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Agilent 6100 Series Quadrupole LCMS Systems Concepts Guide


Agilent 6100 Series Quadrupole LC/MS Systems

Concepts Guide

Agilent Technologies

Notices
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Manual Part Number
G1960-90030

Edition
First Edition, February 2007 Printed in USA Agilent Technologies, Inc. 5301 Stevens Creek Blvd. Santa Clara, CA 95051 Microsoft? is a U.S. registered trademark of Microsoft Corporation.

WA R N I N G
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Software Revision
This guide is valid for the B.03.01 or later revision of the Agilent ChemStation software for the Agilent 6100 Series Quadrupole LC/MS systems, until superseded. If you have any comments about this guide, please send an e-mail to feedback_lcms@agilent.com.

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Agilent 6100 Series Quadrupole LC/MS Systems Concepts Guide

In This Guide...
The Concepts Guide presents an overview of the Agilent 6100 Series Quadrupole LC/MS systems, to help you understand how the hardware and software work. If you have any comments about this guide, please send an e-mail to feedback_lcms@agilent.com.

1

Overview
Learn how the hardware works in the Agilent 6100 Series Quadrupole LC/MS systems, and get a brief introduction to the ChemStation software.

2

Instrument Preparation
Learn the concepts you need to prepare the LC and column for an analysis, and to tune the MS.

3

Data Acquisition
Learn about setting up methods and running samples.

4

Data Analysis
Learn the software concepts you need for qualitative and quantitative data analysis.

5

Reports
Learn about predefined results reports and about setting up custom reports.

6

Verification of Performance
Learn the software concepts for Operational Qualification/ Performance Verification (OQ/PV) and system verification.

7

Maintenance and Troubleshooting
Learn about software tools that help you maintain your system and diagnose and fix problems.

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Agilent 6100 Series Quadrupole LC/MS Systems Concepts Guide

Contents

Contents
1 Overview of Hardware and Software 9 10 How the Agilent quadrupole LC/MS systems work Overview 10 Details 11

Types of data you can acquire 15 Scan versus selected ion monitoring (SIM) 15 Generation of fragment ions: low versus high fragmentor Positive versus negative ions 18 Multiple signal acquisition 19 Ion sources 21 Electrospray ionization (ESI) 21 Atmospheric pressure chemical ionization (APCI) 26 Atmospheric pressure photoionization (APPI) 28 Multimode ionization (MMI) 29 Introduction to the ChemStation software Overview 30 Reviewing data remotely 32 2 Instrument Preparation 33 30

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Preparation of the LC system 34 Purpose 34 Summary of procedures 34 Setting parameters for LC modules 36 Column conditioning and equilibration 39 Monitoring the stability of flow and pressure Preparation of the MS – tuning Overview 42 Ways to tune 44 When to tune – Check Tune
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42

45
5

Contents

Autotune 47 Manual tuning 49 Tune reports 51 Gain calibration 52 3 Data Acquisition 55

Working with methods 56 Method and Run Control View 56 Loading, editing, saving and printing methods More on editing methods 60 Running samples 63 Running a single sample 64 Running a sequence 64 Flow injection analysis 69 Monitoring analyses 73 Online signal plots 73 Quick method overview 74 Logbooks 74 Instrument shutdown 4 Reports 77 76

58

Using predefined reports 78 Generating reports 78 Report styles 79 Defining custom reports 80 Summary of process 80 Example report templates 80 The Report Layout View 81

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Data Analysis

83 84

The Data Analysis View

Loading and manipulating chromatograms 86 Loading signals 87 Removing signals from the chromatogram display 91 Changing how chromatograms are displayed 91 Working with spectra Displaying spectra Peak purity 95 93 94

Performing quantification 96 Integrating peaks 96 Calibration 98 Data review and sequence reprocessing The Navigation Table 100 Batch review 100 6 Verification of Performance The Verification (OQ/PV) View 103 104 100

Instrument verification 105 Setting up and running instrument verification 106 OQ/PV tests that are included with the software 108 Verification logbook 109 System verification 110 Overview 110 Setting up and running system verification

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Contents

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Maintenance and Troubleshooting The Diagnosis View Overview 114 Instrument panel Logbooks 117 114 115

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Maintenance 118 Early maintenance feedback 118 Maintenance logbook 119 Maintenance procedures 119 Venting and pumping down the MS Diagnosing and fixing problems 121 Symptoms and causes 121 Diagnostic tests for the MS 122 Fixing problems 122

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Agilent 6100 Series Quadrupole LC/MS Systems Concepts Guide

Agilent 6100 Series Quadrupole LC/MS Systems Concepts Guide

1 Overview of Hardware and Software
How the Agilent quadrupole LC/MS systems work 10 Overview 10 Details 11 Types of data you can acquire 15 Scan versus selected ion monitoring (SIM) 15 Generation of fragment ions: low versus high fragmentor 16 Positive versus negative ions 18 Multiple signal acquisition 19 Ion sources 21 Electrospray ionization (ESI) 21 Atmospheric pressure chemical ionization (APCI) 26 Atmospheric pressure photoionization (APPI) 28 Multimode ionization (MMI) 29 Introduction to the ChemStation software 30 Overview 30 Reviewing data remotely 32

This chapter provides an overview of the hardware and software that comprises the Agilent 6100 Series Quadrupole LC/MS systems. The family consists of four systems — the 6110, 6120, 6130, and 6140.

Agilent Technologies

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Overview of Hardware and Software How the Agilent quadrupole LC/MS systems work

How the Agilent quadrupole LC/MS systems work
Overview
Mass spectrometry (MS) is based on the analysis of ions moving through a vacuum. The result is mass spectra, which provide valuable information about the molecular weight, structure, identity, quantity, and purity of a sample. MS adds specificity to both qualitative and quantitative analyses. A quadrupole mass analyzer is sometimes called a quadrupole mass filter or a quadrupole. Figure 1 shows a diagram of the Agilent 6100 Series Quadrupole LC/MS systems. The ionization of a sample occurs at atmospheric pressure in the ion source that is shown on the left. The Agilent 6100 Series Quadrupole LC/MS systems are compatible with a number of Agilent atmospheric pressure ionization (API) sources.
HPLC inlet quadrupole mass filter ion optics detector

nebulizer

capillary

ion source

rough pump

split-flow turbo pump

Figure 1 API – atmospheric pressure ionization

Block diagram for an Agilent quadrupole LC/MS system

A common atmospheric sampling interface introduces ions from these ionization sources into the vacuum system of the mass spectrometer. Various ion-optic elements focus and guide the ions through a series of vacuum stages until they reach the quadrupole mass analyzer, which separates the ions. The ions then travel to the detector, where they are recorded as signals.

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Overview of Hardware and Software Details

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Details
Figure 2 and Figure 3 show more detailed schematics of the ion paths in the Agilent 6100 Series Quadrupole LC/MS systems. After the API source forms ions, the ion-optic elements in the ion transport and focusing region of the system direct the ions toward the quadrupole and the detector. During transit, the ions move from atmospheric pressure (760 torr) at the source to a vacuum in the 10-6 torr range at the quadrupole and detector.

Vacuum stage: HPLC inlet nebulizer capillary

1

2

3

4

skimmer octopole 5X10-6 torr detector 3 torr

fragmentation zone (CID) lenses Ion source quadrupole Ion transport and focusing region

Figure 2

Ion path for Agilent 6130 and 6140 Quadrupole LC/MS systems

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Overview of Hardware and Software Details

Vacuum stage: HPLC inlet nebulizer capillary

1

2

3

4

skimmers octopole 6X10-6 torr detector 2 torr

fragmentation zone (CID) lenses Ion source quadrupole Ion transport and focusing region

Figure 3

Ion path for Agilent 6110 and 6120 Quadrupole LC/MS systems

The ion transport and focusing region of the Agilent 6100 Series Quadrupole LC/MS systems is enclosed in a vacuum manifold. The function of the vacuum system is to evacuate regions of ion focusing and transport and keep the quadrupole at low pressure. Because the nebulizer is at a right angle to the inlet capillary, most of the solvent is vented from the spray chamber and never reaches the capillary. Only ions, drying gas, and a small amount of solvent are transmitted through the capillary. By autotuning the instrument, you automatically set most of the voltages for the elements in the ion path. See “Preparation of the MS – tuning” on page 42. The following discussion of the ion optics is organized according to the stages of the ion path and the vacuum stages of the mass spectrometer.

Ion transport and fragmentation (first vacuum stage)
Ions produced in the API source are electrostatically drawn through a drying gas and then through a heated sampling capillary into the first stage of the vacuum system. Near the exit

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of the capillary is a metal skimmer with a small hole. Heavier ions with greater momentum pass through the skimmer aperture. Most of the lighter drying gas (nitrogen) molecules are deflected by the skimmer and pumped away by a rough pump. The ions that pass through the skimmer move into the second stage of the vacuum system. The atmospheric pressure ionization techniques are all relatively “soft” techniques. They generate primarily: ? Molecular ions M+ or M? Protonated molecules [M + H]+ ? Simple adduct ions [M + Na]+ ? Ions representing simple losses, such as the loss of a water molecule [M + H - H2O]+ CID – collision-induced dissociation These types of ions give molecular weight information, but you often need complementary structural information. To gain structural information, you can fragment the analyte ions in the first vacuum stage. To do that, you give them extra energy and collide them with neutral molecules in a process known as collision-induced dissociation (CID). A voltage is applied at the end of the atmospheric sampling capillary to add energy to the collisions and create more fragmentation. For more information, see “Generation of fragment ions: low versus high fragmentor” on page 16.

Ion transport (second and third vacuum stages)
An octopole ion guide is a set of small parallel metal rods with a common open axis through which the ions can pass.

Agilent 6130 and 6140 Quadrupole LC/MS systems

In the second vacuum stage, the ions are immediately focused by an octopole ion guide that traverses two vacuum stages. The ions pass through the octopole ion guide because of the momentum they received from being drawn from atmospheric pressure through the sampling capillary. Radio-frequency voltage applied to the octopole rods repels ions above a particular mass range to the open center of the rod set. The ions exit this ion guide and then pass through two focusing lenses into the fourth stage of the vacuum system.

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Overview of Hardware and Software Details

Agilent 6110 and 6120 Quadrupole LC/MS systems In the second vacuum stage, the ions are transported between skimmer 1 and skimmer 2. They then enter the third vacuum stage, where they pass through the octopole ion guide. The ions exit this ion guide and then pass through two focusing lenses into the fourth stage of the vacuum system.

Ion separation and detection (fourth vacuum stage)
In the fourth vacuum stage, the quadrupole mass analyzer separates the ions by mass-to-charge ratio. An electron multiplier then detects the ions. m/z – mass/charge ratio The quadrupole mass analyzer (Figure 4) consists of four parallel rods to which specific direct-current (DC) and radio-frequency (RF) voltages are applied. The analyte ions are directed down the center of the rods. Voltages applied to the rods generate electromagnetic fields. These fields determine which mass-to-charge ratio of ions can pass through the filter at a given time. The ions that pass through are focused on the detector.

To detector

From ion source

Figure 4

Quadrupole mass analyzer

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Agilent 6100 Series Quadrupole LC/MS Systems Concepts Guide

Overview of Hardware and Software Types of data you can acquire

1

Types of data you can acquire
Scan versus selected ion monitoring (SIM)
You set up a scan or SIM analysis in the Method and Run Control View, described in Chapter 3. As shown in Figure 5, quadrupole mass analyzers can operate in two modes. To get the most from your analysis, it is important to pick the appropriate mode. The discussion below will help you choose.

