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Chromatography

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Mohammed Bahgat Mohammed Sofyan
Mohammed Bahgat Mohammed Sofyan

رايط الشرح علي اليوتيوب https://youtu.be/kxSJPSxA-Yk

Education
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BY dr: Mohammed Bahgat Mohammed Sofyan Assistant lecturer of medical biochemistry, Faculty of medicine, Al-Azhar university (Assiut branch) 1
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‫صورة‬ ‫كل‬ ‫على‬ ‫اضغط‬ ‫الشاش‬ ‫عرض‬ ‫وضع‬ ‫في‬ ‫ة‬ ‫قنواتنا‬ ‫على‬ ‫وادعمنا‬ ‫اإلنترنت‬ ‫على‬
‫على‬ ‫ملفي‬ ‫في‬ ‫هنا‬ ‫أرفعها‬ ‫التي‬ ‫شرائحي‬ ‫ومذاكرة‬ ‫بالتعلم‬ ‫وأسعد‬ ‫وأرحب‬ ‫أسمح‬ Slide share website ‫وال‬ ‫مانع‬ ‫أيضا‬ ‫من‬ ‫نسخ‬ ‫شريحة‬ ‫او‬ ‫اثنين‬ ‫عند‬ ‫الضرورة‬ ، ‫وال‬ ‫مانع‬ ‫من‬ ‫شرح‬ ‫البوربو‬ ‫ينت‬ ‫الخاصة‬ ‫بي‬ ‫للغير‬ ‫بشرط‬ ‫عدم‬ ‫إزالة‬ ‫اسمي‬ ‫من‬ ‫البوربوينت‬ ، ‫فال‬ ‫أسمح‬ ‫أبدا‬ ‫بإزالة‬ ‫اسمي‬ ‫من‬ ‫على‬ ‫الباوربوينت‬ ‫ووضع‬ ‫اسمك‬ ‫بدال‬ ‫منه‬ ‫لتصبح‬ ‫وكأنك‬ ‫من‬ ‫صممتها‬ ‫فهذه‬ ‫سرقة‬ ‫ال‬ ‫أسمح‬ ‫بها‬ ‫وتضييع‬ ‫لحق‬ ‫من‬ ‫تعب‬ ‫في‬ ‫عملها‬ . ‫وفقكم‬ ‫هللا‬ ‫وإياي‬ ‫للتعلم‬ ‫ونفع‬ ‫اآلخرين‬ I allow, welcome, and be happy to learn and study my slides that I upload here in my profile on Slide share website There is also no objection to copying one or two slides when necessary, and there is no objection to explaining my PowerPoint to others on the condition that my name is not removed from the PowerPoint. I never allow my name to be removed from PowerPoint and to replace it with yours, Make it look like you designed it. This is theft that I do not allow and a waste of the right of those who are tired in this work. May God bless you and me for learning and benefiting others 5
DEFINTION It's a powerful analytical tool that is used for the separation, detection and quantitation of a wide variety of analytes. Example of analytes that is separated : Proteins, amino acids, isoenzymes, electrolytes, lipoproteins and hemoglobin variants,..... ‫طريقة‬ ‫تستخدم‬ ‫لفصل‬ ‫المواد‬ ‫عن‬ ‫بعضها‬ ‫سواء‬ ‫كانوا‬ ‫بروتينيات‬ ‫أو‬ ‫أحماض‬ ‫أمينية‬ ‫أو‬ ‫إ‬ ‫لكترونات‬ ‫وغير‬ ‫ذلك‬ ... ‫وبعد‬ ‫فصلهم‬ ‫يمكن‬ ‫التعرف‬ ‫عليهم‬ ‫ومعرفة‬ ‫كميتهم‬ . 6
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Analyze: examine a mixture, its components, and their relations to one another 1 Identify: determine the identity of a mixture or components based on known components 2 Purify: separate components in order to isolate one of interest for further study 3 Quantify: determine the amount of the mixture and/or the components present in the sample 4 8
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Uses for Chromatography Pharmaceutical Company: determine amount of each chemical found in new product Hospital: detect blood or alcohol levels in a patient’s blood stream Law Enforcement : to compare a sample found at a crime scene to samples from suspects Environmental Agency : determine the level of pollutants in the water supply Manufacturing Plant: to purify a chemical needed to make a product 11
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In chromatography a sample is introduced into a flowing stream referred to a mobile phase, which passes through a bed ( layer or column) referred to stationary phase. ‫يعني‬ ‫ببساطة‬ ‫فيه‬ ،‫جزئين‬ ‫الجزء‬ ‫األول‬ ‫متحرك‬ ‫ويحتوي‬ ‫علي‬ ‫المادة‬ ‫المراد‬ ،‫فصلها‬ ‫ويمر‬ ‫علي‬ ‫مخدة‬ ( ‫مفلطحة‬ ‫أ‬ ‫و‬ ‫أنبوبية‬ ) ‫هذه‬ ‫المخدة‬ ‫ثابتة‬ ‫وهي‬ ‫الجزء‬ ‫الثاني‬ . ‫ث‬ ،‫الحقا‬ ‫عنها‬ ‫التحدث‬ ‫سيتم‬ ‫ما‬ ‫بطريقة‬ ‫المخدة‬ ‫الثابت‬ ‫للجزء‬ ‫المتحرك‬ ‫الجزء‬ ‫من‬ ‫فصلها‬ ‫المراد‬ ‫المادة‬ ‫تنتقل‬ ‫أجزاء‬ ‫تعود‬ ‫م‬ ‫تسمي‬ ‫بطريقة‬ ‫منفصلة‬ ‫لكن‬ ‫أخري‬ ‫مرة‬ ‫المادة‬ elution 13
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Explanation •Compound is placed on stationary phase •Mobile phase passes through the stationary phase •Mobile phase solubilizes the components •Mobile phase carries the individual components a certain distance through the stationary phase, depending on their attraction to both of the phases 15
Classification 1_ According to Mobil phase : • ‫المتحركة‬ ‫المادة‬ ‫طبيعة‬ ‫يعني‬ • A _ Liquid chromatography ( LC ) • ‫سائلة‬ ‫المتحركة‬ ‫المادة‬ ‫لو‬ • B _ Gas chromatography ( GC ) • ‫غازية‬ ‫المتحركة‬ ‫المادة‬ ‫لو‬ 2_ According to chromatographic bed (stationary phase) : • ‫الثابت‬ ‫الجزء‬ ‫حسب‬ ‫علي‬ • A _ Planner chromatography • ‫الثابت‬ ‫الجزء‬ ‫لو‬ ( ‫المخدة‬ ) ‫زجاجية‬ ‫او‬ ‫ورقية‬ ‫كانت‬ ‫سواء‬ ‫مفلطح‬ • B _ Column chromatography • ‫الثابت‬ ‫الجزء‬ ‫لو‬ ( ‫المخدة‬ ) ‫أنبوبي‬ 16
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‫الملخص‬ ‫ببساطة‬ ‫فيه‬ ،‫جزئين‬ ‫الجزء‬ ‫األول‬ ‫متحرك‬ Mobile phase ‫ويحتوي‬ ‫علي‬ ‫المادة‬ ‫المراد‬ ،‫فصلها‬ ‫ويمر‬ ‫علي‬ ‫مخدة‬ ( ‫مفلطحة‬ ‫أو‬ ‫أنبوبية‬ ) ‫هذه‬ ‫المخدة‬ ‫ثابتة‬ ‫هي‬ ‫الجزء‬ ‫الثاني‬ Stationary phase ‫تنتقل‬ ‫المادة‬ ‫المراد‬ ‫فصلها‬ ‫من‬ ‫الجزء‬ ‫المتحرك‬ ‫للجزء‬ ‫الثابت‬ ‫المخدة‬ ‫بطريقة‬ ‫ما‬ ‫سيتم‬ ‫التحدث‬ ‫عنها‬ ،‫الحقا‬ ‫ثم‬ ‫تعود‬ ‫أجزاء‬ ‫ا‬ ‫لمادة‬ ‫مرة‬ ‫أخري‬ ‫لكن‬ ‫منفصلة‬ ‫بطريقة‬ ‫تسمي‬ Elution ‫بعد‬ ‫ذلك‬ The mobile phase with its separated solutes zones emerges from a column (stationary phase) and passes through a detector. ‫بعد‬ ‫ما‬ ‫أجزاء‬ ‫المادة‬ ‫المراد‬ ‫فصلها‬ ‫يحصل‬ ‫لها‬ elution ‫تدريجيا‬ ‫وتخرج‬ ‫من‬ ‫األنبوبة‬ ‫منفصلة‬ ‫تمر‬ ‫علي‬ ‫جهاز‬ detector ‫الذي‬ ‫يحول‬ ‫الجزء‬ ‫المفصول‬ ‫منها‬ ‫الي‬ peak 19
‫الملخص‬ ‫بيعمل‬ ‫الجهاز‬ ‫ألن‬ ،‫معا‬ ‫وليسوا‬ ‫تدريجيا‬ ‫معين‬ ‫وقت‬ ‫في‬ ‫بيخرج‬ ‫مفصول‬ ‫جزء‬ ‫كل‬ ‫طبعا‬ Elution at specific pH and specific ionic strength or according to affinity between mobile and stationary phases ‫ال‬ ‫يسمي‬ ‫معروف‬ ‫زمن‬ ‫في‬ ‫يخرج‬ ‫المفصولة‬ ‫األجزاء‬ ‫من‬ ‫كل‬ ‫ولهذا‬ Retention time ‫ب‬ ‫سيفصل‬ ‫جزء‬ ‫كل‬ ‫يعني‬ Specific pH and specific ionic strength or according to affinity between mobile and stationary phases. ‫يسمي‬ ‫معروف‬ ‫معين‬ ‫محدد‬ ‫زمن‬ ‫في‬ ‫ويخرج‬ Retention time ‫مفصول‬ ‫جزء‬ ‫كل‬ ‫علي‬ ‫نتعرف‬ ‫وبهذا‬ . Separation and Detection 20
‫ال‬ ‫الجهاز‬ detector ‫ويرسم‬ ‫المفصول‬ ‫الجزء‬ ‫بهذا‬ ‫يحس‬ peak ‫كل‬ peak ‫لها‬ Hight, width and peak area ‫ال‬ ‫وضعف‬ ‫قوة‬ ‫تكون‬ ‫المادة‬ ‫هذه‬ ‫تركيز‬ ‫حسب‬ ‫علي‬ peak ‫وباستخدام‬ standard ‫المفصول‬ ‫المادة‬ ‫قيمة‬ ‫معرفة‬ ‫نستطيع‬ ‫القيمة‬ ‫معلوم‬ quantitiation ‫نعمل‬ ‫ممكن‬ Calibration curve. ‫بااستخدام‬ ‫ذكرها‬ ‫االتي‬ ‫المعادلة‬ ‫نطبق‬ ‫وممكن‬ 1 standard ‫الملخص‬ 21
