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

Components of HPLC

• Pump• Injector• Column• Detector• Recorder/integrator/data system

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 generatedand 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

HPLC Injector

• Valve injectors

(a) Isolated from the pump eluentStream (LOAD position)

(b) Positioned in it (INJECT position)

Solvent Properties

• Dissolve sample• k’ of 1-10• High purity• Cost, viscosity, toxicity, boiling point

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 stronglyadsorbed sample components; same internal diameter as the analyticalcolumn.• 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.

HPLC Column Packing Materials(1)

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.

Types of packing materials (1)

• Silica-based column packings: particle size and pore distribution- Bonded phase

Si OH + Cl Si R3 -------- Si O Si R3 + HCl

R1

R2 R2

R1

R1 and R2 may be halides or methyl groups; the nature of R3

determines normal-phase, reversed phase, or ion-exchange C;• Main disadvantage: silica skeleton slowly dissolves in aqueoussolutions.

- Pellicular packing materials: deposit a thin layer or coatingonto an inert , nonporous, microparticulate core; inorganic core: like Silica; organic core- poly(styrene-divinylbenzene).

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.

Detectors

• Measure concentration• Ultraviolet

- Fixed wavelength- variable wavelength

- Diode array spectrophotometric detector• Refractive index• Electrochemical detectors

UV Detector RI 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

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 + CH3Cl - 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 (C18) chain [-(CH2)17CH3] or octadecylsilyl (ODS) bonded phase. - Mobile phase: polar solvent, like water mixed with methanol, acetonitrile, or THF.

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

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

SPME Device

SPME Procedure

Extraction

Desorption

Sample Derivatization

Components of GC

• Gas supply and regulators;• Injection port;• Oven;• Column;• Detector;• Electronic;• Recorder/data handling system.

Diagram of GC System