Chromatography is a technique used to separate a mixture into its basic components. Not only does chromatography separate mixtures, it is also used to quantify the components. The HPLC principle to separation apparatus consists of a stationary phase which is characterized by a column and a mobile phase which contains a vehicle that facilitates the movement of sample components.
The principle of HPLC is to separate components depending on their affinity. Components that have an affinity for the mobile phase will move rapidly as compared to components that have an affinity for the stationary phase.
In order to attain a good qualitative analysis ensure that the column used is of the same size, the fillers are the same and the column temperature is constant. Also, ensure that the flow rate and composition of the mobile phase are constant.
When determining the quantitative value of a component, note the height and area of the peak and use the values to draw a calibration curve that you use to determine the concentration. Obtain the calibration curve from a standard than match the value of the sample in question against the standard.
HPLC apparatus has a pumping unit, injection unit, separation unit, detection unit and a data processing unit.
•Pumping unit: The pumping unit is responsible for the flow rate and pressure of the eluent. The eluent is the medium which moves the components through the mobile phase.
• Sample injection unit: This unit is responsible for introducing the sample into the HPLC system for analysis.
• Separation unit: Separation unit includes a column which is chosen based on the sample and purpose of separation. Constant column temperature ensures that elution time is not disrupted therefore leading to an accurate reading. The optimum column temperature is between 25-50 C. If the injected sample has components similar to the filler than the sample will be retained in the filler for longer hence slow movement across the column. On the other hand, if the sample does not have components similar to the filler, it will move rapidly through the HPLC column.
• Detection unit: The separated components are detected and the information is converted into electrical signals. There are different HPLC detectors to suit different samples. A UV detector is used to detect components with an absorption wavelength of 400nm and below. There is another detector, UV-VIS, which detects coloring components such as dyes. A diode array detector is used to detect components in the UV region all the way to the visible light spectrum. The fluorescent detector captures fluorescent substances with high sensitivity. Differential refractive index detector is able to identify components that do not absorb ultraviolet light. Finally, there is the conductivity detector which is able to identify inorganic ions. The Components are further analyzed using a spectrophotometer due to its high sensitivity.
• Data processing unit: This includes a computer which already has data about different components in the study. Once the detection is complete, the computer searches its database until it matches the obtained information to a component in its data bank. The computer will then generate a report stating the component in question and its concentration.