Agilent 6100 User Manual

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Agilent 6100 Series Quadrupole LC/MS Systems

Concepts Guide

Agilent Technologies

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Notices

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G1960-90079

Edition

Revision A, September 2011

Printed in USA

Agilent Technologies, Inc. 5301 Stevens Creek Blvd. Santa Clara, CA 95051

Microsoft® is a U.S. registered trademark of Microsoft Corporation.

Software Revision

This guide is valid for the B.04.02 SPI1 or later revision of the Agilent ChemStation software for the Agilent 6100 Series Quadrupole LC/MS systems, until superseded.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 System Concepts Guide

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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.

1Overview

Learn how the hardware works in the Agilent 6100 Series Quadrupole LC/MS systems, and get a brief introduction to 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 concepts you need for qualitative and quantitative data analysis with ChemStation software.

5Reports

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

6 Verification of Performance

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

7 Maintenance and Troubleshooting

Agilent 6100 Series Quadrupole LC/MS System Concepts Guide 3

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Learn about tools that are proved in ChemStation software to help you maintain your system and diagnose and fix problems.

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1 Overview of Hardware and Software 9

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 19 Multiple signal acquisition 19

Ion sources 22

Electrospray ionization (ESI) 22 Atmospheric pressure chemical ionization (APCI) 28 Atmospheric pressure photoionization (APPI) 30 Multimode ionization (MMI) 31

Introduction to ChemStation software 32

Overview 32 Reviewing data remotely 34

2 Instrument Preparation 35

Preparation of the LC system 36

Purpose 36 Summary of procedures 36 Setting parameters for LC modules 38 Column conditioning and equilibration 39 Monitoring the stability of flow and pressure 41

Preparation of the MS – tuning 42

Agilent 6100 Series Quadrupole LC/MS System Concepts Guide 5

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Contents

Overview 42 Ways to tune 44 When to tune – Check Tune 45 Autotune 47 Manual tuning 49 Tune reports 51 Gain calibration 53

3 Data Acquisition 57

Working with methods 58

Method and Run Control View 58 Loading, editing, saving and printing methods 60 More on editing methods 61

Running samples 64

Running a single sample 65 Running a sequence 66 Flow injection analysis 69

Monitoring analyses 73

Online signal plots 73 Quick method overview 74 Logbooks 74

Instrument shutdown 76

4 Data Analysis 77

The Data Analysis View 78

Loading and manipulating chromatograms 80

Loading signals 81 Removing signals from the chromatogram display 85 Changing how chromatograms are displayed 85

Working with spectra 87

Displaying spectra 88

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Peak purity 89

Performing quantification 90

Integrating peaks 90 Calibration 92

Data review and sequence reprocessing 94

The Navigation Table 94 Batch review 94

5Reports97

Using predefined reports 98

Generating reports 98 Report styles 99

Defining custom reports 101

Summary of process 101 Example report templates 101 The Report Layout View 102

Contents

6 Verification of Performance 105

The Verification (OQ/PV) View 106

Instrument verification 107

Setting up and running instrument verification 108 Available OQ/PV tests 110 Verification logbook 111

System verification 112

Overview 112 Setting up and running system verification 113

7 Maintenance and Troubleshooting 115

The Diagnosis View 116

Overview 116 Instrument panel 117

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Contents

Logbooks 119

Maintenance 120

Early maintenance feedback 120 Maintenance logbook 121 Maintenance procedures 122 Venting and pumping down the MS 122

Diagnosing and fixing problems 124

Symptoms and causes 124 Diagnostic tests for the MS 125 Fixing problems 126

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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 19 Multiple signal acquisition 19

Ion sources 22

Electrospray ionization (ESI) 22 Atmospheric pressure chemical ionization (APCI) 28 Atmospheric pressure photoionization (APPI) 30 Multimode ionization (MMI) 31

Introduction to ChemStation software 32

Overview 32 Reviewing data remotely 34

This chapter provides an overview of the hardware and software that comprises the Agilent 6100 Series Quadrupole LC/MS systems. The family consists of three models: 6120B, 6130B, and 6150B.

Agilent Technologies

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1 Overview of Hardware and Software

capillary

nebulizer

HPLC inlet

ion source

rough pump

split-flow turbo pump

detector

quadrupole mass filter

ion optics

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.

API – atmospheric pressure

ionization

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.

