uhplc-ms basic principles and applications
TRANSCRIPT
UHPLC-MS Basic Principles
and ApplicationsJitnapa Voranitikul
February, 2018 Product Specialist LC/MS
https://www.thermofisher.com/order/catalog/product/IQLAAAGABHFAPUMZZZ?SID=srch-srp-IQLAAAGABHFAPUMZZZ
Fundamental of Liquid
Chromatography
33
HPLC System Range
Basic Automated
§Highly economic & reliable
§620 bar UHPLC compatible
§Flow rates up to 10 mL/min
§100 Hz detector range
§Modular flexibility
Standard
§3rd Generation Modules
§620 bar UHPLC compatible
§Flow rates up to 10 mL/min
§Oven temp. 5 – 80 ºC
§100 Hz DAD, MWD, VWD, FLD, CAD
§Highest flexibility
x2 Dual LC
§Two systems in one
§620 bar UHPLC compatible
§Flow rates up to 10 mL/min
§Oven temp. 5 – 80 ºC
§Automated Application Switching
§Parallel and Tandem LC
§Online SPE-LC
§Automated method scouting
§Turn key Viper kits for ease of use
RSLC
§Binary and Quaternary UHPLCs
§1000 bar up to 5 mL/min
§800 bar up to 8 mL/min
§Oven temp. 5 – 110 ºC
§200 Hz DAD, MWD, VWD, FLD
§Improved sub 2-µm particle column
compatibility
§Ultrafast/ultra resolution system
x2 Dual RSLC
§x2 Dual UHPLC System
§Two systems in one
§1000 bar up to 5 mL/min
§800 bar up to 8 mL/min
§Oven temp. 5 – 110 ºC
§200 Hz DAD, MWD, VWD, FLD
§Parallel and Tandem LC
§Online SPE-LC
§Automated method scouting
§Offline 2D-UHPLC
§Turn key Viper kits for ease of use
RSLCnano
§UHPLC system for Nano/Cap/Micro
§20 nL/min – 50 µL/min up to 800 bar
§Continuous direct flow
§New standard in retention time
precision
§Snap-in valves
§nanoViper fitting system for easy
operation
Basic Standard x2 Dual LC RSLC x2 Dual RSLC RSLCnano
VanquishTM
Max Pressure 1517 bar
Thermo Analytical LC Systems
1.9mm
5mm
u opt
u opt
Linear Velocity (mm/s)
H E
T P
(m
m)
5
10
15
0 61 2 3 4 5
3mm
u opt
Incr
easi
ng C
olum
n Ef
ficie
ncy
Increasing Flowrate
What is the Small Particle Advantage ?
Higher efficiency, independent of flow rate means…
Faster runs without loss of performance
Efficiency is the key!!! Small Particle Advantage
5mm
1.9mm N = 142,000 plates/m
(189% higher)
N = 75,000 plates /m
Higher resolution – narrower peaks
Higher sensitivity – taller peaks
Higher peak capacity (more peaks / unit time) – narrower peaks
( )k
kNRs
+-=
1 1
41
aa
Selectivity Efficiency Retention
Increase Speed, Maintain Resolution 200x2.1mm
0 2 4 6 8 10 12 14 16 18Time (min)
12mm
8mm
5mm
3mm
1.9mm600µl/min 655 bar
400µl/min 190 bar
250µl/min 102 bar
100µl/min 56bar
150µl/min 68 bar
Spee
d
Speeding up analysis with 1.9 mm Hypersil GOLD
The UltiMate™ 3000 LC Systems
Pumps
Autosampler
Column Compartments
Detectors
With ValvesStandard
Quaternary Dual-GradientBinary
VWD MWD/DAD Fluorescence Corona
Standard Thermostatted + FractionationBasic Automated
Isocratic
Coulochem
UHPLC+ Applications
Tandem LC
Online SPEParallel LC
Application Switching
Automated Method Scouting
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Fundamental of Mass Spectrometry
“The basis in mass spectrometry (MS) is the productionof ions, that are subsequently separated or filteredaccording to their mass-to-charge (m/z) ratio, anddetected. The resulting mass spectrum is a plot of the(relative) abundance of the produced ions as a functionof the m/z ratio.”
Niessen, W. M. A.; Van der Greef, J., Liquid Chromatography–Mass Spectrometry:
Principles and Applications, 1992, Marcel Dekker, Inc., New York, p. 29.
What is Mass Spectrometry?
