|
Choosing a Mass Analyser
|
Quadrupole Ion Trap
|
Triple Quadrupole
|
|
Highest full-scan and MS/MS product ion scan sensitivity.
|
Highest SIM and SRM sensitivity.
|
|
Can perform MSn.
|
Capable of common neutral loss scans and precursor ion scans.
|
|
High sensitivity for high masses (>500 Da).
|
Wide linear dynamic range for quantitation.
|
|
High resolution scanning.
|
High sample capacity and throughput.
|
|
Constant resolution across the m/z scale.
|
Ideal for quantitation.
|
|
Lower initial cost.
|
Capable of many SRM transitions in a single segment.
|
|
High sample capacity and throughput.
|
|
|
Ideal for qualitative analysis/structural elucidation.
|
|
Choosing an Interface
|
Compound Parameter
|
APCI
|
ESI
|
|
Water Soluble
|
-
|
X
|
|
Organic Soluble
|
X
|
-
|
|
Ionic
|
-
|
X
|
|
Ionizable
|
X
|
-
|
|
Polar
|
-
|
X
|
|
Polar Non-Ionic
|
X
|
-
|
|
Thermally Stabile
|
X
|
-
|
|
Thermally Labile
|
-
|
X
|
|
High Flow Rates ( ≈ 1mL.min-1)
|
X
|
-
|
|
Low Flow Rates (100-250mL.min-1)
|
-
|
X
|
|
Reduced Ionization Suppression
|
X
|
-
|
|
Production of Multiply Charged Ions
|
-
|
X
|
Choosing Mobile Phase AdditivesBuffers
|
Ionization Mode
|
Buffer
|
pH Range
|
|
Positive
|
Acetic Acid
|
3.8 – 5.8
|
|
Formic Acid
|
- 2.8 – 4.8
|
|
Negative
(Check pH range of column stationary phase)
|
Ammonium Acetate
|
- 8.2 – 10.2
|
|
Ammonium Hydroxide
|
- 8.0 – 10.0
|
|
Ammonium Hydrogen-carbonate
|
- 7.0 – 11.0
|
|
Ammonium Formate
|
- 8.2 – 10.2
|
Chromatography Troubleshooting
|
Symptom
|
Cause
|
Solution
|
|
No Peaks.
|
Injector problem, instrument problem, wrong mobile phase, wrong stationary phase, eluting after segment.
|
- Prime pump and check flow, check proper instrument function, lengthen segment or perform full scan analysis, check sample for degradation.
|
|
Broad Peaks.
|
Sample overload, injection volume too large, poor column efficiency, wrong pH.
|
- Dilute in A solvent and reinject, decrease injection volume, increase column temperature, flush column with strong solvent, check guard and tubing or blockages.
|
|
Ghost Peak.
|
Contamination, peak eluting from previous run.
|
- Flush column, use sample cleanup, replace solvents.
|
|
Peak Doubling or Splitting.
|
Coelution of interfering compound, injection solvent too strong, column overloaded.
|
- Use sample cleanup, flush column, increase column diameter, decrease sample amount, use weaker injection solvent.
|
|
Peak Fronting.
|
Channeling in column, column overloaded.
|
- Replace/repack column, increase column diameter, decrease sample amount.
|
|
Peak Tailing.
|
Degradation at high pH, unswept dead volume, interfering coeluting peak.
|
- Use bas-deactivated or polar-embedded RP column, reduce colmn temperature, use small-diameter tubing, check that tubing is properly cut and connected, change mobile or stationary phase.
|
|
Spikes.
|
Bubbles in mobile phase, column stored without caps.
|
- Degas mobile phase, ensure all fittings are tight, store column tightly capped, flush RP columns with degassed MeOH.
|
|
High Back Pressure.
|
Blockages in tubing, sensor problems.
|
- Check for blockages in tubing and column, use lower viscosity mobile phase, increase column particle size.
