector was named UV with Signal name UV_VIS_1 ; In the Server Configuration the second UV Detector was named UV_2 with Signal name

UV_VIS_1_2 UV.Data_Collection_Rate = 4 [Hz] UV.TimeConstant = 1.0 [s] UV_VIS_1.Wavelength = 220 [nm] UV_VIS_1.Average = On UV_2.Data_Collection_Rate = 4 [Hz] UV_2.TimeConstant = 1.0 [s] UV_VIS_1_2.Wavelength = 220 [nm] UV_VIS_1_2.Average = On UV_VIS_2_2.Wavelength = 280 [nm] UV_VIS_2_2.Average = On



; Settings for ColumnOven ColumnOven.TempCtrl = On ColumnOven.Temperature.Nominal = 50.0 [°C] ColumnOven.Temperature.LowerLimit = 5.0 [°C] ColumnOven.Temperature.UpperLimit = 70.0 [°C] ColumnOven.ReadyTempDelta = 1.0 [°C] ColumnOven_Temp.Step = Auto ColumnOven_Temp.Average = On ColumnOven.ValveLeft = 1_2 ColumnOven.ValveRight = 10_1 FlowSplitter_1.FSControlMode = Off ; Settings for Autosampler ;Sampler.TempCtrl = On ; Valid for AutoSampler with cooling ;Sampler.Temperature.Nominal = 5[°C] ;Sampler.Temperature.UpperLimit = 45 [°C] ;Sampler.Temperature.LowerLimit = 4 [°C] Sampler.DrawSpeed = 200 [nl/s] Sampler.DrawDelay = 5000 [ms] Sampler.DispSpeed = 2000 [nl/s] Sampler.DispenseDelay = 2000 [ms] Sampler.WasteSpeed = 4000 [nl/s] Sampler.WashSpeed = 4000 [nl/s] Sampler.SampleHeight = 4.000 [mm] Sampler.PunctureDepth = 8.000 [mm] Sampler.WashVolume = 25.000 [µl] Sampler.LowDispersionMode = Off Sampler.InjectMode = UserProg Sampler.ReagentAVial= R1 ; Buffer A must be present in the ReagentAVial loacated at position R1 Sampler.ReagentBVial= R2 Sampler.PrepVial= R1 Sampler.UdpDispense To=Drain, Volume=0.000,


SyringeSpeed=GlobalSpeed, SampleHeight=6 Sampler.UdpMixWait Duration=5 Sampler.UdpDraw From=SampleVial, Volume=10.000,



SyringeSpeed=GlobalSpeed, SampleHeight=GlobalHeight Sampler.UdpInjectValve Position=Inject Sampler.UdpInjectMarker ; Settings for Fraction Collection FractionCollection.PumpDevice = "LoadingPump" TubeMaxVolume = Unlimited MaxTubesPerFraction = Unlimited TubeWrapping = No



TubeChangeDuration = 2.0 [s] DelayTime = 5.0 [s] ;============================================================================ ; Definition of triggers for fraction collection starts here. ; ; Vials have to be uncapped (Commands: StartFraction/ChangeTube: VialType= Uncapped) ; Needle remains outside the puncture (e.g. 10 mm -> NeedleExtension) ; during fractionation / vial change ;============================================================================ Trigger FracStart FracStartDetected Sampler.TubePosition = FractionCollection.TubePosition StartFractionUncapped NeedleExtension=10000, fractionheight=8000 EndTrigger Trigger TubeChange FracTubeChange Sampler.TubePosition = FractionCollection.TubePosition ChangeTubeUncapped NeedleExtension=10000, Fractionheight=8000 EndTrigger Trigger FracEnd FracEndDetected EndFraction EndTrigger ;============================================================================ ; Definition of triggers for fraction collection ends here. ;============================================================================ -0.001 Sampler.TubePosition = Gc1 ;Fill in start position for fractionation FractionCollection.TubePosition = Sampler.TubePosition Airdry = off ; Select Airdry = on or Airdry = off 0.000 UV_2.autozero LoadingPump.Flow = 50 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 0.0 [%] wait UV_2.Ready and ColumnOven.Ready and Sampler.Ready Inject ColumnOven_Temp.AcqOn LoadingPump_Pressure.AcqOn UV_VIS_1_2.AcqOn ; Select the correct UV detector UV_VIS_2_2.AcqOn ; Select the correct UV detector LoadingPump.Flow = 50 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 0.0 [%] 6.000 CollectFractions = By_Time CollectPeriod = 60 [s] 15.000 LoadingPump.Flow = 50 [µl/min] LoadingPump.%B = 70.0 [%] LoadingPump.%C = 0.0 [%]