Information Rich Data
Applications of Mass Spectrometry
• Pharmaceutical analysis– Bioavailability studies
– Drug metabolism studies,
pharmacokinetics
– Characterization of potential drugs
– Drug degradation product analysis
– Screening of drug candidates
– Identifying drug targets
• Biomolecule
characterization– Proteins and peptides
– Oligonucleotides
• Environmental analysis– Pesticides on foods
– Soil and groundwater contamination
• Forensic analysis/clinical
Mass Spectrum
(1023.566 x 1) - 1
= 1022.5
(512.287 x 2) - 2
= 1022.5
mass to charge = ( molecular weight + charge ) / charge
Mass spectrometry Characteristics
•Operate at very low pressure (10-5 to 10-7 torr)
(Atmosphere = 760 torr)
•Mass spectrometer work with IONS
•Measure gas-phase ions
•Determine the mass are separated according to their mass-to-charge
(m/z) ratio
Mass Spectrometry – Block Diagram
• ESI
• APCI
• APPI
Liquid Chromatography Very important!
- Many columns
- Many solvent systems
ION SOURCE
IONIZATION TECHNIQUES
Type of Ionization techniques
•Electron impact (EI)
•Chemical Ionization (CI)
•Atmospheric Pressure Ionization (API)
•Electrospray Ionization (ESI)
•Atmospheric Pressure Chemical Ionization (APCI)
•Atmospheric Pressure Photo-Ionization (APPI)
•Matrix Assisted Laser Desorption/Ionization (MALDI)
Electrospray Ionization (ESI)
Three Fundamental Processes:
1. Production of charged droplets.
2. Droplet size reduction, and fission.
3. Gas phase ion formation.
Ion Evaporation Theory
Capillary
+4 kV
Droplet
containing ions
As droplet evaporates, field
increases and ions move to
surface
Raleigh Limit – Droplet
Instability - releases
smaller droplets ions
ESI - Ion Max Source
Electrospray Ionization Atmospheric Pressure
Chemical ionization
Atmospheric Pressure Ionization
APCI:
Ions formed by gas phase chemistry
Good for volatile / thermally stable
Good for non-polar analytes
Good for small molecules (Steroids)
Chemistry Considerations ESI or APCI
ESI:
Ions formed by solution chemistry
Good for thermally labile analytes
Good for polar analytes
Good for large molecules (Proteins / Peptides)
Ion Max Source Design - APCI Probe
• It depends on the exact application.
• Increasing polarity and molecular weight and thermal instability
favors electrospray.
– Most drugs of abuse are highly polar and are easily
analyzed using electrospray.
– High molecular weight proteins also require electrospray
• Lower polarity and molecular weight favors APCI or APPI.
• Lower background, but compounds must be more
thermally stable.
Which is Best?
Mass Spectrometry – Block Diagram
Typical Mass Accuracy and Resolution
Type of MS Mass accuracy Resolution Utility for
Quadrupole 0.1 amu 6,000 Identify
Traps 0.1 amu 8,000 Identify
TOF 0.0001 amu <20,000 TOF
60,000 Q-TOF
Empirical formula/
composition
Sector 0.0001 amu 10,000 Empirical formula/
composition
Orbitrap 0.0001 amu 1,000,000 Empirical formula/
composition
MASS ANALYSER
QUADRUPLE
TSQ Quantiva MS—Powered by AIM Technology
Systematic optimization of all electric fields, in concert, to produce breakthrough performance.
Active Ion Management (AIM)
TSQ Triple Quadrupole (available on YouTube)
http://www.youtube.com/watch?v=LFB14D8pkoc
Scan Modes in Quadrupole
Scan Mode Q1 Q2 Q3 Purpose
Full Scan Scanning Pass All Pass All MW Info.
SIM Fixed m/z Pass All Pass All Quantitation
Product Fixed m/z Pass All (+ CE) Scanning Structural Info.
