{"id":17811,"date":"2021-10-10T13:52:06","date_gmt":"2021-10-10T13:52:06","guid":{"rendered":"https:\/\/antecscientific.com\/techniques\/quest-ce-que-lhplc\/"},"modified":"2023-05-08T15:41:21","modified_gmt":"2023-05-08T15:41:21","slug":"quest-ce-que-lhplc","status":"publish","type":"page","link":"https:\/\/antecscientific.com\/fr\/produits\/techniques\/quest-ce-que-lhplc\/","title":{"rendered":"Qu’est-ce que l’HPLC ?"},"content":{"rendered":"

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Qu’est-ce que l’HPLC ?<\/h2>\n

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La HPLC ou chromatographie liquide \u00e0 haute performance est une technique d’analyse permettant d’identifier et de quantifier des substances dans une solution. Il peut \u00eatre appliqu\u00e9 pour mesurer la concentration de glucides dans les aliments et les boissons, de pesticides dans les eaux de surface pour l’analyse environnementale, ou de cat\u00e9cholamines dans l’urine \u00e0 des fins cliniques, pour n’en citer que quelques-uns.<\/p>\n

L’UHPLC (U pour \u00ab\u00a0ultra\u00a0\u00bb) repose sur le m\u00eame principe, \u00e0 ceci pr\u00e8s que des colonnes chromatographiques plus r\u00e9centes et plus efficaces sont utilis\u00e9es. Les colonnes UHPLC sont remplies de particules plus petites offrant de meilleures performances de s\u00e9paration. En raison des canaux \u00e9troits entre les petites particules, la pression dans un tel syst\u00e8me est plus \u00e9lev\u00e9e. Pour cette raison, des instruments sp\u00e9ciaux sont souvent n\u00e9cessaires.<\/p>\n

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Un syst\u00e8me HPLC avec \u00e0 gauche une pompe avec des flacons de phase mobile, un passeur automatique d’\u00e9chantillons et un d\u00e9tecteur (\u00e0 droite).
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Comment fonctionne la HPLC ?<\/h2>\n

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Un syst\u00e8me HPLC se compose d’une ou plusieurs pompes, d’un passeur d’\u00e9chantillons, d’un d\u00e9tecteur et d’un logiciel d’acquisition de donn\u00e9es. Au c\u0153ur du syst\u00e8me HPLC se trouve une colonne chromatographique, remplie de particules de phase stationnaire, o\u00f9 s’effectue la s\u00e9paration des substances dans un m\u00e9lange. Les instruments sont reli\u00e9s par un tube, appel\u00e9 le chemin d’\u00e9coulement. La ou les pompes fournissent une solution porteuse, ou phase mobile, \u00e0 travers le chemin d’\u00e9coulement du syst\u00e8me. L’\u00e9chantillon \u00e0 analyser est introduit dans le circuit par un passeur automatique d’\u00e9chantillons. Typiquement, le d\u00e9bit dans la plupart des HPLC est compris entre 0,05 et 2 mL\/min. La pression dans le syst\u00e8me d\u00e9pend principalement de la taille des particules de la phase stationnaire, de la longueur de la colonne et du d\u00e9bit. Les pressions typiques en HPLC sont de 100 \u00e0 400 bars.<\/p>\n

Le principe de s\u00e9paration de la HPLC repose sur la r\u00e9partition de l’analyte entre la phase stationnaire (colonne remplie de particules) et la phase mobile (solvant qui la traverse). Dans un syst\u00e8me HPLC qui fonctionne bien, le temps pass\u00e9 dans la colonne est unique pour chaque substance du m\u00e9lange. Ce \u00ab\u00a0temps de r\u00e9tention\u00a0\u00bb permet d’identifier la substance. En chromatographie en phase invers\u00e9e, on utilise des particules de phase stationnaire non polaires. Par cons\u00e9quent, les substances moins polaires ont une interaction plus forte et passent plus de temps dans la colonne que les substances polaires.<\/p>\n

Le d\u00e9tecteur mesure en permanence la phase mobile qui traverse la cellule de d\u00e9tection. Les substances qui s’\u00e9luent de la colonne entra\u00eenent une d\u00e9viation du signal, sous la forme d’un pic gaussien. Le signal est stock\u00e9 et analys\u00e9 dans un chromatogramme par un logiciel d’acquisition de donn\u00e9es sur un PC qui est connect\u00e9 au d\u00e9tecteur.<\/p>\n

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Configuration HPLC sch\u00e9matique, la valve d’injection fait partie d’un passeur d’\u00e9chantillons.
<\/em><\/p>\n