|
Common LCMS Contaminant Peaks
|
m/z
|
Ionization Mode
|
Possible Source
|
|
102
|
(+) ESI
|
Triethylamine (TEA)
|
|
113
|
(-) ESI
|
Trifluoroacetic Acid (TFA)
|
|
114
|
(+) ESI
|
N2 Gas Tubing
|
|
116
|
(+) ESI
|
- Detergent
|
|
149
|
(+) ESI
(+) APCI
|
- Red Bloom Algae(H2O, Purification)
|
|
149
391
|
(+) ESI
(+) APCI
|
- Phthalates (Plastics)
|
|
256
284
|
(+) ESI
|
- Nylon Filters
|
|
311
352
640
|
(+) ESI
|
Column Bleed
|
|
331
|
(+) ESI
|
- Ethylene Polypropylene
|
|
122
123.9
132
134
145
147
258
315
|
(+) ESI
|
- Water (Replace Purification System Filters)
|
|
265
|
(-) ESI
|
- LC Pump Grease
|
Emergency Phone Numbers In Switzerland
|
Emergency Phone Numbers In Switzerland
|
|
Fire
|
118
|
|
Ambulance
|
144
|
|
Intoxication
|
145
|
|
Police
|
117
|
|
Biopack Lab
|
+41 (0)21 213 03 15
|
Fire Extinguishers
|
EU/Australian Fire Class
|
Fuel/Heat Source
|
Fire Extinguisher
|
|
A
|
Ordinary Combustibles
|
- Water (A)
- Water with additives (AB)
- ABC powder
|
|
B
|
Flammable Liquids
|
- CO2
- Water with additives (AB)
- BC or ABC powder
|
|
C
|
Flammable Gases
|
- BC or ABC powder
|
|
D
|
Combustible Metals
|
- D powder
- Sand
|
|
E
|
Electrical Equipment
|
- CO2
|
|
F
|
Cooking Oil or Fat
|
- BC powder
|
HPLC Buffers to Avoid in LCMS
- Phosphate buffers and other nonvolatile salts precipitate and clog MS ionization chambers.
- Salts and buffers may cause ion suppression.
- Sodium and potassium salts cause the formation of nonspecific sodium and potassium adducts (see Ions and Adducts Formed in Electrospray or in APCI).
- Detergents and surfactants in protein analysis can cause ion suppression, surfactant adduct formation and clusters that interfere with mass spectral data.
HPLC Ion Pairing Reagents in LCMS
- Do not use nonvolatile reagents.
- Long-chain reagents may interfere with mass spectra.
- Ion-pairing reagents may compete in ion evaporation.
- TFA/HFBA form strong ion pairs that suppress ionization (unless a post-column infusion of propionic acid is used).
- Recommendation: Do not use ion pairing.
Ions and Adducts Formed in APCI
|
Ion
|
m/z
|
|
[M + H]+
|
M + 1
|
|
[M – H]-
|
M - 1
|
|
[M + NH4]+
|
M + 18
|
|
[M + CH3NH]+
|
M + 42
|
|
[M + CH3OH2]+
|
M + 33
|
|
[M + H3O]+
|
M + 19
|
|
[M + Cl]-
|
M + 35
|
|
[M + CH3COO]-
|
M + 59
|
|
[M + CO2H]-
|
M + 45
|
|
M+.
|
M
|
|
M-.
|
M
|
Maintenance - Troubleshooting
|
Maintenance/Troubleshooting
|
|
Daily or Weekly
|
- - Clean ionization chamber
- - Run autosampler through wash cycle
- - Prime LC pumps
- - Check LC solvents for dust or bacterial growth
- - Rinse capillary with water and methanol 2-3 times
- - Check wash sample level for autosampler (at least 75% methanol or acetonitrile)
- - Check foreline pump oil level
|
|
Monthly
|
- - Replace LC solvents (HPLC grade or better)
- - Check LC flow rate
- - Vent system and clean API plug
- - Check API needle for clogging
|
|
Every 3 Months
|
- - Change foreline pump oil using GP 45 oil and change the oil exhaust filter cartridge
- - Clean autosampler spindle with isopropanol, relubricate
- - Autotune system
- - Run electronic diagnostics
- - Change nitrogen generator filters
|
|
Yearly
|
- - Replace API needle
- - Replace capillary
- - Test system sensitivity
|
Method Development Roadmap
Step 1.
Definition.
- Literature search/gather info.
- Acquire pure standards of target analytes and internal standards for quantification.
- Research previous methods used.
Step 2.
Optimize targets in MS.
- Infuse each target and determine best MS conditions, fragmentation products, and develop SRM transitions.
- Flow inject targets to optimize ionization/spray conditions.
Step 3.
Optimize Chromatography.
- Suspend analytes in the A solvent for injection, or a mixture of A and B that is similar to the starting LC gradient.
- Adjust gradient to separate target analytes.
- If necessary, reoptimize MS or MS/MS conditions with confirmed eluting mobile phase (segment time, dwell time).
Step 4
Evaluate Matrix Effects
- Identify possible matrix interferences leading to ion suppression, false positive peaks, or baseline instability.
- Increase method specificity by adding at least a second SRM transition for each target analyte.
- Confirm identity with ion ratio criteria when necessary.
- Develop sample preparation and separation process to eliminate matrix interference or ion suppression.