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Equation Concentration of sample=(signal of sample / signal of standard) X concentration of standard. Concentration of sample = ( peak height (or area) of sample / peak height (or area) of standard) X concentration of standard. 25
Factors affecting the rate of separation Characteristics of the mobile and stationary phases. Characteristics of the analytes in the mobile phase. Characteristics of the porous medium. The flow dynamic. 26
calibration • Two methods of calibration are used : 1- External calibration, in which solutions of known concentration are prepared and processed in an identical manner as the unknowns. A calibration curve of peak height or peak area versus concentration is constructed, and the concentration of the unknown is obtained by interpolation. 27
• internal calibration, in which analyte solutions of known concentration are prepared, and a constant amount of a second compound, the internal standard, is added to each calibrator and sample. In principle, the size of the internal standard peak should be constant in all calibrators and samples. Any change in the internal standard should be the same as a change in the analyte of interest.. By plotting the ratio of the peak height (or area) of the analyte of interest to the peak height (or area) of the internal standard versus concentration of the analyte of interest, a calibration curve that corrects for systematic losses can be constructed. The concentration of the unknown is then obtained from the curve by interpolation. calibration 28
• In chromatography a sample is introduced into a flowing stream referred to a mobile phase, which passes through a bed ( layer or column) referred to stationary phase. • The sample is attracted to the stationary phase by specific method ( will be mentioned later, God willing) then elute gradually and separately. • The mobile phase with its separated solutes zones emerges from a column (stationary phase) and passes through a detector. • Finally, the separated analytes is detected by detector and analyzed by computer or any thing else. 29
Separation mechanism ‫علي‬ ‫تعدي‬ ‫علشان‬ ‫تاني‬ ‫ترجع‬ ‫وازاي‬ ‫الثابت‬ ‫للجزء‬ ‫المتحرك‬ ‫الجزء‬ ‫من‬ ‫بتنتقل‬ ‫المادة‬ ‫ازاي‬ detector 1_ Adsorption and elution : • ‫واع‬ ‫الساكن‬ ‫الجزء‬ ‫الي‬ ‫المتحرك‬ ‫الجزء‬ ‫من‬ ‫فصلها‬ ‫المراد‬ ‫المادة‬ ‫فصل‬ ‫يتم‬ ‫الخاصيتين‬ ‫هاتين‬ ‫باستخدام‬ ‫بساطة‬ ‫بكل‬ ‫أخري‬ ‫مرة‬ ‫ادتها‬ ‫مفصولين‬ ‫يعودوا‬ ‫تجعلهم‬ ‫بطريقة‬ ‫لكن‬ ‫ال‬ ‫طبعا‬ adsorption ‫ب‬ ‫يكون‬ ‫ممكن‬ • a_ acidic adsorbent ➡ adsorb basic substances • b_ basic adsorbent ➡ adsorb acidi substances ‫حصل‬ adsorption ‫اعمل‬ elution ‫في‬ ‫تمسك‬ ‫انها‬ ‫علي‬ ‫المادة‬ ‫قدرة‬ ‫حسب‬ ‫وعلي‬ adsorbent ‫ماسك‬ ‫فاللي‬ ‫خروجها‬ ‫يتم‬ ‫له‬ ‫يحصل‬ ‫ضعيف‬ elution ‫له‬ ‫يحصل‬ ‫جامد‬ ‫ماسك‬ ‫واللي‬ ،‫اوال‬ elution ‫مؤخرا‬ . The main application of adsorption chromatography is separation of steroids and vitamins. 30
2-Affinity chromatography ‫هنا‬ ‫بقي‬ ‫هرسب‬ ‫المادة‬ ‫المراد‬ ‫فصلها‬ ‫بال‬ affinity ‫يعني‬ ‫شيئين‬ ‫بيحبوا‬ ‫بعض‬ ‫زي‬ Ag Ab, enzyme substrate or hormones receptor. ‫يعني‬ ‫لو‬ ‫عاوز‬ ‫أقيس‬ Ag ‫في‬ ‫الجزء‬ ‫المتحرك‬ ‫احط‬ ‫علي‬ ‫الثابت‬ It's Ab ‫يمسك‬ ‫فيه‬ ‫في‬ ‫ظروف‬ ‫معينة‬ . ‫اعمل‬ ‫ظروف‬ ‫اخري‬ ‫تفكك‬ ‫ال‬ Ag ‫من‬ Ab ‫باني‬ Adjust pH and ionic strength ‫يقوم‬ ‫يحصل‬ elution ‫تدريجيا‬ The main application of adsorption chromatography is separation of glycated Hb, lipoproteins. 31
3_ Ion Exchange chromatography ‫هذه‬ ‫الطريقة‬ ‫بتفصل‬ ‫األحماض‬ ‫األمينية‬ ‫والهيموجلوبين‬ ‫وغيره‬ ... ‫نجعل‬ ‫الوسط‬ ‫حمضي‬ ‫فستصبح‬ ‫األحماض‬ ‫األمينية‬ ‫المراد‬ ‫فصلها‬ ‫موجبة‬ ، ‫ونجعل‬ ‫مثال‬ ‫الصوديوم‬ ‫قليل‬ ( ‫ما‬ ‫دة‬ ‫توجد‬ ‫في‬ ‫الجزء‬ ‫المتحرك‬ ) ‫وبوضع‬ ‫مادة‬ ‫صمغية‬ ‫عليها‬ ‫ايونات‬ ‫سالبة‬ ‫في‬ ‫الجزء‬ ‫الثابت‬ ، ‫فعند‬ ‫مرور‬ ‫األحماض‬ ‫األميني‬ ‫ة‬ ‫الموجية‬ ‫تمسك‬ ‫في‬ ‫األيونات‬ ‫السالبة‬ ‫التي‬ ‫في‬ ‫الجزء‬ ‫الثابت‬ ‫بدال‬ ‫عن‬ ‫الصوديوم‬ ‫ألنه‬ ‫قليل‬ ‫فتزيحه‬ ‫وتمسك‬ ‫باأل‬ ‫يونات‬ ‫السالبه‬ ‫الموجودة‬ ‫علي‬ ‫الجزء‬ ‫الثابت‬ ‫بدال‬ ‫من‬ ‫الصوديوم‬ Ion Exchange ‫ما‬ ‫رأيك‬ ‫لو‬ ‫جعلنا‬ ‫الوسط‬ ‫قلوي‬ ‫بدال‬ ‫من‬ ‫حمضي‬ ‫وزودنا‬ ‫ايونات‬ ‫الصوديوم؟‬ Adjust pH and ionic strength ‫في‬ ‫الوسط‬ ‫القلوي‬ ‫البروتينات‬ ‫ستصبح‬ ‫سالبة‬ ‫وستتنافر‬ ‫مع‬ ‫األيونات‬ ‫السالبه‬ ‫الموجودة‬ ‫علي‬ ‫الجزء‬ ‫ال‬ ،‫ثابت‬ ‫ولو‬ ‫زودنا‬ ‫ايونات‬ ‫الصوديوم‬ ‫ايضا‬ ‫ستحل‬ ‫محل‬ ‫األحماض‬ ‫األمينية‬ ‫وبالتالي‬ ‫يحصل‬ Elution to amino acids gradually The main application of adsorption chromatography is separation of different amino acids hemoglobin variants and isoenzymes. 32
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Chromatography in which separation is based mainly on differences in the ion-exchange affinities of the sample components. Anions like SO3-or cations like N(CH3)3+ are covalently attached to stationary phase, usually a resin. So there are two types : 1_ Cation-exchange resin 2_ Anion-exchange resins 3_ Ion Exchange chromatography 36
4- Partition chromatography ‫ال‬ ‫أحد‬ ‫نجعل‬ ‫الحالة‬ ‫هذه‬ ‫في‬ Mobile and stationary phases polar and the other non polar ‫الفصل‬ ‫ويتم‬ ‫ف‬ ‫الثابت‬ ‫الجزء‬ ‫كان‬ ‫لو‬ ‫مثال‬ polar if a stationary phase is polar ‫كانت‬ ‫ما‬ ‫كل‬ ‫المادة‬ ‫يبقي‬ nonpolar ‫وال‬ ‫بطيئ‬ ‫مشيها‬ ‫ويبقي‬ ‫أكثر‬ ‫فيها‬ ‫تمسك‬ elution ‫كانت‬ ‫لو‬ ‫بخالف‬ ،‫أبطء‬ polar ‫وال‬ ‫أسرع‬ ‫تمشي‬ elution ‫الفصل‬ ‫يتم‬ ‫وبذلك‬ ،‫أسرع‬ ‫يكون‬ ‫ال‬ ‫حسب‬ polarity ‫صحيح‬ ‫والعكس‬ . 37
Partition chromatography Chromatography in which separation is based mainly on differences between the solubility of the sample components in the stationary phase (gas chromatography), or on differences between the solubilities of the components in the mobile and stationary phases (liquid chromatography). 38
others 5_ Stearic exclusion chromatography . 6_ Molecular exclusion chromatography. 7_ Gel-permeation chromatography. 39
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1_ paper chromatography 43
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paper chromatography ‫نادر‬ ‫االستخدام‬ ‫في‬ ‫الحياة‬ ‫العملية‬ ، ‫بنجيب‬ Filter paper ‫ونضع‬ ‫المادة‬ ‫المراد‬ ‫فصلها‬ ‫ونغمسها‬ ‫في‬ Chromatographic solvent which is a mixture of water, alcohol and acid or base. ‫فيتحرك‬ ‫كال‬ ‫من‬ ‫ال‬ Solvent and specimens that needed to separate. For example amino acids with large nonpolar side chains ( as leucine and isoleucine) migrate more slowly than short nonpolar side chain (serine and threonine) in non polar stationary phase ‫نحسب‬ ‫المسافة‬ ‫التي‬ ‫تحركتها‬ ‫المواد‬ ‫المفصولة‬ ‫إلى‬ ‫التي‬ ‫تحركها‬ ‫ال‬ Solvent ‫وعن‬ ‫طريق‬ ‫ذلك‬ ‫نعمل‬ Identification to the separated substances Quantification may be performed by cutting out each spot eluting with suitable solvent and performing quantitative colorimetric assay by dring strip and treated with 0.5% ninhydrin 47
