1. June 10, 20151 Introduction to LC/GC Instrumentation CM3007 Introduction to Separation Science Analytical Chemistry Lecture #1

2. 2 Milestones in Chromatography u 1903 Tswett - plant pigments separated on chalk columns u 1931 Lederer & Kuhn - LC of carotenoids u 1938 TLC and ion exchange u 1950 reverse phase LC u 1954 Martin & Synge (Nobel Prize) u 1959 Gel permeation u 1965 instrumental LC (Waters)

3. 3 Basis of Chromatography u Definition: C s = C m K u Mechanism - selective retardation caused by interactions with bonded phase of stationary phase

4. 4 Chromatogram u Detector signal vs. retention time or volume time or volume Detector Signal 12 Presence of several Peaks separated in Time/volume evidences Separation of components Peak area  Concentration

5. 5 Definitions u Mobile phase - phase that moves through chromatograph –In LC - eluent –In GC - carrier gas u Stationary phase - column; phase that is stationary in chromatograph u Bonded phase - reactive groups imparted to stationary phase in order to achieve selectivity

6. 6 Types of Chromatography u Classification by mobile phase: –Gas - Gas chromatography (GC) »1951 Martin and James (fatty acids) –Liquid - Liquid chromatography (LC) »1964 Horvath (Yale) instrument »1966 Horvath and Lipsky (nucleic acid components) –Supercritical fluid - Supercritical fluid chromatography (SFC) »1958 Lovelock (Yale)

7. 7 Purpose of Chromatography u Identify components in mixtures u Isolate component in mixture u Demonstrate level of purity –Quantitation »internal standard analytical preparative or semi-preparative

8. 8 Instrumentation for LC u Strong/weak eluent u Pump (up to 6000 psi) u Injector u Column u Detector u {Fraction Collector} u Computer

9. 9 Instrumentation for GC u Carrier gas –N 2, He, H 2 u Injector u Column u Detector u Computer oven

10. 10 Modes of LC Separation u Adsorption or normal phase u Reverse phase u Hydrophobic interaction u Size exclusion u Ion Exchange u Affinity

11. 11 Modes of LC Separation u Adsorption or Normal phase –Stationary phase more polar than mobile phase »column: hydroxyapatite (Ca 10 (PO 4 ) 6 (OH) 2 »mobile phase: hexane, CH 2 Cl 2, THF, CH 3 OH »gradient elution

12. 12 Modes of LC Separation (cont’d) u Reverse phase (RPC) –Stationary phase hydrophobic and mobile phase hydrophilic »column: silica, polystyrene covalently modified with alkyl chain 3-18 C’s »mobile phase: buffered water + organic solvent (propanol CH 3 CN, CH 3 OH) »gradient elution u Affinity (AC) –Based on specific and selective interactions between enzymes or antibodies and ligands

13. 13 Modes of LC Separation (cont’d) u Hydrophobic interaction (HIC) –Stationary phase hydrophobic and mobile phase hydrophilic »stationary phase: low density of short alkyl chains or phenyl »mobile phase: decreasing concentration of high salt solution (3M NH 4 SO 4 ) »gradient elution

14. 14 Modes of LC Separation (cont’d) u Size Exclusion (Gel Permeation) (SEC) –Chemical interactions between sample and stationary phase undesirable; separation accomplished by sieving mechanism with porous stationary phase »stationary phase: varies - silica »mobile phase: low ionic strength (100 mM) buffer or dilute acid (0.1% TFA) with CH 3 CN or isopropanol (20-50%) »isocratic elution

15. 15 Modes of LC Separation (cont’d) u Ion-Exchange (IEC) –Ion exchange interactions between cationic or anionic analyte and stationary phase bearing opposite charge »stationary phase: polystryrene, silica modified with functional groups such as quaternary amines »mobile phase: buffer containing increasing concentration of salt (NaCl, MgCl 2, K 3 PO 4, NH 4 SO 4 )

