1 Function Separation of volatile organic compounds Volatile – when heated, VOCs undergo a phase transition into intact gas-phase species Separation occurs as a result of unique equilibria established between the solutes and the stationary phase (the GC column) An inert carrier gas carries the solutes through the column

2 Components Carrier Gas, N 2 or He, 1-2 mL/min Injector Oven Column Detector

3 Gas tank Oven Column Injector Syringe Detector

4 Sample Introduction (continued): – A sample can be introduced in several ways, the most common being with a direct insertion probe or by infusion through a capillary column. Samples are often introduced using a direct insertion probe or a capillary column. The probe and capillary carry the sample into the vacuum of the mass spectrometer. Once inside the mass spectrometer, the sample is exposed to the ionization source

5 Injector A GC syringe penetrates a septum to inject sample into the vaporization camber Instant vaporization of the sample, 280  C Carrier gas transports the sample into the head of the column Purge valve controls the fraction of sample that enters the column

6 Splitless (100:90) vs. Split (100:1) Injector Syringe Injector Syringe Purge valve open Purge valve closed GC column He

7 Split or splitless Usually operated in split mode unless sample limited Chromatographic resolution depends upon the width of the sample plug In splitless mode the purge valve is close for s, which means the sample plug is seconds As we will see, refocusing to a more narrow sample plug is possible with temperature programming

mm ID Liquid Stationary phase Mobile phase (Helium) flowing at 1 mL/min Open Tubular Capillary Column m in length  m

9 Matrice Matrice : la piu’ usata e’ polvere di diatomee impregnata di fase stazionaria Fase stazionaria Fase stazionaria : liquido ad alto punto di ebollizione (ad esempio olio di silicone: di cui si puo’ variare la polarita’ sostituendo ad es. con o con Fase mobile Fase mobile : gas inerte (azoto, elio, argon) in cui sono volatilizzati i composti da separare mantenuti allo stato gassoso dall’ alta temperatura. Nella GLC i coefficienti di partizione sono strettamente dipendenti dalla temperatura. Il sistema di rivelazione piu’ diffuso e’ il rivelatore a ionizzazione di fiamma (FID flame ionisation detector). Anche spettrometro di massa. Prima dell’ HPLC la GLC era la forma di cromatografia piu’ diffusa.

10 Stationary Phases Must have: (1) low volatility(2) thermal stability (4) chemical inertness (5) solvation properties giving suitable values for k’, . Commonest are polysiloxanes: Nature of R varied to give different polarities. e.g. All R = Me : non-polar column. Best for non-polar analytes (hydrocarbons, PAH’s etc.) or R = 50% Me, 50% cyanopropyl - increased polarity - best for alcohols, acids etc. Greater polarity from polyethylene glycols: Detectors will discuss these later (and applications of GC in real analytical problems).

11 GAS CROMATOGRAFIA (GC)

12 RIVELATORI PER GC

13 General Elution Problem in GC (a) - low temperature (45 0 C) - good resolution initially - but too slow later. (b) - higher temperature (145 o C) - much faster but poor resolution for early-eluting species. In general - best results for temperatures near boiling point of analyte. If there is a wide range of boiling points in the sample - then the best results \re obtained by temperature programming as shown in (c), for the same mixture, where the temperature steps are as shown.

14 Oven Programmable Isothermal- run at one constant temperature Temperature programming - Start at low temperature and gradually ramp to higher temperature – More constant peak width – Better sensitivity for components that are retained longer – Much better chromatographic resolution – Peak refocusing at head of column

15 Typical Temperature Program Time (min)  C 220  C 160  C

16 Mass Spectrometry Gas Chromatography-Mass Spectrometry (GC-MS) The computer drives the MS, records the data, and converts the electrical impulses into visual displays and hard copy displays. Identification of a compound based on it's mass spectrum relies on the fact that every compound has a unique fragmentation pattern A large library of known mass spectra is stored on the computer and may be searched using computer algorithms to assist the analyst in identifying the unknown. Sample introduced into GC inlet vaporized at 250 °C, swept onto the column by He carrier gas & separated on column. Sample components emerge from column, flowing into the capillary column interface connecting the GC col-umn and the MS (He removed). interface

