Agilent 6100 User Manual

Agilent 6100 Series Quadrupole LC/MS Systems

Concepts Guide

Agilent Technologies

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G1960-90079

Edition

Revision A, September 2011

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Agilent 6100 Series Quadrupole LC/MS System Concepts Guide

In This Guide...

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

If you have any comments about this guide, please send an e-mail to feedback_lcms@agilent.com.

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

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

4 Agilent 6100 Series Quadrupole LC/MS System Concepts Guide

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

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

6 Agilent 6100 Series Quadrupole LC/MS System Concepts Guide

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

Agilent 6100 Series Quadrupole LC/MS System Concepts Guide 7

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

8 Agilent 6100 Series Quadrupole LC/MS System Concepts Guide

Agilent 6100 Series Quadrupole LC/MS Systems Concepts Guide

1 Overview of Hardware and Software

How the Agilent quadrupole LC/MS systems work 10

Overview 10 Details 11

Types of data you can acquire 15

Scan versus selected ion monitoring (SIM) 15 Generation of fragment ions: low versus high fragmentor 16 Positive versus negative ions 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

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.

10 Agilent 6100 Series Quadrupole LC/MS System Concepts Guide

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

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

12 Agilent 6100 Series Quadrupole LC/MS System Concepts Guide

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.

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

14 Agilent 6100 Series Quadrupole LC/MS System Concepts Guide

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.

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.

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

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

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.

Agilent 6100 Series Quadrupole LC/MS System Concepts Guide 19

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

20 Agilent 6100 Series Quadrupole LC/MS System Concepts Guide

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

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.

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

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

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

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

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

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

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

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

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