15.100 LoadingPump.Flow = 50 [µl/min] LoadingPump.%B = 100.0 [%] LoadingPump.%C = 0.0 [%] 17.000 LoadingPump.Flow = 50 [µl/min] LoadingPump.%B = 100.0 [%] LoadingPump.%C = 0.0 [%] 17.100 LoadingPump.Flow = 50 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 0.0 [%] 20.000 CollectFractions = No 21.000 Sampler.Wash ; This command needs to be programed after the fractionation ; to empty the syringe and prepare for next injection 35.000 ColumnOven_Temp.AcqOff LoadingPump_Pressure.AcqOff UV_VIS_1_2.AcqOff UV_VIS_2_2.AcqOff LoadingPump.Flow = 50 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 0.0 [%] ReleaseExclusiveAccess End



5.6.2 Prepare System for Second Dimension Separation (Wash IEX prepare for RP)

;============================================================================ ; Wash Program for Switching from WAX to RP ; --------------------------------------------------------------------------- ; PGM-Version March 2008 ;============================================================================ AcquireExclusiveAccess ; Settings for Pump LoadingPump.Pressure.LowerLimit = 0 [bar] LoadingPump.Pressure.UpperLimit = 400 [bar] LoadingPump.MaximumFlowRampDown = 100 [µl/min²] LoadingPump.MaximumFlowRampUp = 100 [µl/min²] LoadingPump.%A.Equate = "20 mM Tris-HCl buffer pH 7.6" LoadingPump.%B.Equate = "as A + 1M NaCl" LoadingPump.%C.Equate = "100% water + 0.05% HFBA" MicroPump.Pressure.LowerLimit = 0 [bar] MicroPump.Pressure.UpperLimit = 350 [bar] MasterPressure.LowerLimit = 0 [bar] MasterPressure.UpperLimit = 500 [bar] MicroPump.MaximumFlowRampDown = 5.003 [µl/min²] MicroPump.MaximumFlowRampUp = 5.003 [µl/min²] MicroPump.%A.Equate = "100% water + 0.05% TFA" MicroPump.%B.Equate = "20/80 water/ACN + 0.04% TFA" MicroPump.%C.Equate = "%C" LoadingPump_Pressure.Step = Auto LoadingPump_Pressure.Average = On MicroPump_MasterPressure.Step = Auto MicroPump_MasterPressure.Average = On ; Settings for UV Detector ; no UV detection ; Settings for ColumnOven ColumnOven.TempCtrl = On ColumnOven.Temperature.Nominal = 50.0 [°C] ColumnOven.Temperature.LowerLimit = 50.0 [°C] ColumnOven.Temperature.UpperLimit = 65.0 [°C] ColumnOven.ReadyTempDelta = 1.0 [°C] ColumnOven.ValveLeft = 1_2 ColumnOven.ValveRight = 10_1 FlowSplitter_1.FSControlMode = Off ColumnOven_Temp.Step = Auto ColumnOven_Temp.Average = On ColumnPressure.Step = Auto ColumnPressure.Average = On ColumnOven_FC_BridgeFlow.Step = Auto ColumnOven_FC_BridgeFlow.Average = On ColumnOven_FC_Stepper.Step = Auto ColumnOven_FC_Stepper.Average = On ; Settings for Autosampler



