HPLC – High Performance Liquid Chromatography

1. HPLC – High Performance Liquid Chromatography

2. 1. Solvent reservoir 2. Degasser 7. Detector 8. Computer 6. Column 5. Injector 9. Waste 4. Pump 3. Mixing chamber

3. What is HPLC and what is it used for? High Performance Liquid Chromatography is a widely used analytical separation technique. This form of liquid chromatography or liquid separation is highly sensitive and automated which leads to accurate quantitative determinations. It is used in order to separate and identify the different analytes/compounds within a sample and to find the concentration of these various analytes. This separation technique can be used to analyse – • Proteins • Amino acids • Fats • Carbohydrates • Nucleic acids • Drugs • Pesticides • Antibiotics • Steroids • Foods e.g. amount of caffeine in fizzy drinks

4. HPLC instrument • This technique involves injecting a small amount of a liquid sample into a column which contains the stationary phase. • It is this solid stationary phase that interacts with the sample and causes the different compounds within the sample to separate. • There are various chemical and physical interactions between the sample molecules and the packing particles within the stationary phase that cause the analytes within the sample to separate.

5. The sample ‘moves’ down through the column with a liquid mobile phase. • The sample and mobile phase are both interacting with the stationary phase and are forced through the column at high pressures by the pump. • The individual separated compounds from the sample are then eluted (exit) from the column and are identified by the detector.

6. Components of the HPLC instrument 1 • There are 9 main components within the HPLC instrument • Solvent reservoir • Degasser • Mixing chamber • Pump • Injector • Column • Detector • Computer • Waste 7 8 2 5 6 3 9 4

7. 1. Solvent reservoir 2. Degasser A simply schematic diagram of the HPLC components 3. Mixing chamber 4. Pump 7. Detector 5. Injector 6. Column 8. Computer 9. Waste

8. Here is a fun, easy to remember animation of the HPLC components Mixing Chamber

9. Solvent Reservoir • This is the area at the top of the HPLC system where the mobile phase is placed. • The mobile phase is kept in large glass reagent bottles. • Tubes are placed into the reagent bottles to ‘draw out’ the mobile phase into the system. 2. Degasser • The degasser is like an ultra-sonic bath, it removes any gas (de-fizzes) and air bubbles from the mobile phase. • It is important for the mobile phase to be degassed prior to entering the column as air bubbles in the mobile phase can affect the flow of the mobile phase and can even block the column. The presence of air bubbles means a non-continuous flow of mobile phase through the system.

10. 3. Mixing chamber • Ensures that the mobile phase is thoroughly mixedto provide a homogenous mixture to enter into the system. • Mobile phases are generally made up of more than 1 component i.e. a mixture, e.g. 70:30 mix of methanol and water. • An unmixed mobile phase can affect the interactions between the mobile phase and the stationary phase (column) and therefore can affect the results. 4. Pump • The role of the pump is to force the liquid mobile phase and the liquid sample through the system at a specific flow rate. • The flow rate is a changeable programme feature and allows for a continuous flow of the mobile phase though the system e.g. 2ml/min, 5ml/min.

11. Injector Vials 5. Injector • The role of the injector is to introduce (inject) the liquid sample into the flow stream of the mobile phase which then travels into the column. • Samples are contained within small glass vials . • The injection volumes of the sample are a changeable programme feature, e.g. 5 - 20µl. • Most modern HPLC systems use automated injection systems. This is called an autosampler and is used when many samples require analysis. This automatic system saves time and is more accurate than manual injection.

12. 6. Column • The role of the column is to separate the different components/analytes in samples. • The column is packed with a solid stationary phase. This solid stationary phase is usually silica particles which are coated to the inside of the column. The type of coating used depends on the polarity of the sample and the mobile phase. It is this stationary phase that interacts with the sample and causes the different analytes within the sample to separate. • The pump is required to ‘push hard’ in order for the mobile phase to move through the column. As there is pressure within the column created by the small particles of the stationary phase. • Most columns are 5 – 20 cm in length, have an inside diameter of 3 – 5mm and the particle size of the packing is 3 – 5 µm.

13. 7. Detector • The detector is used to detect or ‘see’ the individual components of the sample as they elute from the column. • The separated components will all elute (leave) from the column at different times. This allows the detector to sense each different one. • The detector measures the amount of each component/analyte so that there is quantitative analysis of the sample components. • The results from the detector are transferred onto the computer programme and are presented in the form of a ‘chromatogram’. • A UV-Vis detector is the type of detector used for the HPLC analysis. Waste

14. 8. Computer • The computer programme is used to control the entire HPLC system. It can ‘stop’ and ‘start’ runs, programme the injection volumes, the number of injections, the flow rate etc. • The results from the detector appear on the computer programme as a ‘chromatogram’. A chromatogram is a graph that represents and illustrates the separated components of the sample and the times at which they separated. Each component/analyte of the sample that has separated is represented as a peak on the chromatogram. 9. Waste • When the analysis is complete and the ‘run’ is over, the used mobile phase and sample is directed into the ‘waste’ reagent bottle.

15. What have I learned? HPLC is used for the separation and identification of the different components within a sample. The column can be said to be the ‘heart’ of the HPLC, as it is the column that separates the sample. UV-Vis detectors are used for the analysis of the individual analytes The column is coated inside with tiny particles (µm)e.g. silica, (SP) which interacts with the sample and MP and causes separation. The size of the stationary phase particles (µm) determines if the sample particles will separate and be eluted from the column. Stationary phase – solid particles inside the column Mobile phase – liquid phase which flows through the system