Figure 1 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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Details

4321Vacuum stage:

Ion source Ion transport and focusing region

quadrupole

capillary

nebulizer

HPLC inlet

fragmentation zone (CID)

detector

skimmer

octopole

lenses

3 torr

5X10

-6

torr

Overview of Hardware and Software 1

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.

Agilent 6100 Series Quadrupole LC/MS System Concepts Guide 11

Figure 2 Ion path for Agilent 6130 and 6150 Quadrupole LC/MS sys-

tems

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1 Overview of Hardware and Software

4321

Vacuum stage:

Ion source Ion transport and focusing region

quadrupole

capillary

nebulizer

HPLC inlet

fragmentation zone (CID)

detector

skimmers

octopole

lenses

2 torr

6X10

-6

torr

Details

Figure 3 Ion path for Agilent 6120 Quadrupole LC/MS system

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

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.

of solvent are transmitted through the capillary.

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 of the capillary is a metal skimmer with a small hole. Heavier

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CID – collision-induced

dissociation

Overview of Hardware and Software 1

Details

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

• Protonated molecules [M + H]

• Simple adduct ions [M + Na]

or M

• Ions representing simple losses, such as the loss of a

water molecule [M + H - H

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 6100 Series Quadrupole LC/MS System Concepts Guide 13

Agilent 6130 and 6150 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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1 Overview of Hardware and Software

From ion source

To d e t ec t or

Details

Agilent 6120 Quadrupole LC/MS system 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.

Figure 4 Quadrupole mass analyzer

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Types of data you can acquire

m/z

1 scan

SIM

scan

discrete masses

mass range

abundance

time

Scan versus selected ion monitoring (SIM)

Overview of Hardware and Software 1

Types of data you can acquire

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.

Figure 5 A quadrupole mass analyzer can operate in either scan mode

or selected ion monitoring (SIM) mode

Scan mode

Agilent 6100 Series Quadrupole LC/MS System Concepts Guide 15

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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1 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.

16 Agilent 6100 Series Quadrupole LC/MS System Concepts Guide

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.

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Overview of Hardware and Software 1

m/z

100

200

300

50000

100000

150000

200000

250000

300000

350000

279.1

301.0

280.0

281.0

[M + Na]

[M + H]

Generation of fragment ions: low versus high fragmentor

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.)

Figure 6 Mass spectrum of sulfamethazine – low fragmentor

Agilent 6100 Series Quadrupole LC/MS System Concepts Guide 17

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m/z

100

200

300

20000

40000

60000

[M + H]

[M + Na]

80000

124.1

186.0

279.1

156.1

108.2

301.0

323.0

213.2

107.1

280.1

125.1

187.0

157.1

m/z

156

m/z

186

m/z

124

m/z

213

m/z

108

1 Overview of Hardware and Software

Generation of fragment ions: low versus high fragmentor

Figure 7 Mass spectrum of sulfamethazine – high fragmentor

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.

FIA – flow injection analysis The ideal fragmentation voltage depends on the structure of

It is possible to produce both molecular ions and fragment ions within the same spectrum by using an intermediate fragmentation voltage.

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

18 Agilent 6100 Series Quadrupole LC/MS System Concepts Guide

(FIA) series. An FIA series allows you to inject the compound multiple times within the same run, and to vary the fragmentor

Page 19

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

Overview of Hardware and Software 1

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.

Multiple signal acquisition

You establish the conditions for multiple signal acquisition 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.

The Agilent 6120, 6130 and 6150 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.

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1 Overview of Hardware and Software

Multiple signal acquisition

Polarity switching

The Agilent 6120, 6130 and 6150 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 6150 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 6150 LC/MS models allow you to alternate between SIM and scan modes, so you can accomplish both goals in a single analysis.

Putting it all together

The 6120, 6130 and 6150 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

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Overview of Hardware and Software 1

Multiple signal acquisition

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.

Agilent 6100 Series Quadrupole LC/MS System Concepts Guide 21

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1 Overview of Hardware and Software

NOTE

Ion sources

The Agilent 6100 Series Quadrupole LC/MS systems operate with the following interchangeable atmospheric pressure ionization (API) sources:

• ESI (electrospray ionization)

• ESI with Agilent Jet Stream technology

• APCI (atmospheric pressure chemical ionization)

• APPI (atmospheric pressure photoionization)

• MMI (multimode ionization)

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.