Sampler.DrawSpeed = 200 [nl/s] Sampler.DrawDelay = 7000 [ms] Sampler.DispSpeed = 2000 [nl/s] Sampler.DispenseDelay = 2000 [ms] Sampler.WasteSpeed = 4000 [nl/s] Sampler.WashSpeed = 4000 [nl/s] Sampler.PunctureDepth = 8.000 [mm] Sampler.WashVolume = 0.000 [µl] Sampler.LowDispersionMode = Off Sampler.InjectMode = UserProg Sampler.SampleHeight = 4.000 [mm] Sampler.ReagentAVial= R1 Sampler.PrepVial= RA1 Sampler.UdpDispense To=Drain, Volume=0.000,





SyringeSpeed=GlobalSpeed, SampleHeight=GlobalHeight Sampler.UdpInjectValve Position=Inject Sampler.UdpInjectMarker 0.000 Wait ColumnOven.Ready and Sampler.Ready Sampler.Inject LoadingPump_Pressure.AcqOn MicroPump_MasterPressure.AcqOn ColumnOven_Temp.AcqOn ColumnPressure.AcqOn ColumnOven_FC_BridgeFlow.AcqOn ColumnOven_FC_Stepper.AcqOn CollectFractions = No LoadingPump.Flow = 50 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 0.0 [%] LoadingPump.Curve = 5 MicroPump.Flow = 0.000 [µl/min] MicroPump.%B = 50.0 [%] MicroPump.%C = 0.0 [%] MicroPump.Curve = 5 2.00 LoadingPump.Flow = 0 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 0.0 [%] LoadingPump.Curve = 5 MicroPump.Flow = 2.500 [µl/min] MicroPump.%B = 50.0 [%] MicroPump.%C = 0.0 [%]



MicroPump.Curve = 5 2.1 ColumnOven.ValveRight = 1_2 ; Switch IEX column outline 2.5 LoadingPump.Flow = 0 [µl/min] ; Switch to loading solvent LoadingPump.%B = 0.0 [%] LoadingPump.%C = 100.0 [%] LoadingPump.Curve = 5 4.000 LoadingPump.Flow = 50 [µl/min] ; Flush lines with loading solvent LoadingPump.%B = 0.0 [%] LoadingPump.%C = 100.0 [%] LoadingPump.Curve = 5 MicroPump.Flow = 2.500 [µl/min] ; Equilibrate column MicroPump.%B = 0.0 [%] MicroPump.%C = 0.0 [%] MicroPump.Curve = 5 FlowSplitter_1.FSControlMode = Auto ; Start column flow 6.00 LoadingPump.Flow = 50 [µl/min] ; Flush lines with loading solvent LoadingPump.%B = 0.0 [%] LoadingPump.%C = 100.0 [%] LoadingPump.Curve = 5 7.000 LoadingPump.Flow = 20 [µl/min] ; Flush lines with loading solvent LoadingPump.%B = 0.0 [%] LoadingPump.%C = 100.0 [%] LoadingPump.Curve = 5 7.500 ColumnOven.ValveLeft = 10_1 ; Switch trapcolumn in-line with loading pump 10.000 LoadingPump_Pressure.AcqOff MicroPump_MasterPressure.AcqOff ColumnOven_Temp.AcqOff ColumnPressure.AcqOff ColumnOven_FC_BridgeFlow.AcqOff ColumnOven_FC_Stepper.AcqOff ReleaseExclusiveAccess End




;============================================================================ ; Second Dimension RP Program ; --------------------------------------------------------------------------- ; PGM-Version March 2008 ; Sample: First Dimension Fractions ;============================================================================ AcquireExclusiveAccess ; Settings for Pump LoadingPump.Pressure.LowerLimit = 0 [bar] LoadingPump.Pressure.UpperLimit = 400 [bar] LoadingPump.MaximumFlowRampDown = 100 [µl/min²] LoadingPump.MaximumFlowRampUp = 100 [µl/min²] LoadingPump.%A.Equate = "20 mM Tris-HCl buffer pH 8" LoadingPump.%B.Equate = "as A + 1M NaCl" LoadingPump.%C.Equate = "100% water + 0.05% HFBA" MicroPump.Pressure.UpperLimit = 350 [bar] MicroPump.Pressure.LowerLimit = 0 [bar] MasterPressure.LowerLimit = 0 [bar] MasterPressure.UpperLimit = 400 [bar] MicroPump.MaximumFlowRampDown = 2.500 [µl/min²] MicroPump.MaximumFlowRampUp = 2.500 [µl/min²] MicroPump.%A.Equate = "100% water + 0.05% TFA" MicroPump.%B.Equate = "20/80 water/ACN + 0.04% TFA" MicroPump.%C.Equate = "%C" LoadingPump_Pressure.Step = Auto LoadingPump_Pressure.Average = On MicroPump_MasterPressure.Step = Auto MicroPump_MasterPressure.Average = On LoadingPump.Flow = 20 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 100.0 [%] ; Settings for UV Detector ; In the Server Configuration the first UV Detector was named UV with Signal name UV_VIS_1 ; In the Server Configuration the second UV Detector was named UV_2 with Signal name