1 scan abundance scan

mass range

m/z

time SIM 1 scan abundance

m/z
discrete masses

m/z

time

m/z

Figure 5

A quadrupole mass analyzer can operate in either scan mode or selected ion monitoring (SIM) mode

Scan mode
In scan mode, a range of m/z values are analyzed, for example, m/z 200 to 1000. The quadrupole sequentially filters one mass after another, with an entire scan typically taking about a second. (The exact time depends on mass range and scan speed.) The MS firmware steps the quadrupole through increasing DC and RF voltages, which sequentially filters the corresponding m/z values across a mass spectrum.

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Overview of Hardware and Software Generation of fragment ions: low versus high fragmentor A full scan analysis is useful because it shows all of the ions in a given mass range that are present in the ion source. Because it provides a complete picture of all the ionized compounds that occur above the detection limit in the chosen mass range, a full scan analysis is often used for sample characterization, structural elucidation, and impurity analysis. It is also the starting point for development of methods for SIM data acquisition (discussed next).

Selected ion monitoring (SIM) mode
To obtain the best sensitivity, the quadrupole is operated in SIM mode. In SIM mode, the quadrupole analyzes the signals of only a few specific m/z values. The required RF/DC voltages are set to filter one mass at a time. Rather than stepping through all the m/z values in a given mass range, the quadrupole steps only among the values that the analyst chooses. Because the quadrupole spends more time sampling each of these chosen m/z values, the system can detect lower levels of sample. SIM mode is significantly more sensitive than scan mode but provides information about fewer ions. Scan mode is typically used for qualitative analyses or for quantitation when analyte masses are not known in advance. SIM mode is used for quantitation and monitoring of target compounds.

Generation of fragment ions: low versus high fragmentor
When you set up a method for data acquisition, you can control the amount of fragmentation with the fragmentor setting. You set up a method in the Method and Run Control View, described in Chapter 3. Fragment ions, also known as product ions, are formed by breaking apart precursor ions. On the Agilent 6100 Series Quadrupole LC/MS systems, the fragmentation region is between the capillary exit and the skimmer, where the gas pressure is about 2 to 3 torr. Depending on the voltage in this region, precursor ions may pass through unchanged or they may be fragmented. When a lower voltage is applied across this region, the ions pass through unchanged. Even if these ions collide with the gas molecules in this region, they usually do not have enough energy to fragment. (See Figure 6.)

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Agilent 6100 Series Quadrupole LC/MS Systems Concepts Guide

Overview of Hardware and Software Generation of fragment ions: low versus high fragmentor

1

CH3 350000 300000 250000 200000 150000 100000 50000 0 100 200 300 H2N O O S NH N N CH3

279.1

[M + H]

+

281.0 280.0

301.0

[M + Na]

+

m/z

Figure 6

Mass spectrum of sulfamethazine – low fragmentor

124.1

m/z156 m/z186
O 186.0 N NH N CH3 O S 279.1 CH3

80000

m/z108
H2N

60000

156.1

[M + H]

+

m/z213 m/z124

40000

108.2

301.0

[M + Na] 323.0

+

107.1

125.1

0 100 200 300

157.1

187.0

280.1

20000

213.2

m/z

Figure 7

Mass spectrum of sulfamethazine – high fragmentor 17

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Overview of Hardware and Software Positive versus negative ions If the voltage is increased, the ions have more translational energy. Then, if the ions collide with gas molecules, the collisions convert the translational energy into molecular vibrations that can cause the ions to fragment. This is called collision-induced dissociation (CID). Figure 7 shows an example. Even though this fragmentation does not occur where the ions are formed at atmospheric pressure, it is a tradition to call this type of fragmentation “in-source CID.” The ions from molecular fragments are used for structural determination or confirmation of the presence of a particular chemical species. It is possible to produce both molecular ions and fragment ions within the same spectrum by using an intermediate fragmentation voltage. FIA – flow injection analysis The ideal fragmentation voltage depends on the structure of the compound and the needs of the analysis. For target compound analysis, it is good practice to determine in advance the compound’s response to fragmentor setting. The fastest way to accomplish this is with a flow injection analysis (FIA) series. An FIA series allows you to inject the compound multiple times within the same run, and to vary the fragmentor setting in different time windows. From the resulting data, you can judge the best fragmentor setting. For more information on FIA, see “Flow injection analysis” on page 69.

Positive versus negative ions
You set the ion polarity when you set up a method in the Method and Run Control View, described in Chapter 3. Atmospheric pressure ionization techniques can produce both positive and negative ions. For any given analysis, the predominant ion type depends on the chemical structure of the analyte and (particularly for electrospray ionization) the pH of the solution. While either or both ion types may be present in the ion source, the polarity of the ion optics in the ion transport and focusing region determines which ion type is detected. Analyses of positive and negative ions require different settings for the ion optics. The software-controlled autotune process optimizes the settings for both positive and negative ions, and stores them in a single tune file. During data acquisition, the software accesses the tune file for the appropriate settings. 18 Agilent 6100 Series Quadrupole LC/MS Systems Concepts Guide

Overview of Hardware and Software Multiple signal acquisition

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Multiple signal acquisition
You establish the conditions for multiple signal acquisition in the Method and Run Control View, described in Chapter 3. The Agilent 6120, 6130 and 6140 LC/MS models allow you to acquire multiple types of data during a single analysis. Within a single analytical run, you can choose alternating positive and negative ionization; alternating high and low fragmentor settings; and alternating scan and SIM modes. Because optimum MS conditions vary from compound to compound, this multisignal capability enables you to analyze more compounds, with greater sensitivity, within a single run.

Polarity switching
The Agilent 6120, 6130 and 6140 LC/MS models allow you to switch from scan to scan between analysis of positive ions and analysis of negative ions. To switch polarities very quickly, these models incorporate fast-switching power supplies for the API source, the lens system, the quadrupole, and the detector. The ability to switch polarities on the chromatographic time scale is very useful for analysis of complete unknowns because it obviates the need to run the sample twice to detect both types of ions.

Alternating high/low fragmentor
With the Agilent 6120, 6130 and 6140 LC/MS models, you can also alternate from scan to scan between high and low fragmentation voltages. This capability allows you to acquire scans at low fragmentor settings for molecular weight information, and high fragmentor settings for structural information.

Alternating SIM/scan
Many analyses require use of SIM mode to monitor and/or quantitate target compounds at very low levels. Sometimes it is also desirable to characterize the other sample components with a scan analysis. The Agilent 6120, 6130 and 6140 LC/MS models allow you to alternate between SIM and scan modes, so you can accomplish both goals in a single analysis.

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Overview of Hardware and Software Multiple signal acquisition

Putting it all together
The 6120, 6130 and 6140 LC/MS models can cycle through four different user-selected acquisition modes on a scan-by-scan basis within a single run. For example, you can set up a single run to do the following: ? Positive ion scan with low fragmentor voltage ? Positive ion scan with high fragmentor voltage ? Negative ion scan with low fragmentor voltage ? Negative ion scan with high fragmentor voltage Such an analysis is ideal for a mixture of compounds where some respond better in positive mode and some respond better in negative mode, and where you need both molecular ions and fragment ions. The time required for one cycle varies depending on the number of modes chosen, the scan range, and the interscan delay required for the switching. For separations with narrow chromatographic peaks, it is important to ensure that total cycle time is short enough that the instrument makes sufficient measurements across the peak.

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Agilent 6100 Series Quadrupole LC/MS Systems Concepts Guide

Overview of Hardware and Software Ion sources

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Ion sources
The Agilent 6100 Series Quadrupole LC/MS systems operate with the following interchangeable atmospheric pressure ionization (API) sources: ? ESI (electrospray ionization) ? APCI (atmospheric pressure chemical ionization) ? APPI (atmospheric pressure photoionization) ? MMI (multimode ionization)

NOTE

The sources that are used on the 6100 Series LC/MS systems are the B-type sources. The 6100 Series LC/MS systems are not compatible with the A-type sources that were used on previous Agilent LC/MS models.

Electrospray ionization (ESI)
You control the spray chamber parameters (nebulizer pressure, drying gas flow and temperature, and capillary voltage) when you set up a method in the Method and Run Control View, described in Chapter 3. Electrospray ionization relies in part on chemistry to generate analyte ions in solution before the analyte reaches the mass spectrometer. As shown in Figure 8, the LC eluent is sprayed (nebulized) into a spray chamber at atmospheric pressure in the presence of a strong electrostatic field and heated drying gas. The electrostatic field occurs between the nebulizer, which is at ground in the Agilent design, and the capillary, which is at high voltage. The spray occurs at right angles to the capillary. This patented Agilent design reduces background noise from droplets, increases sensitivity, and keeps the capillary cleaner for a longer period of time.

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Overview of Hardware and Software Electrospray ionization (ESI)

HPLC inlet

nebulizer

capillary

solvent spray

heated drying gas

Figure 8

Electrospray ion source

Electrospray ionization (ESI) consists of four steps: 1 Formation of ions 2 Nebulization 3 Desolvation 4 Ion evaporation

Formation of ions
Ion formation in API-electrospray occurs through more than one mechanism. If the chemistry of analyte, solvents, and buffers is correct, ions are generated in solution before nebulization. This results in high analyte ion concentration and good API-electrospray sensitivity.

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Agilent 6100 Series Quadrupole LC/MS Systems Concepts Guide

Overview of Hardware and Software Electrospray ionization (ESI)

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Preformed ions are not always required for ESI. Some compounds that do not ionize in solution can still be analyzed. The process of nebulization, desolvation, and ion evaporation creates a strong electrical charge on the surface of the spray droplets. This can induce ionization in analyte molecules at the surface of the droplets.

Nebulization
Nebulization (aerosol generation) takes the sample solution through these steps: a Sample solution enters the spray chamber through a grounded needle called a nebulizer. b For high-flow electrospray, nebulizing gas enters the spray chamber concentrically through a tube that surrounds the needle. c The combination of strong shear forces generated by the nebulizing gas and the strong voltage (2–6 kV) in the spray chamber draws out the sample solution and breaks it into droplets. d As the droplets disperse, ions of one polarity preferentially migrate to the droplet surface due to electrostatic forces. e As a result, the sample is simultaneously charged and dispersed into a fine spray of charged droplets, hence the name electrospray. Because the sample solution is not heated when the aerosol is created, ESI does not thermally decompose most analytes.

Desolvation and ion evaporation
Before the ions can be mass analyzed, solvent must be removed to yield a bare ion. A counter-current of neutral, heated drying gas, typically nitrogen, evaporates the solvent, decreasing the droplet diameter and forcing the predominantly like surface-charges closer together (see Figure 9).

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Overview of Hardware and Software Electrospray ionization (ESI)

evaporation

analyte ion ejected
+

+-+ + +- - + +- -+ + - + ++
Figure 9 Coulomb repulsion – repulsion between charged species of the same sign

++ + +-- -+ + - -- + + ++ +

+ ++- + + + -- --- + +++ + + +--+ +-- + +

Desorption of ions from solution

When the force of the Coulomb repulsion equals that of the surface tension of the droplet, the droplet explodes, producing smaller charged droplets that are subject to further evaporation. This process repeats itself, and droplets with a high density of surface-charges are formed. When charge density reaches approximately 108 V/cm3, ion evaporation occurs (direct ejection of bare ions from the droplet surface). These ions are attracted to and pass through a capillary sampling orifice into the ion optics and mass analyzer.