22 Agilent 6100 Series Quadrupole LC/MS System Concepts Guide

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 1

heated drying gas

capillary

nebulizer

HPLC inlet

solvent spray

Electrospray ionization (ESI)

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.

Agilent 6100 Series Quadrupole LC/MS System Concepts Guide 23

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1 Overview of Hardware and Software

Electrospray ionization (ESI)

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).

24 Agilent 6100 Series Quadrupole LC/MS System Concepts Guide

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Overview of Hardware and Software 1

+++

evaporation analyte ion ejected

Electrospray ionization (ESI)

Figure 9 Desorption of ions from solution

Coulomb repulsion – repulsion

between charged species of the

same sign

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 10 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.

V/cm3, ion

Agilent 6100 Series Quadrupole LC/MS System Concepts Guide 25

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1 Overview of Hardware and Software

Electrospray ionization (ESI)

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.

Agilent Jet Stream Technology

The Agilent Jet Stream technology is supported on compatible Agilent 6100 Series LC/MS system.

Agilent Jet Stream Technology enhances analyte desolvation by collimating the nebulizer spray and creating a dramatically “brighter signal.” The addition of a collinear, concentric, super-heated nitrogen sheath gas (Figure 10) to the inlet assembly significantly improves ion drying from the electrospray plume and leads to increased mass spectrometer signal to noise allowing the triple quadrupole to surpass the femtogram limit of detection. The Agilent Jet Stream Technology is patent pending.

26 Agilent 6100 Series Quadrupole LC/MS System Concepts Guide

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Overview of Hardware and Software 1

Electrospray ionization (ESI)

Figure 10 Electrospray Ion Source with Agilent Jet Stream Technology

Agilent Jet Stream thermal gradient focusing consists of a superheated nitrogen sheath gas that is introduced collinear and concentric to the pneumatically assisted electrospray. Thermal energy from the superheated nitrogen sheath gas is focused to the nebulizer spray producing the most efficient desolvation and ion generation possible. The enhanced molecular ion desolvation results in more ions entering the sampling capillary as shown in Figure 10 and concomitant improved signal to noise. Parameters for the Agilent Jet Stream Technology are the superheated nitrogen sheath gas temperature and flow rate and the nozzle voltage.

Agilent 6100 Series Quadrupole LC/MS System Concepts Guide 27

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1 Overview of Hardware and Software

drying gas

capillary

nebulizer (sprayer)

HPLC inlet

vaporizer

corona

(heater)

discharge needle

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 11).

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.

Figure 11 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.

28 Agilent 6100 Series Quadrupole LC/MS System Concepts Guide

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Overview of Hardware and Software 1

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.

Agilent 6100 Series Quadrupole LC/MS System Concepts Guide 29

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1 Overview of Hardware and Software

drying gas

capillary

nebulizer (sprayer)

HPLC inlet

vaporizer

UV lamp

(heater)

hν

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 12). 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.

Figure 12 Atmospheric pressure photoionization (APPI) source

30 Agilent 6100 Series Quadrupole LC/MS System Concepts Guide

Page 31

Multimode ionization (MMI)

drying gas

capillary

nebulizer

HPLC inlet

APCI

thermal container

ESI zone

zone

corona discharge needle

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.

Overview of Hardware and Software 1

Multimode ionization (MMI)

Figure 13 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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1 Overview of Hardware and Software

Introduction to ChemStation software

Overview

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 14 These buttons allow you to switch among the six ChemSta-

tion views

The following summarizes the ChemStation views and their functionality:

For more information about the Method and Run Control view, see

Chapter 3.

32 Agilent 6100 Series Quadrupole LC/MS System Concepts Guide

Method and Run Control

• Set up methods

• Change setpoints for the Agilent 1100/1200/1260/1290

Series LC 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 1

Overview

For more information about the Data Analysis view, see Chapter 4.

For more information about the Report Layout view, see Chapter 5.

For more information about the Verification view, see Chapter 6.

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

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

Report Layout

• Design custom report templates

Verificat ion (OQ/PV)

• Verify system performance

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.

Agilent 6100 Series Quadrupole LC/MS System Concepts Guide 33

MSD Tune

• Optimize and calibrate the MS

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1 Overview of Hardware and Software

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 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.

Another way is to install the Analytical Studio Reviewer on the remote computer. Analytical Studio Reviewer lets you easily review ChemStation LC and LC/MS data files, but the functionality is different than with the full ChemStation Data Analysis. The Analytical Studio Reviewer software is ideal for synthetic chemists and others who use the LC/MS system for “walk-up” analysis.

