Analytical Chemistry Section D Separation Techniques

Analytical Chemistry

Section D

Separation Techniques

<Instant Notes, Kealey & Haines>

Contents

6. High-Performance Liquid Chromatography: Principles and

Instrumentation

7. High-Performance Liquid Chromatography: Modes,

Procedures and Applications

8. Electrophoresis and Electrochromatography: Principles

and Instrumentation

9. Electrophoresis and Electrochromatography: Procedures

and Applications

Principles High-performance liquid chromatography(HPLC) is a technique for the separation ofcomponents of mixtures by differential migration through a column con-taining a microparticulate solid stationary phase. Solutes are transported through the column by a pressurized flow of liquid mobile phase, and detected as they are eluted. Mobile Phase The mobile phase is either a single solvent or blend of two or more hav-ing theappropriate eluting power for the sample components. It ranges from a nonpolar liquid to aqueousbuffers mixed with an organic solvent. Solvent Delivery system The solvent delivery system comprises a means of degassing, filtering and blending up to four solvents which are them delivered to the top of the column under pressure by a constant flow pump.

6. High-Performance Liquid chromatog-raphy : Principles and Instrumentation

Sample Injection Liquid samples or solutions are introduced into the flowing mobile phase at the top of the column through a constant or variable volume loop and valve injector that is loaded with a syringe. Column and Stationary Phase Columns are straight lengths of stainless steel tubing tightly packed with a microparticulate stationary phase. The column packings are chemically modi-fied silicas, unmodified silica or polymeric resins or gels. Solute Detection Solutes are detected in the mobile phase as they are eluted from the end of column. The detector generates an electrical signal that can be amplified and presented in the form of a chromatogram of solute concentration as a func-tion of time. Instrument Control and Data Processing A dedicated microcomputer is an integral part of a modern High-perfor-mance chromatograph. Software packages facilitate the control and monitor-ing of instrumental parameters, and the display and processing of data.



Principle(1)

- The elution order is depending on the chemical nature of the solutes and the overall polarity of the two phases.

- Very small particles of stationary phase are essential for separation efficiency and resolution.

Principle(2)

Fig. 1. Schematic diagram of a high-performance liq-uid chromatograph


Solvent Deliv-ery System

Sample Injection Valve

Col-umn

Detection and Recording System

Microcomputer with Control and Data-Pro-cessing Software

Mobile Phase(1)

- Unlike a GC carrier gas, Mobile phase influence retention and selectivity.

- The composition of mobile phase is crucial in both re-spects.

→ For normal-phase separation

(Stationary Phase more polar than Mobile Phase.)

→ For reversed-phase separation

(Stationary Phase less Polar than Mobile Phase.)


Mobile Phase(2)

- Elution can be under isocratic conditions or a composition gradient.

→ Isocratic condition is constant mobile phase condition.

→ Gradient can be generated to improve the resolution of

complex mixtures.


Solvent Delivery System

- Solvent Reservoirs

It removes dust and other particulate material.

- De-gassing

It prevents air interfere with detector response by bubbles.

- Gradient Former

It generates binary, ternary or quaternary mixtures of sol-vents.


Sample Injection(1)

- The loop is filled or partially filled with sample from a sy-ringe.

- Disposable guard column

→ It is positioned between the injector and the analytical

column.

→ It can protect the analytical column from particulate matter

and retained matrix components from injected sam-ples.


Sample Injection(2)

Fig. 2. Sample-injection valve and loop(a) Sample-loading position(b) Sample-injection position


Column(1)

- The column is where the separation is occurred.

- There are two types of HPLC column.

→ Conventional column

→ Microbore column


Column(2)

Conventional/Microbore column

- The two kinds of columns are basically same.

- Microbore column has low flow rate, higher operating

pressures and smaller internal diameter .

- Being more sensitive and efficient, the microbore col-umn has

better than conventional column.


Column(3)


Stationary Phase

- Octadecyl silica(ODS or C18) is the most widely used of all the

stationary phases.

→ Octyl and shorter alkyl chains are more suitable for polar

solutes.

- Size exclusion and ion-exchange stationary phases are based

either silica or polymer.


Solute Detection(1)

- Detector is several type

① UV-visible absorbance detector

a. Fixed-wavelength filter-photometer

b. variable-wavelength spectrophotometer

c. Photodiode-array detector

② Fluorescence detector

③ Refractive index

④ Electrochemical detector


Solute Detection(2)

- UV-visible absorbance detector

Fixed-wavelength filter-photometer

① This is the simplest detector.

② It has limited number of wavelength.

③ It has limited use.