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19 Meccanismo di ionizzazione per EI Un fascio di elettroni viene generato da un filamento riscaldato di W o Re ed accelerato da una d.d.p. di ~70V. Il fascio di elettroni urta il fascio di molecole del campione a 90°. I prodotti primari della collisione sono ioni positivi a carica unitaria (ioni molecolari, M + ), che si formano per repulsione elettrostatica: M + e -  M+ + 2e - M+ è uno ione radicale. Il processo di ionizzazione ha efficienza molto bassa (circa 1/10 6 ). M+ è uno ione instabile (ha un numero dispari di elettroni) e può dare reazioni di decomposizione che danno origine agli ioni frammento. Gli ioni generati vengono attirati con una piccola d.d.p. verso la zona di accelerazione, dove sono soggetti ad un potenziale di V per raggiungere le loro velocità finali prima di entrare nell’analizzatore di massa.

20 Meccanismo di ionizzazione chimica Le molecole del campione vengono ionizzate per collisione con gli ioni di un gas reagente, ionizzato per impatto elettronico. È possibile generare anche ioni negativi. Si impiega la stessa strumentazione della EI. Nella zona di ionizzazione il gas reagente viene mantenuto alla pressione di circa 1 torr. Il gas impiegato più comunemente è CH 4 : CH CH 4  CH CH 3 CH CH 4  C 2 H CH 3 CH XH  XH CH 4 C 2 H XH  XH C 2 H 4 C 2 H XH  X + + C 2 H 6 Lo ione generato può avere massa M+1, M+2, M-1 o anche M+29 (+C 2 H 5 ).

21 mass scanning mode m1 m3 m4 m2 m3 m1 m4 m2 single mass transmission mode m2 m3 m1 m4 m2 Quadrupoles have variable ion transmission modes

22 Interpretation of EI Mass Spectra

23 Mass Spectrometry When the electron beam ionizes the molecule, the species that is formed is called a radical cation, and symbolized as M +. The radical cation M + is called the molecular ion or parent ion. The mass of M + represents the molecular weight of M. Because M is unstable, it decomposes to form fragments of radicals and cations that have a lower molecular weight than M +. The mass spectrometer analyzes the masses of cations. A mass spectrum is a plot of the amount of each cation (its relative abundance) versus its mass to charge ratio (m/z, where m is mass, and z is charge). Since z is almost always +1, m/z actually measures the mass (m) of the individual ions. Introduction

24 Mass Spectrometry Consider the mass spectrum of CH 4 below: Introduction The tallest peak in the mass spectrum is called the base peak. The base peak is also the M peak, although this may not always be the case. Though most C atoms have an atomic mass of 12, 1.1% have a mass of 13. Thus, 13 CH 4 is responsible for the peak at m/z = 17. This is called the M + 1 peak.

25 Basic Mechanisms of Fragmentation Mass spectral reactions are unimolecular; the sample pressure in the EI source is kept sufficiently low so that bimolecular (ion-molecule) or other collisions are usually negligible. If sufficiently excited, the M + ions can decompose by a variety of energy dependent mechanisms each of which results in the formation of an ion and a neutral species (radical). This primary product may have sufficient energy to decompose further. In the MS of ABCD, the abundance of BCD + will depend on the average rates of its formation and decomposition, whereas [BC + ] will depend upon the relative rates of several competitive reactions. There are several types of unimolecular reactions that can take place: ABCD ABCD + A + + BCD A + BCD + AD + + B=C D + ABC + A + BC + D + BC + e–e–