2_ Thin layer chromatography (TLC) The same principle as that of paper chromatography, but the stationary phase is made of silica jell or cellulose acetate and liquid with volatile organic solvent is preferred used as mobile phase. • Advantages Cheap Simple The developing can be monitored visually Able to use various chemical as a detector 48
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3_ Gas chromatography Gas chromatography is a process by which a mixture of compounds is separated into its constituent components by flowing a gaseous mixture of analytes and mobile phase through a column containing a stationary phase. Mobile phase ➡ inert gas as helium carry substances that want to be separated. Stationary phase ➡ fixed at column, may be solid or liquid 55
• ‫تكون‬ ‫فصلها‬ ‫المراد‬ ‫المادة‬ ‫الزم‬ Volatile and polar 56
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Instrumentation 1_ carrier gas supply : ‫ال‬ ‫تدفع‬ Mobile phase ‫الهيليوم‬ ‫مثل‬ 2_ sample injector ‫فيه‬ ‫بيبقي‬ ‫بعدها‬،‫فصلها‬ ‫المراد‬ ‫المادة‬ ‫تدفع‬ Vaporisation champer ‫بالتسخين‬ ‫غاز‬ ‫الي‬ ‫المواد‬ ‫تحول‬ 59
Instrumentation 3_ column containing stationary phase , Column may be packed or capillary ‫الثابتة‬ ‫المادة‬ ‫يحمل‬ • Stationary phase may be liquid or solid • Stationary phase may be hydrophilic or hydrophobic 4_ detector • ‫معرفة‬ ‫طريق‬ ‫عن‬ ‫المفصولة‬ ‫المادة‬ ‫بيلقط‬ ( ‫وكميتها‬ ،‫مجيئها‬ ‫وقت‬ ) ‫ل‬ ‫ويترجمها‬ peak 5_ computer • ‫ال‬ ‫ويرسم‬ ‫النتائج‬ ‫يحلل‬ peak 60
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Mechanism of sepration • It depends on the relative solubility and volatility of solutes in the two phases and their interaction with the stationary one. • Or It depends on volatility and polarity. • if a stationary phase is polar • ‫كانت‬ ‫ما‬ ‫كل‬ ‫المادة‬ ‫يبقي‬ polar ‫وال‬ ‫بطيء‬ ‫مشيها‬ ‫ويبقي‬ ‫أكثر‬ ‫فيها‬ ‫تمسك‬ elution ‫كانت‬ ‫لو‬ ‫بخالف‬ ،‫أبطء‬ non polar ‫وال‬ ‫أسرع‬ ‫تمشي‬ elution ‫ال‬ ‫حسب‬ ‫الفصل‬ ‫يتم‬ ‫وبذلك‬ ،‫أسرع‬ ‫يكون‬ polarity ‫صحيح‬ ‫والعكس‬ . ‫في‬ ‫األكثر‬ ‫المادة‬ ‫كذلك‬ volatility ‫األسرع‬ ‫هي‬ ‫بال‬ ‫نفصل‬ ‫ممكن‬ Gas chromatography ‫ونعمل‬ Quantitative by mass spectrometer ‫ساعتها‬ ‫الجهاز‬ ‫ويسمى‬ Gas chromatography mass spectrometer GC/MS It's used primarily to for the analytes of drugs in sport and military medicine. • ‫والعسكريين‬ ‫للرياضيين‬ ‫العقاقير‬ ‫وفصل‬ ‫ومعرفة‬ ‫قياس‬ ‫في‬ ‫تستخدم‬ . 62
Detector a· Electron capture detector b. Flame ionization detector c· Photoionization detector d· Thermal conductivity detector e· Thermionic selective detector f. Mass spectrometers 63
4_ High Performance Liquid Chromatography (HPLC) HPLC is a process by which a mixture of compounds is separated into it's constituent components by pumping a liquid mobile phase through a column containing a stationary phase. Mobile phase ➡ liquid carry substances that want to be separated. Stationary phase ➡ fixed at column may be solid as silica or liquid ‫علشان‬ ‫نفصل‬ ‫المواد‬ ‫بال‬ HPLC ‫بيتم‬ ‫فصلها‬ ‫عن‬ ‫طريق‬ Polarity 64
Instrumentation 1_ solvent reservoir and (pump) : • ‫تدفع‬ ‫ال‬ Mobile phase 2_ sample injector • ‫تدفع‬ ‫المادة‬ ‫المراد‬ ‫فصلها‬ 3 _column containing stationary phase ,Column may be packed or capillary • ‫يحمل‬ ‫المادة‬ ‫الثابتة‬ Stationary phase may be liquid or solid Stationary phase may be hydrophilic silica ➡ normal phase HPLC or hydrophobic silica C 18 ➡ reversed phase HPLC 4_ detector • ‫بيلقط‬ ‫المادة‬ ‫المفصولة‬ ‫عن‬ ‫طريق‬ ‫معرفة‬ ( ‫وقت‬ ،‫مجيئها‬ ‫وكميتها‬ ) ‫ويترجمها‬ ‫ل‬ peak 5_ computer • ‫يحلل‬ ‫النتائج‬ ‫ويرسم‬ ‫ال‬ peak 65
66
67
68
Mechanism of separation • It depends on the relative solubility of solutes in the two phases and their interaction with the stationary one. Also, ion exchange . • if a stationary phase is nonpolar • ‫يبقي‬ ‫المادة‬ ‫كل‬ ‫ما‬ ‫كانت‬ polar ‫تمسك‬ ‫فيها‬ ‫أكثر‬ ‫ويبقي‬ ‫مشيها‬ ‫بطيء‬ ‫وال‬ elution ،‫أبطء‬ ‫بخالف‬ ‫لو‬ ‫كانت‬ nonpolar ‫تمشي‬ ‫أسرع‬ ‫وال‬ elution ‫يكون‬ ،‫أسرع‬ ‫وبذلك‬ ‫يتم‬ ‫الفصل‬ ‫حسب‬ ‫ال‬ polarity • ‫والعكس‬ ‫صحيح‬ . • HPLC may be used for the separation, detection and quantitation of a wide variety of analytes. • Example of analytes that is separated : • Proteins, amino acids, electrolytes and hemoglobin variants. 69
70
71
High Performance Liquid Chromatography (HPLC) for separation of Hb variants ‫ايه‬ ‫فكرة‬ ‫عمل‬ ‫فصل‬ ‫أنواع‬ ‫الهيموجلوبين‬ ‫بجهاز‬ ‫ال‬ HPLC ?? ‫نضع‬ ‫عينة‬ ‫الدم‬ ‫في‬ ‫الجهاز‬ ‫مع‬ ‫ال‬ Mobile phase ‫إيه‬ ‫اللي‬ ‫هيحصل؟‬ ‫ال‬ column that contain stationary phase ‫يمتز‬ ‫الهيموجلوبين‬ ‫ألن‬ ‫الهيموجلوبين‬ positive ‫وال‬ Stationary phase contains negative charge ‫كده‬ ‫الهيموجلوبين‬ ‫ذو‬ ‫الشحنة‬ ‫الموجبة‬ ‫مسك‬ ‫في‬ ‫ال‬ column ‫ذو‬ ‫الشحنة‬ ‫السالبة‬ 72
‫بعد‬ ‫ذلك‬ ‫نزيد‬ ‫من‬ ‫مرور‬ Buffer that contain cations in mobile phase and change pH ‫تقوم‬ ‫ال‬ cations ‫تتنافس‬ ‫مع‬ ‫الشحنات‬ ‫الموجبة‬ ‫الموجودة‬ ‫على‬ ‫الهيموجلوبين‬ ‫على‬ ‫الشحنات‬ ‫السالبة‬ ‫الموجو‬ ‫دة‬ ‫على‬ ‫ال‬ Stationary phase. The cations in the mobile phase compete with the adsorbed proteins ( Hb ) for the anionic binding sites present in the stationary phase. ‫يقوم‬ ‫ال‬ cations ‫تحل‬ ‫الهيموجلوبين‬ ‫على‬ ‫الجزء‬ ‫الثابت‬ ‫ويحصل‬ gradual elution to Hb variants ‫علي‬ ‫حسب‬ Their affinity for the stationary phase ( polarity ) ‫طبعا‬ ‫كل‬ ‫ما‬ ‫زادت‬ ‫الشحنة‬ ‫الموجبة‬ ‫في‬ ‫نوع‬ ‫معين‬ ‫من‬ ‫الهيموجلوبين‬ ‫كل‬ ‫ما‬ ‫مسك‬ ‫أكثر‬ ‫بالجزء‬ ،‫الثابت‬ ‫وبالت‬ ‫الي‬ ‫يحصل‬ ‫له‬ elution ‫أبطأ‬ ‫وبالتالي‬ ‫يصل‬ ‫لل‬ detector ‫متأخرا‬ ‫وبالتالي‬ High retention time. ‫وكل‬ ‫ما‬ ‫زادت‬ ‫الشحنة‬ ‫السالبة‬ ‫في‬ ‫نوع‬ ‫معين‬ ‫من‬ ‫الهيموجلوبين‬ ‫كل‬ ‫ما‬ ‫حصل‬ ‫له‬ elution ‫أسرع‬ ‫وبالتالي‬ ‫يصل‬ ‫لل‬ detector ‫أسرع‬ ‫وبالتالي‬ Low retention time 73
‫طب‬ ‫الجهاز‬ ‫عارف‬ ‫الوقت‬ ‫اللي‬ ‫هيوصل‬ ‫عنده‬ ‫كل‬ ‫نوع‬ ‫من‬ ‫أنواع‬ ‫الهيموجلوبين‬ ‫والذي‬ ‫يس‬ ‫مي‬ ‫ال‬ retention time ‫كل‬ ‫نوع‬ ‫من‬ ‫الهيموجلوبين‬ ‫له‬ Specific retention time ‫محدد‬ ‫الجهاز‬ ‫عارفه‬ ‫وبالتالي‬ ‫الجهاز‬ ‫يعرف‬ ‫ما‬ ‫هو‬ ‫البروتين‬ ‫أو‬ ‫الهيموجلوبين‬ ‫اللي‬ ‫وص‬ ‫ل‬ ‫في‬ ‫هذا‬ ‫الوقت‬ ‫وتركيزه‬ ‫يساوي‬ ‫كم؟‬ . ‫كل‬ ‫ده‬ ‫واضح‬ ‫على‬ ‫الورقة‬ ‫بعد‬ ‫كده‬ ‫يسجل‬ ‫الكالم‬ ‫ده‬ ‫في‬ curves ‫عن‬ ‫طريق‬ ‫جهاز‬ ‫ال‬ densometer ‫أعلم‬ ‫وهللا‬ . 74