SRM Fixed m/z Pass All (+ CE) Fixed m/z Targeted Quantitation
Neutral Loss Scanning Pass All (+ CE) Scanning Analyte Screening
Precursor Scanning Pass All (+ CE) Fixed m/z Analyte Screening
RT: 0.00 - 20.02
0 2 4 6 8 10 12 14 16 18 20Time (min)
05
1015202530
35404550556065
707580859095
100
Relative
Abundan
ce
1.24
4.60
2.62
3.79
3.105.38
7.787.58
18.3416.93 18.9415.958.23 15.0914.058.90 12.739.81
NL:7.35E7TIC MS HS-helin-1024-1
One Click
Chromatogram
SpectrumH +
Base peak
at m/z 240 (MH+)H O
H O
N H tB uO H
60 80 100 120 140 160 180 200 220 240 260 280 3000
100
%
240
241
Full Scan Mode
Full Scan Mode
Purpose: Survey scan of a chromatographic peak
Q1 Scanning RF Only Q3 RF Only
Full scan Q1:
Q1 RF Only RF Only Q3 Scanning
Full Scan Q3:
Full Scan (Q1 or Q3)
SIM Mode
Purpose: Quantitation on a specific m/z range of ions
Selected Ion Monitoring – SIM
Q1 Set RF Only + CE Q3 RF Only
SIM Q1:
Q1 RF Only RF Only + CE Q3 Set
SIM Q3:
SIM is in essence a full scan acquisition on a relatively narrow mass
window (defined as center mass / scan width)
Fixed m/z Pass All Pass All
¨ Advantages
¤ Targeted analyte monitoring
¤ High duty cycle
¨ Disadvantages
¤ Can suffer from interferences
¤ Not as sensitive or selective as SRM
Selected Ion Monitoring – SIM
RT: 0.00 - 75.04 SM: 7G
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75Time (min)
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
52.33
47.88
31.3055.14
34.47 50.24
39.421.00 18.87 23.568.09 24.156.50 17.2211.51 65.28 70.26 72.6363.6542.17 44.24 56.03
31.30
39.8538.39 47.883.23 30.99 40.53 59.413.45 64.64 67.2452.44 73.5755.5327.2610.36 21.9019.6614.03
NL: 2.91E8Base Peak F: + c NSI Full ms [ 400.00-1800.00] MS data14
NL: 7.97E7Base Peak m/z= 1030.90-1031.90 F: + c NSI Full ms [ 400.00-1800.00] MS data14
SIM
Full Scan
Full Scan versus SIM
Fixed m/zPass AllFixed m/z
Q1 Q2 Q3
¨ Advantages
¤ Targeted analyte monitoring
¤ High duty cycle
¤ “Simultaneous” monitoring of
multiple transitions
¨ Disadvantages
¤ No structural information
Selected Reaction Monitoring (SRM)
The Need for True MS/MS
RT: 2.28 - 5.89 SM: 15G
2.5 3.0 3.5 4.0 4.5 5.0 5.5Time (min)
0
20
40
60
80
100
0
20
40
60
80
100
Relat
ive A
bund
ance
0
20
40
60
80
100
RT: 5.37SN: 1093
RT: 5.37SN: 27528
RT: 5.37SN: 201353020
NL: 8.14E7m/z= 191.50-192.50 MS Genesis Full-MS2
NL: 1.38E8m/z= 191.50-192.50 F: + c EI Q1MS [191.945-191.995] MS Genesis SIM-1_111128173605
NL: 3.27E7TIC F: + c EI SRM ms2 191.950 [126.935-126.985] MS Genesis Full-SRM-Survey-1
x300
Full MS Scan
SIM Scan
SRM Scan
Noise Level
SRM Selectivity in Complex Matrices
18 19 20 21 22 23 24 25 26 27Time (min)
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
0
RT: 23.76
25.37
23.5327.3522.93
26.6220.19 23.43 24.55 25.4824.6721.25 26.2622.41 24.0220.57 22.1719.61
RT: 23.76
24.5923.13 24.17 25.18 25.3422.93 27.0926.31 26.4623.37
21.89 22.5221.6721.1520.5719.65 20.15
RT: 23.77
RT: 23.77
NL: 2.67E4m/z= 271.50-272.50 F: + c SIM ms [236.50-237.50, 271.50-272.50, 306.50-307.50] MS probe20f_sim
NL: 9.94E3m/z= 306.50-307.50 F: + c SIM ms [236.50-237.50, 271.50-272.50, 306.50-307.50] MS probe20f_sim
NL: 6.10E5m/z= 207.50-208.50 F: + c EI SRM ms2 237.000 [207.999-208.001] MS Genesis Probe20F
NL: 1.06E6m/z= 236.50-237.50 F: + c EI SRM ms2 272.000 [236.999-237.001] MS Genesis Probe20F
Superior Selectivity
Free from sample matrix
SRM
SIM
Comparison of SIM and SRM
HIGH RESOLUTION MASS ANALYSER
ORBITRAP
Mass Analyzers Parameters
•Nominal Mass
The mass of an ion with a given empirical formula calculated using the
integer mass numbers of the most abundant isotope of each element
Ex : M=249 C20H9+ or C19H7N
+ or C13H19N3O2+
•Exact Mass
The mass of an ion with a given empirical formula calculated using the
exact mass of the most abundant isotope of each element
Ex : M=249 C20H9+ 249.0070
C19H7N+ 249.0580
C13H19N3O2+ 249.1479
MASS RESOLUTION
• Ability of a mass spectrometer to distinguish between ions of
nearly equal m/z ratios (isobars).