34 Agilent 6100 Series Quadrupole LC/MS System Concepts Guide

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Agilent 6100 Series Quadrupole LC/MS Systems Concepts Guide

2 Instrument Preparation

Preparation of the LC system 36

Purpose 36 Summary of procedures 36 Setting parameters for LC modules 38 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 53

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.

Agilent Technologies

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2 Instrument Preparation

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 2

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 f low and pressure are stable.

For more information, see “Monitoring the stability of flow

and pressure” on page 41.

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.

Agilent 6100 Series Quadrupole LC/MS System Concepts Guide 37

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2 Instrument Preparation

Injection

Pump

Mass spectrometer

Diode-array detector

Column thermostat,

Column switching valve

Solvent bottles

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.

Figure 15 Example system diagram (yours may be different)

To access help for any system module, click Help on the module context menu. To access help for a given dialog box, click the Help button on the dialog box.

To set module control parameters

This procedure uses the pump module as an example.

1 Click More Pump > Control HPLC Pump on the

Instrument menu to open the Pump Control dialog box.

2 Set desired control parameters and click OK.

Alternate

method

38 Agilent 6100 Series Quadrupole LC/MS System Concepts Guide

Select the desired control parameter such as Standby from the Pump context menu.

Page 39

Column conditioning and equilibration

To set module setpoint parameters

This procedure uses the binary pump module as an example.

1 Click Set up Instrument Method on the Instrument menu

to open the Setup Method dialog box.

2 Click the BinPump tab.

3 Set desired setpoints and click OK.

To access other instrument parameters

1 Click to open the Instrument menu.

2 Click the desired command such as Select Injection

Source, Columns, or Instrument Configuration.

Column conditioning and equilibration

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

Instrument Preparation 2

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.

Agilent 6100 Series Quadrupole LC/MS System Concepts Guide 39

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2 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.

Column conditioning and equilibration

You can condition and equilibrate a column in one of three ways with 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.

40 Agilent 6100 Series Quadrupole LC/MS System Concepts Guide

• 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.

Page 41

Instrument Preparation 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 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.

Agilent 6100 Series Quadrupole LC/MS System Concepts Guide 41

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2 Instrument Preparation

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.

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Instrument Preparation 2

Overview

Tune files and reports

The product of tuning is a tune file (actually a directory) that contains parameter settings for both positive and negative ionization. When data acquisition uses a tune file, the settings appropriate for the ion polarity specified by the data acquisition method are loaded automatically.

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.

Agilent 6100 Series Quadrupole LC/MS System Concepts Guide 43

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2 Instrument Preparation

NOTE

CAUTION

Ways to tune

Access this functionality via the Tune menu in the MSD Tune view.

ChemStation software provides the following 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.

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.

44 Agilent 6100 Series Quadrupole LC/MS System Concepts Guide

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.

Page 45

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 16 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 53.

Instrument Preparation 2

When to tune – Check Tune

Agilent 6100 Series Quadrupole LC/MS System Concepts Guide 45

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2 Instrument Preparation

All masses pass the mass axis and peak width tests

When to tune – Check Tune

Figure 16 Check Tune report

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Autotune

Instrument Preparation 2

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 up to six choices for running an autotune, depending on 6100 model:

• 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 53.

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2 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.

48 Agilent 6100 Series Quadrupole LC/MS System Concepts Guide

Page 49

Manual tuning

CAUTION

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

• When you want to tune specifically for the very low end

• When you want to tune with a compound other than the

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.

Instrument Preparation 2

Manual tuning

sacrificing some resolution

of the mass range (< 150 u)

standard calibrants.

Agilent 6100 Series Quadrupole LC/MS System Concepts Guide 49

Tips for manual tuning

Ionization

modes

Polarity Separate tuning is required for positive and negative

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.

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. Positive ion and negative ion settings can be updated separately.

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2 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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Tune reports

Instrument Preparation 2

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.

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2 Instrument Preparation

Tune reports

Figure 17 Tune report (page 1 of 4)

52 Agilent 6100 Series Quadrupole LC/MS System Concepts Guide

Page 53

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.

Instrument Preparation 2

Gain calibration

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.

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2 Instrument Preparation

Gain calibration

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 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.

More gain is not always better

In general, it is best to 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.

