1. DR ZIAD W JARADAT PROTEIN BIOTECHNOLOGY BT 452 Chapter 3 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

2. DR. ZIAD W JARADAT Types Adsorption Chromatography; Liquid-solid This chromatographic method is considered one of the most used methods in protein purification and analysis. It is mainly based on the following concepts; – The interactions of the solute or mobile phase molecules at the surface of a solid particle forms the basis of the adsorption mechanism. – The separation happened due to the competition between the analyte and the solvent for the binding site on the stationary phase. – A solvent that elutes compounds more rapidly and therefore, competes better for the chromatographic sites is called strong solvent while the one that can not compete well is called weak solvent.

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4. Example Water is a strong solvent for silica adsorbents while hexane is a weak solvent for silica adsorbents. Thus, solvent strength can be adjusted by using mixtures of these solvents. There are two types of adsorbents; – Polar adsorbents such as silica or alumina – None-polar adsorbents such as hexane and heptane. The polar adsorbents are the most widely used stationary phases in liquid-solid chromatography. Retention of solute (substance to be separated) on these phases increases with increasing polarity and requires displacement of adsorbed solvent molecules from the stationary phase.

5. DR. ZIAD W JARADAT Partition and bonded phase chromatography; Liquid-Liquid This method is based on the separation of solutes by the use of difference in their distribution between a liquid mobile phase and a liquid stationary phase coated onto a solid support. Chemical forces that exist between molecules can be used in this type of separation including hydrogen bonds, van der Waals, ion-ion interactions In classical partition chromatography a polar liquid such as β, β’- oxydipropionitrile (ODPN) was coated onto the support particles and hexane was used as a mobile phase.

6. DR. ZIAD W JARADAT Polarity The role of polarity is central to the interaction of molecules in the liquid or gaseous state and the polarity is a major determinant property in the overall chromatographic process. The physicochemical basis for polarity is the interaction of attractive forces that exist between molecules. These attractive forces are; Dispersive (van der Waals forces); these are important only when other forces are lacking. Dipolar Hydrogen-bonding Dielectric interactions or electrostatic interactions

7. DR. ZIAD W JARADAT The more polar molecules have strong dipoles, an ionic character, which is the ability to form strong hydrogen bonds or a combination of the three forces together. The less polar (non-polar) have dispersive forces as the primary basis for interaction with a very weak ability to interact through dipolar, H-bonding or electrostatic forces.

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9. Solvent polarity and solvent strength Solvent strength in liquid chromatography is a measure of the ability of the mobile phase to compete with the solute molecules for active sites (i.e interaction or attraction sites) on the stationary phase. For instance when the stationary phase is silica gel, the active sites are the highly polar hydroxyl groups (Si-OH). Therefore, in this case solvent strength increases with the solvent or mobile phase polarity. When the stationary phase is non-polar (e.g polydivinyl- benzene) then the solvent strength decreases with the increase in solvent or mobile phase polarity. The strength of a solvent is directly related to its polarity.

10. DR. ZIAD W JARADAT The overall degree of interactive forces of the solvent is quantitated in the polarity index P’. There are some tables available showing polarity index of certain materials. Criteria for selecting an elution solvent – The first important step in solvent selection is to maximize solute solubility in a given solvent i.e the solvent to be a good mobile phase should dissolve the sample to be eluted over a wide range of concentrations. This is highly affected by the solvent polarity. – Polarity can be adjusted by mixing two solutions with different polarities so that we can obtain a mobile phase with the desired strength. Example; 50% of a - 0.4 polar solution mixed with 50 of a 4.3 polar solution would be a 1.95 P.

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12. CHROMATIC PHASES Normal Phase; when the stationary phase is more polar than the mobile phase Reverse Phase; when the mobile phase is more polar than the stationary phase Reverse phase chromatography; The silica gel is polar and to be used for the reverse phase separation, its polar surface has to be changed. This can be done by attaching different functional groups such as hydrocarbons mostly C-8 and C18 (none polar). As a result we create a none polar phase. This type is used more than the Normal Phase, and the reason why it is more popular is that its weak mobile phase is the high polar water, therefore, the samples are applied in this weak mobile phase i.e applied in aqueous status such as biological compounds. This makes it especially attractive in clinical chemistry for drug confirmation, amino acid analysis and hormone separations.

13. DR. ZIAD W JARADAT Normal phase chromatography; although silica is polar, yet some other groups are attached to it to strengthen its polarity these groups are; CN-, NO2-, NH2-. In the above two phases, because the C8 or C18 or the use of CN-, NO2-, NH2- are bonded to the silica gel this is called bonded phase. How can we chose which phase to use for a certain solute? The material of choice should be the one that give high retention of separated components. So if we know that the separated material are polar, we will chose the normal phase because likes attract likes and vice versa. Although the Normal phase was the first type to be used, it is not used as the Reverse Phase because in the normal phase the polar material is used on the stationary phase which is found to retain the compounds most strongly.

14. DR. ZIAD W JARADAT Disadvantages of the partition chromatography; The liquid stationary phase will eventually bleed from the column Due to this bleeding, reproducibility get affected And short column life

15. DR. ZIAD W JARADAT The mobile phase can be; Isocratic system; the use of a constant mobile phase composition to elute solutes. Gradient system; eluting with gradient starting from weakest mobile solution to the strongest mobile phase. This system enhances the resolution especially if the two components to be resolved are close to each other.

16. DR. ZIAD W JARADAT Example Figure 5-14. In this example a mixture of mono, di and triglycerides of lauric acid will be separated using partition chromatography. The silica gel stationary phase has a monolayer of water strongly held by hydrogen bonding. The solute molecules are partitioned between liquid mobile phase (chloroform; methanol) and the water monolayer. The most polar component, the monoglycerides are retained most by the polar stationary phase The intermediate polarity components, the diglycerides are retained to a much lesser degree Non-polar components such as triglycerides are not retained and pass quickly.

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18. Please note that if a none-polar material was attached to the silica gel such as the hydrocarbon octadecyl or octyl groups ( this will be reversed phase). And in this case the mobile phase will be a highly polar material such as water, methanol or a cetonitrile. In this case the most polar elutes first while the less polar retained and the none polar retained the last.

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20. FPLC It is an alternative chromatographic system to the HPLC. It means Fast Protein Liquid Chromatography. This system employes operating pressure significantly lower than those used in conventional HPLC systems. Lower pressure allows the use of matrix beads based on polymers such as agarose. The columns are constructed of inert glass while those for HPLC are made of stainless steel.

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35. End of Chapter