is a process in which a mixture of molecules (such as normal and hemoglobins variant) with a net positive charge is separated into its components by their adsorption onto a negatively charged stationary phase in a chromatography column, followed by their elution by a mobile phase. The mobile phase is a liquid with an increasing concentration of cations flowing through the column; the cations in the mobile phase compete with the adsorbed proteins for the anionic binding sites. 75
Thus, the adsorbed positively charged hemoglobin molecules are eluted from the column into the liquid phase at a rate related to their affinity for the stationary phase. When separated in this way, they can be detected optically in the eluate, provisionally identified by their retention time, and quantified by computing the area under the corresponding peak in the elution profile. The more positively charged hemoglobins (e.g. hemoglobin S and C) have a longer retention time. 76
Variant with weak positive charge will adsorb weakly to the column and is eluted rapidly from the column with the injection of low strength elution buffer. Variant with strong positive charge will adsorb strongly to the column and is eluted slowly from the column with the injection of high strength elution buffer. Procedure: HPLC is fully automated. Samples are loaded in a rack and automatically processed one by one (about 6 minutes/sample). Result: Data are graphically represented as absorbance vs time (peaks). Retention time is the time from injection to the top of the peak. 77
‫له‬ ‫الهيموجلوبين‬ ‫من‬ ‫نوع‬ ‫كل‬ Specific Retention time ‫ال‬ ‫يطلع‬ ‫ولما‬ ،‫الحالة‬ ‫للجهاز‬ ‫تدخل‬ ‫فإنت‬ ،‫عارفه‬ ‫الجهاز‬ Its Retention time ‫ال‬ ‫بمعلومية‬ ‫فورا‬ ‫الهيموجلوبين‬ ‫نوع‬ ‫يقولك‬ ‫يقوم‬ Its retention time. ‫ال‬ ‫نفس‬ ‫لها‬ ‫اللي‬ ‫األنواع‬ ‫من‬ ‫بالك‬ ‫وخلي‬ retention time ‫ال‬ ‫ومشاكل‬ Glycated Hb and HbS with HbA2..... etc. ‫العيوب‬ ‫في‬ ‫الحقا‬ ‫ستذكر‬ ‫كما‬ . 78
79
80
81
Immunochromatography is a combination of chromatography and immunoassay. This technology Is in use for a wide array of tests for clinical, veterinary, and industrial applications. 82
1_ The specimen (e.g., serum, urine) containing the antigen to be detected is placed on the sample pad, which soaks up the specimen fluid. 2_ The fluid then migrates to the conjugate pad, which contains conjugated antibodies (conjugated with gold, colored latex, or a chromophore) directed against the antigen. 83
3_ the antigen-antibody- conjugate complex is formed. Ag-Ab complex continues to migrate across the membrane until it reaches the capture zone where the complex will bind to immobilized antibodies. As more and more Ag-Ab complexes are captured at the “test” line, the line becomes visible on the membrane. 4 _ The sample then migrates further along the strip until it reaches the control zone where excess conjugate binds and produces a second visible line (control line) on the membrane. This control line indicates that the sample has migrated across the membrane as intended. 84
Immunochromatographic methods are widely used in clinical practice for : 1_Detection of toxins and drugs 2_ Pregnancy tests- detection of human chorionic gonadotropin (hCG) 3_ Diagnosis of parasitic infections as : a_ Malaria b_ G. lamblia and Cryptosporidium parvum C. E. histolytica 85
3_ Diagnosis of bacterial infections as :  Mycoplasma pneumoniae  Mycoplasma pneumoniae  H.pylori antigens in stool  V. cholerae O1 and O139 from stool specimens  Streptococcus pneumoniae  antigen detection in CSF or in urine 86
4_ Diagnosis of viral Infections , Antigen detection of :  RSV  Rotavirus  Influenza A/B  Hepatitis B and Hepatitis C 5_ Antibodies detection of : Detection of HIV-1 and HIV-2 antibodies. 87
• In chromatography a sample is introduced into a flowing stream referred to a mobile phase, which passes through a bed ( layer or column) referred to stationary phase. • The sample is attracted to the stationary phase by specific method ( will be mentioned later, God willing) then elute gradually and separately. • The mobile phase with its separated solutes zones emerges from a column (stationary phase) and passes through a detector. • Finally, the separated analytes is detected by detector and analyzed by computer or any thing else. 88
Lecture titles 1_ Definition 2_ uses and application 3_ principle 4_ classification 5_ Calibration 6_ instrumentation 7_ mechanism of separation 8_ types of Chromatography 9_ paper chromatography 10_ thin layer chromatography ( TLC) 11_ Gas chromatography ( GC ) 12_ high performance liquid chromatography (HPLC) 13_ HPLC in hb electrophoresis 14_ Immunochromatography. 89
REFERENCES • _ https://labtestsonline.org • _ https://www.medscape.com • _ https://www.wikipedia.org • _ https ps://www.labcorp.com • _ https://www.uptodate.com • _ https://www.ncbi.nlm.nih.gov Home - PubMed – NCBI • _TIETZ textbook of clinical chemistry and molecular diagnostics, sixth edition 2018. • _Essential of clinical pathology book; 1st edition; Shirish M Kawthalkar; 2010. • _Essential of biochemistry book ;1st edition; 2012. • _ Harper's illustrated biochemistry 30th edition 2015. • _ Lippincott's illustrated review of biochemistry sixth edition 2014. • _ Lecture Notes Clinical Biochemistry, 9th Edition Walker, Simon, 2103. • _Many audios and videos from Well-known, trusted professors who study from accredited books. • _ Clinical chemistry from principles to practice 2nd Edition dr Ola H. Demerdash, second edition 90
‫اليوتيوب‬ ‫علي‬ ‫البحث‬ ‫أو‬ ،‫اليوتيوب‬ ‫علي‬ ‫قناتي‬ ‫دي‬ ‫د‬ ‫باسم‬ . ‫سفيان‬ ‫بهجت‬ ‫محمد‬ ، ‫هللا‬ ‫شاء‬ ‫إن‬ ‫نستمر‬ ‫حتى‬ ‫بالقناة‬ ‫وتشتركوا‬ ‫تتابعوني‬ ‫ياريت‬ https://www.youtube.com/channel/UCaYs1d8s0ntZvteHS3mMmGA ‫ب‬ ‫الفيس‬ ‫في‬ ‫البحث‬ ‫أو‬ ،‫انضمامكم‬ ‫يشرفنا‬ ‫الفيسبوك‬ ‫علي‬ ‫بتاعتي‬ ‫الصفحة‬ ‫دي‬ DMBMS2018 ، ‫هللا‬ ‫شاء‬ ‫إن‬ ‫نستمر‬ ‫حتى‬ ‫تتابعوني‬ ‫ياريت‬ https://www.facebook.com/DMBMS2018/ ‫باسم‬ ‫التلجرام‬ ‫علي‬ ‫قناتي‬ ‫ودي‬ ِ ‫للناس‬ ‫أنفعهم‬ ‫الناس‬ ‫خير‬ 📚 ‫هللا‬ ‫شاء‬ ‫إن‬ ‫نستمر‬ ‫حتى‬ ‫تتابعوني‬ ‫ِريت‬‫ا‬‫ي‬ https://t.me/DMBMS2020 ‫على‬ ‫بروفايلي‬ ‫لينكيدإن‬ ‫وتعملولي‬ ‫تتابعوني‬ ‫ياريت‬ endorsement ‫مهاراتي‬ ‫على‬ https://www.linkedin.com/in/mohammed-bahgat-sofyan-8ba012142/ ‫موقع‬ ‫على‬ ‫بروفايلي‬ ‫وده‬ SlideShare ‫تتابعونا‬ ‫ياريت‬ https://www.slideshare.net/MohammedBahgatMohamm1 ‫وده‬ ‫بروفايلي‬ ‫الشخصي‬ ‫الفيسبوك‬ ‫علي‬ https://www.facebook.com/mohammed.bahgat.165 ‫الفيسبوك‬ ‫على‬ ‫بنا‬ ‫الخاص‬ ‫اللوجوا‬ ‫وده‬ # ‫دمحمد‬ _ ‫بهجت‬ _ ‫سفيان‬ _ medical_biochimestry 91