54 Agilent 6100 Series Quadrupole LC/MS System Concepts Guide

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

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Instrument Preparation 2

Gain calibration

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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2 Instrument Preparation

Gain calibration

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Agilent 6100 Series Quadrupole LC/MS Systems Concepts Guide

3 Data Acquisition

Working with methods 58

Method and Run Control View 58 Loading, editing, saving and printing methods 60 More on editing methods 61

Running samples 64

Running a single sample 65 Running a sequence 66 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.

Agilent Technologies

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3 Data Acquisition

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 18.

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 3

View Selection

Tool Set Selection: Sequence or Single Sample

Message Line

Run Control

System Diagram

Top To ol ba r

Online Plot

Method Overview

File List

Method Sequence

Data Analysis

Status Bar

MS, FIA

Disk Space

Method and Run Control View

Figure 18 In this view, you set up methods, establish instrument settings, and inject samples.

Status and Run Bars: Main Tab Right-click the Method icon to see the following commands:

Agilent 6100 Series Quadrupole LC/MS System Concepts Guide 59

Run Time Checklist, Method Information, Edit Entire Method, Method Audit Trail, Print Method, and Help.

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3 Data Acquisition

Sequence

Single sample

Load a

Save

Edit entire MethodMethod method

Select method from list of recent methods

current

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, which allows you to load,

• Use the Method icon in the Run Control Bar.

• Use the toolbar or method drop-down list.

Figure 19 Toolset selection

edit, save, and print methods.

Right-click the icon to access the context menu.

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 19.

Figure 20 When you click the icon for the Single Sample Toolset, you access tools to work with methods.

• Use the ChemStation Explorer pane on the left side of the

Method and Run Control view.

Click the Method tab at the bottom to display the method files.

Double-click a method to load it.

Right-click in the white area to view the context menu, which contains options such as Load Method.

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More on editing methods

NOTE

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 62.

Editing the entire method

You can edit the entire method from the Method menu or the Edit entire Method icon in the Run Control Bar or the Method icon in the Status Bar. If you elect to edit the entire method, the appropriate dialog boxes are displayed in succession. The first dialog box (Figure 21) lets you select the parts of the method to view and edit. For more information as you edit the method, click the Help button in each dialog box.

Data Acquisition 3

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.

64 Agilent 6100 Series Quadrupole LC/MS System Concepts Guide

FIA icon

Figure 24 To run samples, you first click the appropriate icons.

For FIA, you must modify the method to enable multiple injections in the same run.

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Running a single sample

Single Sample

Click to enter sample information

Click to start the run

After you have set up and saved a method, you are ready to run a single sample interactively as shown below. First you click the icon to display the Single Sample Toolset.

Data Acquisition 3

Running a single sample

Agilent 6100 Series Quadrupole LC/MS System Concepts Guide 65

Figure 25 How to enter sample information and start the run.

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3 Data Acquisition

Sequence

Running a sequence

To run samples in an automated, unattended sequence, you first click the icon to display the Sequence Toolset. The Sequence icon on the left provides access to the Sequence Toolset and the sampling diagram that shows the sample tray.

Figure 26 Sequence Toolset selection icon.

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

sequences.

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Page 67

• Use the toolbar or sequence drop-down list.

Load Save Select sequence from

list of recent sequences

Sequence Sequence

NOTE

Figure 28 Tools to work with sequences

• Use the Method and Run Control file list. If necessary,

• Click the sampling diagram to access the menu.

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 30).

Data Acquisition 3

Flow injection analysis

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3 Data Acquisition

Click to enable FIA or set up FIA series

FIA icon

NOTE

Flow injection analysis

Figure 30 Two ways to enable FIA within a method

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 31. If you decide later to add a setpoint, you can use the insert row/append row functionality.

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.

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Data Acquisition 3

Automatically build an FIA table

Mark check box first

Flow injection analysis

Figure 31 The FIA series is a convenient way to optimize MS parame-

ters. The example shows a test of fragmentor voltage.

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3 Data Acquisition

Add rows manually or automatically

Flow injection analysis

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 only the vial numbers. Alternatively, you can use the insert row/append row functionality to add samples.

Figure 32 shows an example of FIA for a linearity check.

72 Agilent 6100 Series Quadrupole LC/MS System Concepts Guide

Figure 32 For pure samples, the FIA series can be used to check sensi-

tivity or linearity. The example shows a linearity check.

