Mass Directed Purification from 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 TIC - Total 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 compound with single bromine Isotopic pattern for typical organic compound

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

Mass Spectrometer block diagram Source Region Mass Analyzer Detector Inlet 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) Fragment mass analyzer Select ion of interest Collision induced disassociation 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 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)

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 Electron multiplier Used in PurIon Amplifies signal by generating electrons Faraday cup Ions hits cup Enough ions generate a measurable charge

PurIon system Good place to mention that Since the turbo is at 60,000 Rpm unit needs to be in Shutdown 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)

Teledyne Isco Confidential Fluid Interface Flow Diagram Pulse damper Pressure Transducer To Mass Spec Splitter Valve Prime valve Back pressure 40 psi From Rf port C To Rf port D Pump head Teledyne Isco Confidential

Other uses for PurIon- Flow Injection Analysis Not running Not switching for FIA

Teledyne Isco Confidential Flow Injection or Reaction Monitoring Use “Method Development Screen No flow from CombiFlash Useful for reaction monitoring 1 mg/20 mL or less 200 µL/min to ESI/APCI Mass Spectrometer Isocratic make up Pump 200 µL/min Methanol with 0.1% Formic Acid Teledyne Isco Confidential

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-

[M+1] even: odd # of nitrogens [M+1] odd: 0 or even # of nitrogen Nitrogen rule [M+1] even: odd # of nitrogens [M+1] odd: 0 or even # of nitrogen Applies only to the parent ion!! 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!!!

Multiply charged species What m/z would they appear at? Ions Multiply charged species What m/z would they appear at? Probably not common on small molecules 93, 94

Fragmentations, rearrangements Generally, fragments occur near heteroatoms (N, O, S) Also can occur with “good” leaving groups, stable ions

Fragmentation case study Base peak = 168 What drives this reaction?

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

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

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

Key Markets Pharmaceutical Drug Discovery Agricultural Chemistry Petrochemicals Natural Products Foods and Flavors

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