26 EI Fragmentation of CH 3 OH CH 3 OHCH 3 OH + CH 3 OHCH 2 O=H + + H CH 3 OH + CH 3 + OH CHO=H + + HCH 2 O=H +

27 EI Breaks up Molecules in Predictable Ways Molecular ion Electron Impact MS of CH 3 OH

28 relative abundance Mass (mass-to-charge ratio) Unknown m/z Int 1<

29 Mass (mass-to-charge ratio relative abundance m/z Int

30 Mass Spectrometry Introduction

31 Mass Spectrometry Alkyl Halides and the M + 2 Peak (CH 3 ) 2 CH +

32 Mass Spectrometry Alkyl Halides and the M + 2 Peak (CH 3 ) 2 CH +

33 How many peaks will a molecule containing 3 bromine atoms exhibit? 4 peaks at intervals of 2 mass units in the ratio of 1:3:3:1. CHBr 3 What species are responsible for the “four peaks”? 79 Br 79 Br 79 Br = Br 79 Br 81 Br = Br 81 Br 79 Br = Br 79 Br 79 Br = Br 81 Br 79 Br = Br 79 Br 81 Br = Br 81 Br 81 Br = Br 81 Br 81 Br = 243

34 Are A + 2 peaks present? No! What does the fact that the “base peak” = M +. imply?stability How many carbons are implied by the M + 1 peak?6.8/100:- 6 carbons (and 6 hydrogen’s) What does the A + 2 peak at m/z 80 arise from? What is the molecule’s identity?Benzene 13 C 2 12 C 4 H

35 n-decane

36 n-tridecane 184

37 n-pentadecane 212

38 n-eicosane 282

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40 GC/MS di un estatto (SPME) di vino: Phorate Diazinon Methyl-parathion Fenitrithion Malathion Fenthion Ethyl-parathionMethidathion Unknow

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42 Residual Pesticides in Food

43 Selected Applications - PCB Analysis

44 Forensic Mass Spectrometry J. Yinon, Ed., Forensic Applications of Mass Spectrometry, CRC Press, 1995 Analysis of Body Fluids for Drugs of Abuse Analysis of Hair in Drug Testing Sports Testing Analysis of Accelerants in Fire Debris Analysis of Explosives Use of Isotope Ratios

45 Mass Spectrometry Gas Chromatography-Mass Spectrometry (GC-MS) To analyze a urine sample for tetrahydrocannabinol, (THC) the principle psychoactive component of marijuana, the organic compounds are extracted from urine, purified, concentrated and injected into the GC-MS. THC appears as a GC peak, and gives a molecular ion at 314, its molecular weight. To improve GC separa- tions, compounds are often derivatized, e.g. as their trimethylsilyl (TMS) ethers or trifluor- oacetate (TFA) esters. THC TMS ether (l) & TFA ester (r)

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48 Il plasma assume una forma toroidale. l’introduzione di campioni liquidi nebulizzati, solidi particellati o gassosi direttamente nella zona centrale del plasma, quasi istantaneamente avviene volatilizzazione, atomizzazione ed eccitazione.

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51 La sorgente ICP risulta idonea per la spettrometria di emissione atomica, di fluorescenza atomica e di massa. Nel caso della spettrometria di massa, diverse sono le possibili realizzazioni strumentali, sostanzialmente riconducibili a sistemi quadrupolari, a tempo di volo e a settore magnetico.

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60 the mass defect Collision/reaction cell

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62 the mass defect Collision/reaction cell Polyatomic interferenceelement 40 Ar 12 C 52 Cr 40 Ar 35 Cl 75 As 40 Ar 40 Ar 80 Se 14 N 16 O 1 H 31 P monoisotopic Polyatomic ion removal 1.Collision 2.Reaction 3.Energy discrimination Polyatomic ion removal 1.Collision 2.Reaction 3.Energy discrimination

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72 provide quantification and identification MS approaches to metallomics ICP-MS Low detection levels, quantification, element specific screening MALDI, ESI MS, ITMS Structure identification, poorer detection levels than ICP-MS Metallomics characterization all aspects of metals in living systems

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