Ion Exchange Chromatography PDF

IEC : is a separation technique used for purification or analysis of molecules based their charge..‫ب‬ٙ‫ شسٕز‬ٍٝ‫ئبد ثٕب ًء ػ‬٠‫ً اٌدض‬١ٍ‫ رس‬ٚ‫خ أ‬١‫خ فصً رغزخذَ ٌزٕم‬١ٕ‫ رم‬ٟ٘ : IEC the method can be used to separate charged molecules from uncharged ones or it can separate molecules of different...

IEC : is a separation technique used for purification or analysis of molecules based their charge..‫ب‬ٙ‫ شسٕز‬ٍٝ‫ئبد ثٕب ًء ػ‬٠‫ً اٌدض‬١ٍ‫ رس‬ٚ‫خ أ‬١‫خ فصً رغزخذَ ٌزٕم‬١ٕ‫ رم‬ٟ٘ : IEC the method can be used to separate charged molecules from uncharged ones or it can separate molecules of different charge from one another. ً‫ّىٓ فص‬٠ ٚ‫ٔخ أ‬ٛ‫ش اٌّشس‬١‫ئبد غ‬٠‫ٔخ ػٓ اٌدض‬ٛ‫ئبد اٌّشس‬٠‫مخ ٌفصً اٌدض‬٠‫ّىٓ اعزخذاَ ٘زٖ اٌطش‬٠.‫ب اٌجؼض‬ٙ‫ئبد راد اٌشسٕبد اٌّخزٍفخ ػٓ ثؼض‬٠‫اٌدض‬ ionizable chemical groups are immobilized on a solid support such as cellulose or agarose..‫ص‬ٚ‫ االغبس‬ٚ‫ص أ‬ٍٛ١ٍ‫ دػبِخ صٍجخ ِثً اٌغ‬ٍٝ‫ٕخ ػ‬٠‫خ اٌّزأ‬١‫بئ‬١ّ١‫ػبد اٌى‬ّٛ‫ذ اٌّد‬١‫زُ رثج‬٠ The support, or resin, is usually maintained in a column. Molecules of opposite charge can bind the column by electrostatic interaction while uncharged residues will pass through. ‫د‬ّٛ‫ئبد راد اٌشسٕخ اٌّؼبوغخ سثظ اٌؼ‬٠‫ّىٓ ٌٍدض‬٠.‫د‬ّٛ‫ ػ‬ٟ‫ ف‬،‫ اٌشارٕح‬ٚ‫ أ‬،ُ‫ اٌذػ‬ٍٝ‫زُ اٌسفبظ ػ‬٠ ‫ػبدح ِب‬.‫ٔخ‬ٛ‫ش اٌّشس‬١‫ب غ‬٠‫ّٕب رّش اٌجمب‬١‫ ث‬ٟ‫ى‬١‫عزبر‬ٚ‫ش‬ٙ‫ك اٌزفبػً اٌى‬٠‫ػٓ طش‬ Once bound to the column, molecules can be released with salt (NaCl is commonly used, but other salts can be used also). The salt ions compete for interaction for the column, and the molecule of interest is released. Hence the term "ion exchange" ٓ‫ٌى‬ٚ ،‫َ ثشىً شبئغ‬ٛ٠‫د‬ٛ‫ذ اٌص‬٠‫س‬ٍٛ‫ُغزخذَ و‬٠( ‫ئبد ِغ اٌٍّر‬٠‫ّىٓ إطالق اٌدض‬٠ ،‫د‬ّٛ‫ب ثبٌؼ‬ٙ‫ثّدشد سثط‬ ‫ء‬ٞ‫ش اٌدض‬٠‫زُ رسش‬٠ٚ ،‫د‬ّٛ‫ اٌؼ‬ٟ‫ اٌزفبػً ف‬ٍٝ‫ٔبد اٌٍّر ػ‬ٛ٠‫ رزٕبفظ أ‬.)‫ضًب‬٠‫ أ‬ٜ‫ّىٓ اعزخذاَ أِالذ أخش‬٠ "ٟٔٛ٠‫ِٓ ٕ٘ب خبء ِصطٍر "اٌزجبدي األ‬ٚ.