[\/et_pb_text][et_pb_image src=\u00a0\u00bbhttps:\/\/antecscientific.com\/wp-content\/uploads\/2021\/09\/chrom1.jpg\u00a0\u00bb title_text=\u00a0\u00bbchrom1″ _builder_version=\u00a0\u00bb4.10.5″ global_colors_info=\u00a0\u00bb{}\u00a0\u00bb][\/et_pb_image][et_pb_text admin_label=\u00a0\u00bbimage explanation\u00a0\u00bb _builder_version=\u00a0\u00bb4.11.2″ text_font=\u00a0\u00bb||||||||\u00a0\u00bb text_font_size=\u00a0\u00bb13px\u00a0\u00bb text_line_height=\u00a0\u00bb2em\u00a0\u00bb header_font=\u00a0\u00bb||||||||\u00a0\u00bb header_2_font=\u00a0\u00bbRaleway|700|||||||\u00a0\u00bb header_2_font_size=\u00a0\u00bb50px\u00a0\u00bb header_2_line_height=\u00a0\u00bb1.4em\u00a0\u00bb header_6_font=\u00a0\u00bbRaleway|700||on|||||\u00a0\u00bb header_6_font_size=\u00a0\u00bb16px\u00a0\u00bb header_6_letter_spacing=\u00a0\u00bb5px\u00a0\u00bb header_6_line_height=\u00a0\u00bb1.8em\u00a0\u00bb max_width=\u00a0\u00bb700px\u00a0\u00bb module_alignment=\u00a0\u00bbcenter\u00a0\u00bb header_2_font_size_tablet=\u00a0\u00bb35px\u00a0\u00bb header_2_font_size_phone=\u00a0\u00bb25px\u00a0\u00bb header_2_font_size_last_edited=\u00a0\u00bbon|phone\u00a0\u00bb global_colors_info=\u00a0\u00bb{}\u00a0\u00bb]<\/p>\n

Trois instantan\u00e9s dans le temps alors qu’un m\u00e9lange est transport\u00e9 \u00e0 travers une colonne. Au d\u00e9part (\u00e0 gauche), il n’y a pas de s\u00e9paration. Lorsque le bouchon d’\u00e9chantillon se d\u00e9place plus loin dans le chemin d’\u00e9coulement, les substances (\u2206 et \u25cf) sont s\u00e9par\u00e9es.<\/em><\/p>\n

[\/et_pb_text][\/et_pb_column][\/et_pb_row][et_pb_row custom_padding_last_edited=\u00a0\u00bbon|phone\u00a0\u00bb admin_label=\u00a0\u00bbDividerline\u00a0\u00bb _builder_version=\u00a0\u00bb4.5.6″ width=\u00a0\u00bbauto\u00a0\u00bb max_width=\u00a0\u00bbnone\u00a0\u00bb custom_padding=\u00a0\u00bb|0px|||false|false\u00a0\u00bb custom_padding_tablet=\u00a0\u00bb\u00a0\u00bb custom_padding_phone=\u00a0\u00bb|10%|||false|false\u00a0\u00bb collapsed=\u00a0\u00bbon\u00a0\u00bb global_colors_info=\u00a0\u00bb{}\u00a0\u00bb][et_pb_column type=\u00a0\u00bb4_4″ _builder_version=\u00a0\u00bb4.4.8″ global_colors_info=\u00a0\u00bb{}\u00a0\u00bb][et_pb_divider color=\u00a0\u00bb#dee1e6″ _builder_version=\u00a0\u00bb4.4.8″ global_colors_info=\u00a0\u00bb{}\u00a0\u00bb][\/et_pb_divider][\/et_pb_column][\/et_pb_row][et_pb_row column_structure=\u00a0\u00bb2_3,1_3″ custom_padding_last_edited=\u00a0\u00bboff|desktop\u00a0\u00bb _builder_version=\u00a0\u00bb4.5.6″ width=\u00a0\u00bbauto\u00a0\u00bb max_width=\u00a0\u00bbnone\u00a0\u00bb custom_padding=\u00a0\u00bb27px|0|27px|0px|false|false\u00a0\u00bb custom_padding_tablet=\u00a0\u00bb|0px||0px||true\u00a0\u00bb locked=\u00a0\u00bboff\u00a0\u00bb global_colors_info=\u00a0\u00bb{}\u00a0\u00bb][et_pb_column type=\u00a0\u00bb2_3″ _builder_version=\u00a0\u00bb3.25″ custom_padding=\u00a0\u00bb|||\u00a0\u00bb global_colors_info=\u00a0\u00bb{}\u00a0\u00bb custom_padding__hover=\u00a0\u00bb|||\u00a0\u00bb][et_pb_text admin_label=\u00a0\u00bbSubtitle\u00a0\u00bb _builder_version=\u00a0\u00bb4.10.5″ text_font=\u00a0\u00bb||||||||\u00a0\u00bb text_font_size=\u00a0\u00bb16px\u00a0\u00bb text_line_height=\u00a0\u00bb2em\u00a0\u00bb header_font=\u00a0\u00bb||||||||\u00a0\u00bb header_2_font=\u00a0\u00bb|600||on|||||\u00a0\u00bb header_2_text_color=\u00a0\u00bb#df0834″ header_2_font_size=\u00a0\u00bb20px\u00a0\u00bb header_2_line_height=\u00a0\u00bb1.4em\u00a0\u00bb header_6_font=\u00a0\u00bb|700||on|||||\u00a0\u00bb header_6_font_size=\u00a0\u00bb16px\u00a0\u00bb header_6_letter_spacing=\u00a0\u00bb5px\u00a0\u00bb header_6_line_height=\u00a0\u00bb1.8em\u00a0\u00bb max_width=\u00a0\u00bb700px\u00a0\u00bb module_alignment=\u00a0\u00bbcenter\u00a0\u00bb custom_margin=\u00a0\u00bb||0px||false|false\u00a0\u00bb custom_padding=\u00a0\u00bb||0px||false|false\u00a0\u00bb header_2_font_size_tablet=\u00a0\u00bb25px\u00a0\u00bb header_2_font_size_phone=\u00a0\u00bb25px\u00a0\u00bb header_2_font_size_last_edited=\u00a0\u00bbon|phone\u00a0\u00bb global_colors_info=\u00a0\u00bb{}\u00a0\u00bb]<\/p>\n