UV_VIS_1_2 UV.Data_Collection_Rate = 10.0 [Hz] UV.TimeConstant = 0.10 [s] ;UV_2.Data_Collection_Rate = 10.0 [Hz] ;UV_2.TimeConstant = 0.10 [s] UV_VIS_1.Wavelength = 220 [nm] UV_VIS_1.Step = Auto UV_VIS_1.Average = On ;UV_VIS_1_2.Wavelength = 220 [nm] ;UV_VIS_1_2.Step = Auto ;UV_VIS_1_2.Average = On



; Settings for ColumnOven ColumnOven.TempCtrl = On ColumnOven.Temperature.Nominal = 60.0 [°C] ColumnOven.Temperature.LowerLimit = 5.0 [°C] ColumnOven.Temperature.UpperLimit = 85.0 [°C] ColumnOven.ReadyTempDelta = 1.0 [°C] ColumnOven.ValveLeft = 10_1 ColumnOven.ValveRight = 1_2 ColumnOven_Temp.Step = Auto ColumnOven_Temp.Average = On ColumnPressure.Step = Auto ColumnPressure.Average = On ColumnOven_FC_BridgeFlow.Step = Auto ColumnOven_FC_BridgeFlow.Average = On ColumnOven_FC_Stepper.Step = Auto ColumnOven_FC_Stepper.Average = On FlowSplitter_1.FSControlMode = Auto ; Settings for Autosampler Sampler.DrawSpeed = 200 [nl/s] Sampler.DrawDelay = 5000 [ms] Sampler.DispSpeed = 2000 [nl/s] Sampler.DispenseDelay = 2000 [ms] Sampler.WasteSpeed = 4000 [nl/s] Sampler.WashSpeed = 4000 [nl/s] Sampler.SampleHeight = 1.000 [mm] Sampler.PunctureDepth = 1.000 [mm] Sampler.WashVolume = 100.000 [µl] Sampler.LowDispersionMode = Off Sampler.InjectMode = UserProg Sampler.ReagentAVial= R1 Sampler.ReagentBVial= R2 ; loading solvent must be present in the ReagentBVial loacated at position R2 Sampler.PrepVial= R2 Sampler.UdpDispense To=Drain, Volume=0.000,

SyringeSpeed=GlobalSpeed, SampleHeight=GlobalHeight Sampler.UdpSyringeValve Position=Needle Sampler.UdpInjectValve Position=Load Sampler.UdpDraw From=ReagentBVial, Volume=5.000,

SyringeSpeed=GlobalSpeed, SampleHeight=6 Sampler.UdpMixWait Duration=5 Sampler.UdpDraw From=ReagentBVial, Volume=0.000,

SyringeSpeed=GlobalSpeed, SampleHeight=6 Sampler.UdpDispense To=SampleVial, Volume=5.000,




SyringeSpeed=GlobalSpeed, SampleHeight=GlobalHeight Sampler.UdpDraw From=ReagentAVial, Volume=2.500,

SyringeSpeed=GlobalSpeed, SampleHeight=GlobalHeight



Sampler.UdpMixWait Duration=5 Sampler.UdpDraw From=ReagentBVial, Volume=0.000,

SyringeSpeed=GlobalSpeed, SampleHeight=6 Sampler.UdpDispense To=Drain, Volume=0.000,

