Mass Directed Purificationfrom

Teledyne Isco

CombiFlash®

a Name You Can Rely On

Fundamentals of Mass Spectroscopy:

Goals:

• Terminology• Major components of a mass spectrometer• Ionization techniques • Mass analyzers;• PurIon Mass Spectrometer overview• Why use PurIon?• Mass directed purification overview

Terminology

■ daltons (Da) – most commonly used today■ Unified atomic mass unit (u)- same as Da, based on 12C■ amu (atomic mass units)- archaic, technically based on

16O, but people still use it…■ m/z- mass-to-charge ratio, may see as m/q (older

literature)

Mass Spectrometry Simplified

■ Analytes are converted to gas-phase ions (source).■ The ions are separated by their mass-to-charge

ratios (m/z, Analyzer) and are detected (Detector).■ Relative ion current (signal) is plotted versus m/z

to produce a mass spectrum.

Some Common Terms

■ M: mass of a given molecule■ [M+H]+: mass of a molecule

with a proton; carries a positive charge

■ MeOH: methanol■ MeCN, ACN: acetonitrile■ EtOAc: ethyl acetate

■ Base peak: Tallest peak in a mass spectrum

Ion Current

■ XIC- Extracted Ion Current

■ Ion current for a limited mass range (often m/z range of 1 or 2)

■ Used to monitor characterized compounds

■ TIC - Total Ion Current

■ Uses the entire spectral range

■ Useful for natural products, unknown compounds

■ Useful for molecules that generate weak molecular ion peaks

Isotopes

■ Same element (determined by number of protons)

■ Different number of neutrons

■ Changes mass of atom (and molecules containing this atom)

■ Hydrogen (1H, 2H deuterium, 3H tritium)

■ Carbon (12C, 13C, 14C)■ Chlorine (35Cl, 37Cl;

~75:25)■ Bromine (79Br, 81Br;

~50:50)

Isotopic Patterns

Isotopic pattern for typical organic compound

Isotopic pattern for compound withsingle bromine

Mass

■ Masses based on 12C=12.0000

■ Other elements/isotopes do not have integer masses

■ 1H=1.0079■ 16O=15.9949■ 14N=14.00307■ 79Br= 78.9183

Mass

■ Nominal mass= integer mass using most abundant isotope; the number we usually state in conversation

■ Monoisotopic mass: Sum of atomic masses using the most common isotope of each element in a molecule

■ Exact Mass: used by some chemistry software; sum of atomic masses of most common isotope when no isotopic species specified.

■ Average mass: sum of average atomic masses in a molecule

Formula Weight : 194.19Exact Mass : 194.08Formula : C8H10N4O2

N

N

O

N

ON

Mass Spectrometer block diagram

Inlet Source Region Mass Analyzer Detector

Vacuum system

Vacuum system

■ Roughing pump■ Turbo molecular pump■ Vacuum needed:

■ To avoid further reactions (fragmentation, reactions, etc.)

■ Increase mean-free path (maintain ion energy)

Mass Analyzer Types

■ Single quadrupoles (MS)■ Triple quadrupoles (MS/MS)■ Ion traps■ Time-of-flight (TOF)■ Sector analyzers■ Hybrids (ex. QTOF)■ and more hybrids (magnetic

sector TOF)

Quadrupoles (PurIon)

■ Less expensive; compact design; low scan times, very common

■ Limited resolution, not suited for pulsed ionization methods

■ Scan the electric fields/ frequency to scan mass range The correct combination of AC and DC

electric fields allow resolution of ions by their m/z ratio

Triple Quadrupoles (MS-MS)

Select ion of interest

Collision induced disassociation

Fragment mass analyzer

Q1 Q2 Q3

• Select ion of interest in Q1• Fragment this ion in Q2• Scan Q3 for fragment masses; fragmentation pattern used to deduce original ion structure

Ionization Techniques

■ Required to put charge on molecule

■ While ionizing, get molecule into gas phase

■ No ions = no mass spectrometry!!