َ‫ِسً اال٘زّب‬ lon-exchange chromatography preserves analyte molecules on the column based on ionic interactions. ‫خ‬١ٔٛ٠‫ اٌزفبػالد األ‬ٍٝ‫د ثٕب ًء ػ‬ّٛ‫ اٌؼ‬ٟ‫ً ف‬١ٍ‫ئبد اٌزس‬٠‫ خض‬ٍٝ‫ ػ‬ٌٍٟٔٛ‫ب اٌزجبدي ا‬١‫غشاف‬ٛ‫ِبر‬ٚ‫سبفع وش‬٠ Mobile phage - buffer, pH and salt concentration---opposite charged solute ions attracted to the stationary phage by electrostatic force. ‫ٔخ‬ٛ‫ٔبد اٌّزاثخ اٌّشس‬ٛ٠‫ األ‬--- ‫اٌٍّر‬ٚ ‫ضخ‬ّٛ‫دسخخ اٌس‬ٚ ‫ض اٌّخضْ اٌّؤلذ‬١‫ رشو‬- ‫بد اٌّزٕمٍخ‬١‫اٌؼبث‬. ‫خ‬١‫ى‬١‫عزبر‬ٚ‫ش‬ٙ‫ح اٌى‬ٛ‫اعطخ اٌم‬ٛ‫بد اٌثبثزخ ث‬١‫ اٌؼبث‬ٌٝ‫ رٕدزة إ‬ٟ‫اٌّؼبوغخ اٌز‬ Stationary phage - resin is used to covalently attach anions or cations onto it phase.. ‫س‬ٛ‫ اٌط‬ٟ‫ ف‬ّٟ٘‫ٔبد ثشىً رغب‬ٛ١‫ اٌىبر‬ٚ‫ٔبد أ‬ٛ١ٔ‫غزخذَ اٌشارٕح ٌشثظ األ‬٠ Types of IEC The name of the resin refers to the molecules being exchanged, not the molecule bound to the resin..‫ء اٌّشرجظ ثبٌشارٕح‬ٞ‫ظ اٌدض‬١ٌٚ ،‫ب‬ٌٙ‫زُ رجبد‬٠ ٟ‫ئبد اٌز‬٠‫ اٌدض‬ٌٝ‫ٕح إ‬١‫ش اعُ اٌشار‬١‫ش‬٠ anion exchangers ‫خ‬١ٔٛ١ٔ‫ِجبدالد أ‬ cation exchangers ْٛ١‫ِجبدالد اٌىبر‬ Cation exchange chromatography : positively charged molecules are attracted to a negatively charged solid support. Commonly used cation exchange resins are S-resin, sulfate derivatives; and CM resins, carboxylate derived ions. :ٟٔٛ١‫ب اٌزجبدي اٌىبر‬١‫غشاف‬ٛ‫ِبر‬ٚ‫وش‬ ‫ شبئؼخ‬ٟٔٛ١‫ سارٕدبد اٌزجبدي اٌىبر‬.‫ٔخ عبٌجًب‬ٛ‫ ِبدح داػّخ صٍجخ ِشس‬ٌٝ‫خجخ اٌشسٕخ إ‬ٌّٛ‫ئبد ا‬٠‫رٕدزة اٌدض‬.ً١‫وغ‬ٛ‫ٔبد اٌّشزمخ ِٓ اٌىشث‬ٛ٠‫األ‬ٚ ،CM ‫سارٕدبد‬ٚ ‫زبد؛‬٠‫ِشزمبد اٌىجش‬ٚ ،S ‫ سارٕح‬ٟ٘ َ‫االعزخذا‬ Anion exchange chromatography negatively charged molecules is attracted to a positively charged solid support. Commonly used anion exchange resins are Q-resin, a Quaternary amine; and D.AE resin, DiEthylAminoEthane. ‫ شبئؼخ‬ٟٔٛ١ٔ‫ سارٕدبد اٌزجبدي األ‬.‫خجخ اٌشسٕخ‬ِٛ ‫ ِبدح داػّخ صٍجخ‬ٌٝ‫ئبد عبٌجخ اٌشسٕخ إ‬٠‫رٕدزة اٌدض‬.