My logo # ‫دمحمد‬ _ ‫بهجت‬ _ ‫سفيان‬ _ medical_biochimestry 92
THANK YOU Meet on the best later, God willing 93

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Chromatography

  • 1. BY dr: Mohammed Bahgat Mohammed Sofyan Assistant lecturer of medical biochemistry, Faculty of medicine, Al-Azhar university (Assiut branch) 1
  • 2. 2
  • 3. 3
  • 4. ‫صورة‬ ‫كل‬ ‫على‬ ‫اضغط‬ ‫الشاش‬ ‫عرض‬ ‫وضع‬ ‫في‬ ‫ة‬ ‫قنواتنا‬ ‫على‬ ‫وادعمنا‬ ‫اإلنترنت‬ ‫على‬
  • 5. ‫على‬ ‫ملفي‬ ‫في‬ ‫هنا‬ ‫أرفعها‬ ‫التي‬ ‫شرائحي‬ ‫ومذاكرة‬ ‫بالتعلم‬ ‫وأسعد‬ ‫وأرحب‬ ‫أسمح‬ Slide share website ‫وال‬ ‫مانع‬ ‫أيضا‬ ‫من‬ ‫نسخ‬ ‫شريحة‬ ‫او‬ ‫اثنين‬ ‫عند‬ ‫الضرورة‬ ، ‫وال‬ ‫مانع‬ ‫من‬ ‫شرح‬ ‫البوربو‬ ‫ينت‬ ‫الخاصة‬ ‫بي‬ ‫للغير‬ ‫بشرط‬ ‫عدم‬ ‫إزالة‬ ‫اسمي‬ ‫من‬ ‫البوربوينت‬ ، ‫فال‬ ‫أسمح‬ ‫أبدا‬ ‫بإزالة‬ ‫اسمي‬ ‫من‬ ‫على‬ ‫الباوربوينت‬ ‫ووضع‬ ‫اسمك‬ ‫بدال‬ ‫منه‬ ‫لتصبح‬ ‫وكأنك‬ ‫من‬ ‫صممتها‬ ‫فهذه‬ ‫سرقة‬ ‫ال‬ ‫أسمح‬ ‫بها‬ ‫وتضييع‬ ‫لحق‬ ‫من‬ ‫تعب‬ ‫في‬ ‫عملها‬ . ‫وفقكم‬ ‫هللا‬ ‫وإياي‬ ‫للتعلم‬ ‫ونفع‬ ‫اآلخرين‬ I allow, welcome, and be happy to learn and study my slides that I upload here in my profile on Slide share website There is also no objection to copying one or two slides when necessary, and there is no objection to explaining my PowerPoint to others on the condition that my name is not removed from the PowerPoint. I never allow my name to be removed from PowerPoint and to replace it with yours, Make it look like you designed it. This is theft that I do not allow and a waste of the right of those who are tired in this work. May God bless you and me for learning and benefiting others 5
  • 6. DEFINTION It's a powerful analytical tool that is used for the separation, detection and quantitation of a wide variety of analytes. Example of analytes that is separated : Proteins, amino acids, isoenzymes, electrolytes, lipoproteins and hemoglobin variants,..... ‫طريقة‬ ‫تستخدم‬ ‫لفصل‬ ‫المواد‬ ‫عن‬ ‫بعضها‬ ‫سواء‬ ‫كانوا‬ ‫بروتينيات‬ ‫أو‬ ‫أحماض‬ ‫أمينية‬ ‫أو‬ ‫إ‬ ‫لكترونات‬ ‫وغير‬ ‫ذلك‬ ... ‫وبعد‬ ‫فصلهم‬ ‫يمكن‬ ‫التعرف‬ ‫عليهم‬ ‫ومعرفة‬ ‫كميتهم‬ . 6
  • 7. 7
  • 8. Analyze: examine a mixture, its components, and their relations to one another 1 Identify: determine the identity of a mixture or components based on known components 2 Purify: separate components in order to isolate one of interest for further study 3 Quantify: determine the amount of the mixture and/or the components present in the sample 4 8
  • 9. 9
  • 10. 10
  • 11. Uses for Chromatography Pharmaceutical Company: determine amount of each chemical found in new product Hospital: detect blood or alcohol levels in a patient’s blood stream Law Enforcement : to compare a sample found at a crime scene to samples from suspects Environmental Agency : determine the level of pollutants in the water supply Manufacturing Plant: to purify a chemical needed to make a product 11
  • 12. 12
  • 13. In chromatography a sample is introduced into a flowing stream referred to a mobile phase, which passes through a bed ( layer or column) referred to stationary phase. ‫يعني‬ ‫ببساطة‬ ‫فيه‬ ،‫جزئين‬ ‫الجزء‬ ‫األول‬ ‫متحرك‬ ‫ويحتوي‬ ‫علي‬ ‫المادة‬ ‫المراد‬ ،‫فصلها‬ ‫ويمر‬ ‫علي‬ ‫مخدة‬ ( ‫مفلطحة‬ ‫أ‬ ‫و‬ ‫أنبوبية‬ ) ‫هذه‬ ‫المخدة‬ ‫ثابتة‬ ‫وهي‬ ‫الجزء‬ ‫الثاني‬ . ‫ث‬ ،‫الحقا‬ ‫عنها‬ ‫التحدث‬ ‫سيتم‬ ‫ما‬ ‫بطريقة‬ ‫المخدة‬ ‫الثابت‬ ‫للجزء‬ ‫المتحرك‬ ‫الجزء‬ ‫من‬ ‫فصلها‬ ‫المراد‬ ‫المادة‬ ‫تنتقل‬ ‫أجزاء‬ ‫تعود‬ ‫م‬ ‫تسمي‬ ‫بطريقة‬ ‫منفصلة‬ ‫لكن‬ ‫أخري‬ ‫مرة‬ ‫المادة‬ elution 13
  • 14. 14
  • 15. Explanation •Compound is placed on stationary phase •Mobile phase passes through the stationary phase •Mobile phase solubilizes the components •Mobile phase carries the individual components a certain distance through the stationary phase, depending on their attraction to both of the phases 15
  • 16. Classification 1_ According to Mobil phase : • ‫المتحركة‬ ‫المادة‬ ‫طبيعة‬ ‫يعني‬ • A _ Liquid chromatography ( LC ) • ‫سائلة‬ ‫المتحركة‬ ‫المادة‬ ‫لو‬ • B _ Gas chromatography ( GC ) • ‫غازية‬ ‫المتحركة‬ ‫المادة‬ ‫لو‬ 2_ According to chromatographic bed (stationary phase) : • ‫الثابت‬ ‫الجزء‬ ‫حسب‬ ‫علي‬ • A _ Planner chromatography • ‫الثابت‬ ‫الجزء‬ ‫لو‬ ( ‫المخدة‬ ) ‫زجاجية‬ ‫او‬ ‫ورقية‬ ‫كانت‬ ‫سواء‬ ‫مفلطح‬ • B _ Column chromatography • ‫الثابت‬ ‫الجزء‬ ‫لو‬ ( ‫المخدة‬ ) ‫أنبوبي‬ 16
  • 17. 17
  • 18. 18
  • 19. ‫الملخص‬ ‫ببساطة‬ ‫فيه‬ ،‫جزئين‬ ‫الجزء‬ ‫األول‬ ‫متحرك‬ Mobile phase ‫ويحتوي‬ ‫علي‬ ‫المادة‬ ‫المراد‬ ،‫فصلها‬ ‫ويمر‬ ‫علي‬ ‫مخدة‬ ( ‫مفلطحة‬ ‫أو‬ ‫أنبوبية‬ ) ‫هذه‬ ‫المخدة‬ ‫ثابتة‬ ‫هي‬ ‫الجزء‬ ‫الثاني‬ Stationary phase ‫تنتقل‬ ‫المادة‬ ‫المراد‬ ‫فصلها‬ ‫من‬ ‫الجزء‬ ‫المتحرك‬ ‫للجزء‬ ‫الثابت‬ ‫المخدة‬ ‫بطريقة‬ ‫ما‬ ‫سيتم‬ ‫التحدث‬ ‫عنها‬ ،‫الحقا‬ ‫ثم‬ ‫تعود‬ ‫أجزاء‬ ‫ا‬ ‫لمادة‬ ‫مرة‬ ‫أخري‬ ‫لكن‬ ‫منفصلة‬ ‫بطريقة‬ ‫تسمي‬ Elution ‫بعد‬ ‫ذلك‬ The mobile phase with its separated solutes zones emerges from a column (stationary phase) and passes through a detector. ‫بعد‬ ‫ما‬ ‫أجزاء‬ ‫المادة‬ ‫المراد‬ ‫فصلها‬ ‫يحصل‬ ‫لها‬ elution ‫تدريجيا‬ ‫وتخرج‬ ‫من‬ ‫األنبوبة‬ ‫منفصلة‬ ‫تمر‬ ‫علي‬ ‫جهاز‬ detector ‫الذي‬ ‫يحول‬ ‫الجزء‬ ‫المفصول‬ ‫منها‬ ‫الي‬ peak 19
  • 20. ‫الملخص‬ ‫بيعمل‬ ‫الجهاز‬ ‫ألن‬ ،‫معا‬ ‫وليسوا‬ ‫تدريجيا‬ ‫معين‬ ‫وقت‬ ‫في‬ ‫بيخرج‬ ‫مفصول‬ ‫جزء‬ ‫كل‬ ‫طبعا‬ Elution at specific pH and specific ionic strength or according to affinity between mobile and stationary phases ‫ال‬ ‫يسمي‬ ‫معروف‬ ‫زمن‬ ‫في‬ ‫يخرج‬ ‫المفصولة‬ ‫األجزاء‬ ‫من‬ ‫كل‬ ‫ولهذا‬ Retention time ‫ب‬ ‫سيفصل‬ ‫جزء‬ ‫كل‬ ‫يعني‬ Specific pH and specific ionic strength or according to affinity between mobile and stationary phases. ‫يسمي‬ ‫معروف‬ ‫معين‬ ‫محدد‬ ‫زمن‬ ‫في‬ ‫ويخرج‬ Retention time ‫مفصول‬ ‫جزء‬ ‫كل‬ ‫علي‬ ‫نتعرف‬ ‫وبهذا‬ . Separation and Detection 20
  • 21. ‫ال‬ ‫الجهاز‬ detector ‫ويرسم‬ ‫المفصول‬ ‫الجزء‬ ‫بهذا‬ ‫يحس‬ peak ‫كل‬ peak ‫لها‬ Hight, width and peak area ‫ال‬ ‫وضعف‬ ‫قوة‬ ‫تكون‬ ‫المادة‬ ‫هذه‬ ‫تركيز‬ ‫حسب‬ ‫علي‬ peak ‫وباستخدام‬ standard ‫المفصول‬ ‫المادة‬ ‫قيمة‬ ‫معرفة‬ ‫نستطيع‬ ‫القيمة‬ ‫معلوم‬ quantitiation ‫نعمل‬ ‫ممكن‬ Calibration curve. ‫بااستخدام‬ ‫ذكرها‬ ‫االتي‬ ‫المعادلة‬ ‫نطبق‬ ‫وممكن‬ 1 standard ‫الملخص‬ 21
  • 22. 22
  • 23. 23
  • 24. 24
  • 25. Equation Concentration of sample=(signal of sample / signal of standard) X concentration of standard. Concentration of sample = ( peak height (or area) of sample / peak height (or area) of standard) X concentration of standard. 25
  • 26. Factors affecting the rate of separation Characteristics of the mobile and stationary phases. Characteristics of the analytes in the mobile phase. Characteristics of the porous medium. The flow dynamic. 26