Mass Resolution: What is it?
• m - measured mass
• Δm - peak width measured at 50% peak intensity (Full Width
Half Maximum)
- or the mass difference
between two adjacent peaks
of equal intensity, in this case
pw @ 10% valley definition is
used.
Resolution & Peak Width
• At minimum the resolution of the mass analyzer should be
sufficient to separate two ions differing by one mass unit
anywhere in the mass range scanned (unit mass resolution).
• Typical values of resolution for low resolution mass analyzers
(e.g. quadrupoles and ion traps) are below 5000.
• High resolution instruments have a resolution exceeding 15000.
Mass Resolution: What is it?
Commercial High Resolution MS Technology Race
Time progression (year)
Mas
s re
solu
tion
(FW
HM
)
Bendix Tof0
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
1955 1965 1975 1985 1995 2005 2015
Orbitrap Tof / QTof
Ion Trap-Orbitrap
Quad Orbitrap
Tribrid Orbitrap
ORBITRAP’s spectacular climb in performance in a decade!
First Q-Tof
Q-Orbitrap*
New Q-Orbitrap
New Tribrid Orbitrap
Entry Q-Orbitrap
LIT-Orbitrap ETD
Major accurate-mass analyzers for Life Science
Orbitrap Mass Analyzer: Principle of Operation
{ })/ln(2/2
),( 222mm RrRrzkzrU ×+-×=
z
φHyper-logarithmic potential distribution:
“ideal Kingdon trap”
r
12
2
-÷øö
çèæ=
RRmzwwj 2
2
-÷øö
çèæ=
RRm
zr wwqm
kz /
=w
Makarov A. Anal. Chem. 2000, 72, 1156-1162.
§ Characteristic frequencies:• Frequency of rotation ωφ
• Frequency of radial oscillations ωr• Frequency of axial oscillations ωz
Many Ions Generate a Complex “Transient”
Fourier Transform
Frequency DomainTime Domain
Mass Spectrum
http://planetorbitrap.com/
UHPLC with Q Exactive Mass Spectrometer
Applications of Triple
Quadrupole LC-MS/MS
TSQ Quantis Sensitivity Study: Pesticides
TSQ Quantis Sensitivity Study: Pesticides
Quantitation of more than 250 Pesticides below MRLs in Leek
LC: Vanquish Flex Binary System
Column: Accucore aQ (2.1 × 100 mm, 2.6 µm)
Column Temperature: 25ºC
Injection Volume: 1 µL
Mobile Phase: A) 98% water with 2% methanol; B) 98% methanol with 2% water— Both containing 0.1% formic acid and 5 mMammonium formate
Flow Rate: 300 µL/min
Run Time: 15 min
Thermo Scientific Vanquish Flex UHPLC
• Maximize UHPLC separation with 1034 bar (15,000 psi) pump pressure limit
• Viper-based, tool-free fluidic connections• Biocompatible, Iron-free flow path• Sample pre-compression for better
injection reproducibility and longer column lifetimes
• Standard Autosamper capacity: 4 racks (216 vials)
• New column thermostatting technology• Removable doors for easy access
TSQ Quantis Parameter
Sample Preparation
LC-MS/MS chromatogram of more than 250 pesticides in leek extract at 100 μg/kg.
Chromatogram of more than 250 pesticides
the number of transitions per unit time
Azoxystrobin elutes at 8.69 min
Panels B and C show the reproducibility of azoxystrobin comparing the 10th injection to410th injection
Panels A the number of transitions per unit time
Azoxystrobin
TSQ Quantis MS VS TSQ Endura MS
Differences in performance are shown in peak area and peak height.
Negative mode Positive mode Negative mode
Positive mode Positive mode
• Red lines represent ―20% of Atrazine response at 10 μg/kg.• Yellow lines show the exact moment the system was placed in
standby mode for 12 h (no maintenance was performed).• The data shows that the response was within the expected ―20%
range for at least 400 injections of 10 ppb QC in leek.
Reliable Performance when Starting from Standby mode
• rapid and robust quantitation of more than 250 pesticides in leek at or below their respective MRLs.
• selectivity and sensitivity enabled analysis of only 1 μL sample
• without need for dispersive SPE sample cleanup or sample dilution.
Conclusion
Thank You for Your Attention
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