The importance of solution chemistry
The choice of solvents and buffers is a key to successful ionization with electrospray. Solvents like methanol that have lower heat capacity, surface tension, and dielectric constant, promote nebulization and desolvation. For best results in electrospray mode: ? Adjust solvent pH according to the polarity of ions desired and the pH of the sample. ? To enhance ion desorption, use solvents that have low heats of vaporization and low surface tensions. ? Select solvents that do not neutralize ions through gas-phase reactions such as proton transfer or ion pair reactions. ? To reduce the buildup of salts in the ion source, select more volatile buffers.

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Overview of Hardware and Software Electrospray ionization (ESI)

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Multiple charging
Electrospray is especially useful for analyzing large biomolecules such as proteins, peptides, and oligonucleotides, but can also analyze smaller molecules like drugs and environmental contaminants. Large molecules often acquire more than one charge. Because of this multiple charging, you can use electrospray to analyze molecules as large as 150,000 u even though the mass range (or more accurately mass-to-charge range) for a typical quadrupole LC/MS instrument is around 3000 m/z. For example: 100,000 u / 10 z = 1,000 m/z The optional Agilent LC/MSD Deconvolution & Bioanalysis Software performs the calculations to accomplish deconvolution. When a large molecule acquires many charges, a mathematical process called deconvolution is used to determine the actual molecular weight of the analyte.

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Overview of Hardware and Software Atmospheric pressure chemical ionization (APCI)

Atmospheric pressure chemical ionization (APCI)
APCI is a gas-phase chemical ionization process. The APCI technique passes LC eluent through a nebulizing needle, which creates a fine spray. The spray is passed through a heated ceramic tube, where the droplets are fully vaporized (Figure 10). The resulting gas/vapor mixture is then passed over a corona discharge needle, where the solvent vapor is ionized to create reagent gas ions. These ions in turn ionize the sample molecules via a chemical ionization process. The sample ions are then introduced into the capillary.
HPLC inlet nebulizer (sprayer)

vaporizer (heater) drying gas

+ + + +
corona discharge needle

+ +

+
capillary

Figure 10

Atmospheric pressure chemical ionization (APCI) source

APCI requires that the analyte be in the gas phase for ionization to occur. To vaporize the solvent and analyte, the APCI source is typically operated at vaporizer temperatures of 400 to 500 °C.

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Agilent 6100 Series Quadrupole LC/MS Systems Concepts Guide

Overview of Hardware and Software Atmospheric pressure chemical ionization (APCI) APCI is applicable across a wide range of molecular polarities. It rarely results in multiple charging, so it is typically used for molecules less than 1,500 u. Because of this molecular weight limitation and use of high-temperature vaporization, APCI is less well-suited than electrospray for analysis of large biomolecules that may be thermally unstable. APCI is well suited for ionization of the less polar compounds that are typically analyzed by normal-phase chromatography.

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Overview of Hardware and Software Atmospheric pressure photoionization (APPI)

Atmospheric pressure photoionization (APPI)
With the APPI technique, LC eluent passes through a nebulizing needle to create a fine spray. This spray is passed through a heated ceramic tube, where the droplets are fully vaporized. The resulting gas/vapor mixture passes through the photon beam of a krypton lamp to ionize the sample molecules (Figure 11). The sample ions are then introduced into the capillary. APPI and APCI are similar, with APPI substituting a lamp for the corona needle for ionization. APPI often also uses an additional solvent or mobile phase modifier, called a “dopant”, to assist with the photoionization process. APPI is applicable to many of the same compounds that are typically analyzed by APCI. APPI has proven particularly valuable for analysis of nonpolar compounds.
HPLC inlet nebulizer (sprayer)

vaporizer (heater)
drying gas



+ + + +

+

+

+
capillary

UV lamp

Figure 11

Atmospheric pressure photoionization (APPI) source

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Overview of Hardware and Software Multimode ionization (MMI)

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Multimode ionization (MMI)
The multimode source is an ion source that can operate in three different modes—APCI, ESI or simultaneous APCI/ESI. The multimode source incorporates two electrically separated, optimized zones—one for ESI and one for APCI. During simultaneous APCI/ESI, ions from both ionization modes enter the capillary and are analyzed simultaneously by the mass spectrometer.

HPLC inlet

nebulizer

ESI zone

APCI zone
thermal container

corona discharge needle

capillary

drying gas

Figure 12

Multimode source

Multimode ionization (MMI) is useful for screening of unknowns, or whenever samples contain a mixture of compounds where some respond by ESI and some respond by APCI. In these cases, the multimode source obviates the need to run the samples twice to accomplish a complete analysis. Unlike the APCI and APPI sources where the temperature of the vaporizer is monitored, in the multimode source the actual vapor temperature is monitored. As a result, the vaporizer is typically set to between 200 and 250 °C.

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Overview of Hardware and Software Introduction to the ChemStation software

Introduction to the ChemStation software
Overview
The ChemStation software for the Agilent 6100 Series Quadrupole LC/MS systems is organized into views. Each view allows you to do a specific set of tasks. The menus and toolbars change with each view.

Figure 13

These buttons allow you to switch among the six ChemStation views

The following summarizes the ChemStation views and their functionality: For more information about the Method and Run Control View, see Chapter 3.

Method and Run Control
? Set up methods ? Change setpoints for the Agilent 1100 Series LC or Agilent 1200 Series LC modules, including the Chip Cube ? Change setpoints for the Agilent 6100 Series Quadrupole LC/MS systems ? Change setpoints for the Agilent API sources ? Run single samples ? Run automated sequences ? Run an FIA series ? View data in real time, as it is acquired

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Overview of Hardware and Software Overview For more information about the Data Analysis View, see Chapter 5.

1

Data Analysis
? View chromatograms and spectra from the MS and UV detectors ? Integrate chromatographic peaks ? Perform quantitation ? Check peak purity ? Deconvolute multiply charged spectra ? Generate reports ? Reprocess data from sequences

For more information about the Report Layout View, see Chapter 4. For more information about the Verification View, see Chapter 6.

Report Layout
? Design custom report templates

Verification (OQ/PV)
? Verify system performance ? Perform tests to demonstrate compliance with regulatory requirements (Operational Qualification/Performance Verification, or OQ/PV)

For more information about the Diagnosis View, see Chapter 7.

Diagnosis
? Learn possible causes of instrument problems ? Run tests to diagnose instrument problems ? Receive notification when it is time to perform system maintenance ? Pump down and vent the system

For more information about the MSD Tune View, see Chapter 2.

MSD Tune
? Optimize and calibrate the MS

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1

Overview of Hardware and Software Reviewing data remotely

Reviewing data remotely
There are two ways to set up a computer so you can review ChemStation data remotely. One way is to install a Data Analysis-only version of the ChemStation software on the remote computer. This installation provides the same Data Analysis functionality that you have on the ChemStation that controls your Agilent 6100 Series LC/MS system. It is ideal if you need full features for in-depth data analysis. You can find the ChemStation Data Browser software on your LC/MS ChemStation installation CD. Another way is to install the ChemStation Data Browser on the remote computer. The ChemStation Data Browser allows you to easily review ChemStation LC and LC/MS data files, but the functionality is different than with the full ChemStation Data Analysis. The Data Browser software is ideal for synthetic chemists and others who use the LC/MS system for “walk-up” analysis.

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Agilent 6100 Series Quadrupole LC/MS Systems Concepts Guide

2 Instrument Preparation
Preparation of the LC system 34 Purpose 34 Summary of procedures 34 Setting parameters for LC modules 36 Column conditioning and equilibration 39 Monitoring the stability of flow and pressure 41 Preparation of the MS – tuning 42 Overview 42 Ways to tune 44 When to tune – Check Tune 45 Autotune 47 Manual tuning 49 Tune reports 51 Gain calibration 52

In this chapter, you learn the concepts that help you prepare the instrument for an analysis. This chapter assumes that the hardware and software are installed, the instrument is configured and the performance verified. If this has not been completed, see the Agilent 6100 Series Single Quad LC/MS System Installation Guide.

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2

Instrument Preparation Preparation of the LC system

Preparation of the LC system
Purpose
To achieve good sensitivity, it is important to properly prepare the LC and column prior to an LC/MS analysis. For best signal-to-noise, the entire LC system must be free of contamination from salts (such as nonvolatile buffers) and unwanted organic compounds. Some contaminants that are not bothersome for a UV detector can cause problems for the MS. Contaminants may cause ion suppression and/or high background, and these problems can seriously degrade sensitivity. To achieve a smooth baseline with little noise, the LC flow must also be very stable.

Summary of procedures
The exact LC preparation steps depend on how the LC was used previously and the type of analysis to be performed. The following provides guidelines:

Typical preparation
Before beginning an analysis, the entire LC path should be contaminant-free and the flow should be stable. Usually, you can accomplish these goals by doing the following: 1 Purge the pump to remove air bubbles. Purge each channel that you plan to use. For instructions to purge the pump, search the online help for the keyword “purge” and scroll down the list of topics until you see entries that begin with the word “purge.” 2 Condition the column to remove impurities or residual sample.

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Instrument Preparation Summary of procedures For more information, see “Column conditioning and equilibration” on page 39. 3 Equilibrate the column at the initial mobile phase composition. For more information, see “Column conditioning and equilibration” on page 39. 4 Ensure that the system flow and pressure are stable. For more information, see “Monitoring the stability of flow and pressure” on page 41.

2

More extensive preparation
While the four-step procedure that is outlined above works well on a day-to-day basis, more extensive LC/column flushing may be necessary if any of the following are true: ? You have not used this LC for MS. ? The column is new. ? You are changing to a different mobile phase composition. ? The LC was used to analyze dirty samples. ? The next analysis requires ultimate sensitivity. A protocol for more thorough LC cleaning is given in the Agilent 6100 Single Quad System Installation Guide. See the section on conditioning the LC in the chapter on system verification. When you flush the LC, remember to flush all channels that you plan to use. Also, flush the injector by making several injections of the same solvent(s) that you use to flush the system.

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Instrument Preparation Setting parameters for LC modules

Setting parameters for LC modules
You set up the LC modules in the Method and Run Control View. Within the system diagram, click each module to set parameters.