Neutral Commonly Added to Ions in Electrospray and APCI
|
Ion
|
m/z
|
|
Mn+ + xCH3CN
|
(M + 41x)/n
|
|
Mn+ + xCH3CO2H
|
(M + 60x)/n
|
|
Mn+ + xCO2H2
|
(M + 46x)/n
|
|
Mn+ + xCH3CH2OH
|
(M + 46x)/n
|
|
Mn+ + xCH3OH
|
(M + 32x)/n
|
Optimizing LC Gradients
|
Problem
|
Solution
|
|
Peaks resolved but run time too long.
|
Increase starting %B, decrease gradient time (maintain slope).
|
|
Poor resolution of initial peaks.
|
Decrease solvent strength.
|
|
Wide gaps with initial peaks.
|
Increase starting %B, maintain slope (shorten total gradient).
|
|
Final peaks elute after gradient.
|
Select stronger solvent B, or increase starting %B.
|
|
Poor resolution of final peaks.
|
Decrease final %B and/or slope.
|
|
Wide gaps between peaks.
|
Increase slope (shorten gradient time).
|
PDA versus MS Detection
|
Criteria
|
LC
|
LC/MS
|
|
Method Specificity.
|
Derived from separation.
|
Derived from mass analyzer.
|
|
Method Specificity.
|
Derived from separation.
|
- Derived from separation and mass analyzer.
|
|
Quantification.
|
Baseline separations are required.
|
- No separation is required, but some separation is recommended.
|
|
Standardization.
|
Usually use external standards.
|
- Usually use internal standards.
|
|
Calibration.
|
Single point calibration is typical.
|
- Multi-point calibration is typical.
|
|
Buffers.
|
Added to suppress ionization and enhance solubility.
|
- Ionization suppression for vaporization, ionization enhancement for desorption.
|
|
Effects from Chemical Interferences.
|
Baseline separations are required.
|
- Labeled IS may compensate for interference.
|
pH Ranges for Column Stationary Phases
|
Stationary Phase
|
pH Range
|
|
C18
|
1.5 - 10.0
|
|
C8
|
1.5 - 10.0
|
|
Diphenyl
|
1.5 - 7.5
|
|
Pentafluorophenyl (PFP)
|
3.0 - 7.5
|
Preparing Liquid Samples
- Add Internal Standard.
- Remove particles.
Filtration.
Centrifugation.
SEC.
Dialysis.
Precipitation.
- Concentrate the sample and isolate target analytes.
Liquid-liquid extraction.
Solid-phase extraction.
Evaporation.
Lyophilization.
Supercritical fluid extraction.
- Analyze Sample.
Reversed Phase LC Mobile Phases
|
Solvents
|
Solvent Strength (P’)
|
|
Water/Buffers
|
Varies
|
|
Water (Weakest)
|
10.2
|
|
Acetonitrile
|
5.8
|
|
Methanol (Strongest)
|
5.1
|
Sorbent Specifications (Sample Preparation)
|
Sorbent Phase
|
Category
|
Bonded Functional Group / Base Material
|
Mean Pore Size
|
|
AccuCat
|
Mixed Mode
|
Sulfonic Acid (SCX) and Quaternary Amine (SAX) / Silica Based
|
60Å
|
|
Alumina (AL-A)
|
Polar
|
Aluminium Oxide – Acidic
|
|
|
Alumina (AL-B)
|
Polar
|
Aluminium Oxide – Basic
|
|
|
Alumina (AL-N)
|
Polar
|
Aluminium Oxide – Neutral
|
|
|
Aminopropyl (NH2)
|
Polar / Anion Exchanger
|
Aminopropyl / Silica Based
|
60Å
|
|
SPEC Aminopropyl (NH2)
|
Polar / Anion Exchanger
|
Aminopropyl / Silica Based
|
70Å
|
|
Atrazine
|
Application Specific
|
Trifunctional Octadecyl / Silica Based
|
60Å
|
|
BioBond Elut
|
Non Polar
|
Trifunctional Octadecyl / Silica Based
|
500Å
|
|
C1
|
Non Polar
|
Methyl / Silica Based
|
60Å
|
|
C2
|
Non Polar
|
