High Performance Liquid Chromatography (HPLC)

What is HPLC? Types of Separations Columns and Stationary Phases Mobile Phases and Their Role in

Separations Injection in HPLC Detection in HPLC

Variations on Traditional HPLC Ion Chromatography Size Exclusion Chromatography

What is HPLC?HPLC is really the automation of traditional liquid chromatography under conditions which provide for enhanced separations during shorter periods of time!

Probably the most widely practiced form of quantitative, analytical chromatography practiced today due to the wide range of molecule types and sizes which can be separated using HPLC or variants of HPLC!!

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Partitioning

Separation is based on the analyte’s relative solubility between two liquid phases

Stationary PhaseMobile Phase

Solvent Bonded Phase

Instrumentation

Pump

Injector

ColumnDetector

Mobile Phases

Gradient Controller

•

What does the analyst do?

Select the correct type of separation for the analyte(s) of interest, based on the sample type (among other factors).

Select an appropriate column (stationary phase) and mobile phase

Select an appropriate detector based on whether universal or compound-specific detection is required or available

Optimize the separation using standard mixtures

Analyze the standards and sample

What does the analyst do?

Select the correct type of separation for the analyte(s) of interest, based on the sample type (among other factors).

HPLC MethodsParameter Group Method Compounds

• SDW05.23000’s EPA 555 Cl-PhenoxyAcids

• WPP05.13000’s EPA 610 PAHs

• WPP05.06000’s EPA 605 Benzidines

• SHW06.26000’s SW-846 8316 Acrylics

• SHW06.28000’s SW-846 8330’s Explosives

• SHW07.06000’s SW-846 8325 Benzidines and N- Pesticides

Compounds

2,4,5-T

Benzidine

Fluorene

Compounds

TNT (2,4,6-Trinitrotoluene)

H2C=CH-CN Acrylonitrile

Carbaryl

HPLC - ModesNormal Phase.- Polar stationary phase and non-polar solvent.

• Reverse Phase.- Non-polar stationary phase and

a polar solvent.

Common Reverse Phase SolventsMethanol CH3OH

Acetonitrile CH3CN

Tetrahydrofuran

Water H2O

What does the analyst do?

Select an appropriate column (stationary phase) and mobile phase

Columns and Stationary PhasesHPLC is largely the domain of packed columns:

some research into microbore/capillary columns is going on.

Molecules move too slowly to be able to reach and therefore “spend time in” the stationary phase of an open tubular column

in HPLC. In solution, not the gas phaseLarger molecules in HPLC vs. GC (generally)

Columns and Stationary Phases

Stationary phases are particles which are usually about 1 to 20 m in average diameter (often irregularly shaped):

In Adsorption chromatography, there is no additional phase on the stationary phase particles (silica, alumina, Fluorosil).

In Partition chromatography, the stationary phase is coated on to (often bonded)

a solid support (silica, alumina, divinylbenzene resin)

Columns Solid Support - Backbone for bonded phases.

Usually 10µ, 5µ or 3µ silica or polymeric particles.

Bonded Phases - Functional groups firmly linked (chemically bound) to the solid support.Extremely stableReproducible

Guard - Protects the analytical column:ParticlesInterferencesProlongs the life of the analytical column

Bonded Phases

• C-2 Ethyl Silyl -Si-CH2-CH3

•CN Cyanopropyl Silyl -Si-(CH2)3-CN

• C-18C-18 Octadecyl SilylOctadecyl Silyl -Si-(CH -Si-(CH22))1717-CH-CH33

•C-8 Octyl Silyl -Si-(CH2)7-CH3

Stationary Phases Polar (“Normal” Phase):

Silica, alumina Cyano, amino or diol terminations on the bonded phase

Non-Polar (“Reversed Phase”) C18 to about C8 terminations on the bonded phase Phenyl and cyano terminations on the bonded phase

Mixtures of functional groups can be used!!

