Agilent Technologies 1260 Infinity User manual

Agilent 1260 Infinity
HPLC-Chip Cube
Interface (G4240A )
User Manual

HPLC-Chip Cube Interface User’s Guide
Notices
© Agilent Technologies, Inc. 2010, 2012
No part of this manual may be reproduced in
any form or by any means (including elec-
tronic storage and retrieval or translation
into a foreign language) without prior agree-
ment and written consent from Agilent
Technologies, Inc. as governed by United
States and international copyright laws.
Manual Part Number
G4240-90006 Rev. B
Edition
05/2012
Printed in Germany
Agilent Technologies
Hewlett-Packard-Strasse 8
76337 Waldbronn
Warranty
The material contained in this docu-
ment is provided “as is,” and is sub-
ject to being changed, without notice,
in future editions. Further, to the max-
imum extent permitted by applicable
law, Agilent disclaims all warranties,
either express or implied, with regard
to this manual and any information
contained herein, including but not
limited to the implied warranties of
merchantability and fitness for a par-
ticular purpose. Agilent shall not be
liable for errors or for incidental or
consequential damages in connec-
tion with the furnishing, use, or per-
formance of this document or of any
information contained herein. Should
Agilent and the user have a separate
written agreement with warranty
terms covering the material in this
document that conflict with these
terms, the warranty terms in the sep-
arate agreement shall control.
Technology Licenses
The hardware and/or software described in
this document are furnished under a license
and may be used or copied only in accor-
dance with the terms of such license.
Restricted Rights Legend
If software is for use in the performance of a
U.S. Government prime contract or subcon-
tract, Software is delivered and licensed as
“Commercial computer software” as
defined in DFAR 252.227-7014 (June 1995),
or as a “commercial item” as defined in FAR
2.101(a) or as “Restricted computer soft-
ware” as defined in FAR 52.227-19 (June
1987) or any equivalent agency regulation or
contract clause. Use, duplication or disclo-
sure of Software is subject to Agilent Tech-
nologies’ standard commercial license
terms, and non-DOD Departments and
Agencies of the U.S. Government will
receive no greater than Restricted Rights as
defined in FAR 52.227-19(c)(1-2) (June
1987). U.S. Government users will receive
no greater than Limited Rights as defined in
FAR 52.227-14 (June 1987) or DFAR
252.227-7015 (b)(2) (November 1995), as
applicable in any technical data.
Safety Notices
CAUTION
A CAUTION notice denotes a haz-
ard. It calls attention to an operat-
ing procedure, practice, or the like
that, if not correctly performed or
adhered to, could result in damage
to the product or loss of important
data. Do not proceed beyond a
CAUTION notice until the indicated
conditions are fully understood and
met.
WARNING
A WARNING notice denotes a
hazard. It calls attention to an
operating procedure, practice, or
the like that, if not correctly per-
formed or adhered to, could result
in personal injury or death. Do not
proceed beyond a WARNING
notice until the indicated condi-
tions are fully understood and
met.

HPLC-Chip Cube Interface User’s Guide 3
In This Guide…
This manual gives an introduction to HPLC-Chip technology
and explains how to install, maintain and troubleshoot you
HPLC-Chip Cube.
1Introduction to HPLC-Chip Technology
2Introduction to the Chip Cube
3Site Preparation
4Installation
5Operation
6Maintenance Procedures
7Consumables and Maintenance Parts
Appendix A Appendix A Safety Information

4 HPLC-Chip Cube Interface User’s Guide

Contents
HPLC-Chip Cube Interface User’s Guide 5
Content
1 Introduction to HPLC-Chip Technology 9
Overview 10
Chip Types 13
The MS Calibration and Diagnosis Chip, Part No. G4240-61001 13
The Protein ID Chip, Part No. G4240-62001 14
Modes of Operation 14
More Chips 18
2 Introduction to the Chip Cube 19
Intended Use 20
Introduction 21
Power Switch 22
Safety Features 23
Main Assemblies 24
Principle of Operation 26
Flow Connections 29
3 Site Preparation 31
Site Requirements for the G4240A Chip Cube 32
Power Considerations 32
Bench Space 32
Environment 33
Sound Emission 34
Specifications of the G4240A HPLC-Chip Cube 34
4 Installation 37
Unpacking the Agilent G4240A HPLC-Chip MS Interface 38
Damaged Packaging 38
Delivery Checklist 38

6 HPLC-Chip Cube Interface User’s Guide
Contents
Accessory Kit 40
System Setup Considerations 42
Single Stack Setup 43
Tw i n S t a c k s 45
Preparation of Existing Systems 47
Chip Cube Hardware Installation 49
5Operation53
Chip Loading and Unloading 54
Configuration 55
Configure Loading Pump and Analytical Pump 55
Injection Flush Volume 56
Injector Flush Volume and the use of Injector Programs 57
Operational Parameters 60
Setting up Chip Cube Parameters 61
Chip (Valve Positions / Tip) 61
Time 62
Time Table 63
Instrument Actuals 63
Chip Details Screen 64
Spray Ignition 66
Conditions to avoid 69
6 Maintenance Procedures 71
Maintenance Overview 72
Replacing Capillaries 73
Cleaning the Valve Rotors (quick procedure) 76
Updating the Chip Cube Firmware 79

