Electrospray and Atmospheric Electrospray and Atmospheric Pressure Chemical Ionization Pressure Chemical Ionization
Quadrupole MS for Small MoleculesQuadrupole MS for Small Molecules
Jonathan A. Karty, Ph.D.Jonathan A. Karty, Ph.D.
LC-MS PictureLC-MS Picture
General Sample GuidelinesGeneral Sample Guidelines Purify analyte if possiblePurify analyte if possible
Analyte should be 1-100 mg/L in concentrationAnalyte should be 1-100 mg/L in concentration Colored samples should be translucentColored samples should be translucent
Sample must have no particulates or precipitatesSample must have no particulates or precipitates
Use only volatile solvents/buffersUse only volatile solvents/buffers MeOH, HMeOH, H22O, acetone, CHO, acetone, CH33CN, THF, CHClCN, THF, CHCl33, etc., etc.
HOAc, HCOOH, NHHOAc, HCOOH, NH44OAc, NHOAc, NH33, etc. (weak, volatile), etc. (weak, volatile) Ionic strength < 20 mM is best (e.g. 0.1% v/v HOAc)Ionic strength < 20 mM is best (e.g. 0.1% v/v HOAc) 1 – 5 1 – 5 μμM NaOAc for acid/base labile samplesM NaOAc for acid/base labile samples
Need at least 50 Need at least 50 µµL for loop injectionL for loop injection If you need non-polar solvent for APCI, see Dr. If you need non-polar solvent for APCI, see Dr.
Karty firstKarty first
Agilent 6130 System DescriptionAgilent 6130 System Description Inlet is an Agilent 1200 HPLC SystemInlet is an Agilent 1200 HPLC System
Binary gradient (4 solvent bottles, only 2 can be used in an Binary gradient (4 solvent bottles, only 2 can be used in an analysis (A1 or A2 with B1 or B2, not A1 and A2)analysis (A1 or A2 with B1 or B2, not A1 and A2)
50-2,500 uL/min flow rates 50-2,500 uL/min flow rates Autosampler 0.5-100 Autosampler 0.5-100 μμL injection volumeL injection volume
6-column selection valve (loop or 1 of 5 HPLC columns)6-column selection valve (loop or 1 of 5 HPLC columns) CC1818 reversed phase is default column (H reversed phase is default column (H22O-CHO-CH33OH solvents)OH solvents)
Agilent 1200 DAD detectorAgilent 1200 DAD detector 190-800 nm UV-VIS detector190-800 nm UV-VIS detector Can record entire UV-VIS spectrum throughout a runCan record entire UV-VIS spectrum throughout a run
Electrospray Ionization-Atmospheric Pressure Chemical Electrospray Ionization-Atmospheric Pressure Chemical Ionization/Quadrupole (ESI/APCI-Q) mass spectrometerIonization/Quadrupole (ESI/APCI-Q) mass spectrometer 50 – 3,000 m/z range50 – 3,000 m/z range Can make ions by ESI, APCI, or bothCan make ions by ESI, APCI, or both Can alternate positive and negative ion modes during a runCan alternate positive and negative ion modes during a run
Air Sensitive MSAir Sensitive MS Source on 6130 MS is flushed constantly with Source on 6130 MS is flushed constantly with
nitrogen from a dewarnitrogen from a dewar Enables walk-up air senstive ESI/APCI MSEnables walk-up air senstive ESI/APCI MS
A syringe pump with air-tight “Sample-Lock” A syringe pump with air-tight “Sample-Lock” syringe can be used for air-sensitive analysessyringe can be used for air-sensitive analyses Requires special training from Dr. KartyRequires special training from Dr. Karty A separate syringe is needed for each sampleA separate syringe is needed for each sample
A “dummy” sample must still be placed in the A “dummy” sample must still be placed in the autosamplerautosampler
Remember to keep concentrations around 20 mg/LRemember to keep concentrations around 20 mg/L Colored solutions should be translucentColored solutions should be translucent
HPLC-MS vs. Loop InjectionHPLC-MS vs. Loop Injection MSF uses the 1200 autosampler for nearly all MSF uses the 1200 autosampler for nearly all
samplessamples Must inject something, even if syringe pump is usedMust inject something, even if syringe pump is used
Sample can go through an empty loopSample can go through an empty loop Solution must contain only sample and volatile Solution must contain only sample and volatile
componentscomponents Allows 2.5 minute analysis timeAllows 2.5 minute analysis time
Sample can also be separated by HPLCSample can also be separated by HPLC Up to 5 columns are availableUp to 5 columns are available Currently only CCurrently only C1818 reversed phase available for general reversed phase available for general
useuse