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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Monitoring analyses

Online signal plots

Data Acquisition 3

Monitoring analyses

During an analysis, you can view online signals, as shown in

Figure 33. (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 33 Online plot

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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 34 Quick method overview, showing LC settings

Logbooks

Several logbooks are important in the Method and Run Control view. You can view logbooks from the View menu or the logbook icons as show in Figure 35.

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.

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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Data Acquisition 3

Current logbook icon

Sequence logbook icon

Logbooks

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.

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).

Figure 35 How to open logbooks with icons.

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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 Data Analysis

The Data Analysis View 78 Loading and manipulating chromatograms 80

Loading signals 81 Removing signals from the chromatogram display 85 Changing how chromatograms are displayed 85

Working with spectra 87

Displaying spectra 88 Peak purity 89

Performing quantification 90

Integrating peaks 90 Calibration 92

Data review and sequence reprocessing 94

The Navigation Table 94 Batch review 94

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, double-click User Interface Reference, then double-click Toolbars in online Help.

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4 Data Analysis

The Data Analysis View

The Data Analysis view, shown in Figure 36, 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

• Integrate peaks and perform quantification

• Check peak purity

• Perform deconvolution (with the optional Agilent LC/MSD

• Generate reports from predefined report templates

• Reprocess data from sequences.

detectors

Deconvolution & Bioanalysis Software)

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Data Analysis 4

Title bar

LC/MS signal

Message line

Toolset selection

Top toolbar

DAD signal

File list (ChemStation Explorer)

Cursor tools

View selection

Navigation Ta bl e

Signal view selection

Navigation toolbar Graphics tools

The Data Analysis View

Figure 36 The Data Analysis view provides menus and tools for qualitative and quantitative analysis.

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Loading and manipulating chromatograms

In ChemStation software, chromatograms are called signals.

TIC – total ion chromatogram

EIC – extracted ion chromatogram

BPC – base peak chromatogram

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. 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.

Specialized chromatograms available for MS data 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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Loading signals

View file information Select instrument curves to display

Specify integration

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

Ways to load signals

You can load chromatograms (signals) in the following ways:

• Select from the File menu.

Data Analysis 4

Loading signals

Figure 37 The Load Signal dialog box lets you select which data files and signals to load.

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4 Data Analysis

Load Signals

Load signals and overlay with current signals

Batch

Single runs

Sequence

Loading signals

• Use the top toolbar.

Figure 38 Tools to load signals.

• Use the Data file list in the ChemStation Explorer.

(If necessary, first click the Data tab at the bottom.)

Figure 39 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 4

Double-click a signal to view it

Click overlay signals to view signals from multiple data files

Click the “+” to expand

Loading signals

Refining your selection

Once you have loaded signals for a data file or files, you can refine your selection as shown in Figure 40 and Figure 41.

Figure 40 You can view specific signals by double-clicking a signal (on

left) or by right-clicking to display the menu at right.

Figure 41 You can view specific signals by selecting from the signal list.

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4 Data Analysis

Overlay signal(s) – can be from different data files

Load signal(s) from a data file

Display extracted ion chromatograms

Overlay a base peak chromatogram

Subtract background spectrum from every point in the data file (see online Help for BSB)

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.

Figure 42 The File menu provides ways to load chromatograms and per-

form background subtraction.

In 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.)

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Display separate

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Data Analysis 4

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. If so, you can remove signals from the display in either of the following ways:

• 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.

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4 Data Analysis

Define how chromatograms are displayed

Zoom in and out

Add and edit annotations

Changing how chromatograms are displayed

The chromatogram display can be customized as follows:

• Select from the Graphics menu.

Figure 43 The Graphics menu provides ways to change how the chro-

matograms are displayed.

• 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

Data Analysis 4

Working with spectra

With ChemStation software, you can display both UV and mass spectra.

In 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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4 Data Analysis

Select spectra

Select background spectra for subtraction (background subtraction must also be enabled in Spectra Options)

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

Specify ion labeling

Examine peak purity by MS and/or UV

Displaying spectra

You can display spectra in the following different ways:

• Select from the Spectra menu.

Figure 44 The Spectra menu provides ways to select and display spec-

tra.

• 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.

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Peak purity

Data Analysis 4

Peak purity

For many analyses, it is important to know whether a chromatographic peak represents a single component or multiple components. 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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Performing quantification

ESTD – external standard

calibration

ISTD – internal standard calibration

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

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 generate a calibration curve for each analyte, and to determine the amount of each analyte in your sample, using ChemStation software.