Contents
HPLC-Chip Cube Interface User’s Guide 7
7 Consumables and Maintenance Parts 81
HPLC-Chips 82
Capillaries 83
Fittings 84
Infusion Spare Parts 85
Valve Rotor Cleaning Tools and Replacement Parts 86
8 Appendix A
Safety Information 87
Safety Information 88
Power Cords 88
Operation 89
Safety Symbols 90
Chemical and Biological Safety 91
Lithium Batteries Information 92
The Waste Electrical and Electronic Equipment (WEEE) Directive
(2002/96/EC) 93
Legal Notice 94

8 HPLC-Chip Cube Interface User’s Guide
Contents

9
Agilent 1260 Infinity HPLC-Chip Cube MS Interface (G4240A)
Chip Cube User’s Guide
Agilent Technologies
1
Introduction to HPLC-Chip Technology
Overview 10
Chip Types 13
The MS Calibration and Diagnosis Chip, Part No. G4240-61001 13
The Protein ID Chip, Part No. G4240-62001 14
This chapter will provide you with a basic understanding of Agilent’s
HPLC-Chip technology and an overview of the currently available chips.

10 HPLC-Chip Cube Interface User’s Guide
1 Introduction to HPLC-Chip Technology
Overview
Overview
Traditional nanospray mass spectrometry has proven it's potential as a
cost-effective, sensitive and reproducible technique for the identification of
peptides at femtomol to atomol levels. However, connecting nano capillaries,
columns and valves frequently is a tedious procedure and requires user skills
and routine. When handled incorrectly, nano flow connections are prone to
leakage which are difficult to detect and result in poor system performance
and extended downtime of the complete system. Quartz nano spray needles
are prone to blockages and require frequent replacement.
With the invention of HPLC-Chip technology, Agilent is significantly reducing
the need for user interaction and making nanospray a rugged state-of-the-art
technology.
The cornerstone of chip technology is the HLPC-Chip, a 3-dimensional
structure made of sandwiched polyimide films. Grooves of specific dimensions
are laser-ablated into a layer of polyimide film. The lamination of a top and
bottom layer then forms channels of trapezoidal or triangular shape inside the
chip which can either be used as capillaries or, if filled with packing material,
as nanocolumns. One end of the chip tapers off into a polyimide nanospray
emitter.

HPLC-Chip Cube Interface User’s Guide 11
Introduction to HPLC-Chip Technology 1
Overview
Figure 1 Principle of HPLC-Chip Fabrication
By ablating small holes into the top and bottom layers, the chip can be used as
a self-connecting and sealing microvalve. The unique rotor-in-rotor microvalve
in the Chip Cube moves two concentric valve rotors from one side towards the
chip while a 6-port stainless steel stator face closes in from the other side,
forming a pressure tight connection without any user interaction.
Top film with holes
Biocompatible,
polyimide film
UV Laser
2. Clean
3. Laminate
1. Ablate
Bottom film with channels
Finished HPLC-Chip
X-Y Stage
4. Trim
5. Metallize
CAD Drawing
HV-ESI contact
Top film with holes
Biocompatible,
polyimide film
UV Laser
2. Clean
3. Laminate
1. Ablate
Bottom film with channels
Finished HPLC-Chip
X-Y Stage
4. Trim
5. Metallize
CAD Drawing
HV-ESI contact

12 HPLC-Chip Cube Interface User’s Guide
1 Introduction to HPLC-Chip Technology
Overview
Figure 2 Schematic of Chip Cube Valve (Side View)
Once the valve is closed, both rotors can rotate in either direction. The use of
extremely smooth, hard ceramic rotors guarantees chemical inertness and
pressure tightness with virtually no wear on the rotors and very little wear of
the chip.
Figure 3 Rotor-in-rotor design of the Chip Cube valve
Summary
The very flexible HPLC-Chip concept can combine multiple columns, the valve
plumbing and the nanospray emitter on one chip. A rugged chip holder
protects the chip and retracts the fragile tip whenever the HPLC-Chip is
removed from the Chip Cube.
outer rotor
inner rotor
Stator
Rotor

HPLC-Chip Cube Interface User’s Guide 13
Introduction to HPLC-Chip Technology 1
Chip Types
Chip Types
The MS Calibration and Diagnosis Chip, Part No. G4240-61001
This chip has two functions:
Infusion
The main purpose of this chip is to allow for easy infusion of MS tuning mix or
samples that do not require chromatographic separation.The chip uses a
dedicated valve port (no. 3) with a pre-installed 75 µm capillary and
connection fittings to a 100 µL syringe, thus infusion is possible at any time
without hardware modifications by simply inserting the MS Calibration and
Diagnosis Chip in the Chip Cube.
HPLC-Valve Calibration
The second function of this chip is to calibrate the Chip Cube valve (Agilent
1260 Infinity HPLC-Chip Cube - Service Manual, Valve Calibration).
Make sure that port 2 is connected to the nano pump and port 6 to waste. Port
5 should be open or connected to the µ-Well-plate Sampler.
CAUTION
Before running any systems test with the MS Calibration & Diagnosis chip, make sure
that the 100 µm ID waste capillary (black) is connected to port 6. Otherwise the test
will error.

