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