16. 16 Ion Exchange u Strong - charge of strong ion exchanger is essentially independent of pH –sulfonic acids (cation) –quaternary amines (anion) u Weak - charge of weak ion exchanger is pH dependent –carboxymethyl (CM) (cation) –primary, secondary, and tertiary amines, e.g., diethylaminoethyl (DEAE) (anion)

17. 17 Anion Exchange - Proteins u Anion exchange - protein is acidic bonded phase either weak - DEAE strong - quaternary amine

18. 18 Cation Exchange - Proteins u Cation exchange - protein is basic bonded phase either weak - CM strong - sulfonic

19. 19 Elution Approaches u Isocratic - constant mobile phase composition u Gradient - variable mobile phase composition –step - change accomplished sharply at a defined point in time –continuous - change accomplished gradually over time

20. 20 Modes of GC Separation u Packed –earliest –Solid particles either porous or non-porous coated with thin (1  m) film of liquid –1 - 8 mm ID; 1 - 10 m length

21. 21 Modes of GC Separation u Capillary (open tubular) –modern –Inner wall modified with thin (1  m) film of liquid –0.1 - 0.5 mm i.d.; 10 - 50 m length –types: –porous layer open tubular (PLOT) –wall-coated open tubular (WCOT) »liquid coating

22. 22 GC Liquid Phase u Low volatility u High bp u Chemically unreactive u Examples: –1-squalene –Tetrahydroxyethylenediamine –Carbowax (polyethylene glycol)

23. 23 Elution Analogies: GC vs. LC u Isothermal (GC)  Isocratic (LC) u Programmed temperature (GC)  Gradient (LC) –Raising column temperature (GC) »Decreases retention time »Sharpens peaks

24. 24 5 Properties of a Good Detector u Sensitivity –  Response/  C u Selectivity –Universal or selective response »selectivity - ability to distinguish between species u Rapid response u Linearity - concentration range over which signal proportional to concentration u Stability with respect to noise (baseline noise) and time (drift)

25. 25 Detectors for LC u UV-vis –PMT (single ) –PDA (simultaneous multi- monitoring) u Fluorescence u Electrochemical –Amperometry u NMR –microcoil NMR: J. Sweedler (U. Illinois)

26. 26 Detectors for GC u Electron capture (ECD) –radioactive –good for X -, NO 2 - and conjugated u Thermal conductivity (TCD) –change in resistance of heated wire u Flame ionization (FID) –destruction of combustible sample in flame produces measurable current u Fourier transform infrared (FTIR) u Mass spectrometry (MS)

27. 27 MS Components u Ionization source u Analyzer –Differentiating characteristic m/z u Ion detector

28. 28 Ionization Methods - Examples u Electron capture (EC) –70 eV e - + neutral molecule  energetic molecular ion –hard; fragmentation u Chemical ionization (CI) –Reagent ion + molecule  molecular ion + reagent ion –Reagent ion = He, OH - (water), CH 5 + or CH 3 + (CH 4 ) –soft; less fragmentation

29. 29 Ionization Methods - Examples (cont’d) u Electrospray (ESI) –generation of ions by desolvation or desorption of charged liquid droplets u Matrix Assisted Laser Desorption (MALDI) –ionization facilitated by laser irradiation of sample dissolved in an organic matrix –EX: sinapinic acid

30. 30 Types of MS Analyzers u Quadrupole - most common u Ion trap u Time of Flight (TOF)

31. 31 Two Operational Modes u Scan –Collect mass data over known range –Slow u Selective ion monitoring (SIM) –Sample mass at predetermined values –Fast

32. 32 Chromatogram Retention Time Detector Response time of injection trtrtrtr

33. 33 Chromatogram - GC-MS vs. LC (UV) u x-axis –GC-MS - m/z –LC - retention time or volume u y-axis - detector response –GC-MS - % abundance –LC - Abs