1. By: Thilag.k & Stephen

2. What is Hpcl??? Hplc or high performance liquid chromatography is the most widely used analytical separation technique. The difference between this technique and others is that it uses a liquid mobile phase to separate components of mixtures. Another difference is that it utilizes high pressure to push the mixture through the columns. Hplc is pretty much a column chromatography but the difference is that instead of allowing the solvent to drip through the column it is forced through under high pressure ( abt 400 atmospheres) which makes the process much faster. Which is the main reason why this is the most popular form of column chromatography. Another reason for this however is that the detection methods in this is highly automated and sensitive.

3. Normal phase of HPLC In this phase the column is filled with tiny silica particles and the solvent used will be non- polar. A commonly used column would be about 4.6 mm in diameter and about 150 – 250mm in length. Polar compounds in the mixture being analysed will take a longer time to pass through than non- polar compounds as they will stick to the silica particles longer than the non – polar compounds. Although this process is known as the normal phase, it is not the most common use of HPLC.

4. Reverse phase of HPLC This method of HPLC is the most commonly used form of the process. In this process the dimensions of the tube does not change but the silica particles in them are made to be polar by attaching long hydrocarbon chains to them. This is usually done with attaching about 8 to 18 carbon atoms to them. The solvent used will also be polar (methanol). This means that the polar molecules will spend more time moving around with the solvent. The non- polar compounds will however form bonds with the hydrocarbon chains meaning that it will become less soluble in the solvent as it will need to break its bonds to do so. It will also mean that will become slower to pass through the tube than the polar compuonds.

5. flow scheme for HPLC

6. The injection of sample is automatic and is pushed through under real high pressure. Then the retention time is the time taken for the sample to travel through the columns and into the detector. This time starts when it gets injected till the display shows a maximum peak height. Some factors that determine a compounds retention time includes things such as what it is made up of, its particle size, the temperature of the tube. To find out when a substance has passed through the column and about to enter the detector a UV light and detector gets used commonly. The UV light will be on 1 side of the tube and a detector will be on the opposite side. The amount of light getting through to the detector will show how much substance is actually flowing through at that time.

7. The display will show the results of the analysis with a number of peaks on the screen. These peaks are a result of what and how much a particular compound is passing through the detector at that time. The area under the peak shows how much substance is actually passing through at that time. If the concentration of a substance was however less, the area under the peak would also be smaller even though the retention time will be the same. Sometimes a HPLC can be used together with a mass spectrometer by diverting the sample from the HPLC into it. From this you will get a fragmentation pattern which can be compared to some known patterns. This enables the composition of some compound being found without knowing their retention times.