ْ‫ثب‬٠‫ إ‬ٕٛ١ِ‫ً أ‬١‫ث‬٠‫ إ‬ٟ‫ ثٕبئ‬،D.AE ‫سارٕح‬ٚ ‫؛‬ٟ‫ٓ سثبػ‬١ِ‫ أ‬ٛ٘ٚ ،Q ‫ سارٕح‬ٟ٘ َ‫االعزخذا‬  Proteins will bind to an ion exchanger with different affinities..‫ اسرجبطبد ِخزٍفخ‬ٞ‫ ر‬ٟٔٛ٠‫ٕبد ثّجبدي أ‬١‫ر‬ٚ‫ف رشرجظ اٌجش‬ٛ‫ع‬  As the column is washed with buffer, those proteins relatively low affinities for the ion exchange resin will move through the column faster than the proteins that bind to the column. ‫ًب‬١‫ٕبد راد االسرجبطبد إٌّخفضخ ٔغج‬١‫ر‬ٚ‫ فئْ رٍه اٌجش‬،ُ‫ي إٌّظ‬ٍٛ‫د ثبٌّس‬ّٛ‫زُ غغً اٌؼ‬٠ ‫ػٕذِب‬.‫د‬ّٛ‫ رشرجظ ثبٌؼ‬ٟ‫ٕبد اٌز‬١‫ر‬ٚ‫د ثشىً أعشع ِٓ اٌجش‬ّٛ‫ف رزسشن ػجش اٌؼ‬ٛ‫ ع‬ٟٔٛ٠‫ٌشارٕح اٌزجبدي األ‬  The greater the binding affinity of a protein for the ion exchange column, the more it will be slowed in eluting off the column..‫د‬ّٛ‫خ خبسج اٌؼ‬١‫ اٌزصف‬ٟ‫ وٍّب صاد رجبطؤٖ ف‬،ٟٔٛ٠‫د اٌزجبدي األ‬ّٛ‫ٓ ثؼ‬١‫ر‬ٚ‫وٍّب صاد اسرجبط اٌجش‬  Proteins can be eluted by changing the elution buffer to one with a higher salt concentration and/or a different pH (stepwise elution or gradient elution). ‫ض ٍِر‬١‫ رشو‬ٍٝ‫ ػ‬ٞٛ‫سز‬٠ ‫ي‬ٍٛ‫ ِس‬ٌٝ‫ي اٌشطف إٌّظُ إ‬ٍٛ‫ش ِس‬١١‫ك رغ‬٠‫ٕبد ػٓ طش‬١‫ر‬ٚ‫ّىٓ شطف اٌجش‬٠.)‫ شطف ِزذسج‬ٚ‫ضخ ِخزٍفخ (شطف ِزذسج أ‬ّٛ‫ دسخخ ز‬ٚ‫أ‬/ٚ ٍٝ‫أػ‬  Cation exchangers bind to proteins with positive charges..‫خجخ‬ٌّٛ‫ٕبد راد اٌشسٕبد ا‬١‫ر‬ٚ‫ْ ثبٌجش‬ٛ١‫رشرجظ ِجبدالد اٌىبر‬  Anion exchangers bind to proteins with negative charges ‫ٕبد راد اٌشسٕبد اٌغبٌجخ‬١‫ر‬ٚ‫خ ثبٌجش‬١ٔٛ١ٔ‫رشرجظ اٌّجبدالد األ‬  In this experiment, you will separate adenosine 5'-monophosphate (AMP) and adenosine 5'-triphosphate (ATP). These compounds will be separated by chromatography on diethylaminoethyl (DEAE) cellulose. The mixture of compounds will be loaded onto the column, and eluted with a NH4C|/NH3 gradient. -'5 ٓ١‫ع‬ٕٛ٠‫األد‬ٚ )AMP( ‫عفبد‬ٛ‫ اٌف‬ٞ‫أزبد‬-'5 ٓ٠‫ص‬ٕٛ٠‫َ ثفصً األد‬ٛ‫ف رم‬ٛ‫ ع‬،‫ ٘زٖ اٌزدشثخ‬ٟ‫ف‬ ٟ‫ص ثٕبئ‬ٍٛ١ٍ‫ اٌغ‬ٍٝ‫ ػ‬ٌٍٟٔٛ‫ك