Injection Pump Solvent bottles

Column thermostat, Diode-array detector Column switching valve

Mass spectrometer

Figure 14

Example system diagram (yours may be different)

Table 1 shows the tasks that you may perform to set up the LC modules and the menu items that allow you to do the tasks. To access all the Instrument menu items, be sure to enable full menus. (Select View > Full Menu.) To access help for any system module, click the module, then the Help menu item. To access help for a given dialog box, click the Help button within the dialog box. Table 1 Tasks to set up the LC modules
And you want to: Click the module icon and select this menu item: Set up Injector Configuration Click little button to lower right of injection icon Or choose the following from the Instrument menu Set up Injector More Injector > Configuration

If you have this module: Autosampler

Set autosampler parameters Change volumes for the installed syringe Move the gripper arm to home position before removing the tray

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Instrument Preparation Setting parameters for LC modules Table 1 Tasks to set up the LC modules
And you want to: Click the module icon and select this menu item: Set up injector Configuration Configuration Or choose the following from the Instrument menu Set up injector More Injector > Configuration More Injector > Configuration More Injector > Sample Thermostat Preconfigured Wellplate Types Configure Wellplate Types Set up Pump Set up Pump

2

If you have this module: Well-Plate Sampler (WPS) or ?WPS

Set autosampler parameters Change volumes for the installed syringe Select tray type and its position Set the sample thermostat View a list of pre-configured well-plates Configure a new type of well-plate

Binary, isocratic, and quaternary pumps

Set pump parameters

Turn the pump off or on

Click little button to lower right of solvent delivery (pump) icon Auxiliary Data Curves

More Pump > Control

Set compressibility for a unique solvent Choose which run-time-based pump information you want to store on disk Have the software monitor solvent levels Set date and time for automatic pump turnon Select method to use when there is an instrument error

More Pump > Auxiliary More Pump > Data Curves

Click solvent bottles icon and select Filling Levels

More Pump > Bottle filling More Pump > Control More Pump > Control

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Instrument Preparation Setting parameters for LC modules Tasks to set up the LC modules
And you want to: Click the module icon and select this menu item: Same as binary pump Or choose the following from the Instrument menu Same as binary pump More Pump > Purge Configuration Configuration Set up Column Thermostat More Pump > Configuration More Pump > Configuration Set up Column Thermostat Set up Column Thermostat, then click the More>> button

Table 1

If you have this module: Capillary pump and nanopump

Do same tasks as binary pump Purge the pump Set mixer and filter volumes Enable a fast change in solvent composition

Thermostatted Column Compartment (TCC)

Set the temperature for left column compartment Set the temperature for right column compartment Activate temperature control Choose which run-time-based column temperature information you want to store on disk Set up a column temperature time table

Set up Column Thermostat, then click the More>> button Click little button to lower right of column icon Set up Column Thermostat, then click the More>> button Set up Column Thermostat, then click the More>> button Set up Column Thermostat Set up Column Thermostat, then click the More>> button

Set up Column Thermostat, then click the More>> button Set up Column Thermostat, then click the More>> button Set up Valve Set up Valve

Column Switching Valve

Change the valve position to redirect flow Change the valve position during an analytical run

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Instrument Preparation Column conditioning and equilibration Table 1 Tasks to set up the LC modules
And you want to: Click the module icon and select this menu item: Set up DAD Signals Or choose the following from the Instrument menu Set up DAD Signals More DAD > Control Click little button to lower right of DAD icon Set up DAD Signals Set up DAD Signals More DAD > Control Set up DAD Signals Set up DAD Signals More DAD > Control

2

If you have this module: Diode Array Detector (DAD)

Set detector parameters Turn UV and visible lamps off or on Bring the baseline of the plot to zero Bring the baseline of the plot to zero before each analysis Choose which spectra to store Select method to use when there is an instrument error

Column conditioning and equilibration
There are several ways to set parameters to condition and equilibrate a column.

Conditioning
Column conditioning eliminates any previously separated compounds or impurities from the column, particularly after runs with solvent of a single composition (isocratic runs). There are a number of ways to condition a column before a sample run. One way is to pump the organic solvent that you intend to use (100% solvent B) through the column for a period of time. Another way is to run the gradient that you intend to use, then extend the time at the final composition until no further peaks elute. When a column is new, “conditioning” may include injecting a few samples or high-level standards until peak area and retention time are stable.

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Instrument Preparation Column conditioning and equilibration

Equilibration
Column equilibration returns column characteristics to their initial state after a gradient run. To equilibrate a column before a sample run, you pass the solvent of initial composition through the column for a period of time.

Using the software to condition and equilibrate a column
You can condition and equilibrate a column in one of three ways with the ChemStation software. ? Interactively You set the pump to the solvent composition for the end of the run and higher-than-normal flow rates. You can then immediately apply these setpoints to the pump. After you pump about three column-volumes of solvent, then set the pump to the solvent composition and flow rate for the beginning of the run. With this procedure, you do not store a data file. If you use this procedure, you can tune the MS while you condition and equilibrate the column. When you tune the MS, the MS stream selection valve automatically diverts the LC effluent to waste. For information on tuning, see “Preparation of the MS – tuning” on page 42. You set up a method or sequence in the Method and Run Control View, described in Chapter 3. ? With a method in an interactive run You set up a method for your analysis and then run a solvent blank. The run uses the method stop time. You can also use a post-run time within the method to equilibrate the column. With this procedure, you store a data file. ? With a sequence You set up a method for your analysis and then set up a solvent blank as the first run in a sequence. The method includes a post-run time to equilibrate the column. With this procedure, you store a data file.

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Instrument Preparation Monitoring the stability of flow and pressure

2

Monitoring the stability of flow and pressure
The LC solvent flow and the system backpressure must be stable to ensure a quiet baseline and best results for API-MS. The best time to monitor the stability of flow and pressure is after you have equilibrated the column, and before you start the analysis. Chapter 3 provides more information about online signals, which are also called online plots. You can measure stability with the ChemStation software. To do this, you set up an isocratic method with the same solvent composition as the initial composition you intend to use for your analysis. During the run, you monitor the online signals for flow and pressure.

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Instrument Preparation Preparation of the MS – tuning

Preparation of the MS – tuning
Overview
Use the MSD Tune View for all tasks that relate to tuning. Tuning is the process of adjusting MS parameters to generate high quality, accurate mass spectra. During tuning, the MS is optimized to: ? Maximize sensitivity ? Maintain acceptable resolution ? Ensure accurate mass assignments

Parameters that are adjusted
The Agilent 6100 Series Quadrupole LC/MS systems have two sets of parameters that can be adjusted. One set of parameters is associated with the formation of ions. These parameters control the spray chamber (for example, electrospray or APCI) and fragmentor. The other set of parameters is associated with the transmission, filtering, and detection of ions. These parameters control the skimmer, octopole, lenses, quadrupole mass filter, and high-energy dynode (HED) electron multiplier (detector). Tuning is primarily concerned with finding the correct settings for the parameters that control the transmission, filtering, and detection of ions. It is accomplished by introducing a calibrant into the MS and generating ions. Using these ions, the tune parameters are then adjusted to achieve sensitivity, resolution, and mass assignment goals. With a few exceptions, the parameters that control ion formation are not adjusted. They are set to fixed values known to be good for generating ions from the calibrant solution.

Tune files and reports
The product of tuning is a tune file (actually a directory) that contains two collections of parameter settings: one for positive ionization and one for negative ionization. Whenever data

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Instrument Preparation Overview

2

acquisition uses a tune file, the software automatically loads the settings appropriate for the ion polarity specified by the data acquisition method. Autotune, the automated tuning program, also generates a report. See page 51.

Use of tune files during data acquisition
During data acquisition, the parameters associated with ion formation are controlled by the data acquisition method. The parameters associated with ion transmission are controlled by the tune file assigned to the data acquisition method.

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Instrument Preparation Ways to tune

Ways to tune
Access this functionality via the Tune menu in the MSD Tune View. The software provides two ways to tune the MS: ? Autotune is an automated tuning program that tunes the MS for good performance over the entire mass range. It uses known compounds in a standard calibration mixture that is introduced via the Calibrant Delivery System (CDS). This is the tuning method that you use in most cases. ? Manual Tune allows you to tune the MS by adjusting one parameter at a time until you achieve the desired performance. Manual tuning is most often used when you need maximum sensitivity, when your analysis targets a restricted mass range, or when you need a tuning compound other than the standard calibrants. In addition, a Check Tune program allows you to determine whether you need to tune. Check Tune, Autotune, and Manual Tune are discussed in more detail in the next sections.

NOTE

Frequent tuning is not required for normal operation. Once tuned, the LC/MS is very stable. Tuning is generally not needed more often than monthly, or at most weekly. If you suspect problems related to tuning, use the Check Tune program to confirm that the MS is out of adjustment before you retune it.

CAUTION

Wait at least four hours after pump-down before tuning or operating your Agilent 6100 Series LC/MS system. Ceramic components in the quadrupole mass filter can absorb moisture from the air. Tuning or acquiring data too soon may result in arcing. Further, it takes the analyzer at least nine hours to reach thermal equilibrium. Tune files created or data acquired before the MS is at thermal equilibrium may have incorrect mass assignments and other inaccuracies.

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Instrument Preparation When to tune – Check Tune

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When to tune – Check Tune
Check Tune allows you to quickly determine whether the MS is correctly tuned without performing a complete autotune. It performs a single profile scan of the tune masses and compares the peak widths and mass axes with target values. If the values obtained by Check Tune are within acceptable ranges, the tune report indicates that Check Tune passes. (See Figure 15 on page 46.) If the values are outside of acceptable ranges, Check Tune suggests that you adjust peak widths or calibrate the mass axis. A third parameter, gain, may be added to the parameters checked by Check Tune. If so, Check Tune compares the current gain value with the gain value from the most recent autotune. For a discussion of gain, see page 52.

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Instrument Preparation When to tune – Check Tune

All masses pass the mass axis and peak width tests

Figure 15

Check Tune report

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Instrument Preparation Autotune

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Autotune
Autotune is a program that adjusts the MS for good performance over the entire mass range.

Autotune files
A tune file, ATUNES.TUN is provided as a starting point for autotune. The results of autotune are saved to the current autotune file. This makes autotune faster because each autotune begins from the most recent good tune parameters. This means, however, that previous tune values are lost. You may want to occasionally save the autotune file to a different file name before using autotune. A single autotune file contains the results of both positive ionization and negative ionization autotunes.

Autotune polarity and scan speed
You have six choices for running an autotune: ? Dual Polarity ? Positive Polarity ? Negative Polarity ? Dual Polarity Fast Scan ? Positive Polarity Fast Scan ? Negative Polarity Fast Scan The Dual Polarity modes perform an autotune for both positive ionization and negative ionization. Positive Polarity and Negative Polarity modes perform autotunes only for the specified polarity.

Gain checking in autotune
Gain checking occurs only in the positive polarity autotune or the positive portion of a dual polarity autotune. For more information on gain checking, see “Gain calibration” on page 52.

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Instrument Preparation Autotune

Spray chamber parameters
Autotune adjusts the parameters that affect ion transmission. The spray chamber parameters, which affect ion formation, are not adjusted by autotune. They are set to default values known to be good for generating ions of the calibrant solution. Because there may be slight variations from spray chamber to spray chamber, the default parameters may not be optimum for a particular spray chamber. Changes such as adjustment of the nebulizer and normal aging of the LC/MS system components can also result in a system where the default values are not optimum. Therefore, it is possible to manually set the spray chamber parameters. Typically, this is necessary only when the instrument fails autotune or when the results of autotune show a significant decrease in system performance. Be aware that other factors can also decrease system performance.

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Instrument Preparation Manual tuning

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Manual tuning
The autotune program adjusts the MS for good performance over the entire mass range, and this tune program is sufficient for most applications. There are times, however, when it is advantageous to tune the MS manually. These include: ? When you want to achieve maximum sensitivity by sacrificing some resolution ? When you want to tune specifically for the very low end of the mass range (< 150 u) ? When you want to tune with a compound other than the standard calibrants.

CAUTION

The calibrant delivery system (CDS) is designed specifically for precise delivery of the Agilent calibrants in a 90:10 acetonitrile:water solvent mix. Other solvents and mixes may have different flow rates, which may not be optimum for tuning. Further, the O-ring seals in the CDS are ethylene-propylene. They can be damaged by aliphatic, aromatic, or halogenated hydrocarbons. Never use these types of solvents in the CDS. We recommend delivering non-standard calibrants through the LC.

Tips for manual tuning
Ionization modes Separate tuning is not required for electrospray, APCI/APPI and multimode-ESI+APCI. Tuning involves adjusting the parameters that control the transmission of ions. It does not matter how those ions are generated. A tune file created in electrospray mode often will provide good results for APCI/APPI samples. The reverse is also true. Separate tuning is required for positive and negative ionization. The MS must, at some time, be tuned twice — once with positive ions and once with negative ions. The two tunes can be stored under the same tune file name; the software keeps separate the positive ion and negative ion settings. Positive ion and negative ion settings can be updated separately.

Polarity

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Instrument Preparation Manual tuning Mass range Tuning is almost always done over the entire mass range. The exception is if all the ions of interest are smaller than approximately 150 u. Correct manual tuning over a reduced mass range can improve transmission and detection of these very low mass ions.