Electrospray Ionization (ESI)Electrospray Ionization (ESI) Dilute solution of analyte (1 mg/L) infused Dilute solution of analyte (1 mg/L) infused
through a fine needle in a high electric fieldthrough a fine needle in a high electric field
Very small, highly charged droplets are createdVery small, highly charged droplets are created
Solvent evaporates, droplets split and/or ions Solvent evaporates, droplets split and/or ions ejected to lower charge/area ratio ejected to lower charge/area ratio Warm nebulizing gas accelerates dryingWarm nebulizing gas accelerates drying Basic sites are eventually protonated (M+H)Basic sites are eventually protonated (M+H)++
Ketones, esters, and alcohols can sodiate (M+Na)Ketones, esters, and alcohols can sodiate (M+Na)++
Free ions are directed into the vacuum chamberFree ions are directed into the vacuum chamber
ESI PictureESI Picture
http://newobjective.com/images/electro/spraytip_bw.jpg
Advantages of ESIAdvantages of ESI
Gentle ionization processGentle ionization processHigh chance of observing intact molecular ionHigh chance of observing intact molecular ionVery labile analytes can be ionizedVery labile analytes can be ionizedNon-covalent complexes can be studied by ESINon-covalent complexes can be studied by ESI
Molecule need not be volatileMolecule need not be volatileProteins/peptides easily analyzed by ESIProteins/peptides easily analyzed by ESISalts can be analyzed by ESISalts can be analyzed by ESI
Easily coupled with HPLCEasily coupled with HPLC Both positive and negative ions can be Both positive and negative ions can be
generated by the same sourcegenerated by the same source
Characteristics of ESI IonsCharacteristics of ESI Ions ESI is a thermal process (1 atm in source)ESI is a thermal process (1 atm in source)
Little fragmentation due to ionization (cf EI) Little fragmentation due to ionization (cf EI) Solution-phase ions are often preservedSolution-phase ions are often preserved
e.g. organometallic saltse.g. organometallic salts ESI ions are generated by ion transferESI ions are generated by ion transfer
(M+H)(M+H)++, (M+Na), (M+Na)++, or (M-H), or (M-H)--, rarely M, rarely M++•• or M or M--••
ESI often generates multiply charged ionsESI often generates multiply charged ions (M+2H)(M+2H)2+2+ or (M+10H) or (M+10H)10+10+
Most ions are 500-1500 m/zMost ions are 500-1500 m/z ESI spectrum x-axis must be mass/charge (m/z or Th, ESI spectrum x-axis must be mass/charge (m/z or Th,
not amu or Da)not amu or Da)
ESI DisadvantagesESI Disadvantages Analyte must have an acidic or basic siteAnalyte must have an acidic or basic site
Hydrocarbons and steroids not readily ionized by ESIHydrocarbons and steroids not readily ionized by ESI
Analyte must be soluble in polar, volatile solventAnalyte must be soluble in polar, volatile solvent ESI is less efficient than other sourcesESI is less efficient than other sources
Most ions don’t make it into the vacuum systemMost ions don’t make it into the vacuum system
ESI is very sensitive to contaminantsESI is very sensitive to contaminants Solvent clusters can dominate spectraSolvent clusters can dominate spectra
Distribution of multiple charge states can make Distribution of multiple charge states can make spectra of mixtures hard to interpretspectra of mixtures hard to interpret e.g. polymer mass spectrae.g. polymer mass spectra
What Samples Can Be Analyzed by What Samples Can Be Analyzed by ESI-QMS?ESI-QMS?
Non-volatile organic moleculesNon-volatile organic molecules Should be soluble in polar, volatile solventsShould be soluble in polar, volatile solvents Molecule must be ionizable (MS detects ions)Molecule must be ionizable (MS detects ions)
R-NHR-NH22, R-CO, R-CO22H, R-HSOH, R-HSO33, R-OH, R-H, R-OH, R-H22POPO33 work best for ESI work best for ESI Aromatics, ketones, protected heteroatoms work well by APCIAromatics, ketones, protected heteroatoms work well by APCI
Organometallic complexesOrganometallic complexes Organometallic salts work especially wellOrganometallic salts work especially well Source is flooded with NSource is flooded with N22 gas from a dewar gas from a dewar
HPLC-MSHPLC-MS With appropriate mobile phasesWith appropriate mobile phases
What Samples Are Inappropriate What Samples Are Inappropriate for ESI-QMS analysis?for ESI-QMS analysis?
