WIXLIB

Overview of Hardware and SoftwareDetails1aperture. Most of the lighter drying gas (nitrogen) molecules aredeflected by the skimmer and pumped away by a rough pump.The ions that pass through the skimmer move into the secondstage of the vacuum system.The atmospheric pressure ionization techniques are allrelatively “soft” techniques. They generate primarily: Molecular ions M or M Protonated molecules [M H] Simple adduct ions [M Na] Ions representing simple losses, such as the loss of a watermolecule [M H - H2O] CID – collision-induceddissociationThese types of ions give molecular weight information, but youoften need complementary structural information. To gainstructural information, you can fragment the analyte ions in thefirst vacuum stage. To do that, you give them extra energy andcollide them with neutral molecules in a process known ascollision-induced dissociation (CID). A voltage is applied at theend of the atmospheric sampling capillary to add energy to thecollisions and create more fragmentation. For moreinformation, see “Generation of fragment ions: low versus highfragmentor” on page 16.Ion transport (second and third vacuum stages)An octopole ion guide is a set ofsmall parallel metal rods with acommon open axis through whichthe ions can pass.LC/MSD, LC/MSD XT, 6130 and 6150 In the second vacuumstage, the ions are immediately focused by an octopole ion guidethat traverses two vacuum stages. The ions pass through theoctopole ion guide because of the momentum they receivedfrom being drawn from atmospheric pressure through thesampling capillary. Radio-frequency voltage applied to theoctopole rods repels ions above a particular mass range to theopen center of the rod set. The ions exit this ion guide and thenpass through two focusing lenses into the fourth stage of thevacuum system.Agilent InfinityLab LC/MSD Series and 6100 Series LC/MS Concepts Guide13

1Overview of Hardware and SoftwareDetails6120 In the second vacuum stage, the ions are transportedbetween skimmer 1 and skimmer 2. They then enter the thirdvacuum stage, where they pass through the octopole ion guide.The ions exit this ion guide and then pass through two focusinglenses into the fourth stage of the vacuum system.Ion separation and detection (fourth vacuum stage)In the fourth vacuum stage, the quadrupole mass analyzerseparates the ions by mass-to-charge ratio. An electronmultiplier then detects the ions.m/z – mass/charge ratioThe quadrupole mass analyzer (Figure 4) consists of fourparallel rods to which specific direct-current (DC) andradio-frequency (RF) voltages are applied. The analyte ions aredirected down the center of the rods. Voltages applied to therods generate electromagnetic fields. These fields determinewhich mass-to-charge ratio of ions can pass through the filter ata given time. The ions that pass through are focused on thedetector.To detectorFrom ionsourceFigure 414Quadrupole mass analyzerAgilent InfinityLab LC/MSD Series and 6100 Series LC/MS Concepts Guide

Overview of Hardware and SoftwareTypes of data you can acquire1Types of data you can acquireScan versus selected ion monitoring (SIM)As shown in Figure 5, quadrupole mass analyzers can operatein two modes. To get the most from your analysis, it isimportant to pick the appropriate mode. The discussion belowwill help you choose.1 scanm/zscanabundanceYou set up a scan or SIM analysisin the Method and Run Controlview, described in Chapter 3.m/ztime1 scanabundancediscrete massesm/zSIMtimeFigure 5mass rangem/zA quadrupole mass analyzer can operate in either scan modeor selected ion monitoring (SIM) modeScan modeIn scan mode, a range of m/z values are analyzed, for example,m/z 200 to 1000. The quadrupole sequentially filters one massafter another, with an entire scan typically taking about asecond. (The exact time depends on mass range and scanspeed.) The MS firmware steps the quadrupole throughincreasing DC and RF voltages, which sequentially filters thecorresponding m/z values across a mass spectrum.Agilent InfinityLab LC/MSD Series and 6100 Series LC/MS Concepts Guide15

