INTRODUCTION

TO

HPLC

Modes of HPLC
Adsorption Chromatography is another name for liquid-solid chromatography. The solid phase is usually silica or alumina, which have highly polar surfaces. The mobile phases are commonly some of the less polar solvents. Normal-Phase Chromatography is based on a polar liquid phase coated or bonded onto a silica support. For historical reasons, this is called normal phase because the stationary phase is polar and the mobile phase is typically a nonpolar solvent such as hexane or isopropylether. In normal phase, the least polar component is eluted first. Reversed-Phase Chromatography uses a non-polar stationary phase (also coated or bonded onto silica or another support) and polar solvents such as water, acetonitrile or methanol. In reversed-phase chromatography, the most polar component is eluted first. Ion-Pair Chromatography is one of the methods used to separate ions. It uses regular reversed phase columns, and can separate acids, bases and neutral compounds during the same chromatographic run. Ion-Exchange Chromatography is based on the use of ion-exchange resins as the stationary phase. Ion Chromatography was developed to separate the ions of strong acids and bases. The equipment used is different from that of ion-exchange chromatography.

Solvent Handling
Degassing
1) Dissolved gasses such as nitrogen and oxygen that can cause problems by forming bubbles in the column or the detector. 2) Oxygen from the air is chemically reactive and may react with the sample. 3) Dissolved oxygen also strongly absorbs at low UV wavelengths

Methods of Degassing Solvents

1) 2) 3) 4) Filtration Sonication under vacuum Sparging with Helium Interal Degassing

Solvent Handling
Inlet Filters
Particulate matter may damage the pumping or injection system by wearing out valves and seals.

Evaporation
The composition of a mixture of solvents can change over time as evaporation depleted the most volatile solvent from the mix. Place sparge tube above the liquid to reduce this problem.

Bacteria Control
- Make up new buffer solutions daily - Use sodium azide

Corrosion
PTFE(Teflon) - Mostly resistant to HPLC Solvents

PEEK - Strong organic solvents may give problems

Stainless Steel - hydrochloride salts and hydrochloric
salts are the most aggressive towards stainless steel. Acidic samples may damage injector systems.

Long-Term Solvent Storage

Alcohols, alkanes, toluene, xylenes, acetonitrile and ethyl acetate are chemically stable for more than a year. Ethers form peroxides which are very reactive compounds that can seriously affect chromatography. Peroxides can be explosive when they build up and therefore pose a safety hazard. Chlorinated solvent degrade to form reactive free-radical by-products and hydrochloric acid. The acid can alter the pH of the mobile phase and seriously affect the chromatography.

Requirements of HPLC Pumps

1) should be able to generate pressures up to 5000 psi. 2) should provide uniform pulse free flow. 3) should be able to provide accurate flow rates from 0.1 to 10 mL/min. 4) should have accurate control of flow rate changes (0.5% or less relative error). 5) should contain corrosion- and leak- resistant components.

HPLC Pump Design

Chamber Filling: This valve closed This valve open

Pumping Stroke: This valve open This valve closed Chamber

HPLC Pump Design

Gradient Elution
One of the most important methods of improving separation of components of complex samples is to change the mobile phase composition during the run. The change of mobile phase composition is called gradient elution and it can be accomplished either by high-pressure mixing or low-pressure mixing.

Solvent Mixing Systems

High-Pressure Injection

HPLC Column Packing

The stationary phase is most commonly made of porous spherical particles with diameters from 310 mm.

The typical porous particles used in analysis of small (non-protein) molecules have 7-12 nm pores. The total surface area of such particles ranges from 150 400 m2/g.

Micropellicular Particles
Micropellicular particles have a stationary phase coating only on the outside surface are useful for rapid separation of macromolecules such as proteins. Because the mobile phase does not diffuse into the particles, the mass transfer kinetics allow for very sharp peaks but since the total surface area of these particles is small sample volume is very limited.

The Surface of Silica

For adsorption chromatography, the silica is used without modification, but for most other types of HPLC, the silica is coated with one of many types of stationary phases. More commonly, in modern chromatography columns, the stationary phase is chemically bonded to the silica

Bonding Phases onto the Silica Support

Silica OH

CH3 | Cl -Si- CH3 | CH3

CH3 | Silica -0-Si- CH3 | CH3

HCl
Could be many different functional groups here

The Surface of Silica Bonded with C18

End Capping
Usually half or more of the silanol groups remain unreacted after bonding with C18. One method used to reduce the effects of these residual silanol groups is a process called endcapping.

End Capping
After bonding with C18. use small silane molecules such as trimethylchlorosilane to react some of the remaining OH groups

Steric Protection
CH3 | CH2 | Silica -0-Si- CH3 | CH2

Hydrolysis may degrade the column by breaking off the bonded phase.

| CH3

All C18 Columns Are Not Created Equal

See Handout..

Differences in:

Particle Size Pore Size Surface Area Carbon Load End-Capping Silica Type *Bonding Density

Comparison of Different C18 Columns

Column: Symmetry C18

Hypersil HyPURITY C18

Mobile Phase: 60% CH3CN 40% 50mM KH2PO4, pH 3.2

1. 2. 3. 4. 5. 6. 7.

Sample: Uracil Pyridine Phenol Dimethyl phthalate N,N-Dimethylaniline 4-Butylbenzoic acid Toluene

C18 Phases Ranked According to Retention for Toluene

Other Bonded Phases

Phenyl phases show weak dipole - induced dipole interactions with polar analytes. Usually this type of bonded phase is used for separating closely related groups of molecules.

Other Bonded Phases

Cyano columns are very slightly polar and are useful for fast separations of mixtures consisting of very different components that might have a very broad range of retention times on the usual columns. Cyano columns can be used with both normal- and reversed-phase HPLC.

Other Bonded Phases

The amino-phase is the most polar, and it can also act as weak anion-exchanger, (protonated at low pH). Amino columns are mainly used in normal-phase mode, especially for selective retention of aromatic compounds.

Other Bonded Phases

Diol columns are slightly polar and are used for normal-phase separations. Diols are useful for samples containing many compounds with different polarities , and which usually have strong retention on unmodified silica.

Non-Silica Supports
The most common polymer support material for reversed-phase separation is made of divinylbenzene cross-linked polystyrene The main advantage of porous polymers is that they can be used in the pH range from 1 to 13. (silica supports tend to dissolve at pH greater than 9).

Because of the surface acidity of silica supports, polymer supports can be a better choice for separating basic compounds.

Column Improperly Installed in the Lab..