Qu’est-ce qu’un chromatogramme ?<\/h2>\n

[\/et_pb_text][et_pb_text admin_label=\u00a0\u00bbParagraph\u00a0\u00bb _builder_version=\u00a0\u00bb4.11.3″ text_font=\u00a0\u00bb||||||||\u00a0\u00bb text_font_size=\u00a0\u00bb18px\u00a0\u00bb text_line_height=\u00a0\u00bb2em\u00a0\u00bb header_font=\u00a0\u00bb||||||||\u00a0\u00bb header_2_font=\u00a0\u00bbRaleway|700|||||||\u00a0\u00bb header_2_font_size=\u00a0\u00bb50px\u00a0\u00bb header_2_line_height=\u00a0\u00bb1.4em\u00a0\u00bb header_6_font=\u00a0\u00bbRaleway|700||on|||||\u00a0\u00bb header_6_font_size=\u00a0\u00bb16px\u00a0\u00bb header_6_letter_spacing=\u00a0\u00bb5px\u00a0\u00bb header_6_line_height=\u00a0\u00bb1.8em\u00a0\u00bb text_orientation=\u00a0\u00bbjustified\u00a0\u00bb max_width=\u00a0\u00bb700px\u00a0\u00bb module_alignment=\u00a0\u00bbcenter\u00a0\u00bb custom_margin=\u00a0\u00bb0px||||false|false\u00a0\u00bb custom_padding=\u00a0\u00bb0px||||false|false\u00a0\u00bb header_2_font_size_tablet=\u00a0\u00bb35px\u00a0\u00bb header_2_font_size_phone=\u00a0\u00bb25px\u00a0\u00bb header_2_font_size_last_edited=\u00a0\u00bbon|phone\u00a0\u00bb global_colors_info=\u00a0\u00bb{}\u00a0\u00bb]<\/p>\n

Un chromatogramme est un graphique qui contient les donn\u00e9es recueillies \u00e0 partir d’un d\u00e9tecteur HPLC par un logiciel d’acquisition de donn\u00e9es. Les donn\u00e9es sont stock\u00e9es dans un fichier, elles contiennent le signal et le temps de r\u00e9tention d’un passage chromatographique. Souvent, on obtient toute une s\u00e9rie de chromatogrammes dans une s\u00e9quence, contenant les analyses d’\u00e9chantillons et de standards pour la calibration. Ces informations sont utilis\u00e9es pour l’identification et la quantification, le but ultime de l’analyse. En utilisant des normes d’\u00e9talonnage, la hauteur du signal est li\u00e9e \u00e0 la concentration et le temps de r\u00e9tention permet d’identifier une substance dans des \u00e9chantillons inconnus.<\/p>\n

Il faut veiller \u00e0 ce que le pic mesur\u00e9 dans le chromatogramme de l’\u00e9chantillon ne soit pas co-\u00e9lu\u00e9 avec d’autres substances ayant le m\u00eame temps de r\u00e9tention. Cette v\u00e9rification, ainsi que d’autres, font partie de la validation d’un essai.<\/p>\n

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Un chromatogramme avec des standards de calibration de plusieurs neurotransmetteurs.<\/em><\/p>\n

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Qu’est-ce que l’HPLC ?La HPLC ou chromatographie liquide \u00e0 haute performance est une technique d’analyse permettant d’identifier et de quantifier des substances dans une solution. 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