The image on the left shows the flow chart for HPLC.
It starts with the injection of the sample which is an automated process. The retention time is the time taken for the compound to travel through the column to the detector. The column consists of a stationary phase which separates the sample into the different chemical and physical elements It is measured from once when the sample is injected to the point at which the display shows a maximum peak height for the compound. Every compound has a different retention time and this can be dependent on many factors such as the pressure used as it can affect the flow rate of the compound, the composition of the solvent, the temperature of the column and the stationary phase. If retention times is the way that you will be identifying compounds make sure that the conditions stated above are tightly controlled. It then goes through to the detector. There are many ways the detector can detect a substance passed through the column, one of the most common ways in the UV absorption. Many organic compounds absorb UV wavelengths so when a beam of the UV light is shining through the stream of the liquid leaving the column the detector on the opposite side can provide a direct reading of the amount of light absorbed. The light absorbed will depend on the amount of a particular compound that will pass through the beam at the time. The detector now provides an out put recorded as a series of peaks, each represents a compound in the mixture as it passes through the detector absorbing the UV light. The retention values calculated will then help identify the compounds present as long as there are readings of the pure samples. The peaks from the output can also be used to measure the amount fo the compound present.
HPLC can be used in different industries such as the pharmaceutical industry where specific drugs are produced. HPLC is very useful here as it is able to analyse specific molecules of a compound which makes it a key process in steps such as quality control. HPLC allows for the sample produced to be compared against the standard sample ensuring the batch produced meets the requirements. It allows for the individual measurements of the components in the mixtures allowing for higher quality control. In the food industry HPLC is also used to analyse samples of food to ensure that they can be consumed. HPLC in this case specifically analyses different components such as additives, nutrients and even contaminates present in the food sample; it is vital as it can select individual components and test for them.
When using HPLC to identify organic compounds many factors must be considered before the chromatography is carried out. Factors such as the ability of many organic compounds to absorb UV light as mentioned above. As the beam of light passes through the sample and is detected by the UV detector; allows for amount of light absorbed by the compound; this information will allow to identify individual components as each component will have a different absorption. The identification of the compound can also be verified by the utilisation of a mass spectrometer ass it will allow for the substance to pass through the mass spectrometer and produced a fragmentation pattern which allows for it to be compared to a database consisting of the readings for different components and will aid in identifying the specific compound detected.
As gas chromatography and HPLC analyse substances by the differentiation of their separation times, it is highly important that the separation takes place accurately. This will then allow for the detector to precisely detect individual components. The temperature at the time of the procedure allows for the control of individual components to be separated. Temperature is an important factor; if the temperature is too high components with high boiling points will be left behind and separate at higher rates, whereas components with lower boiling points may boil of quickly leading to no separation taking place. In HPLC we can use different columns to match the analytes as well as different solvents which are more efficient in the separation of components. Different wavelengths can also be used to suit the solution as well as the detector can be optimised for the solution. In gas chromatography different columns can be used specifically for their analyses alongside specific detectors in accordance to the components.