Solute Detection(3)


variable-wavelength spectrophotometer

① This is much more versatile.

② The detector allows monitoring at any wavelength

within the working range of the detector.


Solute Detection(4)

Fig. 3. UV-visible variable-wavelength spectrophotometric detector


Solute Detection(5)


Photodiode-array detector

① The spectra and the developing chromatogram at any

wavelength can be displayed on VDU screen in real time.

② The data comparisons made with library spectra for

identification purpose.


Solute Detection(6)

Fig. 4. Diode-array detector


Solute Detection(7)

Fig. 5. 3-D display mode for a diode-array detector


Solute Detection(8)

Fluorescence detector

- The detector is based on filter-fluorimeters.

- The detector can be three orders of more sensitive and

selective than UV absorbance.

- The detector responds to fluorescing solutes, for ex-ample

Polynuclear aromatics, quinoline, steroids, etc.


Solute Detection(9)

Refractive index

- It is several orders of magnitude less sensitive than UV

absorbance detector.

- It is highly temperature sensitive.

- It is very difficult to use with gradient elution because the

sample cell and reference cell can’t be matched.


Solute Detection(10)

- Electrochemical detector

It is based on measuring by following two things. ① The conductance of an aqueous mobile phase

② The current generated by the electrochemical reduction or

oxidation of solutes.


Modes of HPLC Modes of HPLC are defined by the nature of the stationary phase, the mechanism of interaction with solutes, and the relative polarities of the stationary and mobile phase.

Optimization of Separations After selection of an appropriate mode, column and detector for the solutes to be separated, the composition of the mobile phase must be optimized to achieve the required separation. A trial and error approach or a computer aided investigation can be adopted.

Qualitative Analysis Unknown solutes can be identified by comparisons of retention factors or times, spiking samples with Known substances or through spectro-metric data.

Quantitative Analysis Quantitative information is obtained from peak area or peak height measurements and calibration addition or internal normalization.

7.HPLC : Modes, Procedures and Applica-tions

Mode of HPLC(1)

- According to stationary phase, the modes of HPLC is de-fined.

Adsorption chromatography

Modified partition chromatography

Ion-exchange chromatography

Ion chromatography

Size exclusion chromatography

Chiral chromatography


Mode of HPLC(2)

Adsorption chromatography

- This mode is not used extensively.

- It is suitable for mixtures of structural isomer and different

functional group.

- Stationary phase is consist of silica gel(Si-OH).

- Non polarity mobile phase is blended with polarity solvents.


Mode of HPLC(3)

Modified partition chromatography

- The most popular stationary phase is octadecyl(C18 or ODS).

- The mobile phase is blended of methanol or acetonitrile with

water or an aqueous buffer.

- The ionized or protonated forms have a much lower affinity

for the ODS than neutral species.


Mode of HPLC(4)

Ion-exchange chromatography

- This mode is suitable for ionic solutes.

- It has sulfonic acid cation-exchange sites(-SO3-H+) or

quaternary ammonium anion-exchange sites(-N+R3OH-)


Mode of HPLC(5)

Ion chromatography

- This is a form of ion-exchange chromatography for the

separation of inorganic and some organic.

- The stationary phase is a pellicular material.

- The mobile phase is electrolytes. ① Anion : NaOH, NaCO3, NaHCO3

② Cation : HCl, CH3SO3H


Mode of HPLC(4)

Size exclusion chromatography

- This is suitable for mixtures of solutes with relative molecular

masses.

- This is used in biological macromolecules

- There are three groups. ① Pore < Solute

② Pore = Solute

③ Pore > Solute


Mode of HPLC(5)

Chiral chromatography

- Cyclodextrins bonded to silica is useful stationary phase.

- Steric repulsion, pH, Ionic strength and temperature of the

mobile phase affect resolution.

- The enantiomer can bond cyclodextrins because of H-bond,

π-π dipolar interaction which can make different retention

time and be resolved.


Optimization of separations

- The mode of HPLC most suited to the structures and properties of the solutes to be separated is selected.

- The shortest column and the smallest particle size of stationary phase consistent with adequate resolution should be used.

- Mobile phase composition is optimized by obtaining and evaluating a number of trial chromatograms.


Qualitative Analysis

- Comparisons of retention factors or retention times with those of known solutes under identical conditions.

- Comparisons of chromatograms of samples spiked with known solutes with the chromatogram of the unspiked sample.


Quantitative Analysis

- Peak areas are more reliable than peak heights.

- Most HPLC detectors have a wide linear dynamic range that helps to calculate quantity of sample.


Documents

Analytical Chemistry Section D Separation Techniques