Ethyl / Silica Based
|
60Å
|
|
SPEC C2
|
Non Polar
|
Dimethyl / Silica Based
|
70Å
|
|
C8
|
Non Polar
|
Octyl / Silica Based
|
60Å
|
|
SPEC C8
|
Non Polar
|
Octyl / Silica Based
|
|
|
C18
|
Non Polar
|
Trifunctional Octadecyl / Silica Based
|
60Å
|
|
SPEC C18
|
Non Polar
|
Monofunctional Octadecyl / Silica Based
|
70Å
|
|
SPEC C18 AR
|
Non Polar
|
Trifunctional Octadecyl / Silica Based
|
70Å
|
|
C18 EWP
|
Non Polar
|
Trifunctional Octadecyl / Silica Based
|
500Å
|
|
C18 INT
|
Non Polar
|
Trifunctional Octadecyl / Silica Based
|
60Å
|
|
C18 LO
|
Non Polar
|
Trifunctional Octadecyl / Silica Based
|
60Å
|
|
C18 OH
|
Non Polar
|
Monofunctional Octadecyl / Silica Based
|
150Å
|
|
CBA
|
Cation Exchanger
|
Carboxylic Acid / Silica Based
|
60Å
|
|
Certify
|
Mixed Mode
|
Octyl and Benzenesulfonic Acid (SCX) / Silica Based
|
60Å
|
|
Certify II
|
Mixed Mode
|
Octyl and Quaternary Acid (SAX) / Silica Based
|
60Å
|
|
CH
|
Non Polar
|
Cyclohexyl / Silica Based
|
60Å
|
|
Cyano (CN-E)
|
Non Polar
|
Cyanopropyl / Silica Based
|
60Å
|
|
Cyano (CN-U)
|
Polar
|
Cyanopropyl / Silica Based
|
60Å
|
|
SPEC Cyano
|
Polar
|
Cyanopropyl / Silica Based
|
70Å
|
|
SPEC DAU
|
Application Specific
|
Silica Based
|
70Å
|
|
DEA
|
Anion Exchanger
|
Diethylaminopropyl / Silica Based
|
60Å
|
|
Diol (2OH)
|
Polar
|
Diol / Silica Based
|
60Å
|
|
ENV
|
Non Polar
|
Styrene Divinyl / Silica Based
|
450Å
|
|
EnvirElut 1664
|
Application Specific
|
Trifunctional Octadecyl / Silica Based
|
60Å
|
|
FL
|
Polar
|
Florisil
|
|
|
FOCUS
|
Polar-enhanced
|
Polar Functionalized Styrene Divinylbenzene
|
120Å
|
|
LMS
|
Non Polar
|
Styrene Divinylbenzene
|
300Å
|
|
SPEC MP1
|
Mixed Mode
|
Nonpolar and Benzenesulfonic Acid (SCX) / Silica Based
|
70Å
|
|
SPEC MP3
|
Mixed Mode
|
Slightly Polar and Benzenesulfonic Acid (SCX) / Silica Based
|
70Å
|
|
NEXUS
|
Mixed Mode
|
Mixed Mode Copolymer
|
100Å / 450Å Bimodal
|
|
PBA
|
Covalent
|
Phenylboronic Acid
|
60Å
|
|
PCB
|
Application Specific
|
Layered Phase
|
|
|
PH
|
Non Polar
|
Phenyl / Silica Based
|
60Å
|
|
SPEC PH
|
Non Polar
|
Phenyl / Silica Based
|
70Å
|
|
PPL
|
Non Polar
|
Functionalized Styrene Divinylbenzene
|
150Å
|
|
PRS
|
Cation Exchanger
|
Propylsulfonic Acid / Silica Based
|
60Å
|
|
PSA
|
Anion Exchanger
|
Ethylenediamine-N-propyl / Silica Based
|
60Å
|
|
SPEC PSA
|
Anion Exchanger
|
Ethylenediamine-N-propyl / Silica Based
|
70Å
|
|
SAX
|
Anion Exchanger
|
Trimethylaminopropyl / Silica Based
|
60Å
|
|
SPEC SAX
|
Anion Exchanger
|
Trimethylaminopropyl / Silica Based
|
70Å
|
|
SCX
|
Cation Exchanger
|
Benzenesulfonic Acid / Silica Based
|
60Å
|
|
SPEC SCX
|
Cation Exchanger
|
Benzenesulfonic Acid / Silica Based
|
70Å
|
|
SI
|
Polar
|
Silica
|
60Å
|
|
SPEC SI
|
Polar
|
Silica
|
70Å
|
|
TCA
|
Application Specific
|
Ethyl / Silica Based
|
60Å
|
|
TOP
|
Application Specific
|
Functionalized Styrene Divinylbenzene
|
150Å
|
Typical Flow Rates Versus Column I.D.
|
Column I.D.
|
Flow Rate
|
|
3 - 4.6 mm
|
0.4 - 2.0 mL.min-1
|
|
2 - 2.1 mm
|
100 - 400 μL.min-1
|
|
1 mm
|
50 - 100 μL.min-1
|
|
0.36 - 0.64 mm
|
0.1 - 20 μL.min-1
|
|