  • 27. calibration • Two methods of calibration are used : 1- External calibration, in which solutions of known concentration are prepared and processed in an identical manner as the unknowns. A calibration curve of peak height or peak area versus concentration is constructed, and the concentration of the unknown is obtained by interpolation. 27
  • 28. • internal calibration, in which analyte solutions of known concentration are prepared, and a constant amount of a second compound, the internal standard, is added to each calibrator and sample. In principle, the size of the internal standard peak should be constant in all calibrators and samples. Any change in the internal standard should be the same as a change in the analyte of interest.. By plotting the ratio of the peak height (or area) of the analyte of interest to the peak height (or area) of the internal standard versus concentration of the analyte of interest, a calibration curve that corrects for systematic losses can be constructed. The concentration of the unknown is then obtained from the curve by interpolation. calibration 28
  • 29. • In chromatography a sample is introduced into a flowing stream referred to a mobile phase, which passes through a bed ( layer or column) referred to stationary phase. • The sample is attracted to the stationary phase by specific method ( will be mentioned later, God willing) then elute gradually and separately. • The mobile phase with its separated solutes zones emerges from a column (stationary phase) and passes through a detector. • Finally, the separated analytes is detected by detector and analyzed by computer or any thing else. 29
  • 30. Separation mechanism ‫علي‬ ‫تعدي‬ ‫علشان‬ ‫تاني‬ ‫ترجع‬ ‫وازاي‬ ‫الثابت‬ ‫للجزء‬ ‫المتحرك‬ ‫الجزء‬ ‫من‬ ‫بتنتقل‬ ‫المادة‬ ‫ازاي‬ detector 1_ Adsorption and elution : • ‫واع‬ ‫الساكن‬ ‫الجزء‬ ‫الي‬ ‫المتحرك‬ ‫الجزء‬ ‫من‬ ‫فصلها‬ ‫المراد‬ ‫المادة‬ ‫فصل‬ ‫يتم‬ ‫الخاصيتين‬ ‫هاتين‬ ‫باستخدام‬ ‫بساطة‬ ‫بكل‬ ‫أخري‬ ‫مرة‬ ‫ادتها‬ ‫مفصولين‬ ‫يعودوا‬ ‫تجعلهم‬ ‫بطريقة‬ ‫لكن‬ ‫ال‬ ‫طبعا‬ adsorption ‫ب‬ ‫يكون‬ ‫ممكن‬ • a_ acidic adsorbent ➡ adsorb basic substances • b_ basic adsorbent ➡ adsorb acidi substances ‫حصل‬ adsorption ‫اعمل‬ elution ‫في‬ ‫تمسك‬ ‫انها‬ ‫علي‬ ‫المادة‬ ‫قدرة‬ ‫حسب‬ ‫وعلي‬ adsorbent ‫ماسك‬ ‫فاللي‬ ‫خروجها‬ ‫يتم‬ ‫له‬ ‫يحصل‬ ‫ضعيف‬ elution ‫له‬ ‫يحصل‬ ‫جامد‬ ‫ماسك‬ ‫واللي‬ ،‫اوال‬ elution ‫مؤخرا‬ . The main application of adsorption chromatography is separation of steroids and vitamins. 30
  • 31. 2-Affinity chromatography ‫هنا‬ ‫بقي‬ ‫هرسب‬ ‫المادة‬ ‫المراد‬ ‫فصلها‬ ‫بال‬ affinity ‫يعني‬ ‫شيئين‬ ‫بيحبوا‬ ‫بعض‬ ‫زي‬ Ag Ab, enzyme substrate or hormones receptor. ‫يعني‬ ‫لو‬ ‫عاوز‬ ‫أقيس‬ Ag ‫في‬ ‫الجزء‬ ‫المتحرك‬ ‫احط‬ ‫علي‬ ‫الثابت‬ It's Ab ‫يمسك‬ ‫فيه‬ ‫في‬ ‫ظروف‬ ‫معينة‬ . ‫اعمل‬ ‫ظروف‬ ‫اخري‬ ‫تفكك‬ ‫ال‬ Ag ‫من‬ Ab ‫باني‬ Adjust pH and ionic strength ‫يقوم‬ ‫يحصل‬ elution ‫تدريجيا‬ The main application of adsorption chromatography is separation of glycated Hb, lipoproteins. 31
  • 32. 3_ Ion Exchange chromatography ‫هذه‬ ‫الطريقة‬ ‫بتفصل‬ ‫األحماض‬ ‫األمينية‬ ‫والهيموجلوبين‬ ‫وغيره‬ ... ‫نجعل‬ ‫الوسط‬ ‫حمضي‬ ‫فستصبح‬ ‫األحماض‬ ‫األمينية‬ ‫المراد‬ ‫فصلها‬ ‫موجبة‬ ، ‫ونجعل‬ ‫مثال‬ ‫الصوديوم‬ ‫قليل‬ ( ‫ما‬ ‫دة‬ ‫توجد‬ ‫في‬ ‫الجزء‬ ‫المتحرك‬ ) ‫وبوضع‬ ‫مادة‬ ‫صمغية‬ ‫عليها‬ ‫ايونات‬ ‫سالبة‬ ‫في‬ ‫الجزء‬ ‫الثابت‬ ، ‫فعند‬ ‫مرور‬ ‫األحماض‬ ‫األميني‬ ‫ة‬ ‫الموجية‬ ‫تمسك‬ ‫في‬ ‫األيونات‬ ‫السالبة‬ ‫التي‬ ‫في‬ ‫الجزء‬ ‫الثابت‬ ‫بدال‬ ‫عن‬ ‫الصوديوم‬ ‫ألنه‬ ‫قليل‬ ‫فتزيحه‬ ‫وتمسك‬ ‫باأل‬ ‫يونات‬ ‫السالبه‬ ‫الموجودة‬ ‫علي‬ ‫الجزء‬ ‫الثابت‬ ‫بدال‬ ‫من‬ ‫الصوديوم‬ Ion Exchange ‫ما‬ ‫رأيك‬ ‫لو‬ ‫جعلنا‬ ‫الوسط‬ ‫قلوي‬ ‫بدال‬ ‫من‬ ‫حمضي‬ ‫وزودنا‬ ‫ايونات‬ ‫الصوديوم؟‬ Adjust pH and ionic strength ‫في‬ ‫الوسط‬ ‫القلوي‬ ‫البروتينات‬ ‫ستصبح‬ ‫سالبة‬ ‫وستتنافر‬ ‫مع‬ ‫األيونات‬ ‫السالبه‬ ‫الموجودة‬ ‫علي‬ ‫الجزء‬ ‫ال‬ ،‫ثابت‬ ‫ولو‬ ‫زودنا‬ ‫ايونات‬ ‫الصوديوم‬ ‫ايضا‬ ‫ستحل‬ ‫محل‬ ‫األحماض‬ ‫األمينية‬ ‫وبالتالي‬ ‫يحصل‬ Elution to amino acids gradually The main application of adsorption chromatography is separation of different amino acids hemoglobin variants and isoenzymes. 32
  • 33. 33
  • 34. 34
  • 35. 35
  • 36. Chromatography in which separation is based mainly on differences in the ion-exchange affinities of the sample components. Anions like SO3-or cations like N(CH3)3+ are covalently attached to stationary phase, usually a resin. So there are two types : 1_ Cation-exchange resin 2_ Anion-exchange resins 3_ Ion Exchange chromatography 36
  • 37. 4- Partition chromatography ‫ال‬ ‫أحد‬ ‫نجعل‬ ‫الحالة‬ ‫هذه‬ ‫في‬ Mobile and stationary phases polar and the other non polar ‫الفصل‬ ‫ويتم‬ ‫ف‬ ‫الثابت‬ ‫الجزء‬ ‫كان‬ ‫لو‬ ‫مثال‬ polar if a stationary phase is polar ‫كانت‬ ‫ما‬ ‫كل‬ ‫المادة‬ ‫يبقي‬ nonpolar ‫وال‬ ‫بطيئ‬ ‫مشيها‬ ‫ويبقي‬ ‫أكثر‬ ‫فيها‬ ‫تمسك‬ elution ‫كانت‬ ‫لو‬ ‫بخالف‬ ،‫أبطء‬ polar ‫وال‬ ‫أسرع‬ ‫تمشي‬ elution ‫الفصل‬ ‫يتم‬ ‫وبذلك‬ ،‫أسرع‬ ‫يكون‬ ‫ال‬ ‫حسب‬ polarity ‫صحيح‬ ‫والعكس‬ . 37
  • 38. Partition chromatography Chromatography in which separation is based mainly on differences between the solubility of the sample components in the stationary phase (gas chromatography), or on differences between the solubilities of the components in the mobile and stationary phases (liquid chromatography). 38
  • 39. others 5_ Stearic exclusion chromatography . 6_ Molecular exclusion chromatography. 7_ Gel-permeation chromatography. 39
  • 40. 40
  • 41. 41
  • 42. 42
  • 44. 44
  • 45. 45
  • 46. 46
  • 47. paper chromatography ‫نادر‬ ‫االستخدام‬ ‫في‬ ‫الحياة‬ ‫العملية‬ ، ‫بنجيب‬ Filter paper ‫ونضع‬ ‫المادة‬ ‫المراد‬ ‫فصلها‬ ‫ونغمسها‬ ‫في‬ Chromatographic solvent which is a mixture of water, alcohol and acid or base. ‫فيتحرك‬ ‫كال‬ ‫من‬ ‫ال‬ Solvent and specimens that needed to separate. For example amino acids with large nonpolar side chains ( as leucine and isoleucine) migrate more slowly than short nonpolar side chain (serine and threonine) in non polar stationary phase ‫نحسب‬ ‫المسافة‬ ‫التي‬ ‫تحركتها‬ ‫المواد‬ ‫المفصولة‬ ‫إلى‬ ‫التي‬ ‫تحركها‬ ‫ال‬ Solvent ‫وعن‬ ‫طريق‬ ‫ذلك‬ ‫نعمل‬ Identification to the separated substances Quantification may be performed by cutting out each spot eluting with suitable solvent and performing quantitative colorimetric assay by dring strip and treated with 0.5% ninhydrin 47
