Chromatographie Liquide a Haute Performance CLHP PDF

# Chromatographie Liquide a Haute Performance CLHP ## Introduction - La chromatographie liquide haute performance, souvent désignée par son abréviation CLHP - HPLC en anglais, constitue une technique analytique très générale d'emploi. - Elle correspond à une évolution de la chromatographie sur col...

# Chromatographie Liquide a Haute Performance CLHP ## Introduction - La chromatographie liquide haute performance, souvent désignée par son abréviation CLHP - HPLC en anglais, constitue une technique analytique très générale d'emploi. - Elle correspond à une évolution de la chromatographie sur colonne amélioration de la sélectivité et de la résolution. - Technique d'analyse pour séparer les constituants d'un mélange non volatile thermosensible, de polarité élevée en phase liquide ## Principe - Injection du mélange à séparer - La phase mobile transporte le mélange au travers la colonne. - Les composés en solution se répartissent alors suivant leur affinité entre la phase mobile et la phase stationnaire. - En sortie de colonne les différents solutés sont caractérisés par un pic chromatographique grâce à un détecteur approprié. # Schéma de principe d'une chaine d'HPLC - This is a diagram of the principle of HPLC, showing the different parts involved. - You can't see the actual parts, but the diagram represents the overall flow of the process. - The main parts include the solvent reservoir, indicator of pressure and debit, injection system, pump, column, integration and printing system, and the detector. # L'Appareillage - Typical configuration of a High Pressure Liquid Chromatograph - Shows the different parts of the apparatus, each with its own function: - Eluent reservoir bottles - Solvent Cabinet - Vacuum - Degasser - Binary Pump - Autosampler - Column Compartment - Detector ## L'Appareillage 1. **Un réservoir de solvant (éluant)** - Le plus souvent ce réservoir est une bouteille en verre contenant la phase mobile. 2. **Dégazeur** - Permet de retirer le gaz (oxygène) présent dans le(s) solvant(s) afin d'éviter d'endommager les échantillons ou la phase stationnaire. 3. **La pompe** - Elle délivre en continu la phase mobile. - Pression maximale > 400 bars - Débit: quelques l à plusieurs ml/min - Elle permet de travailler: - en **mode isocratique**: c'est-à-dire avec 100% d'un même éluant tout au long de l'analyse. - en **mode gradient**: c'est-à-dire avec une variation de la concentration des constituants du mélange 4. **L'injecteur** - Le type d'injecteur le plus couramment utilisé comporte une vanne à boucle d'échantillonnage. - This boucle permet d'introduire l'échantillon sans modifier la pressior dans la colonne. ## 4.1 Vanne d'injection: - C'est un injecteur à boucles d'échantillonnage. Il existe des boucles de différents volumes. - Le choix du volume de la boucle se fait en fonction de la taille de la colonne et de la concentration supposée des produits à analyser. - The system of the injection loop allows for a constant volume of injection, which is important for quantitative analysis. - **En position (a) remplissage de la colonne:** - the column (port 6) is directly connected to the pump (port 1) and is therefore fed with mobile phase. - In parallel, the solution to be chromatographed is injected into port 4 in a loop until it overflows (exit through port 3). - **En position (b) l'injection dans la colonne:** - The lever is turned to connect the loop to the mobile phase circuit. - This will then cause the solution present in the loop to be carried towards the column. ## 5. La Colonne - Est un tube souvent acier inoxydable, inerte aux produits chimiques. - The columns can withstand high pressures. - The flow rate of the mobile phase cannot exceed a few mL/min. - These columns have the advantage of being fast, and therefore, require less solvent and lead to better resolution in the analysis. - The column is often preceded by a pre-column, called a guard column, short (0.4 to 1 cm), filled with the same stationary phase, which serves to retain certain impurities. ## 6. Le Détecteur - The most commonly used detector in HPLC is a UV-visible spectrophotometer (190-800 nm) connected to the column outlet. - There are other detectors: differential refractometer, diode array UV, electrochemical, fluorimetric... ## 7. Les Phases Stationnaires - **Phase stationnaire polaire:** - **Phase polaire normale**: - The stationary phase is polar (silica gel is polar), therefore it is necessary to use an apolar eluent. - Polar products are retained in the column, whereas apolar products are eluted first. - **Phase inverse (apolar)**: - The stationary phase is mostly composed of bonded silica, which is apolar and therefore requires a polar eluent. - In this case, it is the polar compounds that are eluted first. # Domaines d'application de HPLC - The applications of HPLC are numerous, but it is particularly used in: - **Biochimie** for the analysis of the constituents of a compound. - **Separation and identification** of amino acids, nucleic acids, proteins, hydrocarbons, pesticides, carbohydrates, antibiotics, steroids and countless other organic and inorganic substances. - **Quantification** of the analytes present. - **Determination of the purity** of the sample. - **Quality assurance and control.