Steps to manual tune
The online help includes step-by-step instructions for manual tuning. The instructions assume the use of one of the standard calibrants and tuning over the entire mass range, but they can be altered for non-standard calibrants or narrower mass ranges. To access the manual tuning instructions: 1 Open the online help. 2 Click to expand “How to Work with Your ChemStation.” 3 Click to expand “Basic Tasks.” 4 Click to expand “Method and Run Control.” 5 Click to expand “MSD Tune.” 6 Click the link to “Manually Tuning the LC/MSD or CE/MSD.” 7 Scroll down until you see “Steps.”

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Instrument Preparation Tune reports

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Tune reports
At the end of every autotune, the system prints a tune report. You can also manually print a tune report from the File menu.

Figure 16

Tune report (page 1 of 4)

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Instrument Preparation Gain calibration

Gain calibration
Gain calibration sets the electron multiplier voltage in a way that simplifies method development and portability. Gain calibration is performed automatically during autotune. You can also check and adjust the gain calibration during manual tune.

What is gain?
The HED electron multiplier receives an input current that is generated by the ions that strike it, amplifies that current, and generates a proportional output current. Gain = Output current / input current Gain is controlled by the electron multiplier voltage (EMV). The higher the EMV, the higher the gain. The relationship between EMV and gain is log linear. This linear relationship is common for all multipliers. Since the slope of this line is constant, to set the gain, the software needs only to adjust the intercept. The gain calibration routine simply adjusts the value of the intercept for the specific instrument. The instrument-specific gain curve coefficients are then stored in the MS.

Gain calibration curve
A gain calibration curve is generated by taking readings of the ion current generated at discreet multiplier settings across the range of the multiplier. Separate gain calibration curves are generated for positive and negative polarities, since the ion current generated in these two modes varies to some extent. A gain of 1 is set to be a multiplier value of 1e6. That is, one electron striking the input side of the detector will generate 1e6 on the output side. A gain check can be generated on both polarities or on the current polarity.

Gain and method portability
The use of gain enables the distribution of methods from instrument to instrument. In principle, using the same gain on two different instruments should result in the same signal response, thereby easing method development and instrument 52 Agilent 6100 Series Quadrupole LC/MS Systems Concepts Guide

Instrument Preparation Gain calibration portability. Because the relationship between EMV and gain is log linear, a gain of 2.0 should give twice the abundance of a gain of 1.0.

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More gain is not always better
In general, you should run the detector at the lowest gain that still produces adequate abundance. High gains increase noise as well as signal and often result in poorer signal-to-noise ratios. Increasing the gain increases the EMV, which shortens the life-span of the electron multiplier. The maximum EMV is 3000 eV no matter how high you set the gain; a gain of 70 or higher elicits the 3000 eV setting. As an electron multiplier ages, it slowly becomes less efficient. For a given ion current input, it generates a smaller and smaller output current (abundance). Low abundance caused by an aging electron multiplier is not easily distinguished from low abundance caused by poor ion generation or transmission (low ion current input). It may be tempting to compensate for poor ion generation or transmission by increasing the gain (thereby increasing the EMV) even though the electron multiplier is actually performing correctly at its previous gain. The increased gain will improve abundance, but may decrease the signal-to-noise ratio and will shorten the life-span of the electron multiplier.

Check Gain Calibration
Access this functionality via the Tune menu in the MSD Tune View. During a Gain Calibration check, a reading of the ion current is taken and checked against the current gain calibration curve for the current polarity. If it is within acceptable limits, no change is made to the curve for that polarity. If the reading falls outside the limits, a new gain curve is generated. A gain of 1 is set to be a multiplier of 1e6. That is, one electron striking the input side of the detector will generate 1e6 on the output side. If that multiplier setting exceeds 2600 volts, which indicates decreasing electron multiplier performance or instrument performance, you are warned that system maintenance may be required.

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Instrument Preparation Gain calibration

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3 Data Acquisition
Working with methods 56 Method and Run Control View 56 Loading, editing, saving and printing methods 58 More on editing methods 60 Running samples 63 Running a single sample 64 Running a sequence 64 Flow injection analysis 69 Monitoring analyses 73 Online signal plots 73 Quick method overview 74 Logbooks 74 Instrument shutdown 76

In this chapter, you learn the concepts that help you run samples and acquire data. This chapter assumes that the hardware and software are installed, the instrument is configured and the performance verified. If this has not been completed, see the Agilent 6100 Series Single Quad LC/MS System Installation Guide.

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Data Acquisition Working with methods

Working with methods
Method and Run Control View
You set up methods and run analyses from the Method and Run Control View, shown in Figure 17. ChemStation methods control the instrument during data acquisition. The easiest way to prepare a method is to load a similar method (or load DEF_LC.M), save it with a new name, modify (edit) it, and save it again. Then you can use the method to run a single sample or multiple samples via a sequence. You can also set up a method for flow injection analysis (FIA), where you make multiple injections in a single run.

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Data Acquisition Method and Run Control View

3

Title bar Toolset selection

Method

Sequence

Top toolbar

System diagram

Status bar Start/stop run File list

Sampling diagram Online plot

View selection Method overview

Message line

Figure 17

In this view, you set up methods, establish instrument settings, and inject samples.

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Data Acquisition Loading, editing, saving and printing methods

Loading, editing, saving and printing methods
When you need to work with methods, there are several ways to access the necessary functionality: ? Select from the Method menu.

Figure 18

The Method menu allows you to load, edit, save, and print methods.

? Use the system diagram.

Method icon

Figure 19

The Method icon allows you to edit or print methods. Click it to access the menu.

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Data Acquisition Loading, editing, saving and printing methods ? Use the toolbar or method drop-down list. When you want to view all the tools for methods (or to run a single sample interactively), you first click the icon for the Single Sample Toolset, as shown in Figure 20.

3

Sequence
Single sample

Figure 20

Toolset selection

Load a Method

Save current method

Select method from list of recent methods

Edit entire Method

Figure 21

When you click the icon for the Single Sample Toolset, you access tools to work with methods. ? Use the Method and Run Control file list. (If necessary, first click the Method tab at the bottom.)

Figure 22

You can right-click in the white area to view the menu, or double-click a method to load it.

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Data Acquisition More on editing methods

More on editing methods
There are a number of ways to edit a method. If you need to create a new method for a new analysis, then it is best to review and edit the entire method, as described just below. If you want to make only a few changes to an existing method, then it is easiest to edit only the settings that need to change, as described in “Editing a portion of the method” on page 61.

Editing the entire method
You can edit the entire method from the Method menu or the Edit entire Method icon in the toolbar or the Method icon in the instrument diagram. If you elect to edit the entire method, the software displays all the appropriate dialog boxes in succession. The first dialog box (Figure 23) lets you limit which parts of the method you wish to view and potentially modify. If you need detailed information as you edit the method, click the Help button in each dialog box.

Figure 23

Parts of the method that you can display and modify

The Run Time Checklist, the last item listed in Figure 23, controls which parts of the method the software executes when you run your analysis. As shown in Figure 24, a complete method can include both data acquisition and data analysis.

NOTE

Be sure to save the method after you edit it.

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Data Acquisition More on editing methods

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Run a command or macro prior to an analysis in a sequence

Save a checksum-protected register file for Good Laboratory Practices (GLP) Run a command or macro at the end of an analysis (for example, run an Excel macro) Save a complete record of the analysis conditions

Figure 24

Run Time Checklist

Editing a portion of the method
If you need to change only a few settings in a data acquisition method, you can edit a subset of the method. You can do that either from the system diagram (Figure 25) or the Instrument menu (Figure 26). External valves, if present, do not appear in the system diagram. You must edit their settings from the Instrument menu.

Injection

Pump

Variable wavelength detector Column thermostat, Mass spectrometer Column switching valve

Figure 25

You can click a module in the system diagram to access menus that allow you to change settings.

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Data Acquisition More on editing methods

Figure 26

The Instrument menu also allows you to edit parameters for a specific module in your system.

NOTE

Be sure to save the method after you change the instrument settings.

For details on setting LC parameters, see Table 1 on page 36. For details on setting MS parameters, refer to the table below. Table 2 Tasks for setting up the MS
Click the MS icon and select this menu item: Set up MSD Signals Set up MSD Signals Spray Chamber Spray Chamber Data Curves Or choose the following from the Instrument menu Set up MSD Signals Set up MSD Signals More MSD > Spray Chamber More MSD > Spray Chamber More MSD > Data Curves

If you want to: Set scan range or SIM ions Set instrument polarity Set drying gas flow or temperature or nebulizer pressure Set the capillary voltage Select MSD parameters to store during a run

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Data Acquisition Running samples

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Running samples
The LC/MS ChemStation provides three ways to run samples: ? Run a single sample interactively and create a single data file. ? Run sample(s) via a sequence and create a data file for each sample. FIA = flow injection analysis ? Run multiple injections within a single data file, via FIA. You can use the same method to run either a single sample or samples in a sequence. For FIA, you must modify the method to enable multiple injections in the same run.

Display Single Sample Toolset

Enable FIA within method

Display Sequence Toolset

Figure 27

To run samples, you first click the appropriate icon.

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Data Acquisition Running a single sample

Running a single sample
After you have set up and saved a method, you are ready to run a single sample interactively. As shown in Figure 28, the software provides more than one way to enter sample information and start the run.

Click to start the run

Click to enter sample information

Button in Sample Info dialog box

Figure 28

Multiple ways to enter sample information and start the run

Running a sequence
To run samples in an automated, unattended sequence, you first click the icon to display the Sequence Toolset.
Sequence

Figure 29

The Sequence icon on the left provides access to the Sequence Toolset and the sampling diagram that shows the sample tray.

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Data Acquisition Running a sequence

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Loading, editing, saving and printing sequences
When you need to load, edit, save, and print sequences, there are several ways to access the necessary functionality: ? Select from the Sequence menu.

Figure 30

The Sequence menu allows you to load, edit, save, and print sequences.

? Use the toolbar or sequence drop-down list.

Load Sequence

Save Sequence

Select sequence from list of recent sequences

Figure 31

Tools to work with sequences

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Data Acquisition Running a sequence ? Use the Method and Run Control file list. (If necessary, first click the Sequence tab at the bottom.)

Figure 32

You can right-click in the white area to view the menu, or double-click a sequence to load it.

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Data Acquisition Running a sequence ? Use the sampling diagram.

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Figure 33

Click the sampling diagram to access the menu.

More on editing sequences
When you edit a sequence, the following dialog boxes are applicable: ? Sequence Parameters – allows you to enter information like data file path, parts of the method to run, and a post-sequence command/macro to shut down the system ? Sequence Table – includes the sample list and any calibration information ? Sequence Output – controls printing of reports (sample results and the sequence summary) ? Import Sequence – allows you to import a sample table from a text (.txt) file or a delimited file, such as a comma-separated value (.csv) file created in Microsoft? Excel

NOTE

After you have edited your sequence, be sure to save it.

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Data Acquisition Running a sequence

Starting the sequence
After you have set up and saved your sequence and any methods that it uses, you are ready to run the sequence. As shown in Figure 34, the software provides more than one way to start the sequence.

Button in Sequence Table and Partial Sequence dialog boxes

Click one of these to start the sequence

Figure 34

Multiple ways to start the sequence

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Flow injection analysis
Flow injection analysis (FIA) is injection of multiple samples within the same run, and is performed without a column. FIA is very useful when you want to optimize MS parameters such as: ? Drying gas flow and temperature (which depend on the mobile phase and flow rate, as well as the ionization mode). ? Nebulizer pressure (which depends on the mobile phase and flow rate, as well as the ionization mode). ? Fragmentor voltage (which depends on the structure of the compound). FIA is also useful to check quickly for compound sensitivity or linearity, or any time you want to perform an analysis without a separation.