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

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Data Analysis 4

Perform integration

Establish integration events with an events table

Display tabular integration results

Establish manual integration events

Use manual integration events in method

NOTE

Integrating peaks

refine your integration settings. You can then save the integration parameters with the method and apply them to the higher-level standards and the samples.

Set integration parameters and perform peak integration in the following different ways:

• Select from the Integration menu.

Figure 45 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.

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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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 area responses are used to generate a calibration curve.

In 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 46.)

Figure 46 The highlighted features make it faster to add levels to a cali-

bration curve.

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Data Analysis 4

Set up a new calibration table

Establish general settings like curve fit, ISTD/ESTD

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

Add a level to the calibration table

Set detailed options for the calibration table

NOTE

Calibration

Access to calibration tools and commands are provided in the following ways in ChemStation software:

• Select from the Calibration menu.

Figure 47 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.

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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4 Data Analysis

Sequence reprocessing toolsData review tools

Choose method for data review

Click “+” for sample details (signals, etc.)

Click for help with Navigation Table

Data review and sequence reprocessing

ChemStation software gives you two ways to quickly review multiple data files: the Navigation Table and Batch review described below.

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.

Figure 48 Navigation Table, shown with columns for sequence runs

Batch – a user-selected series of

data files from a sequence that is

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Batch review

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, a batch file (with a .b extension) is generated and saved 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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Data Analysis 4

Preview and output report of results for the batch

Load batch file and select method and data files to be included in batch review

View log of all actions related to the batch

Sort samples

Click for help

Batch table

Compound table

Batch review

You load a batch through the Batch menu (Figure 49). 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.

Figure 49 The Batch menu

When you load a batch, the Batch Review toolbar, the batch table, and the compound list are displayed automatically. For help, click the question mark on the toolbar.

Figure 50 Batch Review toolbar, batch table, and compound list

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Batch review

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Agilent 6100 Series Quadrupole LC/MS Systems Concepts Guide

5 Reports

Using predefined reports 98

Generating reports 98 Report styles 99

Defining custom reports 101

Summary of process 101 Example report templates 101 The Report Layout View 102

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 online Help double-click User Interface Reference, then double-click Toolbars.

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5Reports

Specify report style, destination, etc.

Edit parameters for system performance reports Specify how library search is done

Calculate and print report

Calculate and preview report

Specify report

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. ChemStation software includes a number of predefined report styles, and you can generate new styles for custom reports.

Generating reports

Ways to generate reports in Data Analysis view

• Select from the Report menu.

Figure 51 The Report menu provides ways to print reports and set prop-

erties 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.

Figure 52 Tools to work with reports

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Report styles

NOTE

Reports 5

Report styles

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.

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

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)

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5Reports

Report styles

• Extracted ion chromatograms and spectra for each

integrated peak.

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Agilent 6100 User Manual

Specifications and Main Features

Frequently Asked Questions

User Manual

Contents

How the Agilent quadrupole LC/MS systems work

Overview

Details

Types of data you can acquire

Scan versus selected ion monitoring (SIM)

Generation of fragment ions: low versus high fragmentor

Positive versus negative ions

Multiple signal acquisition

Ion sources

Electrospray ionization (ESI)

Atmospheric pressure chemical ionization (APCI)

Atmospheric pressure photoionization (APPI)

Multimode ionization (MMI)

Introduction to ChemStation software

Overview

Reviewing data remotely

Preparation of the LC system

Purpose

Summary of procedures

Setting parameters for LC modules

Column conditioning and equilibration

Monitoring the stability of flow and pressure

Preparation of the MS – tuning

Overview

Ways to tune

When to tune – Check Tune

Autotune

Manual tuning

Tune reports

Gain calibration

Working with methods

Method and Run Control View

Loading, editing, saving and printing methods

More on editing methods

Running samples

Running a single sample

Running a sequence

Flow injection analysis

Monitoring analyses

Online signal plots

Quick method overview

Logbooks

Instrument shutdown

The Data Analysis View

Loading and manipulating chromatograms

Loading signals

Removing signals from the chromatogram display

Changing how chromatograms are displayed

Working with spectra

Displaying spectra

Peak purity

Performing quantification

Integrating peaks

Calibration

Data review and sequence reprocessing

The Navigation Table

Batch review

Using predefined reports

Generating reports

Report styles