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

https://www.thermofisher.com/order/catalog/product/TSQ02-10001?SID=srch-srp-TSQ02-10001

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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