Setting up FIA within a method
You can add FIA to a method either through the Method menu or the FIA icon (see Figure 35).

To optimize settings with a single sample The Edit FIA Series dialog box has an autofill function so you can automatically build an FIA table. You can select up to two MSD parameters, and then automatically increment their settings. This feature makes it easy to set up FIA to optimize settings, as shown with the example in Figure 36. If you decide later to add a setpoint, you can use the insert row/append row functionality.

NOTE

Some parameters, such as gas temperature, take time to reach their setpoints. In these cases, perform multiple injections at each setpoint and allow more time between injections.

To inject multiple samples

If you need to inject multiple samples to check for sensitivity or linearity, you can also use the autofill functionality within the Edit FIA Series dialog box. In this situation, you keep the MS parameters constant and increment

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Data Acquisition Flow injection analysis only the vial numbers. Alternatively, you can use the insert row/append row functionality to add samples. Figure 37 shows an example of FIA for a linearity check.

FIA icon

Click to enable FIA or set up FIA series

Figure 35

Two ways to enable FIA within a method

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Mark check box first Automatically build an FIA table

Figure 36

The FIA series is a convenient way to optimize MS parameters. The example shows a test of fragmentor voltage.

Add rows manually or automatically

Figure 37

For pure samples, the FIA series can be used to check sensitivity or linearity. The example shows a linearity check.

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Data Acquisition Flow injection analysis

Running multiple FIA methods
If you have multiple compounds to test or you need to perform more complex method development, you can run a succession of FIA methods, analogous to a sequence. You access that capability via the RunControl menu.

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Data Acquisition Monitoring analyses

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Monitoring analyses
Online signal plots
During an analysis, you can view online signals, as shown in Figure 38. (Select View > Online Signals.) You can click the little box with the arrows in the lower left of the signal display to display the online plot as a separate window. This feature is useful if you wish to make the window larger so you can view more signals. For the online plots, you can choose which signals to display, and you can adjust the scales of the x and y axes.

Figure 38

Online plot

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Data Acquisition Quick method overview

Quick method overview
Just to the right of the online plot, you can get a quick overview of your method parameters. You can select which type of settings to view, including LC, MS, and data analysis settings. You can click each parameter to display the dialog box to change that parameter.

Figure 39

Quick method overview, showing LC settings

Logbooks
Several logbooks are important in the Method and Run Control View:

Current logbook
The current logbook stores all error, system, and event messages, and is helpful for general information and troubleshooting. It logs events such as ChemStation startup and shutdown, loading of sequences and methods, running of sequences and methods, etc. Figure 40 shows how to view the current logbook. The log file exists as INSTRx.LOG (Where x is the instrument number), in the temporary folder under the instrument folder in CHEM32 (for example, C:\CHEM32\1\TEMP).

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Run logbook
RUN.LOG stores all messages related to analysis of a particular sample. It resides in the data file folder. You can use it to check that the sample was processed correctly.

Sequence logbook
The sequence logbook shows what happened as a sequence ran. You can use it to troubleshoot errors that occurred during unattended operation. Figure 40 shows how to view the sequence logbook. The log file exists as sequencename.LOG (where sequencename is the name of the ChemStation sequence). Each time a sequence runs, the software produces a log file, usually in the DATA folder within the instrument folder (for example, C:\CHEM32\1\DATA).

Current logbook icon

Sequence logbook icon

Click to open logbooks

Figure 40

You can view logbooks from the View menu or the logbook icons.

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Data Acquisition Instrument shutdown

Instrument shutdown
At the end of your analysis, if you do not plan to run more samples until the next day, it is important to: ? Flush the system with a mobile phase without buffers. ? Put the system into standby mode. For details, see the Basic Operation section of the Agilent 6100 Series Quad LC/MS Systems Quick Start Guide.

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4 Reports
Using predefined reports 78 Generating reports 78 Report styles 79 Defining custom reports 80 Summary of process 80 Example report templates 80 The Report Layout View 81

In this chapter, you learn the concepts that help you generate reports of results. For more concepts related to reports, see these two manuals: ? Agilent ChemStation: Understanding your ChemStation ? Agilent ChemStation for LC 3D Systems: Understanding Your Spectra Module For in-depth task-related information and specifics about dialog boxes and toolbars, see the online help. For descriptions of all buttons on toolbars, in the help double-click User Interface Reference, then double-click Toolbars.

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Reports Using predefined reports

Using predefined reports
A report provides qualitative or quantitative information about a sample you have analyzed. You can print a report, display it on your computer screen, or send it to a file. The ChemStation software includes a number of predefined report styles, and you can generate new styles for custom reports.

Generating reports
Ways to generate reports
The ChemStation software gives several ways to generate reports in the Data Analysis View: ? Select from the Report menu.

Specify report style, destination, etc. Edit parameters for system performance reports Specify how library search is done

Figure 41

The Report menu provides ways to print reports and set properties of reports.

? Use the report tools in the Integration Toolset or the Calibration Toolset. You access these toolsets by clicking one of the buttons on the right.

Specify report

Calculate and preview report

Calculate and print report

Figure 42

Tools to work with reports

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Reports Report styles

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System suitability reports
In the Report menu, System Suitability allows you to check the performance of your system before and during your analyses. This report style allows you to set up high and low limits for retention time, peak height, peak width, theoretical plates, resolution, library match, peak purity, and other properties of calibrated peaks. When you choose one of the Performance report styles, the report indicates when any of the parameters are out of specification.

NOTE

When you are done, be sure to save the report parameters with your method.

Report styles
The ChemStation software provides a wide variety of report styles. Most of them are described in one of these two manuals: ? Agilent ChemStation: Understanding your ChemStation ? Agilent ChemStation for LC 3D Systems: Understanding Your Spectra Module Two report styles are specific to LC/MS, and are not discussed in the above manuals. ? LC/MS – includes: ? Header information from the data file ? A total ion chromatogram (optional) ? Tabulated and graphical spectra for each integrated peak ? Deconvolution results (available with the optional Agilent LC/MSD Deconvolution & Bioanalysis Software). ? LC/MS Qualitative – includes: ? Header information from the data file ? A total ion chromatogram (optional) ? Extracted ion chromatograms and spectra for each integrated peak.

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Reports Defining custom reports

Defining custom reports
The ChemStation software provides the capability to set up custom report layouts that can include text, chromatograms, tables, and custom elements such as company logos. The text can be user-defined text or information stored within ChemStation, such as sample name, operator, and data file name.

Summary of process
The overall process to create and use a custom report is as follows: 1 Create a custom report layout using the Report Layout View, described on page 81. 2 Save the custom layout as a report template. 3 Test the report template by loading a data file and generating a report. 4 Add the report template to the list of available report styles. 5 Add the new report style to a method and save the method. For details, see the Report Layout tutorial within the ChemStation Tutorial section of the online help.

Example report templates
The ChemStation software includes example report layouts. They are located in the chem32\repstyle folder. For descriptions of these layouts, see the Agilent 6100 Series Quad LC/MS System Quick Start Guide.

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Reports The Report Layout View

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The Report Layout View
The Report Layout View, shown in Figure 43, provides all the necessary menus and tools to create a template for a custom report.
Title bar Report layout tools Calculation technique Report template Top toolbar

New section menu Report section

Insertion triangle
(Click a blue triangle to activate the New section menu (triangle turns red)

Method list (ChemStation Explorer)

View selection Message line

Figure 43

The Report Layout View provides a graphical interface to set up custom reports. The file menu, shown in Figure 44, provides the functionality to initiate, open, and save report templates. You can also access this functionality from the top toolbar. (See Figure 43.) After you add a new section, you can use the report layout tools shown in Figure 43 to add text, chromatograms, and tables.

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Reports The Report Layout View

Prepare a new report layout Save a new report layout Load data to test a new report layout

Preview a new report layout with loaded data Add new template to available report styles

Figure 44

The File menu within the Report Layout View

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5 Data Analysis
The Data Analysis View 84 Loading and manipulating chromatograms 86 Loading signals 87 Removing signals from the chromatogram display 91 Changing how chromatograms are displayed 91 Working with spectra 93 Displaying spectra 94 Peak purity 95 Performing quantification 96 Integrating peaks 96 Calibration 98 Data review and sequence reprocessing 100 The Navigation Table 100 Batch review 100

In this chapter, you learn the concepts that help you analyze data. For more information on the concepts related to data analysis, see these two manuals: ? Agilent ChemStation: Understanding your ChemStation ? Agilent ChemStation for LC 3D Systems: Understanding Your Spectra Module For more in-depth task-related information and specifics about dialog boxes and toolbars, see the online help. For descriptions of all buttons on toolbars, in the help double-click User Interface Reference, then double-click Toolbars.

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Data Analysis The Data Analysis View

The Data Analysis View
The Data Analysis View, shown in Figure 45, provides all the tools for qualitative and quantitative data analysis. In this view, you can: ? Evaluate chromatograms and spectra ? View and compare signals from both the MS and UV detectors ? Integrate peaks and perform quantification ? Check peak purity ? Perform deconvolution (with the optional Agilent LC/MSD Deconvolution & Bioanalysis Software) ? Generate reports from predefined report templates ? Reprocess data from sequences.

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Data Analysis The Data Analysis View

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Title bar

Navigation toolbar

Top toolbar

Cursor tools

Graphics tools

File list (ChemStation Explorer) Navigation Table Toolset selection Signal view selection DAD signal

LC/MS signal

View selection Message line

Figure 45

The Data Analysis View provides menus and tools for qualitative and quantitative analysis.

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Data Analysis Loading and manipulating chromatograms

Loading and manipulating chromatograms
In the ChemStation software, chromatograms are called signals. The ChemStation software stores multiple signals in the same data file. Examples of signals are MS chromatograms and UV chromatograms. For many analyses, it is important to be able to compare results from different detectors or different instrument settings, or to compare results from various samples. The ChemStation software allows you to load and compare signals within a data file and between data files. For example, within a data file, you can simultaneously load and evaluate UV and MS data, or MS data at two different fragmentor settings or ion polarities. You can load data files from multiple samples to compare retention times and peak heights. TIC – total ion chromatogram EIC – extracted ion chromatogram BPC – base peak chromatogram The software also allows you to load chromatograms that are specialized for MS. These include: ? Total ion chromatogram (TIC) – chromatogram that sums all the ions at each time point ? Extracted ion chromatogram (EIC) – chromatogram of a chosen m/z value or range of values ? Base peak chromatogram (BPC) – chromatogram that represents the abundance of the largest peak of each spectrum in the data file Total ion chromatograms and base peak chromatograms are useful to find all the chromatographic peaks in the sample. Because of their specificity, extracted ion chromatograms are ideal for quantification. You can also use them to check for purity of chromatographic peaks. When chromatographic peaks are composed of more than one component, the EICs often fail to align.

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Data Analysis Loading signals

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Loading signals
Ways to load signals
The ChemStation software provides several ways to load chromatograms (signals): ? Select from the File menu.

Specify integration View file information

Select which signals to load (CTRL-click for multiple signals) Select instrument curves to display

Figure 46

The Load Signal dialog box lets you select which data files and signals to load.

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Data Analysis Loading signals ? Use the top toolbar.

Load Signals

Load signals and overlay with current signals

Figure 47

Tools to load signals.

? Use the Data file list in the ChemStation Explorer. (If necessary, first click the Data tab at the bottom.)

Batch Sequence Single runs

Figure 48

You can right-click the vials to view the menu, or double-click the vials to load multiple data files.