اٌفصً ا‬٠‫زُ فصً ٘زٖ اٌّشوجبد ػٓ طش‬١‫ ع‬.)ATP( ‫عفبد‬ٛ‫ اٌف‬ٟ‫ثالث‬ ‫شطفٗ ثزذسج‬ٚ ،‫د‬ّٛ‫ اٌؼ‬ٍٝ‫ظ اٌّشوجبد ػ‬١ٍ‫ً خ‬١ّ‫زُ رس‬١‫ ع‬.)DEAE( ً١‫ث‬٠‫ إ‬ٕٛ١ِ‫ً أ‬١‫ث‬٠‫إ‬.NH4C|/NH3 Reagents and Materials: Compound mixture: AMP and ATP , Buffer: 0.25 M NH4CI/NH3, pH 9.0. ،M NH4CI/NH3 0.25 :‫ اٌّخضْ اٌّؤلذ‬،ATPٚ AMP :‫ظ ِشوت‬١ٍ‫ خ‬:‫اد‬ٌّٛ‫ا‬ٚ ‫اشف‬ٛ‫اٌى‬.9.0 ٟٕ١‫خ‬ٚ‫ذس‬١ٌٙ‫اٌشلُ ا‬ Experimental Procedure: 1. Prepare a column of DEAE-cellulose by placing a filter paper at the bottom of a column to serve as a plug. Add a slurry of DEAE-cellulose equilibrated in 0.05M buffer. The final height of DEAE-cellulose in the column should be between 7 to 8 cm. ‫د‬ّٛ‫ أعفً اٌؼ‬ٟ‫ر ف‬١‫سلخ رشش‬ٚ ‫ضغ‬ٚ ‫ك‬٠‫ص ػٓ طش‬ٍٛ١ٍ‫ اٌغ‬DEAE ‫د ِٓ ِبدح‬ّٛ‫ش ػ‬١‫لُ ثزسض‬.M0.05 ‫ اٌّخضْ اٌّؤلذ‬ٟ‫ب ف‬ٙ‫شر‬٠‫ص ِؼب‬ٍٛ١ٍ‫ اٌغ‬DEAE ِٓ ٓ١‫ إضبفخ اٌط‬.‫ْ ثّثبثخ عذادح‬ٛ‫ٌزى‬.ُ‫ ع‬8 ٌٝ‫ إ‬7 ٓ١‫د ث‬ّٛ‫ اٌؼ‬ٟ‫ ف‬DEAE ‫ص‬ٍٛ١ٍ‫ ٌٍغ‬ٟ‫بئ‬ٌٕٙ‫ْ االسرفبع ا‬ٛ‫ى‬٠ ْ‫دت أ‬٠ 2. Prepare 10ml solutions of luting buffer from the stock 0.25M solution. The concentrations should range from 0.05M to 0.25M in increments of 0.05M. َ 0.05 ِٓ ‫ضاد‬١‫ذ اٌزشو‬ٚ‫دت أْ رزشا‬٠.ْٚ‫ ًِ ِٓ اٌّخض‬0.25 ‫ي‬ٍٛ‫ً ِٓ ِس‬١ٌ‫ ًِ ِٓ اٌّسب‬10 ‫ش‬١‫لُ ثزسض‬.َ 0.05 ‫بداد لذس٘ب‬٠‫ َ ثض‬0.25 ٌٝ‫إ‬ 3. Drain the column to just above the top of the resin (do not let resin go dry). Add 1 ml of the compound mixture and allow it to run into the column. ِٓ ًِ 1 ‫ أضف‬.)‫دف‬٠ ‫ٕح‬١‫ٕح ِجبششحً (ال رذع اٌشار‬١‫ ِٓ اٌشار‬ٍٞٛ‫ اٌدضء اٌؼ‬ٍٝ‫ أػ‬ٌٝ‫د إ‬ّٛ‫ف اٌؼ‬٠‫لُ ثزصش‬.‫د‬ّٛ‫ اٌؼ‬ٌٝ‫زذفك إ‬٠ ٗ‫ارشو‬ٚ ‫ظ اٌّشوت‬١ٍ‫اٌخ‬ 4. Cap the column, and start collecting effluent..‫بد اٌغبئٍخ‬٠‫ خّغ إٌفب‬ٟ‫اثذأ ف‬ٚ ،‫د‬ّٛ‫خ اٌؼ‬١‫لُ ثزغط‬  5. Have a test-tube rack ready with 20 numbered test-tubes. Collect ~3 ml of effluent in each tube. ٟ‫بد اٌغبئٍخ ف‬٠‫ ًِ ِٓ إٌفب‬3 ~ ‫ خّغ‬.‫ة اخزجبس ِشل ًّب‬ٛ‫ أٔج‬20 ‫دًا ثـ‬ٚ‫ت اخزجبس ِض‬١‫ض زبًِ أٔبث‬ٙ‫خ‬.