Packed particles in a column require: Frits at the ends of the column to keep the particles in Filtering of samples to prevent clogging with debris High pressure pumps and check-valves Often a “Guard Column” to protect the analytical column

The Mobile Phase in HPLC... Must do the following: solvate the analyte molecules and the solvent they are in

be suitable for the analyte to transfer “back and forth” between during the separation process

Must be: compatible with the instrument (pumps, seals, fittings, detector, etc)

compatible with the stationary phase readily available (often use liters/day) of adequate purity

spectroscopic and trace-composition usually! Not too compressible (causes pump/flow problems)

Free of gases (which cause compressability problems)

Typical HPLC Pump (runs to 4,000+ psi)

Polarity Index for Mobile Phases….. The polarity index is a measure of the relative polarity of a solvent. It is used for identifying suitable mobile phase solvents. The more polar your solvent is, the higher the index. You want to try to choose a polarity index for your solvent (or solvent mixture) that optimizes the separation of analytesusually the index is a starting pointthe polarity of any mixture of solvents to make a mobile phase can be modeled to give a theoretical chromatogram

Usually, optimization of solvent composition is experimental

A similar number is the Eluent Strength (Eo] Increasing eluent strength or polarity index values mean increasing solvent polarity.

Remember, the analyte(s) and samples must be mobile phase and stationary phase compatible!

Optimization of Mobile Phase Polarity…

Changing the mobile phase composition alters the separation.

Isocratic versus Gradient Elution Isocratic elution has a constant mobile phase composition

Can often use one pump! Mix solvents together ahead of time! Simpler, no mixing chamber required Limited flexibility, not used much in research

mostly process chemistry or routine analysis.

Gradient elution has a varying mobile phase composition Uses multiple pumps whose output is mixed together

often 2-4 pumps (binary to quarternary systems) Changing mobile phase components changes the polarity index

can be used to subsequently elute compounds that were previously (intentionally) “stuck” on the column

Some additional wear on the stationary phase Column has to re-equiluibrate to original conditions after each run (takes additional time).

Injection in HPLC Usually 5 to 1000 L volumes, all directly onto the column not much worry about capacity since the columns have a large volume (packed).

Injector is the last component before the column(s) A source of poor precision in HPLC

errors of 2-3 %RSD are due just to injection other errors are added to this due to capillary action and the small dimensions/cavities inside the injector

6-PORT Rotary Valve is the standard manual injector Automatic injectors are available Two positions, load and inject in the typical injector Injection loop internal volume determines injection volume.

LOAD (the sample loop)

Inject (move the sampleloop into the mobilephase flow)

What does the analyst do?

Select an appropriate detector based on whether universal or compound-specific detection is required or available

DetectorsUVSingle wavelength (filter) [610, 8330]

Variable wavelength (monochromator) [8316, 8325]

Multiple wavelengths (PDA) [555]Fluorescence [610]Electrochemical [605] Mass Spectrometric [8325]

Chromatograms

Restek® ULTRA C-18 Column (250mm x 4.6mm, 5µ), Mobile Phase: (1:1 Methanol:Water), 1.5 mL/min.

Chromatograms

Restek® CN Column (250mm x 4.6mm, 5µ), Mobile Phase: (1:1 Methanol:Water), 1.5 mL/min.

Chromatograms

Supelcosil LC-PAH Columns. Conditions: A: 150mm x 4.6mm, 5µ.Flow Rate: 1.5 mL/min

Chromatograms

Supelcosil LC-PAH Columns.

Conditions: B: 50mm x 4.6mm, 3µ.Flow Rate: 3.0 mL/min

Detection in HPLC Numerous Types (some obscure) Original HPLC Detectors were common laboratory instruments such as spectrophotometers, etc. you can even use a SPEC 20! Usually a narrow linear range (1E3, usually)

Must be solvent -compatible, stable, etc. Universal

respond to all analytes Analyte Specific

respond to specific properties of analytes Non-destructive

most Destructive

ELSD, MS and a few others.