Samples in non-polar or non-volatile Samples in non-polar or non-volatile solvent:solvent:Hexane, benzene, CHHexane, benzene, CH22ClCl22, DMSO, etc., DMSO, etc.Run these in APCI or mixed modeRun these in APCI or mixed mode
Buffer systems incompatible with ESIBuffer systems incompatible with ESI6M urea, 10% glycerol, 0.1 M NaH6M urea, 10% glycerol, 0.1 M NaH22POPO44, TBAF, , TBAF,
(involatile)(involatile)Strong acid solutions (too conductive)Strong acid solutions (too conductive)detergentsdetergents
Molecules that have no ionizable groupsMolecules that have no ionizable groups
O
O
O
O
OTBDPS
C
O
O
Me
OTIPS
OMOM
Me
Me
OTIPS
OTBS
Me
Na+
O
O
O
HO
OTBDPS
C
O
O
Me
OTIPS
OMOM
Me
Me
OTIPS
OTBS
Me
Na+
OH
Chemical Formula: C77H136NaO13Si4+
Exact Mass: 1403.9m/z: 1404.9 (100.0%), 1403.9 (94.7%), 1405.9 (67.1%), 1406.9 (33.1%), 1407.9 (13.1%),
1408.9 (4.3%), 1409.9 (1.2%)
Chemical Formula: C77H134NaO12Si4+
Exact Mass: 1385.9m/z: 1386.9 (100.0%), 1385.9 (94.7%), 1387.9 (66.9%), 1388.9 (32.8%), 1389.9 (12.9%), 1390.9 (4.2%), 1391.9 (1.2%)
H2O
ESI ExampleESI Example
O
O
O
HO
OTBDPS
C
O
O
Me
OTIPS
OMOM
Me
Me
OTIPS
OTBS
Me
Na+
OH
O
O
O
O
OTBDPS
C
O
O
Me
OTIPS
OMOM
Me
Me
OTIPS
OTBS
Me
Na+
78%22%
Atmospheric Pressure Chemical Atmospheric Pressure Chemical Ionization (APCI)Ionization (APCI)
APCI uses a corona discharge to generate acidic APCI uses a corona discharge to generate acidic solvent cations from a vaporsolvent cations from a vapor Ionizing reagent often CHClIonizing reagent often CHCl33++ or CH or CH33OHOH++
These solvent cations can protonate hydrophobic These solvent cations can protonate hydrophobic species not amenable to ESIspecies not amenable to ESI APCI can be done from hexane or THFAPCI can be done from hexane or THF Often used to study lipids and steroidsOften used to study lipids and steroids In MSF, completely protected macrocycles are routinely In MSF, completely protected macrocycles are routinely
studied by APCIstudied by APCI
APCI is a little harsher than ESIAPCI is a little harsher than ESI Labile molecules might fragmentLabile molecules might fragment
APCI DiagramAPCI Diagram
http://imaisd.usc.es/riaidt/masas/imagenes/apci1.jpg
APCI ExampleAPCI Example
Agilent 6130 Multi-mode SourceAgilent 6130 Multi-mode Source
http://www.chem.agilent.com/Library/Images1/MMS_schematic_300dpi_039393.jpg
Cluster and Background IonsCluster and Background Ions The low m/z (<300) region of the mass spectrum is The low m/z (<300) region of the mass spectrum is
often dominated by solvent cluster ionsoften dominated by solvent cluster ions These are distinguished by low mass defects due to NaThese are distinguished by low mass defects due to Na++
and Kand K++ incorporation incorporation Masses are XXX.0 or XXX.9Masses are XXX.0 or XXX.9
Compounds from previous samples can coat the Compounds from previous samples can coat the source leading to persistent “background” ionssource leading to persistent “background” ions e.g. 242.1 for tetrabutylammoniume.g. 242.1 for tetrabutylammonium Might see large ion from previous person’s runMight see large ion from previous person’s run
To confirm an ion is “background” try injecting a To confirm an ion is “background” try injecting a blank of ESI solution with your solvent systemblank of ESI solution with your solvent system
About the Fragmentor…About the Fragmentor… There is a region in the source where ion-There is a region in the source where ion-
nitrogen collisions can cause fragmentationnitrogen collisions can cause fragmentation This occurs between the end of the heated capillary This occurs between the end of the heated capillary
and the first skimmer coneand the first skimmer cone
The voltage difference between capillary and The voltage difference between capillary and skimmer is called the “Fragmentor” voltageskimmer is called the “Fragmentor” voltage
Fragmentor voltage has 2 postive main effectsFragmentor voltage has 2 postive main effects It increases the number of ions that make it to the quadIt increases the number of ions that make it to the quad It can knock non-covalent attached neutralsIt can knock non-covalent attached neutrals
However, a high fragmentor value can cause However, a high fragmentor value can cause labile groups to dissociatelabile groups to dissociate Phospho-diesters can be rather vulnerable to thisPhospho-diesters can be rather vulnerable to this
A Word About QuantificationA Word About Quantification LC-MS is a quantitative techniqueLC-MS is a quantitative technique Use only 1 m/z when quantifying a compoundUse only 1 m/z when quantifying a compound Intensity is proportional to concentrationIntensity is proportional to concentration
I I αα [X] [X] α is unique to each compoundα is unique to each compound The more two compounds differ chemically, The more two compounds differ chemically,
the more careful one must be when comparing the more careful one must be when comparing their intensitiestheir intensities
Ideally a calibration curve is constructed using Ideally a calibration curve is constructed using multiple solutions of pure analyte at varying multiple solutions of pure analyte at varying concentrationsconcentrations
Hands-on TrainingHands-on Training
Training starts AFTER 11/7/10Training starts AFTER 11/7/10Groups of no more than threeGroups of no more than threeOne hour or so to completeOne hour or so to completeNo charge for first sessionNo charge for first sessionAfter training, students must demonstrate After training, students must demonstrate
competency by running their own samples competency by running their own samples prior to being granted after-hours accessprior to being granted after-hours access