UHPLC-MS Basic Principles and Applications Jitnapa Voranitikul - - PowerPoint PPT Presentation

UHPLC-MS Basic Principles and Applications

Jitnapa Voranitikul

February, 2018 Product Specialist LC/MS

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

Fundamental of Liquid Chromatography

3

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

• peration

Basic Standard x2 Dual LC RSLC x2 Dual RSLC RSLCnano

VanquishTM Max Pressure 1517 bar Thermo Analytical LC Systems

1.9µm 5µm

u opt u opt Linear Velocity (mm/s) H E T P (µm)

5 10 15 6 1 2 3 4 5

3µm

u opt

Increasing Column Efficiency 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

5µm 1.9µm

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 k N Rs + − = 1 1 4 1 α α

Selectivity Efficiency Retention

Increase Speed, Maintain Resolution 200x2.1mm

2 4 6 8 10 12 14 16 18 Time (min)

12µm 8µm 5µm 3µm 1.9µm 600µl/min 655 bar 400µl/min 190 bar 250µl/min 102 bar 100µl/min 56bar 150µl/min 68 bar

Speed

Speeding up analysis with 1.9 µm Hypersil GOLD

The UltiMate™ 3000 LC Systems

Pumps Autosampler Column Compartments Detectors

With Valves Standard Quaternary Dual-Gradient Binary VWD MWD/DAD Fluorescence Corona Standard Thermostatted + Fractionation Basic Automated Isocratic Coulochem

UHPLC+ Applications

Tandem LC Online SPE Parallel 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 production

• f ions, that are subsequently separated or filtered

according to their mass-to-charge (m/z) ratio, and

• detected. The resulting mass spectrum is a plot of the

(relative) abundance of the produced ions as a function

• f 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 2 4 6 8 10 12 14 16 18 20 Time (min) 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Relative Abundance 1.24 4.60 2.62 3.79 3.10 5.38 7.78 7.58 18.34 16.93 18.94 15.95 8.23 15.09 14.05 8.90 12.73 9.81 NL: 7.35E7 TIC MS HS-helin- 1024-1

One Click

Chromatogram Spectrum

H +

Base peak at m/z 240 (MH+)

H O H O N H tB u O H

60 80 100 120 140 160 180 200 220 240 260 280 300 100 %

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 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Time (min) 10 20 30 40 50 60 70 80 90 100 Relative Abundance 10 20 30 40 50 60 70 80 90 100 Relative Abundance 52.33 47.88 31.30 55.14 34.47 50.24 39.42 1.00 18.87 23.56 8.09 24.15 6.50 17.22 11.51 65.28 70.26 72.63 63.65 42.17 44.24 56.03 31.30 39.85 38.39 47.88 3.23 30.99 40.53 59.41 3.45 64.64 67.24 52.44 73.57 55.53 27.26 10.36 21.90 19.66 14.03 NL: 2.91E8 Base Peak F: + c NSI Full ms [ 400.00-1800.00] MS data14 NL: 7.97E7 Base 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/z Pass All Fixed 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 5.5 Time (min) 20 40 60 80 100 20 40 60 80 100 Relative Abundance 20 40 60 80 100 RT: 5.37 SN: 1093 RT: 5.37 SN: 27528 RT: 5.37 SN: 201353020

NL: 8.14E7 m/z= 191.50-192.50 MS Genesis Full-MS2 NL: 1.38E8 m/z= 191.50-192.50 F: + c EI Q1MS [191.945-191.995] MS Genesis SIM-1_111128173605 NL: 3.27E7 TIC 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 27 Time (min) 20 40 60 80 100 Relative Abundance 20 40 60 80 100 Relative Abundance 20 40 60 80 100 Relative Abundance 20 40 60 80 100 Relative Abundance RT: 23.76 25.37 23.53 27.35 22.93 26.62 20.19 23.43 24.55 25.48 24.67 21.25 26.26 22.41 24.02 20.57 22.17 19.61 RT: 23.76 24.59 23.13 24.17 25.18 25.34 22.93 27.09 26.31 26.46 23.37 21.89 22.52 21.67 21.15 20.57 19.65 20.15 RT: 23.77 RT: 23.77

NL: 2.67E4 m/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: 9.94E3 m/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: 6.10E5 m/z= 207.50-208.50 F: + c EI SRM ms2 237.000 [207.999-208.001] MS Genesis Probe20F NL: 1.06E6 m/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

+

• r

C19H7N+

• r

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

• f 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) Mass resolution (FWHM)

Bendix Tof 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 ) , (

2 2 2 m m

R r R r z k z r U ⋅ + − ⋅ =

z φ

Hyper-logarithmic potential distribution: “ideal Kingdon trap”

r 1 2

2

−       = R Rm

z

ω ωϕ 2

2

−       = R Rm

z r

ω ω

q m k

z

/ = ω

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 Domain Time Domain Mass Spectrum

http://planetorbitrap.com/

UHPLC with Q Exactive Mass Spectrometer

Applications of Triple Quadrupole LC-MS/MS

TSQ Q Qu Quanti tis Sens ensit itiv ivity S y Stud udy: P : Pes esticid ides

TSQ Q Qu Quanti tis Sens ensit itiv ivity S y Stud udy: P : Pes esticid ides

Quantit itatio ion of

• f mor
• re th

than 2 250 P Pes esticid icides below M MRLs i in Leek

LC LC: Vanquish Flex Binary System Colum umn: n: Accucore aQ (2.1 × 100 mm, 2.6 µm) Colum umn T n T emperatur ure: 25ºC Inj njectio ion V Volume: 1 µL Mobile le Pha Phase: A) 98% water with 2% methanol; B) 98% methanol with 2% water— Both containing 0.1% formic acid and 5 mM ammonium formate Fl Flow Ra Rate: 300 µL/ min Run T Run Time: 15 min

The hermo S Sci cien entif ific V ic Van anquis ish Fl Flex U x UHPLC

• M aximize 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 Q Qu Quanti tis Param ameter er

Sam ample P le Prep eparatio ion

LC-M S/ M S chromatogram of more than 250 pesticides in leek extract at 100 μg/ kg.

Chr Chromatogram o

• f more tha

han 2 250 pes pesticid icides

the number of transitions per unit time

Azoxystrobin elutes a at 8. 8.69 69 min

Pane nels B B and nd C show the reproducibility of azoxystrobin comparing the 10th injection to 410th injection Pa Panels A A the number of transitions per unit time Azoxystrobin

TSQ Q Qu Quanti tis MS VS VSTSQ Q Endura MS

Differences i in n pe performance are sho hown i in n pe peak area a and pe nd peak he height.

Negative mode Positive mode Negative mode Positive mode Positive mode

• Red lines represent ―20% of Atrazine response at 10 μg/kg.
• Y

ellow 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.

Reliab able P e Performan ance e when en Star artin ing fro rom Standby m mode

• rapid and robust quantitation of

more than 250 pesticides in leek at or below their respective M RLs.

• selectivity and sensitivity enabled

analysis of only 1 μL sample

• without need for dispersive SPE

sample cleanup or sample dilution.

Con Conclusion

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