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    Chromatography; It is based on the principle that various substances move at different speeds through a stationary phase with the help of a mobile phase. HPLC; It uses liquid mobile phase to separate components in a mixture. These components are first dissolved in the solvent and then forced through the chromatography column under high pressure. In chromatographic separation, substances are dispersed between two immiscible phases. One of the phases is called the mobile phase and the other is called the stationary phase.
    The movement speed of each substance in the mixture is determined by the affinity of the substance to the mobile or mobile phase. Substances that have more affinity for the mobile phase move faster. The concentration profile of each substance leaving the column is called a peak. The table formed by the peaks is called chromatogram.

  • HPLC sistemi genel olarak 5 kısımdan oluşur
    • Pump: The duty of the pump is to circulate the liquid in the system. Flow per minute is displayed in mL. • Syringe: Typically injected sample volumes range from 0.1 to 20 microliters (µL). It must be resistant to high pressure in the system. Autosamplers are designed to minimize analyzers' time loss.
    • Column: It can be adjusted between 5 - 100 °C. It is considered the heart of the chromatographic system. Separation takes place in the column. The stationary phase consists of mm-sized porous particles, so high pressure pumps are needed to pass the mobile phase through the column. The separation occurs based on the physical or chemical properties of the components. It is very important to choose the right column for analysis.
    • Detector: It allows us to see the components leaving the column and determine the amount of separated molecules. The substances passing through the detector are recorded with the help of a recorder, creating a graph of the detector response against time, which is called a chromatogram. The most used detectors; Spectroscopic Fluorescence Refractive index.
    • Recorder
    Some of these parts may vary depending on the component to be worked on.
    Working Principle
    Chromatographic process;
    Step 1: Dissolving the sample in the appropriate solvent
    Step 2: Separation, Separation of the components takes place in the column. The column consists of a structure consisting of a steel pipe filled with silica gel. The solute mixture is injected into the column. A pressure is applied to move the liquid through the column, this liquid mixture is called the mobile phase. Step 3: Determination of the amounts of the components, determination of a sample of unknown concentration in solution; In general, this is possible by introducing different known amounts of the standard solution into the column and correlating the obtained values. Generally, correlation is made using peak areas or peak heights.

    Analyzes That Can Be Done with HPLC

    In HPLC, if a column and detector suitable for analysis are available, various analyzes are performed, representing the following typical applications.
    Biochemicals; amino acids, proteins, lipids, carbohydrates (fructose, glucose, sucrose, turanose, maltose)
    foodstuffs; Artificial sweeteners, antioxidants, some mycotoxins such as aflatoxin, naphthalene, hydroxymethylfurfural,
    Medicines; antibiotics, steroids, sedaphytes.