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Data Analysis Loading signals

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Refining your selection
Once you have loaded signals for a data file or files, the software allows you to refine your selection. See Figure 49 and Figure 50.

Click the “+” to expand Click overlay signals Double-click a to view signals from signal to view it multiple data files

Figure 49

You can view specific signals by double-clicking a signal (on left) or by right-clicking to display the menu at right.

Figure 50

You can view specific signals by selecting from the drop-down menu.

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Data Analysis Loading signals

More about signals
In addition to loading signals from a single data file, the File menu allows you to load signals from multiple files and to display extracted ion chromatograms and backgroundsubtracted chromatograms.

Load signal(s) from a data file Overlay signal(s) – can be from different data files Display extracted ion chromatograms Overlay a base peak chromatogram Subtract background spectrum from every point in the data file (see online help for BSB)

Figure 51

The File menu provides ways to load chromatograms and perform background subtraction.

In the ChemStation software, “overlay” means to simultaneously load more than one signal.

In the File menu, Overlay Signal allows you to compare signals from different samples or injections so you can examine fine differences. Note that while the term “overlay” may imply that you display the signals one on top the other in the same window, you actually control the display using buttons that you access from the Signal Toolset, the Purify Toolset, or the Graphics Toolset. These buttons are:
Display overlaid (In the Graphics Toolset, this button toggles between overlaid and separate.) Display separate

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Data Analysis Removing signals from the chromatogram display

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Removing signals from the chromatogram display
If you display too may signals at the same time, the individual displays can become too small or difficult to view. The software provides two ways to remove signals from the display: ? Select View > Window Functions > Delete Window. ? Use the tool within the Signal Toolset and the Purify Toolset that allows you to delete signals from your chromatogram display.

Changing how chromatograms are displayed
When you analyze data, it is very helpful to have control over the data display. For example, you may want to display chromatograms in separate windows or overlaid in a single window. Separate windows are often the best choice if you wish to view UV and MS chromatograms from the same analysis. Overlaid windows may be better if you need to compare fine differences between two complex samples. It is also helpful to be able to zoom in and out, to add annotations, to align chromatograms (for example, UV and MS chromatograms), and to control whether or not retention times and integration baselines are displayed. To accommodate these needs, the ChemStation software provides ways to customize the chromatogram display:

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Data Analysis Changing how chromatograms are displayed ? Select from the Graphics menu.

Define how chromatograms are displayed

Zoom in and out

Add and edit annotations

Figure 52

The Graphics menu provides ways to change how the software displays chromatograms.

? Use the toolsets that are described below. ? Graphics Toolset The graphics tools provide ways to change your signal display. You can add annotations and change whether compound names, retention times, baselines, titles, and axes are displayed. You can print windows and copy them to the clipboard. The icon to display the Graphics Toolset is located near the middle of the Data Analysis window. ? Signal Toolset The signal tools let you change the chromatogram display. You can change from a separate display to overlaid, align x- and y-axes, display signals full-scale or on the same scale, smooth chromatograms, subtract chromatograms (for example, subtract a blank run) and perform other operations on chromatograms.

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Working with spectra
With the ChemStation software, you can display both UV and mass spectra. In the ChemStation software, background spectra are called reference spectra. With MS data, each point in a chromatogram has an associated mass spectrum. You can display single spectra, averaged spectra, and background-subtracted spectra. For LC/MS data, you generally subtract a background spectrum from each analyte spectrum. When you subtract a background spectrum, you remove the ions that are attributable to mobile phase and other background. For UV signals, depending on the storage mode, there may be spectra only for the peak apexes or across the peaks. There is a spectrum for each point in the chromatogram only when you set Store to All when you set up the UV detector portion of the method.

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Data Analysis Displaying spectra

Displaying spectra
The ChemStation software provides several ways to display spectra: ? Select from the Spectra menu.

Select spectra Select background spectra for subtraction (background subtraction must also be enabled in Spectra Options) Examine peak purity by MS and/or UV Specify ion labeling

Set options for spectral display and specify how background subtraction is done (MS Reference)

Figure 53

The Spectra menu provides ways to select and display spectra.

? Use the toolsets that are described below. ? Spectrum Toolset The spectrum tools help you to quickly display and evaluate spectra. You can select spectra (including background spectra), zoom in and out, save spectra to a library, search a spectral library, set spectral options, select a chromatographic peak for purity analysis, and perform other tasks related to spectra. ? Calculation Toolset The calculation tools allow you to multiply, add, and subtract spectra. You access them from the icon shown here, which is part of the Spectrum Toolset. 94 Agilent 6100 Series Quadrupole LC/MS Systems Concepts Guide

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Peak purity
For many analyses, it is important to know whether a chromatographic peak represents a single component or multiple components. The ChemStation software allows you to evaluate peak purity using UV and/or MS data. To start, you select Spectra > Select Peak Purity. For detailed concepts about peak purity, see Agilent ChemStation for LC 3D Systems: Understanding Your Spectra Module.

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Data Analysis Performing quantification

Performing quantification
ESTD – external standard calibration ISTD – internal standard calibration Quantification is the process of determining how much of a compound is present in a sample. You perform quantification by comparing the peak areas or heights of analytes in your sample with those in standards of known concentration. To each standard and sample, you may add a constant amount of an internal standard that you can use to correct for slight variations in injection volume, detector sensitivity, etc. For quantification, it is best to use SIM mode for data acquisition because it generates more data points across a peak, which provides the best precision and accuracy. When you perform quantification, you typically analyze standards at concentrations that bracket those of your samples. Then you use the ChemStation software to generate a calibration curve for each analyte, and to determine the amount of each analyte in your sample.

A calibration curve is a plot of peak area or height on the y-axis versus concentration on the x-axis.

Integrating peaks
Integration – the process of measuring peak areas Qualifier ions – ions that are used to verify that the ion used for quantification is attributable to the analyte rather than an interference Determination of peak areas is a basic component of quantification. To avoid interferences, you use the peak areas from extracted ion chromatograms (EICs) rather than total ion chromatograms. You can use the EIC of the molecular ion for quantification and the EIC(s) of one or more fragment ions for confirmation. The latter are called qualifier ions, and they must be present in the proper ratio to confirm the presence of the analyte. In the ChemStation software, the Signal Details specify which extracted ion signals are used for quantification and which (if any) are used for qualifier ions. You can access the Signal Details under the Calibration menu, and you can save the Signal Details within the method. To ensure that peaks are properly integrated, you need to establish the appropriate integration parameters. The standard of lowest concentration usually presents the greatest challenge for integration, so it is best to use it to refine your integration 96 Agilent 6100 Series Quadrupole LC/MS Systems Concepts Guide

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settings. You can then save the integration parameters with the method and apply them to the higher-level standards and the samples. The ChemStation software provides more than one way to set integration parameters and perform peak integration: ? Select from the Integration menu.

Perform integration Establish integration events with an events table Display tabular integration results

Establish manual integration events

Use manual integration events in method

Figure 54

The Integration menu

? Use the Integration Toolset. The integration tools help you perform integration and generate reports. You can define integration events, perform automatic and manual integration, draw baselines, zoom in and out, specify a report style, and view and print reports.

NOTE

When you are done, be sure to save the integration settings with your method.

For more information about integration concepts, see Agilent ChemStation: Understanding your ChemStation.

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Data Analysis Calibration

Calibration
A calibration curve is a plot of peak area or height on the y-axis versus concentration on the x-axis. When you perform calibration, you insert the peak integrations from standards into a calibration table. The software uses the areas to generate a calibration curve. In the ChemStation software, each concentration of standard is called a “level.” After you integrate the standard of lowest concentration, you add it to the calibration table as level 1. You then add the standard of second-lowest concentration as level 2, and so on, until you have added all standards. Tip: If you have saved the Signal Details (see the Calibration menu) as part of the method, you can automatically load and integrate the extracted ion chromatograms from each standard. (See Figure 55.)

Figure 55

The highlighted features make it faster to add levels to a calibration curve.

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Data Analysis Calibration The ChemStation software provides the following ways to accomplish calibration: ? Select from the Calibration menu.

5

Set up a new calibration table Add a level to the calibration table Establish general settings like curve fit, ISTD/ESTD Set detailed options for the calibration table

Define the signals (such as EICs) to use for quantification

Figure 56

The Calibration menu

? Use the Calibration Toolset. The calibration tools provide a quick way to accomplish calibration tasks and generate reports. You can create a new calibration table, add levels and peaks to the table, recalibrate using the current chromatogram, and perform other calibration tasks. You can also specify a report style, and view and print reports.

NOTE

When you are done, be sure to save the calibration table with your method.

For more information about calibration concepts, see Agilent ChemStation: Understanding your ChemStation.

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Data Analysis Data review and sequence reprocessing

Data review and sequence reprocessing
The ChemStation software gives you two options to quickly review multiple data files. You can use either the Navigation Table or the batch review capabilities.

The Navigation Table
The Navigation Table shows the data files from a given folder and provides a convenient way to navigate between samples. You can use it for data review or to reprocess sequences.

Choose method for data review Click “+” for sample details (signals, etc.)

Data review tools

Sequence reprocessing tools

Click for help with Navigation Table

Figure 57

Navigation Table, shown with columns for sequence runs

Batch review
Batch – a user-selected series of data files from a sequence that is processed using a single user-defined method Batch review enables fast review of sequences or selections of analyses from a sequence. You can use batch review for either quantitative or qualitative analysis. Whenever you run a sequence, the software generates a batch file (with a .b extension) and places it in the same folder as the data files. This batch file contains pointers to the data files in the batch review itself.

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You load a batch through the Batch menu (Figure 58). When you load a batch, you have the opportunity to select the method to use for the batch, and to individually select the desired data files.

Load batch file and select method and data files to be included in batch review Preview and output report of results for the batch

Sort samples

View log of all actions related to the batch

Figure 58

The Batch menu

When you load a batch, the software displays (near the bottom of the Data Analysis window) the Batch Review toolbar, the batch table, and the compound list. For help, click the question mark on the toolbar.
Click for help

Compound table Batch table

Figure 59

Batch Review toolbar, batch table, and compound list

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Data Analysis Batch review

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6 Verification of Performance
The Verification (OQ/PV) View 104 Instrument verification 105 Setting up and running instrument verification 106 OQ/PV tests that are included with the software 108 Verification logbook 109 System verification 110 Overview 110 Setting up and running system verification 111

In this chapter, you learn the concepts that help you verify the performance of the hardware and software in your system. The ChemStation software includes tests and analytical methods that help you verify that your system is working properly. This chapter provides an introduction to that functionality.

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Verification of Performance The Verification (OQ/PV) View

The Verification (OQ/PV) View
Operational qualification/performance verification (OQ/PV) verifies and documents your instrument's ability to meet predefined performance criteria. You determine acceptance limits according to the specific needs of your lab. In general, an OQ/PV procedure performs several system tests, reports the results, and documents whether the system met the acceptance criteria. The Verification View, shown in Figure 60, allows you or an Agilent service engineer to accomplish OQ/PV.
Title bar Verification toolbar System diagram

Status bar Start/stop run Task selection menu Sampling diagram

Online plot View selection

Method overview Message line

Figure 60

The Verification View enables you to verify system performance. The frequency of OQ/PV depends on the specific needs of your lab. In general, you should perform OQ/PV after any major service. Your system may require OQ/PV more frequently if you need to meet regulations such as Good Laboratory Practice (GLP) and Good Manufacturing Practice (GMP), or quality or accreditation standards such as ISO 9000.