Standard Absorbance Detector…. Single Beam UV-VIS instrument with a flow-through cell

(cuvette) Can use any UV-VIS with a special flow cell

Extra connections lead to band-broadening if UV-VIS is far from HPLC column exit.

Usually utilize typical UV-VIS lamps and 254 nm default wavelenth Can be set to other wavelengths (most) Simple filter detectors no longer widely used

adjustable wavelength units are cost-effective Non-destructive, not-universal

not all compounds absorb light can pass sample through several cells at several different

wavelenghts Usually zeroed at the start of each run using an

electronic software command. You can have real-time zeroing with a reference cell.

Diode Array Detector (DAD)The more common tool for research-grade HPLC

instrumentsquite versatile...

Advances in computer technology since ~1985 or so have lead to the development of Diode Array instruments

Non-destructive, non-universalDAD scans a range of wavelengths every second or few seconds. At each point in the chromatogram one gets a complete UV-VIS spectrum!Huge volumes of dataDetailed spectra for each peak and each region of each peak

Refractive Index DetectorOne of a very few Universal HPLC detectors. Non-destructive

Responds to analytes changing the RI of the mobile phaserequires a separate reference flow of mobile phase

Extremely temperature sensitive, usually heatedsensitive to temp changes of +/- 0.001 °C

No longer really widely usedAbsorbance detectors are relatively cheap.

Useful for process work, on-line monitoring, etc.

ELSD (Evaporative Light Scattering Detector) Universal, destructive Useful for very large molecules, and a wide linear range

Analytes are de-solvated in the detector Molecules pass through what is essentially a large cuvette for a UV-VIS instrument

The reduction in light intensity detected (due to scattering by the analytes) is measured

The larger and more concentrated a particular molecule is, the greater the scattering.

What does the analyst do?

Optimize the separation using

standard mixtures and then for

samples.

Optimization of Separations in HPLC Correct choice of column so the above equilibrium has some

meaningful (non-infinity, non-zero) equilibrium constants. Correct choice of mobile phase Decision on the type of mobile phase composition

constant composition = isocraticvarying composition = gradient elution

Determination if flow rate should be constantusually it is

Decision on heating the column heating HPLC columns can influence the above equilibrium….

Types of HPLC Separations (partial list) Normal Phase: Separation of polar analytes by partitioning onto a polar, bonded stationary phase.

Reversed Phase: Separation of non-polar analytes by partitioning onto a non-polar, bonded stationary phase.

Adsorption: In Between Normal and Reversed. Separation of moderately polar analytes using adsorption onto a pure stationary phase

(e.g. alumina or silica)

Types of HPLC Separations (partial list)

Ion Chromatography: Separation of organic and inorganic ions by their partitioning onto ionic stationary phases bonded to a solid support.

Size Exclusion Chromatography: Separation of large molecules based in the paths they take through a “maze” of tunnels in the stationary phase.

Advantages of LC compared to GC:1.) LC can be applied to the separation of any compound that is soluble in a liquid phase.

‚LC more useful in the separation of biological compounds, synthetic or natural polymers, and inorganic compounds

2.) Liquid mobile phase allows LC to be used at lower temperatures than required by GC

‚ LC better suited than GC for separating compounds that may be thermally labile

3.) Retention of solutes in LC depend on their interaction with both the mobile phase and stationary phase.

‚ GC retention based on volatility and interaction with stationary phase

‚ LC is more flexible in optimizing separations change either stationary or mobile phase

4.) Most LC detectors are non-destructive‚ most GC detectors are destructive‚ LC is better suited for preparative or process-scale

separations

Disadvantage of LC compared to GC: 1.) LC is subject to greater peak or band-broadening.

‚ much larger diffusion coefficients of solutes in gases vs. liquids