  • 48. 2_ Thin layer chromatography (TLC) The same principle as that of paper chromatography, but the stationary phase is made of silica jell or cellulose acetate and liquid with volatile organic solvent is preferred used as mobile phase. • Advantages Cheap Simple The developing can be monitored visually Able to use various chemical as a detector 48
  • 49. 49
  • 50. 50
  • 51. 51
  • 52. 52
  • 53. 53
  • 54. 54
  • 55. 3_ Gas chromatography Gas chromatography is a process by which a mixture of compounds is separated into its constituent components by flowing a gaseous mixture of analytes and mobile phase through a column containing a stationary phase. Mobile phase ➡ inert gas as helium carry substances that want to be separated. Stationary phase ➡ fixed at column, may be solid or liquid 55
  • 56. • ‫تكون‬ ‫فصلها‬ ‫المراد‬ ‫المادة‬ ‫الزم‬ Volatile and polar 56
  • 57. 57
  • 58. 58
  • 59. Instrumentation 1_ carrier gas supply : ‫ال‬ ‫تدفع‬ Mobile phase ‫الهيليوم‬ ‫مثل‬ 2_ sample injector ‫فيه‬ ‫بيبقي‬ ‫بعدها‬،‫فصلها‬ ‫المراد‬ ‫المادة‬ ‫تدفع‬ Vaporisation champer ‫بالتسخين‬ ‫غاز‬ ‫الي‬ ‫المواد‬ ‫تحول‬ 59
  • 60. Instrumentation 3_ column containing stationary phase , Column may be packed or capillary ‫الثابتة‬ ‫المادة‬ ‫يحمل‬ • Stationary phase may be liquid or solid • Stationary phase may be hydrophilic or hydrophobic 4_ detector • ‫معرفة‬ ‫طريق‬ ‫عن‬ ‫المفصولة‬ ‫المادة‬ ‫بيلقط‬ ( ‫وكميتها‬ ،‫مجيئها‬ ‫وقت‬ ) ‫ل‬ ‫ويترجمها‬ peak 5_ computer • ‫ال‬ ‫ويرسم‬ ‫النتائج‬ ‫يحلل‬ peak 60
  • 61. 61
  • 62. Mechanism of sepration • It depends on the relative solubility and volatility of solutes in the two phases and their interaction with the stationary one. • Or It depends on volatility and polarity. • if a stationary phase is polar • ‫كانت‬ ‫ما‬ ‫كل‬ ‫المادة‬ ‫يبقي‬ polar ‫وال‬ ‫بطيء‬ ‫مشيها‬ ‫ويبقي‬ ‫أكثر‬ ‫فيها‬ ‫تمسك‬ elution ‫كانت‬ ‫لو‬ ‫بخالف‬ ،‫أبطء‬ non polar ‫وال‬ ‫أسرع‬ ‫تمشي‬ elution ‫ال‬ ‫حسب‬ ‫الفصل‬ ‫يتم‬ ‫وبذلك‬ ،‫أسرع‬ ‫يكون‬ polarity ‫صحيح‬ ‫والعكس‬ . ‫في‬ ‫األكثر‬ ‫المادة‬ ‫كذلك‬ volatility ‫األسرع‬ ‫هي‬ ‫بال‬ ‫نفصل‬ ‫ممكن‬ Gas chromatography ‫ونعمل‬ Quantitative by mass spectrometer ‫ساعتها‬ ‫الجهاز‬ ‫ويسمى‬ Gas chromatography mass spectrometer GC/MS It's used primarily to for the analytes of drugs in sport and military medicine. • ‫والعسكريين‬ ‫للرياضيين‬ ‫العقاقير‬ ‫وفصل‬ ‫ومعرفة‬ ‫قياس‬ ‫في‬ ‫تستخدم‬ . 62
  • 63. Detector a· Electron capture detector b. Flame ionization detector c· Photoionization detector d· Thermal conductivity detector e· Thermionic selective detector f. Mass spectrometers 63
  • 64. 4_ High Performance Liquid Chromatography (HPLC) HPLC is a process by which a mixture of compounds is separated into it's constituent components by pumping a liquid mobile phase through a column containing a stationary phase. Mobile phase ➡ liquid carry substances that want to be separated. Stationary phase ➡ fixed at column may be solid as silica or liquid ‫علشان‬ ‫نفصل‬ ‫المواد‬ ‫بال‬ HPLC ‫بيتم‬ ‫فصلها‬ ‫عن‬ ‫طريق‬ Polarity 64
  • 65. Instrumentation 1_ solvent reservoir and (pump) : • ‫تدفع‬ ‫ال‬ Mobile phase 2_ sample injector • ‫تدفع‬ ‫المادة‬ ‫المراد‬ ‫فصلها‬ 3 _column containing stationary phase ,Column may be packed or capillary • ‫يحمل‬ ‫المادة‬ ‫الثابتة‬ Stationary phase may be liquid or solid Stationary phase may be hydrophilic silica ➡ normal phase HPLC or hydrophobic silica C 18 ➡ reversed phase HPLC 4_ detector • ‫بيلقط‬ ‫المادة‬ ‫المفصولة‬ ‫عن‬ ‫طريق‬ ‫معرفة‬ ( ‫وقت‬ ،‫مجيئها‬ ‫وكميتها‬ ) ‫ويترجمها‬ ‫ل‬ peak 5_ computer • ‫يحلل‬ ‫النتائج‬ ‫ويرسم‬ ‫ال‬ peak 65
  • 66. 66
  • 67. 67
  • 68. 68
  • 69. Mechanism of separation • It depends on the relative solubility of solutes in the two phases and their interaction with the stationary one. Also, ion exchange . • if a stationary phase is nonpolar • ‫يبقي‬ ‫المادة‬ ‫كل‬ ‫ما‬ ‫كانت‬ polar ‫تمسك‬ ‫فيها‬ ‫أكثر‬ ‫ويبقي‬ ‫مشيها‬ ‫بطيء‬ ‫وال‬ elution ،‫أبطء‬ ‫بخالف‬ ‫لو‬ ‫كانت‬ nonpolar ‫تمشي‬ ‫أسرع‬ ‫وال‬ elution ‫يكون‬ ،‫أسرع‬ ‫وبذلك‬ ‫يتم‬ ‫الفصل‬ ‫حسب‬ ‫ال‬ polarity • ‫والعكس‬ ‫صحيح‬ . • HPLC may be used for the separation, detection and quantitation of a wide variety of analytes. • Example of analytes that is separated : • Proteins, amino acids, electrolytes and hemoglobin variants. 69
  • 70. 70
  • 71. 71
  • 72. High Performance Liquid Chromatography (HPLC) for separation of Hb variants ‫ايه‬ ‫فكرة‬ ‫عمل‬ ‫فصل‬ ‫أنواع‬ ‫الهيموجلوبين‬ ‫بجهاز‬ ‫ال‬ HPLC ?? ‫نضع‬ ‫عينة‬ ‫الدم‬ ‫في‬ ‫الجهاز‬ ‫مع‬ ‫ال‬ Mobile phase ‫إيه‬ ‫اللي‬ ‫هيحصل؟‬ ‫ال‬ column that contain stationary phase ‫يمتز‬ ‫الهيموجلوبين‬ ‫ألن‬ ‫الهيموجلوبين‬ positive ‫وال‬ Stationary phase contains negative charge ‫كده‬ ‫الهيموجلوبين‬ ‫ذو‬ ‫الشحنة‬ ‫الموجبة‬ ‫مسك‬ ‫في‬ ‫ال‬ column ‫ذو‬ ‫الشحنة‬ ‫السالبة‬ 72
  • 73. ‫بعد‬ ‫ذلك‬ ‫نزيد‬ ‫من‬ ‫مرور‬ Buffer that contain cations in mobile phase and change pH ‫تقوم‬ ‫ال‬ cations ‫تتنافس‬ ‫مع‬ ‫الشحنات‬ ‫الموجبة‬ ‫الموجودة‬ ‫على‬ ‫الهيموجلوبين‬ ‫على‬ ‫الشحنات‬ ‫السالبة‬ ‫الموجو‬ ‫دة‬ ‫على‬ ‫ال‬ Stationary phase. The cations in the mobile phase compete with the adsorbed proteins ( Hb ) for the anionic binding sites present in the stationary phase. ‫يقوم‬ ‫ال‬ cations ‫تحل‬ ‫الهيموجلوبين‬ ‫على‬ ‫الجزء‬ ‫الثابت‬ ‫ويحصل‬ gradual elution to Hb variants ‫علي‬ ‫حسب‬ Their affinity for the stationary phase ( polarity ) ‫طبعا‬ ‫كل‬ ‫ما‬ ‫زادت‬ ‫الشحنة‬ ‫الموجبة‬ ‫في‬ ‫نوع‬ ‫معين‬ ‫من‬ ‫الهيموجلوبين‬ ‫كل‬ ‫ما‬ ‫مسك‬ ‫أكثر‬ ‫بالجزء‬ ،‫الثابت‬ ‫وبالت‬ ‫الي‬ ‫يحصل‬ ‫له‬ elution ‫أبطأ‬ ‫وبالتالي‬ ‫يصل‬ ‫لل‬ detector ‫متأخرا‬ ‫وبالتالي‬ High retention time. ‫وكل‬ ‫ما‬ ‫زادت‬ ‫الشحنة‬ ‫السالبة‬ ‫في‬ ‫نوع‬ ‫معين‬ ‫من‬ ‫الهيموجلوبين‬ ‫كل‬ ‫ما‬ ‫حصل‬ ‫له‬ elution ‫أسرع‬ ‫وبالتالي‬ ‫يصل‬ ‫لل‬ detector ‫أسرع‬ ‫وبالتالي‬ Low retention time 73
  • 74. ‫طب‬ ‫الجهاز‬ ‫عارف‬ ‫الوقت‬ ‫اللي‬ ‫هيوصل‬ ‫عنده‬ ‫كل‬ ‫نوع‬ ‫من‬ ‫أنواع‬ ‫الهيموجلوبين‬ ‫والذي‬ ‫يس‬ ‫مي‬ ‫ال‬ retention time ‫كل‬ ‫نوع‬ ‫من‬ ‫الهيموجلوبين‬ ‫له‬ Specific retention time ‫محدد‬ ‫الجهاز‬ ‫عارفه‬ ‫وبالتالي‬ ‫الجهاز‬ ‫يعرف‬ ‫ما‬ ‫هو‬ ‫البروتين‬ ‫أو‬ ‫الهيموجلوبين‬ ‫اللي‬ ‫وص‬ ‫ل‬ ‫في‬ ‫هذا‬ ‫الوقت‬ ‫وتركيزه‬ ‫يساوي‬ ‫كم؟‬ . ‫كل‬ ‫ده‬ ‫واضح‬ ‫على‬ ‫الورقة‬ ‫بعد‬ ‫كده‬ ‫يسجل‬ ‫الكالم‬ ‫ده‬ ‫في‬ curves ‫عن‬ ‫طريق‬ ‫جهاز‬ ‫ال‬ densometer ‫أعلم‬ ‫وهللا‬ . 74
  • 75. is a process in which a mixture of molecules (such as normal and hemoglobins variant) with a net positive charge is separated into its components by their adsorption onto a negatively charged stationary phase in a chromatography column, followed by their elution by a mobile phase. The mobile phase is a liquid with an increasing concentration of cations flowing through the column; the cations in the mobile phase compete with the adsorbed proteins for the anionic binding sites. 75