Ionization Techniques

■ Atmospheric Pressure Ionization (API)■ Electron and Chemical Ionization (EI/CI)■ Photo-ionization■ Matrix-assisted laser desorption (MALDI)■ Fast atom bombardment (FAB)

Atmospheric Pressure Ionization

■ “Soft” techniques- reduced fragmentation, more molecular ions

■ Useful for liquid chromatography

■ ESI, APCI

■ Ionize compounds■ Remove solvents■ Get compounds into

analyzer

Electrospray ionization (ESI)- Used on PurIon

ESI Solvents & Additives

■ Solvents■ Water

■ Acetonitrile

■ Methanol

■ Ethanol

■ Propanol

■ 2-propanol

■ Additives■ Acetic Acid

■ Formic Acid

■ Ammonium hydroxide

■ Ammonium formate*

■ Ammonium acetate*

* <= 10 mM

What happened here?!

What’s with the solvent additives?

■ Help charge the analyte■ Acids add protons

(positive charge)■ Bases remove protons

(negative charge)R

O–

R

O H

O–

O

RO H

O

R

R

O H

R

O H 2+

R

N H 3+

R

NH 2

O H–O H

–

H +H +

ESI Solvents- Use with care

■ Trifluoroacetic acid■ Strong ion pair causes neutral

molecule?

■ Still, commonly used for LC-MS, some TFA runs seem Ok

■ Triethylamine- may suppress less basic compounds

Atmospheric Pressure Chemical Ionization (APCI)- Used on PurIon as an Option

■ Heated probe evaporates solvent

■ Corona discharge places charge on molecules

■ Tetrahydrofuran- very flammable when used for APCI (spark)

Compound Ionization Technique Map

Detectors■ Faraday cup■ Ions hits cup■ Enough ions generate a

measurable charge

■ Electron multiplier■ Used in PurIon■ Amplifies signal by

generating electrons

PurIon system

Good place to mention thatSince the turbo is at 60,000Rpm unit needs to be inShutdown to prevent pump destruction

Get Sample into Mass Spectrometer

■ Split/dilute sample■ Solvent good for

ionization (generally methanol)

■ Consistent delivery

■ Tubing with restrictions■ “MRA” valve (used in

PurIon)■ Both use “make-up” or

“carrier solvent” pump

Fluid Interface

Why use Mass Directed Purification?

Traditional open-access LC/MS workflow

• Save steps • Save time• Move right into

the next step

Advantages for the chemist

■ Collect only the desired compound(s)

■ Verification the correct compound is being collected

■ Ignore previously known compounds (natural products, reverse engineering)

Pump head

Prime valve Back pressure 40 psi

Pulse damper

To Mass Spec

To Rf port DFrom Rf port C

Pressure Transducer

SplitterValve

Fluid Interface Flow Diagram

Teledyne Isco Confidential

Other uses for PurIon- Flow Injection Analysis

Not switching for FIA

Not running

Flow Injection or Reaction Monitoring

Teledyne Isco Confidential

33

Isocratic make up Pump

200 µL/min Methanol with 0.1% Formic Acid

200 µL/minto ESI/APCI Mass Spectrometer

• Use “Method Development Screen

• No flow from CombiFlash

• Useful for reaction monitoring

• 1 mg/20 mL or less

Chemistry

■ Isotopes■ Nitrogen Rule■ Fragmentations/

rearrangements■ Adducts■ Multiply charged ions

■ Note: Many of the rules written for electron ionization Parent peak is M+

■ ESI parent is usually [M+1]

■ What does this mean?!

What are ions?

■ Charged molecule■ Shown as M+

■ Na+

■ Cl-

Nitrogen rule

■ [M+1] even: odd # of nitrogens

■ [M+1] odd: 0 or even # of nitrogen

■ Applies only to the parent ion!!

Formula Weight : 194.19Exact Mass : 194.08Formula : C8H10N4O2

N

N

O

N

ON

What is the [M+1] ?Is it even or odd?

Nitrogen rule

■ Synthesized compound has 2 nitrogens

■ See peak at m/z=168 using ESI+

■ Is this a fragment or a parent ion?

■ Why?

Mass Spectroscopy Chemistry

■ Ions- what we produce■ Rearrangements- move the

charge someplace else■ Adducts- “share the charge”■ Fragments- make both

charged and uncharged stable pieces

■ Lower the energy of the molecule!!!

Ions

■ Multiply charged species

■ What m/z would they appear at?