14 HPLC-Chip Cube Interface User’s Guide
1 Introduction to HPLC-Chip Technology
Chip Types
The Protein ID Chip, Part No. G4240-62001
This HPLC-Chip is the standard chip for one-dimensional separations. It is
comprised of a 40 nL enrichment column and a 75 µm x 43mm separation
column which is packed with Zorbax C18-SB 5 µm material. The functionality
of this chip is equivalent to conventional nanospray LC/MS.
Modes of Operation
There are two ways to load the enrichment column. Each has advantages and
disadvantages. It depends on the application which one is preferable. Agilent
recommends to use Forward Flush mode since this mode of operation is less
susceptible to clogging of the enrichment column.
Forward Flush mode
Figure 4 Loading the sample in forward flush mode
Port 6 of the HPLC-Chip is connected to the µ-well-plate sampler. When the
valve is in the loading position, the sample is transferred at a flow of 4 µL/min
onto the enrichment column. Since the loading solvent composition is highly
aqueous, the peptides are retained at the front end of the column. The loading
solvent is directed to waste through port 5. A filter at the back of the
enrichment column retains the packing material.
from µ-WPS
to waste
enrichment column
analytical column
from nano pump

HPLC-Chip Cube Interface User’s Guide 15
Introduction to HPLC-Chip Technology 1
Chip Types
When sample loading is finished, the valve switches to the Analyze position.
The front end of the enrichment column is now connected to the nano pump
via port 2. The outlet end of the enrichment column now connects to the
analytical column.
Figure 5 Analyzing in forward-flush mode
As the nanoflow gradient starts, the retained peptides are eluted onto the
analytical column where they are further separated by reversed phase
chromatography. The analytical column connects directly to the nanospray
emitter, which is grounded using an embedded strip of noble metal that makes
contact with the solvent just before the tip.
to waste
from µ-WPS
analytical column
from nano pump
enrichment column

16 HPLC-Chip Cube Interface User’s Guide
1 Introduction to HPLC-Chip Technology
Chip Types
Backflush mode
Figure 6 Loading the sample in backflush mode
Port 5 of the HPLC-Chip is connected to the µ-well-plate sampler. When the
valve is in the loading position, the sample is transferred at a flow of 4 µL/min
onto the enrichment column. Since the loading solvent composition is highly
aqueous, the peptides are retained at the front end of the column. The loading
solvent is directed to waste through port 6. A filter at the back of the
enrichment column retains the packing material.
When sample loading is finished, the valve switches over to the Analyze
position. The front end of the enrichment column is now connected to the
nano pump via port 2. The back end of the enrichment column connects to the
analytical column.
to waste
from µ-WPS
enrichment column
analytical column
from nano pump

HPLC-Chip Cube Interface User’s Guide 17
Introduction to HPLC-Chip Technology 1
Chip Types
Figure 7 Analysis in backflush mode
As the nanoflow gradient starts, the retained peptides are eluted onto the
analytical column where they are separated by reversed phase
chromatography. The analytical column connects directly to the nanospray
emitter, which is grounded using an embedded strip of noble metal that makes
contact with the solvent just before the tip.
The compact design of the chip significantly reduces delay volumes between
enrichment and analytical columns, thereby increasing efficiency compared to
conventional nano LC/MS systems.
Typical analytical flow rates are 300 nL/min. Preferred solvents are
water/acetonitrile gradients with 0.1% formic acid.
However, most common HPLC solvents can be used as well.
to waste
from µ-WPS
from nano pump
enrichment column
analytical column
CAUTION
Solvents with pH > 8.5 must not be used as they dissolve the quartz capillaries in the
nano LC system and Chip Cube.

18 HPLC-Chip Cube Interface User’s Guide
1 Introduction to HPLC-Chip Technology
Chip Types
More Chips
Chips with different packing materials and layouts will become available in
future.

19
Agilent 1260 Infinity HPLC-Chip Cube MS Interface (G4240A)
Chip Cube User’s Guide
Agilent Technologies
2
Introduction to the Chip Cube
Intended Use 20
Introduction 21
Main Assemblies 24
Principle of Operation 26

20 HPLC-Chip Cube Interface User’s Guide
2 Introduction to the Chip Cube
Intended Use
Intended Use
The G4240A Chip Cube is designed and tested as “Controlled Office electrical
equipment for laboratory use”.
CAUTION
The operator of this instrument is advised that if the equipment is used in a manner
not specified in this manual, the protection provided by the equipment may be
impaired.