1. Food Analysis Lecture 23 (4/19/2005) HPLC (2) and Gas Chromatography (1) Qingrong Huang Department of Food Science Read Material: Chapter 29, page 479 Final Exam: April 29

2. Components of HPLC Pump Injector Column Detector Recorder/integrator/data system

3. HPLC Pump Requirements Two types of pumps: constant pressure and constant volume; Flow rate ~1 mL/min; Typically stainless steel which can withstand the pressure generated and is resistant to corrosion by oxidizing agents; Sensitive to dust and particulate matter, mobile phase needs to be Filtered. Chemically inert High pressure (5,000 psi) Flow rate (0.5-10 mL/min) Pulse free or damped Flow reproducibility < 1% Gradient; recycle; rapid change

4. HPLC Injector Valve injectors (a)Isolated from the pump eluent Stream (LOAD position) (b)Positioned in it (INJECT position)

5. Solvent Properties Dissolve sample k’ of 1-10 High purity Cost, viscosity, toxicity, boiling point

6. HPLC Column Typically stainless steel, connected between the injector and detector; Precolumn: auxiliary column that precedes the analytical column; - guard column: used to protect the analytical column from strongly adsorbed sample components; same internal diameter as the analytical column. Analytical column:typically 10, 15, 25 cm long with internal diameter (ID) of 4.6 or 5 mm; - Generally packed with 3, 5 10  m particles, flow rate 1-2 mL/min; - Smaller diameter column is used. - Advantages: (1) decreased consumption of both mobile and stationary phase; (2) decreased peak volume; (3) increased resolution (with long column); (4) reduced equilibration time.

7. HPLC Column Packing Materials (1)

8. HPLC Column Packing Materials (2) Microbore: Column with ID 0.5-2.0 mm; Microcolumn: Column with ID <0.5 mm; Functions of packing materials: - In L-L C, only support the stationary; - In adsorption, ion-exchange, and affinity C, serves as support and stationary phase. Requirements of packing materials: - Availability in a well-defined particle size with narrow size distribution; - Sufficient mechanical strength to withstand pressure generated during packing and use; - Good chemical stability.

9. Types of packing materials (1) Silica-based column packings: particle size and pore distribution - Bonded phase Si OH + Cl Si R 3 --------  Si O Si R 3 + HCl R1R1 R2R2 R2R2 R1R1 R 1 and R 2 may be halides or methyl groups; the nature of R 3 determines normal-phase, reversed phase, or ion-exchange C; Main disadvantage: silica skeleton slowly dissolves in aqueous solutions. - Pellicular packing materials: deposit a thin layer or coating onto an inert, nonporous, microparticulate core; inorganic core: like Silica; organic core- poly(styrene-divinylbenzene).

10. Types of packing materials (2) Polymeric column packings: - Synthetic organic resins; - Good chemical stability, possible for chemical modofication. Microporous resins– crosslinked copolymer gel resins, apparent porosity is determined by the degree of crosslinking; swelling and contraction in mobile phases. Macroporous resins – highly crosslinked, consisting of a network of microspheric gel beads joined together to form a large bead; - large, permanent pores from 10-400 nm in diameter; - Rigid, microparticulate poly(styrene-divinyl benzene) packing materials are popular; - stable from pH 1 to 14.

11. Detectors Measure concentration Ultraviolet - Fixed wavelength - variable wavelength - Diode array spectrophotometric detector Refractive index Electrochemical detectors UV DetectorRI Detector Good sensitivity 10 -6 – 10 -10 g Universal response Selective Poor sensitivity ~10 -6 g Gradient elution possible No gradients Variable cost Temperature sensitive Most widely used Standard for GPC Used in Prep scale work

12. Separation Methods Normal Phase HPLC: - Stationary phase: polar adsorbant, such as bare silica or polar nonionic functional group modified silica; - Mobile phase: nonpolar solvent like hexane + CH 3 Cl - Solvent strength: weak solvent increases the retention while strong solvent decreases the retention. - best for separation of compounds that are highly soluble in organic solvents. Reversed Phase: - Stationary phase: nonpolar, typically octadecyl (C 18 ) chain [-(CH 2 ) 17 CH 3 ] or octadecylsilyl (ODS) bonded phase. - Mobile phase: polar solvent, like water mixed with methanol, acetonitrile, or THF.

13. Gas Chromatography (1) First introduced in 1952, first commercial instrument: 1956; good for thermally stable volatile compounds; Sample preparation: (1) Headspace methods: one of the simplest methods, only good for volatiles in headspace at C>10 -7 g/L; - Direct headspace sampling; - Headspace concentration techniques or purge and trap methods: (2) Distillation methods: (3) Solvent extraction: preferred for the recovery of volatiles from food; use of two immiscible phases (water and an organic solvent) (4) Solid-phase microextraction (SPME): new and rapidly growing

14. SPME Microextraction technique which employs a thin film of sorptive polymer on a glass fiber Fiber Having varying film thickness of polymer Coatings are based on both absoption and adsorption mechanisms Convenient, solventless extraction technique Used to extract analytes from both liquid and solid matrices

15. SPME Device

16. SPME Procedure ExtractionDesorption

17. Sample Derivatization

18. Components of GC Gas supply and regulators; Injection port; Oven; Column; Detector; Electronic; Recorder/data handling system.

19. Diagram of GC System