‫ة‬ٛ‫وً أٔج‬ 6. Measure the absorbance of each tube at 260 nm..‫ِزش‬ٛٔ‫ ٔب‬260 ‫ة ػٕذ‬ٛ‫خ ٌىً أٔج‬١‫بط االِزصبص‬١‫لُ ثم‬ Data Analysis: 1. Tabulate absorbance of column eluent at 280 nm vs fraction number..‫ِزش ِمبثً سلُ اٌىغش‬ٛٔ‫ ٔب‬280 ‫د ػٕذ‬ّٛ‫خ ٌشبطف اٌؼ‬١‫ي االِزصبص‬ٚ‫خذ‬ 2. Graph your data, plotting absorbance vs fraction number..‫خ ِمبثً سلُ اٌىغش‬١‫ ِغ سعُ االِزصبص‬،‫ًب‬١ٔ‫ب‬١‫بٔبره ث‬١‫لُ ثشعُ ث‬ 3. Draw the structural formulae of the predominant chemical species for the compounds separated in this experiment. lonic charges in the species must be clearly labeled. ‫ف‬١ٕ‫زُ رص‬٠ ْ‫دت أ‬٠.‫ ٘زٖ اٌزدشثخ‬ٟ‫خ اٌغبئذح ٌٍّشوجبد إٌّفصٍخ ف‬١‫بئ‬١ّ١‫اع اٌى‬ٛٔ‫خ ٌأل‬١‫غ اٌجٕبئ‬١‫اسعُ اٌص‬.‫اضر‬ٚ ً‫اع ثشى‬ٛٔ‫ األ‬ٟ‫اٌشسٕبد اٌّضػدخ ف‬ Advantages It is a non-denaturing technique. It can be used at all stages and scales of purification ‫خ‬١‫ظ اٌزٕم‬١٠‫ِمب‬ٚ ً‫غ ِشاز‬١ّ‫ خ‬ٟ‫ّىٓ اعزخذاِٗ ف‬٠ٚ.‫ؼخ‬١‫ش طج‬١١‫ش رغ‬١‫خ غ‬١ٕ‫ب رم‬ٙٔ‫إ‬ An EX separation can be controlled by changing pH, salt concentration and/or the ion exchange media ٟٔٛ٠‫عبئظ اٌزجبدي األ‬ٚ ٚ‫أ‬/ٚ ‫ض اٌٍّر‬١‫رشو‬ٚ ٟٕ١‫خ‬ٚ‫ذس‬١ٌٙ‫ش اٌشلُ ا‬١١‫ك رغ‬٠‫ ػٓ طش‬EX ً‫ فص‬ٟ‫ّىٓ اٌزسىُ ف‬٠ It can serve as a concentrating step. A large volume of dilute sample can be applied to a media, and the adsorbed protein subsequently luted in a smaller volume. ‫زُ دِح‬٠ ُ‫ ث‬،‫عبئظ‬ٌٛ‫ ا‬ٍٝ‫ٕخ اٌّخففخ ػ‬١‫شح ِٓ اٌؼ‬١‫خ وج‬١ّ‫ك و‬١‫ّىٓ رطج‬٠.‫ض‬١‫ح اٌزشو‬ٛ‫ْ ثّثبثخ خط‬ٛ‫ّىٓ أْ رى‬٠.‫ زدُ أصغش‬ٟ‫ٓ اٌّّزض الزمًب ف‬١‫ر‬ٚ‫اٌجش‬ It offers high selectivity; it can resolve molecules with small differences in charge..‫ اٌشسٕخ‬ٟ‫شح ف‬١‫ئبد راد االخزالفبد اٌصغ‬٠‫ً اٌدض‬١ٍ‫ّىٕٗ رس‬٠.‫خ‬١ٌ‫خ ػب‬١‫فش أزمبئ‬ٛ٠ Disadvantages costly equipment and more expensive chemicals ‫خ أوثش رىٍفخ‬١‫بئ‬١ّ١‫اد و‬ِٛٚ ّٓ‫ِؼذاد ثب٘ظخ اٌث‬ - turbidity should be below 10P M..P M10 ِٓ ً‫ْ ٔغجخ اٌزؼىش أل‬ٛ‫دت أْ رى‬٠