1Overview of Hardware and SoftwareGeneration of fragment ions: low versus high fragmentorA full scan analysis is useful because it shows all of the ions in agiven mass range that are present in the ion source. Because itprovides a complete picture of all the ionized compounds thatoccur above the detection limit in the chosen mass range, a fullscan analysis is often used for sample characterization,structural elucidation, and impurity analysis. It is also thestarting point for development of methods for SIM dataacquisition (discussed next).Selected ion monitoring (SIM) modeTo obtain the best sensitivity, the quadrupole is operated in SIMmode. In SIM mode, the quadrupole analyzes the signals of onlya few specific m/z values. The required RF/DC voltages are setto filter one mass at a time. Rather than stepping through all them/z values in a given mass range, the quadrupole steps onlyamong the values that the analyst chooses. Because thequadrupole spends more time sampling each of these chosenm/z values, the system can detect lower levels of sample.SIM mode is significantly more sensitive than scan mode butprovides information about fewer ions. Scan mode is typicallyused for qualitative analyses or for quantitation when analytemasses are not known in advance. SIM mode is used forquantitation and monitoring of target compounds.Generation of fragment ions: low versus high fragmentorWhen you set up a method fordata acquisition, you can controlthe amount of fragmentation withthe fragmentor setting. You set upa method in the Method and RunControl view, described inChapter 3.Fragment ions, also known as product ions, are formed bybreaking apart precursor ions. On the InfinityLab LC/MSDSeries and 6100 Series LC/MS instruments, the fragmentationregion is between the capillary exit and the skimmer, where thegas pressure is about 2 to 3 torr. Depending on the voltage inthis region, precursor ions may pass through unchanged or theymay be fragmented.When a lower voltage is applied across this region, the ions passthrough unchanged. Even if these ions collide with the gasmolecules in this region, they usually do not have enoughenergy to fragment. (See Figure 6.)16Agilent InfinityLab LC/MSD Series and 6100 Series LC/MS Concepts Guide

1279.1Overview of Hardware and SoftwareGeneration of fragment ions: low versus high fragmentorCH3350000O300000S [M H]ONNHNCH3250000H2N200000281.0 280.010000050000301.0150000 [M Na]0100m/z300Mass spectrum of sulfamethazine – low fragmentorm/z156124.1Figure 6200CH3m/z186OO S186.080000279.1156.1CH3m/z213m/z124 323.0[M Na]280.1213.2187.0157.1125.1107.120000NH2N[M H]301.040000 NHm/z108108.260000N0100Figure 7200300m/zMass spectrum of sulfamethazine – high fragmentorAgilent InfinityLab LC/MSD Series and 6100 Series LC/MS Concepts Guide17

1Overview of Hardware and SoftwarePositive versus negative ionsIf the voltage is increased, the ions have more translationalenergy. Then if the ions collide with gas molecules, thecollisions convert the translational energy into molecularvibrations that can cause the ions to fragment. This is calledcollision-induced dissociation (CID). Figure 7 shows anexample. Even though this fragmentation does not occur wherethe ions are formed at atmospheric pressure, it is a tradition tocall this type of fragmentation “in-source CID.” The ions frommolecular fragments are used for structural determination orconfirmation of the presence of a particular chemical species.It is possible to produce both molecular ions and fragment ionswithin the same spectrum by using an intermediatefragmentation voltage.FIA – flow injection analysisThe ideal fragmentation voltage depends on the structure of thecompound and the needs of the analysis. For target compoundanalysis, it is good practice to determine in advance thecompound’s response to fragmentor setting. The fastest way toaccomplish this is with a flow injection analysis (FIA) series. AnFIA series allows you to inject the compound multiple timeswithin the same run, and to vary the fragmentor setting indifferent time windows. From the resulting data, you can judgethe best fragmentor setting. For more information on FIA, see“Flow injection analysis” on page 67.Positive versus negative ionsYou set the ion polarity when youset up a method in the Method andRun Control view, described inChapter 3.Atmospheric pressure ionization techniques can produce bothpositive and negative ions. For any given analysis, thepredominant ion type depends on the chemical structure of theanalyte and (particularly for electrospray ionization) the pH ofthe solution. While either or both ion types may be present inthe ion source, the polarity of the ion optics in the ion transportand focusing region determines which ion type is detected.Analyses of positive and negative ions require different settingsfor the ion optics. The software-controlled autotune processoptimizes the settings for both positive and negative ions, and18Agilent InfinityLab LC/MSD Series and 6100 Series LC/MS Concepts Guide