SyringeSpeed=GlobalSpeed, SampleHeight=GlobalHeight Sampler.UdpInjectValve Position=Inject Sampler.UdpInjectMarker 0.000 MicroPump.Flow = 2.500 [µl/min] UV.Autozero ;UV_2.Autozero MicroPump.%B = 10.0 [%] MicroPump.%C = 0.0 [%] Wait UV.Ready and ColumnOven.Ready and Sampler.Ready Sampler.Inject LoadingPump_Pressure.AcqOn MicroPump_MasterPressure.AcqOn ColumnOven_Temp.AcqOn ColumnPressure.AcqOn ColumnOven_FC_BridgeFlow.AcqOn ColumnOven_FC_Stepper.AcqOn CollectFractions = No UV_VIS_1.AcqOn ; Select the correct UV detector ;UV_VIS_1_2.AcqOn ; Select the correct UV detector MicroPump.Flow = 2.500 [µl/min] MicroPump.%B = 10.0 [%] MicroPump.%C = 0.0 [%] 1.0 sampler.wash 2.000 MicroPump.Flow = 2.5 [µl/min] MicroPump.%B = 10.0 [%] MicroPump.%C = 0.0 [%] 5.000 ColumnOven.ValveLeft = 1_2 6.00 FlowSplitter_1.FSControlMode = Auto 17.000 MicroPump.Flow = 2.5 [µl/min] MicroPump.%B = 70.0 [%] MicroPump.%C = 0.0 [%] 17.100 MicroPump.Flow = 2.5 [µl/min] MicroPump.%B = 80.0 [%] MicroPump.%C = 0.0 [%] 20.000 MicroPump.Flow = 2.5 [µl/min] MicroPump.%B = 80.0 [%] MicroPump.%C = 0.0 [%] 20.100 MicroPump.Flow = 2.5 [µl/min] MicroPump.%B = 10.0 [%] MicroPump.%C = 0.0 [%] 28.000 ColumnOven.ValveLeft = 10_1 29.000 FlowSplitter_1.FSControlMode = Auto 30.000 ColumnOven_Temp.AcqOff



ColumnPressure.AcqOff ColumnOven_FC_BridgeFlow.AcqOff ColumnOven_FC_Stepper.AcqOff LoadingPump_Pressure.AcqOff MicroPump_masterPressure.AcqOff UV_VIS_1.AcqOff ;UV_VIS_1_2.AcqOff ; Select the correct UV detector MicroPump.Flow = 2.5 [µl/min] MicroPump.%B = 10.0 [%] MicroPump.%C = 0.0 [%] ReleaseExclusiveAccess End



5.6.4 Prepare System for First Dimension Separation (Wash RP prepare for IEX)

;============================================================================ ; Wash Program for Switching from RP to WAX ; --------------------------------------------------------------------------- ; PGM-Version March 2008 ;============================================================================ AcquireExclusiveAccess ; Settings for Pump LoadingPump.Pressure.LowerLimit = 0 [bar] LoadingPump.Pressure.UpperLimit = 400 [bar] LoadingPump.MaximumFlowRampDown = 100 [µl/min²] LoadingPump.MaximumFlowRampUp = 100 [µl/min²] LoadingPump.%A.Equate = "20 mM Tris-HCl buffer pH 7.6" LoadingPump.%B.Equate = "as A + 1M NaCl" LoadingPump.%C.Equate = "100% water + 0.05% HFBA" MicroPump.Pressure.LowerLimit = 0 [bar] MicroPump.Pressure.UpperLimit = 350 [bar] MasterPressure.LowerLimit = 0 [bar] MasterPressure.UpperLimit = 500 [bar] MicroPump.MaximumFlowRampDown = 5.003 [µl/min²] MicroPump.MaximumFlowRampUp = 5.003 [µl/min²] MicroPump.%A.Equate = "100% water + 0.05% TFA" MicroPump.%B.Equate = "20/80 water/ACN + 0.04% TFA" MicroPump.%C.Equate = "%C" LoadingPump_Pressure.Step = Auto LoadingPump_Pressure.Average = On MicroPump_MasterPressure.Step = Auto MicroPump_MasterPressure.Average = On ; Settings for UV Detector ; no UV detection ; Settings for ColumnOven ColumnOven.TempCtrl = On ColumnOven.Temperature.Nominal = 50.0 [°C] ColumnOven.Temperature.LowerLimit = 50.0 [°C] ColumnOven.Temperature.UpperLimit = 65.0 [°C] ColumnOven.ReadyTempDelta = 1.0 [°C] ColumnOven.ValveLeft = 10_1 ColumnOven.ValveRight = 1_2 FlowSplitter_1.FSControlMode = Auto ColumnOven_Temp.Step = Auto ColumnOven_Temp.Average = On ColumnPressure.Step = Auto ColumnPressure.Average = On ColumnOven_FC_BridgeFlow.Step = Auto ColumnOven_FC_BridgeFlow.Average = On ColumnOven_FC_Stepper.Step = Auto ColumnOven_FC_Stepper.Average = On ; Settings for Autosampler Sampler.DrawSpeed = 200 [nl/s] Sampler.DrawDelay = 7000 [ms]