** # Chromatographie Liquide en Phase Gazeuse ## Introduction - Method of separation of gaseous compounds or susceptible to be vaporized by heating without decomposition. - It constitutes the most powerful and the finest method to separate, identify and quantify gaseous or volatile substances. ## Principe - The mixture to be eluted is injected using a syringe. - Once vaporized by the injector, the compounds are carried through the column by the carrier gas. - Depending on the affinity with the stationary phase, the compounds are separated before being detected at the column outlet. - When a constituent of the mixture arrives at the detector level, a peak appears on the recorder. # Appareillage - **Carrier Gas:** - The mobile phase that circulates inside the chromatograph, carrying the analytes through the column, from the injector to the detector. - There is no interaction between the gas and the stationary phase. - Helium is the most common carrier gas, but argon, nitrogen and hydrogen are also used. - The choice of gas depends on the detector used. - **Injector:** - Allows for the introduction of the sample into the chromatograph, as well as the volatilization of the analytes. - The injector temperature must be adjusted to vaporize all of the analytes in the sample. - This temperature is generally maintained at 50°C above the boiling point of the least volatile analyte. - The injection is done using a microsyringe for liquids and solutions, or a loop valve for gaseous mixtures. - Generally, the injection chamber should be at a higher temperature than the column to facilitate the evaporation of the samples. - The choice of injector is dictated by the type of column used (packed or capillary) and the nature of the products to be separated (their resistance to decomposition when subjected to high temperatures). - **Oven:** - The column is contained in a fan-type oven to maintain a sufficient temperature to keep the solutes in the gas phase during the analysis. - The temperature is precisely adjustable (typically between 20°C and 450°C) and programmable. # Les Colonnes - Generally rolled in spirals, there are two types: ## Colonnes remplies These are less and less used (the old ones): they are metallic or glass columns (inert to the components to be analyzed) with a diameter of a few mm and a length of 1 to a few meters (in a spiral winding). - Each column is filled with the desired granular support such as: - Silica if adsorption gas chromatography occurs. - Inactive product called impregnated support with a thin film of heavy liquid in gas chromatography with partition, for example, polyethylene glycol impregnating silica. ## Colonnes capillaires - These columns are made of a metal, glass, fused silica or quartz tube, rolled in a spiral. - These columns are rendered less fragile by external coating with a heat-resistant polymer. - The stationary phase coats the inner wall with a controlled thickness of 0.05 to 5 mm. - Capillary columns are now favored by analysts. - A vast variety of grafted phases are available on the market. # Les Echantillons - The analytes must absolutely be injected at the column entrance in the gas state (it is fundamental). - For this reason, the sample is injected into an injection chamber at a temperature that causes the analytes to vaporize. - The temperature of the injection chamber is therefore, in principle, always higher than that of the column. - Anything that is not vaporized in the injection chamber will not enter the column. - The limits of sensitivity depend on the apparatus and range from ng to pg. - When the solutes are directly volatile, the substances are dissolved in a solvent and chromatographed. - When the solutes are not volatile at the temperature of the chromatograph, or are decomposed at this temperature, they must be converted into stable volatile derivatives. - For example, amino acids are esterified by butanol, fatty acids are esterified by methanol, sugars are reduced to alditols, then acetylated. # Détecteurs - The chromatograph gas detector is located in a compartment immediately after the column outlet. - Like the injection chamber, the detection chamber can be heated to very high temperatures. - The detector aims to detect the compounds at the column outlet. - A detector is characterized mainly by its sensitivity and its ability to give a response proportional to the concentration of the compound detected. - The main detectors are: - Catharometer. - Flame ionization detector (FID). - Thermoionic detector. - Electron capture dectector. - Mass spectrometry. # Applications - Analysis of petroleum products and fuels. - Determination of the composition of essential oils and perfumes. - Control in the pharmaceutical industry of medicines. - Analysis of pesticides and insecticides. - Organic research and synthesis. - Toxicology and drug research.