Overview of Hardware and SoftwareMultiple signal acquisition1stores them in a single tune file. During data acquisition, theChemStation Edition program accesses the tune file for theappropriate settings.Multiple signal acquisitionYou establish the conditions formultiple signal acquisition in theMethod and Run Control view,described in Chapter 3.The InfinityLab LC/MSD Series and 6100 Series LC/MS modelsallow you to acquire multiple types of data during a singleanalysis. Within a single analytical run, you can choosealternating positive and negative ionization; alternating highand low fragmentor settings; and alternating scan and SIMmodes. Because optimum MS conditions vary from compoundto compound, this multisignal capability enables you to analyzemore compounds, with greater sensitivity, within a single run.Polarity switchingThe InfinityLab LC/MSD Series and 6100 Series LC/MS modelsallow you to switch from scan to scan between analysis ofpositive ions and analysis of negative ions. To switch polaritiesvery quickly, these models incorporate fast-switching powersupplies for the API source, the lens system, the quadrupole,and the detector. The ability to switch polarities on thechromatographic time scale is very useful for analysis ofcomplete unknowns because it obviates the need to run thesample twice to detect both types of ions.Alternating high/low fragmentorWith the InfinityLab LC/MSD Series and 6100 Series LC/MSmodels, you can also alternate from scan to scan between highand low fragmentation voltages. This capability allows you toacquire scans at low fragmentor settings for molecular weightinformation, and high fragmentor settings for structuralinformation.Agilent InfinityLab LC/MSD Series and 6100 Series LC/MS Concepts Guide19

1Overview of Hardware and SoftwareMultiple signal acquisitionAlternating SIM/scanMany analyses require use of SIM mode to monitor and/orquantitate target compounds at very low levels. Sometimes it isalso desirable to characterize the other sample componentswith a scan analysis. The InfinityLab LC/MSD Series and 6100Series LC/MS models allow you to alternate between SIM andscan modes, so you can accomplish both goals in a singleanalysis.Putting it all togetherThe InfinityLab LC/MSD Series and 6100 Series LC/MS cancycle through four different user-selected acquisition modes ona scan-by-scan basis within a single run. For example, you canset 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 voltageSuch an analysis is ideal for a mixture of compounds wheresome respond better in positive mode and some respond betterin negative mode, and where you need both molecular ions andfragment ions.The time required for one cycle varies depending on the numberof modes chosen, the scan range, and the interscan delayrequired for the switching. For separations with narrowchromatographic peaks, it is important to ensure that totalcycle time is short enough that the instrument makes sufficientmeasurements across the peak.The system issues a warning message when parameter settingsresult in a cycle time that is longer than recommended foroptimum performance. You can fix the problem by selectingFast Scan or Ultrafast scan or by increasing the Peakwidth.20Agilent InfinityLab LC/MSD Series and 6100 Series LC/MS Concepts Guide

Overview of Hardware and SoftwareIon sources1Ion sourcesThe InfinityLab LC/MSD Series and 6100 Series LC/MSinstruments operate with the following interchangeableatmospheric pressure ionization (API) sources: ESI (electrospray ionization) Agilent Jet Stream (LC/MSD XT, 6130 and 6150 only) APCI (atmospheric pressure chemical ionization) APPI (atmospheric pressure photoionization) MMI (multimode ionization)N O TEThe sources that are used on the InfinityLab LC/MSD Series and 6100Series LC/MS systems are the B-type sources. The InfinityLab LC/MSDSeries and 6100 Series LC/MS systems are not compatible with theA-type sources that were used on previous Agilent LC/MS models.Electrospray ionization (ESI)You control the spray chamberparameters (nebulizer pressure,drying gas flow and temperature,and capillary voltage) when youset up a method in the Method andRun Control view, described inChapter 3.Electrospray ionization relies in part on chemistry to generateanalyte ions in solution before the analyte reaches the massspectrometer. As shown in Figure 8, the LC eluent is sprayed(nebulized) into a spray chamber at atmospheric pressure in thepresence of a strong electrostatic field and heated drying gas.The electrostatic field occurs between the nebulizer, which is atground in the Agilent design, and the capillary, which is at highvoltage.The spray occurs at right angles to the capillary. This patentedAgilent design reduces background noise from droplets,increases sensitivity, and keeps the capillary cleaner for alonger period of time.Agilent InfinityLab LC/MSD Series and 6100 Series LC/MS Concepts Guide21