  • 76. Thus, the adsorbed positively charged hemoglobin molecules are eluted from the column into the liquid phase at a rate related to their affinity for the stationary phase. When separated in this way, they can be detected optically in the eluate, provisionally identified by their retention time, and quantified by computing the area under the corresponding peak in the elution profile. The more positively charged hemoglobins (e.g. hemoglobin S and C) have a longer retention time. 76
  • 77. Variant with weak positive charge will adsorb weakly to the column and is eluted rapidly from the column with the injection of low strength elution buffer. Variant with strong positive charge will adsorb strongly to the column and is eluted slowly from the column with the injection of high strength elution buffer. Procedure: HPLC is fully automated. Samples are loaded in a rack and automatically processed one by one (about 6 minutes/sample). Result: Data are graphically represented as absorbance vs time (peaks). Retention time is the time from injection to the top of the peak. 77
  • 78. ‫له‬ ‫الهيموجلوبين‬ ‫من‬ ‫نوع‬ ‫كل‬ Specific Retention time ‫ال‬ ‫يطلع‬ ‫ولما‬ ،‫الحالة‬ ‫للجهاز‬ ‫تدخل‬ ‫فإنت‬ ،‫عارفه‬ ‫الجهاز‬ Its Retention time ‫ال‬ ‫بمعلومية‬ ‫فورا‬ ‫الهيموجلوبين‬ ‫نوع‬ ‫يقولك‬ ‫يقوم‬ Its retention time. ‫ال‬ ‫نفس‬ ‫لها‬ ‫اللي‬ ‫األنواع‬ ‫من‬ ‫بالك‬ ‫وخلي‬ retention time ‫ال‬ ‫ومشاكل‬ Glycated Hb and HbS with HbA2..... etc. ‫العيوب‬ ‫في‬ ‫الحقا‬ ‫ستذكر‬ ‫كما‬ . 78
  • 79. 79
  • 80. 80
  • 81. 81
  • 82. Immunochromatography is a combination of chromatography and immunoassay. This technology Is in use for a wide array of tests for clinical, veterinary, and industrial applications. 82
  • 83. 1_ The specimen (e.g., serum, urine) containing the antigen to be detected is placed on the sample pad, which soaks up the specimen fluid. 2_ The fluid then migrates to the conjugate pad, which contains conjugated antibodies (conjugated with gold, colored latex, or a chromophore) directed against the antigen. 83
  • 84. 3_ the antigen-antibody- conjugate complex is formed. Ag-Ab complex continues to migrate across the membrane until it reaches the capture zone where the complex will bind to immobilized antibodies. As more and more Ag-Ab complexes are captured at the “test” line, the line becomes visible on the membrane. 4 _ The sample then migrates further along the strip until it reaches the control zone where excess conjugate binds and produces a second visible line (control line) on the membrane. This control line indicates that the sample has migrated across the membrane as intended. 84
  • 85. Immunochromatographic methods are widely used in clinical practice for : 1_Detection of toxins and drugs 2_ Pregnancy tests- detection of human chorionic gonadotropin (hCG) 3_ Diagnosis of parasitic infections as : a_ Malaria b_ G. lamblia and Cryptosporidium parvum C. E. histolytica 85
  • 86. 3_ Diagnosis of bacterial infections as :  Mycoplasma pneumoniae  Mycoplasma pneumoniae  H.pylori antigens in stool  V. cholerae O1 and O139 from stool specimens  Streptococcus pneumoniae  antigen detection in CSF or in urine 86
  • 87. 4_ Diagnosis of viral Infections , Antigen detection of :  RSV  Rotavirus  Influenza A/B  Hepatitis B and Hepatitis C 5_ Antibodies detection of : Detection of HIV-1 and HIV-2 antibodies. 87
  • 88. • In chromatography a sample is introduced into a flowing stream referred to a mobile phase, which passes through a bed ( layer or column) referred to stationary phase. • The sample is attracted to the stationary phase by specific method ( will be mentioned later, God willing) then elute gradually and separately. • The mobile phase with its separated solutes zones emerges from a column (stationary phase) and passes through a detector. • Finally, the separated analytes is detected by detector and analyzed by computer or any thing else. 88
  • 89. Lecture titles 1_ Definition 2_ uses and application 3_ principle 4_ classification 5_ Calibration 6_ instrumentation 7_ mechanism of separation 8_ types of Chromatography 9_ paper chromatography 10_ thin layer chromatography ( TLC) 11_ Gas chromatography ( GC ) 12_ high performance liquid chromatography (HPLC) 13_ HPLC in hb electrophoresis 14_ Immunochromatography. 89
  • 90. REFERENCES • _ https://labtestsonline.org • _ https://www.medscape.com • _ https://www.wikipedia.org • _ https ps://www.labcorp.com • _ https://www.uptodate.com • _ https://www.ncbi.nlm.nih.gov Home - PubMed – NCBI • _TIETZ textbook of clinical chemistry and molecular diagnostics, sixth edition 2018. • _Essential of clinical pathology book; 1st edition; Shirish M Kawthalkar; 2010. • _Essential of biochemistry book ;1st edition; 2012. • _ Harper's illustrated biochemistry 30th edition 2015. • _ Lippincott's illustrated review of biochemistry sixth edition 2014. • _ Lecture Notes Clinical Biochemistry, 9th Edition Walker, Simon, 2103. • _Many audios and videos from Well-known, trusted professors who study from accredited books. • _ Clinical chemistry from principles to practice 2nd Edition dr Ola H. Demerdash, second edition 90
  • 91. ‫اليوتيوب‬ ‫علي‬ ‫البحث‬ ‫أو‬ ،‫اليوتيوب‬ ‫علي‬ ‫قناتي‬ ‫دي‬ ‫د‬ ‫باسم‬ . ‫سفيان‬ ‫بهجت‬ ‫محمد‬ ، ‫هللا‬ ‫شاء‬ ‫إن‬ ‫نستمر‬ ‫حتى‬ ‫بالقناة‬ ‫وتشتركوا‬ ‫تتابعوني‬ ‫ياريت‬ https://www.youtube.com/channel/UCaYs1d8s0ntZvteHS3mMmGA ‫ب‬ ‫الفيس‬ ‫في‬ ‫البحث‬ ‫أو‬ ،‫انضمامكم‬ ‫يشرفنا‬ ‫الفيسبوك‬ ‫علي‬ ‫بتاعتي‬ ‫الصفحة‬ ‫دي‬ DMBMS2018 ، ‫هللا‬ ‫شاء‬ ‫إن‬ ‫نستمر‬ ‫حتى‬ ‫تتابعوني‬ ‫ياريت‬ https://www.facebook.com/DMBMS2018/ ‫باسم‬ ‫التلجرام‬ ‫علي‬ ‫قناتي‬ ‫ودي‬ ِ ‫للناس‬ ‫أنفعهم‬ ‫الناس‬ ‫خير‬ 📚 ‫هللا‬ ‫شاء‬ ‫إن‬ ‫نستمر‬ ‫حتى‬ ‫تتابعوني‬ ‫ِريت‬‫ا‬‫ي‬ https://t.me/DMBMS2020 ‫على‬ ‫بروفايلي‬ ‫لينكيدإن‬ ‫وتعملولي‬ ‫تتابعوني‬ ‫ياريت‬ endorsement ‫مهاراتي‬ ‫على‬ https://www.linkedin.com/in/mohammed-bahgat-sofyan-8ba012142/ ‫موقع‬ ‫على‬ ‫بروفايلي‬ ‫وده‬ SlideShare ‫تتابعونا‬ ‫ياريت‬ https://www.slideshare.net/MohammedBahgatMohamm1 ‫وده‬ ‫بروفايلي‬ ‫الشخصي‬ ‫الفيسبوك‬ ‫علي‬ https://www.facebook.com/mohammed.bahgat.165 ‫الفيسبوك‬ ‫على‬ ‫بنا‬ ‫الخاص‬ ‫اللوجوا‬ ‫وده‬ # ‫دمحمد‬ _ ‫بهجت‬ _ ‫سفيان‬ _ medical_biochimestry 91
  • 93. THANK YOU Meet on the best later, God willing 93