Sampler.DispSpeed = 2000 [nl/s] Sampler.DispenseDelay = 2000 [ms] Sampler.WasteSpeed = 4000 [nl/s] Sampler.WashSpeed = 4000 [nl/s] Sampler.PunctureDepth = 8.000 [mm] Sampler.WashVolume = 0.000 [µl] Sampler.LowDispersionMode = Off Sampler.InjectMode = UserProg Sampler.SampleHeight = 4.000 [mm] Sampler.ReagentAVial= R2 Sampler.PrepVial= RA1 Sampler.UdpDispense To=Drain, Volume=0.000,





SyringeSpeed=GlobalSpeed, SampleHeight=GlobalHeight Sampler.UdpInjectValve Position=Inject Sampler.UdpInjectMarker 0.000 Wait ColumnOven.Ready and Sampler.Ready Sampler.Inject LoadingPump_Pressure.AcqOn MicroPump_MasterPressure.AcqOn ColumnOven_Temp.AcqOn ColumnPressure.AcqOn ColumnOven_FC_BridgeFlow.AcqOn ColumnOven_FC_Stepper.AcqOn CollectFractions = No LoadingPump.Flow = 20 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 100.0 [%] LoadingPump.Curve = 5 MicroPump.Flow = 2.500 [µl/min] MicroPump.%B = 10.0 [%] MicroPump.%C = 0.0 [%] MicroPump.Curve = 5 2.00 LoadingPump.Flow = 0 [µl/min] LoadingPump.%B = 0.0 [%] LoadingPump.%C = 100.0 [%] LoadingPump.Curve = 5 MicroPump.Flow = 0.000 [µl/min] ; Switch off MicroPump MicroPump.%B = 10.0 [%] MicroPump.%C = 0.0 [%] MicroPump.Curve = 5



2.1 ValveLeft = 1_2 ; Switch RP trap column outline 2.5 LoadingPump.Flow = 0 [µl/min] ; Switch to IEX buffer A LoadingPump.%B = 0.0 [%] LoadingPump.%C = 0.0 [%] LoadingPump.Curve = 5 4.000 LoadingPump.Flow = 50 [µl/min] ; Flush lines with IEX buffer A LoadingPump.%B = 0.0 [%] LoadingPump.%C = 0.0 [%] LoadingPump.Curve = 5 FlowSplitter_1.FSControlMode = Off 7.500 ValveRight = 10_1 ; Switch IEX column in-line with loading pump buffer A 10.000 LoadingPump_Pressure.AcqOff MicroPump_MasterPressure.AcqOff ColumnOven_Temp.AcqOff ColumnPressure.AcqOff ColumnOven_FC_BridgeFlow.AcqOff ColumnOven_FC_Stepper.AcqOff ReleaseExclusiveAccess End

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Micro-Fraction Collection Option for the UltiMate 3000 ...tools.thermofisher.com/content/sfs/manuals/Man... · The Micro Fraction Collection option of the UltiMate 3000 Series Nano/Cap