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Instrument verification
Instrument verification tests are used to test the performance of your LC/MS system. To qualify a complete LC system, you must perform a series of tests. The specific tests depend on which LC modules you have configured. The tests may include: ? Carryover, linearity and reproducibility of the injector ? Gradient performance of the pump ? Temperature accuracy and precision of the column compartment ? Noise, drift, linearity and wavelength accuracy of the UV detector ? Linearity of the MS detector ? Others, as required. You use the ChemStation to define which tests to run and the acceptance criteria for each. You obtain the appropriate standards from Agilent or your lab and load the autosampler with vials as specified by the software. Then the ChemStation runs the tests and calculates and prints a report that compares the actual results with specified acceptance criteria. Each report includes a pass/fail designation.

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Verification of Performance Setting up and running instrument verification

Setting up and running instrument verification
The ChemStation software provides several ways to set up and run instrument verification: ? Select File > New or File > Open. The software displays the dialog box shown in Figure 62. (Until you set up the first instrument verification with File > New, there are no files to open with File > Open.) ? Use the Verification Toolbar, shown in Figure 61.
Toggle display of logbooks Verification Current Sequence

New

Open Save Edit

Run

View report

Print report

Figure 61

Tools to work with instrument verification

When you set up a new instrument verification, open an existing instrument verification, or edit the current instrument verification, the software displays the Edit Instrument Verification dialog box shown in Figure 62.

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Available tests

Run verification

Selected tests

Set up parameters for selected test

Figure 62

The Edit Instrument Verification dialog box lets you determine the specific tests and their order.

The Setup button in Figure 62 is very important. When you click a scheduled test and click Setup, the software displays a dialog box that is specific to that test. Within the dialog box, you have the opportunity to: ? Set up specific conditions for the test (for example, which detector or flow channel to test) ? Establish acceptance limits for the test ? Click the Help button to get: ? General information about the test ? Specific test conditions, such as the required standards and mobile phase.

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Verification of Performance OQ/PV tests that are included with the software

OQ/PV tests that are included with the software
The ChemStation software incorporates a wide variety of verification tests. Some of the tests are: ? Flow accuracy and precision ? Injection precision and carryover ? Response linearity The Options menu, shown in Figure 63, determines the specific set of tests that are available at any given time.

Displays verification logbook

Determine which tests are available for selection

Figure 63

These options control the types of tests that you can select for instrument verification.

The online help includes complete lists of available tests, along with a description of each one. To access this information: 1 Open the help. 2 In the Contents tab within the tree view on the left, double-click User Interface Reference. 3 Double-click Menu Structure. 4 Double-click Verification (OQ/PV). 5 Click Options. 6 In the window on the right, click the link for enhanced test suite, capillary test suite, or preparative test suite. 7 Click any of the tests to view details.

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Verification logbook
The verification logbook records all events related to instrument verification. It contains a table that lists all the events, along with the date and time they occurred. The Instrument Verification menu gives you the opportunity to add notes to this logbook. You can open the verification logbook from either the Verification Toolbar or the Options menu, as shown in Figure 61 and Figure 63.

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Verification of Performance System verification

System verification
Overview
The system verification tests enable you to verify the correct and consistent operation of your ChemStation software. You can do this by reprocessing data files according to specific methods, and then comparing the results with a pre-defined standard. The verification tests are particularly important to prove the integrity of the integration and quantification results. You can use the standard Agilent verification test, or define your own tests using different method and data files. The Agilent verification test is a protected file and cannot be changed or deleted from outside the ChemStation software. If your system is equipped with an Agilent 1100/1200 Series Diode-Array Detector, the system verification tests also allow you to test the detector electronics.

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Setting up and running system verification
As shown in Figure 64, there are two ways to access the functionality to perform a system verification.

System verification menu

System verification task selection

Figure 64

The software gives two locations to set up and run system verification.

You use the Define Test function to define your own verification test. You specify both a data file and a method file, which will both be stored in a protected register. When you run the test, the software processes the data with the test method and compares the results to the original results. The software designates the results as either Pass or Fail.

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Agilent 6100 Series Quadrupole LC/MS Systems Concepts Guide

7 Maintenance and Troubleshooting
The Diagnosis View 114 Overview 114 Instrument panel 115 Logbooks 117 Maintenance 118 Early maintenance feedback 118 Maintenance logbook 119 Maintenance procedures 119 Venting and pumping down the MS 120 Diagnosing and fixing problems 121 Symptoms and causes 121 Diagnostic tests for the MS 122 Fixing problems 122

In this chapter, you learn the concepts that help you maintain and troubleshoot your Agilent 6100 Series LC/MS system. The Diagnosis View within the ChemStation software includes software tools for both maintenance and troubleshooting.

Agilent Technologies

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Maintenance and Troubleshooting The Diagnosis View

The Diagnosis View
Overview
The Diagnosis View provides the functionality to: ? Learn possible causes of system problems ? Perform diagnostic tests ? Pump down and vent the MS ? Get early maintenance feedback so you can perform maintenance before problems occur ? Record maintenance in a maintenance logbook ? Get details about modules in your system, such as serial numbers, firmware revisions, and hours of operation.
Title bar Observed symptoms Possible causes Cause information pad

Task selection menu Instrument panel Early maintenance feedback

View selection

Variables display Message line

Figure 65

The Diagnosis View helps you to troubleshoot and maintain your system.

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Instrument panel
Top-level display
The instrument panel at the center of the Diagnosis View shows all the modules in your system. (See Figure 66.) You can click each module to display a menu that enables you to do the following: ? Update Variables Display – Show module information, such as serial number, firmware revision, and LAN address, in the space just below the instrument panel. ? Show Module Details – Display a detailed schematic of the module, as shown in Figure 67 on page 116. ? Show Module Tests – Display a list of diagnostic tests for that module, as shown in Figure 71 on page 122.

Figure 66

Instrument panel – top-level display

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Maintenance and Troubleshooting Instrument panel

Module details
More menus Logbooks Tune Electronics Vacuum Get details for sub-module

Switch to top-level instrument panel

Figure 67

Instrument panel – module details for the MS.

As you move your mouse across the diagram shown in Figure 67, whenever the cursor changes from a pointer to a hand, you can click to display a menu that gives access to details for a sub-module. For example, you can click the icon for the turbomolecular pump to view vacuum readings, or the icon for the quadrupole to view its temperature. You can also click the icons in the white area on the right to display their menus.

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Logbooks
The Diagnosis View provides access to a number of logbooks that help with troubleshooting and maintenance. ? Error logbook – view recent errors for the module ? Runs logbook – view start and stop times of the most recent runs ? Maintenance logbook – view the most recent maintenance events. For more about this logbook, see “Maintenance logbook” on page 119. ? Date change logbook – view date changes You can access these logbooks several ways: ? The File menu (File > Open > Logbook) ? The Logbooks icon, shown in Figure 67 on page 116 (click the icon and select Update Variables Display) ? The Open icon on the top toolbar.

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Maintenance and Troubleshooting Maintenance

Maintenance
Proper maintenance of your LC/MS system helps to ensure the quality of your analyses and avoids expensive failures and unexpected downtime. The ChemStation software includes features that assist with a regular maintenance program.

Early maintenance feedback
Early maintenance feedback (EMF) alerts you to necessary maintenance so you can prevent problems from occurring. The software allows you to use your past experience to predict when each type of maintenance will be needed. You can enter the limits for the maintenance intervals within the Diagnosis View. Within both the Method and Run Control View and the Diagnosis View, when a maintenance limit is reached, the green EMF checkmark changes to a yellow question mark. At that point, you can click the EMF icon in the Diagnosis View to view the limits in the EMF Info Pad and to find out which module needs service. The limit for that module is highlighted in yellow.

Figure 68

EMF information pad

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Maintenance logbook
Maintenance records help to ensure that your system is kept in excellent condition, and are especially important when you have multiple operators. The ChemStation maintenance logbook provides a convenient place to record any maintenance that you perform on your LC modules or your MS. You access the maintenance logbook via the Logbooks icon, shown in Figure 67 on page 116. (Click the icon and select Maintenance Logbook Entry.) Then you manually enter the information via the ChemStation software. Rather than being stored on your computer disk, the maintenance logbook is stored in the memory within the LC or MS module. That way, if you move the module to another system, the maintenance record moves with the module.

Maintenance procedures
When you need to perform maintenance, you can find instructions for common procedures in the Agilent 6100 Series Single Quad LC/MS System Maintenance Guide, or in the manuals for the LC modules. You can conduct some maintenance procedures, such as wavelength calibration for a DAD detector, from the Maintenance menu in the Diagnosis View.

Figure 69

The Maintenance menu contains software-driven maintenance tasks for the MS and the LC modules.

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Maintenance and Troubleshooting Venting and pumping down the MS

Venting and pumping down the MS
Venting
Sometimes you need to perform maintenance on MS components that lie within the vacuum system. Examples of such maintenance include changing the electron multiplier and cleaning the ion optics. Before you can perform maintenance on these components, you need to vent the MS. Venting releases the vacuum in a controlled fashion. You use the Maintenance menu within the Diagnosis View to initiate the vent. (See Figure 69 on page 119.) The software executes a sequence of steps that safely vents the instrument. A dialog box allows you to view progress of the vent, and to reverse the procedure if necessary.

CAUTION

Do not vent the instrument by simply turning off the power button on the MS. Doing so will cause excessive wear on the turbomolecular pump.

Pumping down
After you have completed maintenance, you pump down the system. When you pump down, the vacuum pumps turn on in the correct order to bring the system safely under vacuum. To pump down, you simply turn on the MS power switch, which initiates the correct sequence of events. You can monitor and reverse the pump-down process from a dialog box that you access from the Maintenance menu.

Frequency of venting and pumping down
For details on putting the MS into Standby Mode, see the Basic Operation section of the Agilent 6100 Series Quad LC/MS Systems Quick Start Guide. The MS should normally be left under vacuum, even when not in use. Repeated venting and pump-down causes unnecessary wear on the turbomolecular pump. When you keep the system under vacuum, you maintain the level of cleanliness that you need for sensitive analyses. If you do not plan to use the MS for a period of time, leave it in Standby Mode rather than venting it.

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Diagnosing and fixing problems
Symptoms and causes
Figure 70 shows components of the software that help you troubleshoot an instrument problem in a step-by-step fashion. 1 You click either OQ/PV for a failed OQ/PV test or Analytic for a symptom that you observed during an analysis. 2 You select the specific symptom. 3 The software displays a list of possible causes. You select one of the causes. 4 The memo pad displays instrument data and tests you can run to confirm or refute that cause of the symptom. You click Start to display the dialog box for a test. 5 The software displays the dialog box for the test. You click Start to initiate the test, or Explain to view a detailed explanation of the test.
1 2 3 4

5

Figure 70

Overview of process to discover the root cause of instrument problems 121

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Maintenance and Troubleshooting Diagnostic tests for the MS

Diagnostic tests for the MS
There are a number of preprogrammed diagnostic tests that you can access for each module in your system. You can display a complete list of tests for each module by clicking the module icon in the instrument panel and selecting Show Module Tests. Figure 71 shows the tests for the MS.

Figure 71

Diagnostic tests for the MS

Fixing problems
When you have diagnosed a problem and need to fix it, you can find instructions for common procedures in the Agilent 6100 Series Single Quad LC/MS System Maintenance Guide. More serious problems may require that you call an Agilent customer engineer for service.

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www.agilent.com

In This Book
The Concepts Guide presents an overview of the Agilent 6100 Series Quadrupole LC/MS systems, to help you to understand how the hardware and software work.

? Agilent Technologies, Inc. 2007
Printed in USA First Edition, February 2007

*G1960-90030*
G1960-90030

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