■ Probably not common

on small molecules

93, 94

Exact Mass : 186.11

Exact Mass : 94.06

N+

H 3

N H 3+

N+

H 3

N H 2NH 2

Fragmentations, rearrangements

■ Generally, fragments occur near heteroatoms (N, O, S)

■ Also can occur with “good” leaving groups, stable ions

N H 2NH 2

Fragmentation case study

■ [M+1]+ = 185■ Base peak = 168■ What drives this

reaction?

+N H 2C+

N H 2N+

H 3N H 3

Rearrangement case Study

■ λmax 200-220 nm

■ [M+1] = 141 Da expected■ Solvent system

Hexane/EtOAc■ User advised to use a range

of masses, would get weak peak @ m/z 123

O

O H

Rearrangement case Study

Not charged- not seen. Major product (loss of another hydrogen between methyls

Very minor product- charged, m/z=122 or 123 (depending on loss of H)

H+ binds to non-bonding electrons on oxygen

H+

+ H2O

O

O

OH

O

+

Good leaving groups

■ -NH2 (leaves as ammonia)

■ -OH (leaves as water)

■ COOH (leaves as CO2)

■ Look for increase in conjugation

■ Look for easily formed, stable molecules

Adducts

■ Bond to the molecule- usually detected as [M+H+Adduct]

■ May be more intense than [M+1]

■ May occasionally see dimers [M+H+M]+

Adduct List

Most common adducts seen:

■ Methanol■ Acetonitrile■ Sodium■ Potassium

Adduct example & sources

■ Solvent■ Glassware■ Syringe

Adducts- potential confusion

■ Sample dissolved in methanol- use method development, [M+MeOH+H] observed. Purification in hexane/ethyl acetate- will you see adduct?

■ Sample run on LC-MS mobile phase water/MeCN, [M+MeCN+H] observed. Purification in hexane/ethyl acetate- will you see adduct?

■ Use a range that covers [M] through [M+adduct]

Another Adduct Example

■ Solvent system Hexane/EtOAc

■ m/z=141 expected■ Carrier = MeOH/0.1%

formic acid

O

O H

Key Markets

PharmaceuticalDrug Discovery Natural

Products

AgriculturalChemistry Foods

and Flavors

Petrochemicals

Types of Mass Analyzers: Sector

■ Separate ions by charge-to-mass ratio (m/z; m/q)

■ Generally have a electrical field sector (not shown) to focus ion energies

■ Classical mass spectrometer■ Good resolution, dynamic range■ Large, higher cost

Types of Mass Analyzers: Time-Of-Flight (TOF)

■ High mass range■ Well suited for pulsed

ionization methods (MALDI)

■ Requires pulsed ion injection or ion beam switching

“Drift” area

Fourier Transform analyzers■ Ions drift into area of

constant magnetic field- ions move in circular motion

■ Use oscillating electric field to “excite” ions

■ Detect ions by their cyclotron radiation, use Fourier transform to obtain masses

• Highest mass resolution• Expensive analyzer

Ion traps

■ Trap ions for other purposes

■ Quadrupoles■ Linear ion traps■ Orbitraps■ Some used for analyzers

as well as traps

Hybridized techniques

■ Two or more m/z analyzers of different types

■ QTOF: Triple quadrupole, but the last quadrupole replaced by a TOF analyzer (Quadrupole TOF)

■ Provide different information on a molecule

■ Improve signal-to-noise

Other Ionization- APPI

■ Atmospheric Pressure Photo-Ionization■ Light “kicks off” an electron, charging molecule

Other ionization- Electron Ionization

■ Formerly known as “electron impact”■ Molecule charged in vacuum■ Not compatible

with LC■ Fragments

molecules

Other ionization- Chemical Ionization

■ Primary ionization:■ CH4 + e- CH4

+ + 2e-

■ Secondary reagent ion■ CH4 + CH3

+ CH5+ + CH3

■ Product Ion Formation■ M + CH5

+ CH4 + [M+H]+ (protonation)

■ M + CH3+ CH4 + [M-H]+ (proton abstraction)

■ M + CH4+ CH4 + [M]+ (charge exchange)

Other ionization- MALDI

■ Matrix Assisted Laser Desorption/Ionization

■ UV LASER ablates matrix & compound

■ Matrix transfer proton(s) to molecule■ Commonly used with TOF (pulsed

LASER)■ Used with macromolecules, proteins,

bacteria, viruses

Other ionization-DESI, DART

■ Desorption ESI■ Direct Analysis in Real

Time■ Useful for QC,

forensic analysis