1Overview of Hardware and SoftwareElectrospray ionization (ESI)HPLC inletnebulizercapillarysolventsprayheated drying gasFigure 8Electrospray ion sourceElectrospray ionization (ESI) consists of four steps:1 Formation of ions2 Nebulization3 Desolvation4 Ion evaporationFormation of ionsIon formation in API-electrospray occurs through more thanone mechanism. If the chemistry of analyte, solvents, andbuffers is correct, ions are generated in solution beforenebulization. This results in high analyte ion concentration andgood API-electrospray sensitivity.22Agilent InfinityLab LC/MSD Series and 6100 Series LC/MS Concepts Guide

Overview of Hardware and SoftwareElectrospray ionization (ESI)1Preformed ions are not always required for ESI. Somecompounds that do not ionize in solution can still be analyzed.The process of nebulization, desolvation, and ion evaporationcreates a strong electrical charge on the surface of the spraydroplets. This can induce ionization in analyte molecules at thesurface of the droplets.NebulizationNebulization (aerosol generation) takes the sample solutionthrough these steps:a Sample solution enters the spray chamber through agrounded needle called a nebulizer.b For high-flow electrospray, nebulizing gas enters the spraychamber concentrically through a tube that surrounds theneedle.c The combination of strong shear forces generated by thenebulizing gas and the strong voltage (2–6 kV) in the spraychamber draws out the sample solution and breaks it intodroplets.d As the droplets disperse, ions of one polaritypreferentially migrate to the droplet surface due toelectrostatic forces.e As a result, the sample is simultaneously charged anddispersed into a fine spray of charged droplets, hence thename electrospray.Because the sample solution is not heated when the aerosol iscreated, ESI does not thermally decompose most analytes.Desolvation and ion evaporationBefore the ions can be mass analyzed, solvent must be removedto yield a bare ion.A counter-current of neutral, heated drying gas, typicallynitrogen, evaporates the solvent, decreasing the dropletdiameter and forcing the predominantly like surface-chargescloser together (see Figure 9).Agilent InfinityLab LC/MSD Series and 6100 Series LC/MS Concepts Guide23

1Overview of Hardware and SoftwareElectrospray ionization (ESI)evaporation -- - - - - Figure 9Coulomb repulsion – repulsionbetween charged species of thesame signanalyte ion ejected -- - - - - - -- --- - - - - Desorption of ions from solutionWhen the force of the Coulomb repulsion equals that of thesurface tension of the droplet, the droplet explodes, producingsmaller charged droplets that are subject to furtherevaporation. This process repeats itself, and droplets with ahigh density of surface-charges are formed. When chargedensity reaches approximately 108 V/cm3, ion evaporationoccurs (direct ejection of bare ions from the droplet surface).These ions are attracted to and pass through a capillarysampling orifice into the ion optics and mass analyzer.The importance of solution chemistryThe choice of solvents and buffers is a key to successfulionization with electrospray. Solvents like methanol that havelower heat capacity, surface tension, and dielectric constant,promote nebulization and desolvation. For best results inelectrospray mode: Adjust solvent pH according to the polarity of ions desiredand the pH of the sample. To enhance ion desorption, use solvents that have low heatsof vaporization and low surface tensions. Select solvents that do not neutralize ions through gas-phasereactions such as proton transfer or ion pair reactions. To reduce the buildup of salts in the ion source, select morevolatile buffers.24Agilent InfinityLab LC/MSD Series and 6100 Series LC/MS Concepts Guide

Overview of Hardware and SoftwareAgilent Jet Stream1Multiple chargingElectrospray is especially useful for analyzing largebiomolecules such as proteins, peptides, and oligonucleotides,but can also analyze smaller molecules like drugs andenvironmental contaminants. Large molecules often acquiremore than one charge. Because of this multiple charging, youcan use electrospray to analyze molecules as large as 150,000 ueven though the mass range (or more accurately mass-to-chargerange) for a typical q

Contents Agilent InfinityLab LC/MSD Series and 6100 Series LC/MS Concepts Guide 7 Integrating peaks 86 Calibration 88 Data review and sequence reprocessing 90