Lecture 3 - Normal Phase Reverse Phase PDF

09/10/2023 Normal & Reverse Phase Chromatography vevox.app 126-545-180 Nikola P. Chmel L3 CH3F2 1 Outline Lecture 1 What is Analytical Science Errors and units What is chromatography? Terminology Classification History Theoretical plate model Rate theory Kinetic theory Normal phase chromatography (c...

09/10/2023 Normal & Reverse Phase Chromatography vevox.app 126-545-180 Nikola P. Chmel L3 CH3F2 1 Outline Lecture 1 What is Analytical Science Errors and units What is chromatography? Terminology Classification History Theoretical plate model Rate theory Kinetic theory Normal phase chromatography (column, TLC) Reverse phase chromatography (HPLC) Gas chromatography Other chromatographic techniques Lecture 2 Lecture 3 Lecture 4 2 1 09/10/2023 Paper chromatography liquid-liquid partition SP = H2O bound to cellulose. MP = organic solvents Imagine paper divided into compartments. In each compartment partition between SP & MP occurs. Assume it is at equilibrium. Filter paper Capillary action Spots allowed to dry 3 Paper chromatography liquid-liquid partition Mobile phase Sample mixture Stationary phase Equilibrium established at each point (ideally) On leaving compartment MP carries residual solute to next compartment. New solvent enters the compartment & redistribution occurs so get more molecules in MP. Higher efficiency requires more compartments. 4 2 09/10/2023 Thin-Layer Chromatography (TLC) Substances tentatively identified by TLC may be further characterised by other analytical techniques such as NMR, mass spectrometry or other analytical procedures. Provides for separation and tentative identification of substances from milligram to picogram range. MP: liquid – solvent or mixture of solvents SP: active solid – silica, cellulose, alumina, polyamides, ion exchangers, numerous other organic and inorganic sorbents 5 Thin-Layer Chromatography (TLC) Movement of substances during TLC is the result of two opposing forces: Driving force of the mobile phase tends to move the substances from the origin in the direction of the mobile phase flow. Retarding action of sorbent impedes the movement by dragging them out of the mobile phase onto the sorbent. Each molecule alternates between a sorbed and unsorbed condition following a stop-and-go process through the sorbent. Substances that move slowly are attracted more strongly to the sorbent layer whereas those that move quickly spend a smaller fraction of their time in the layer because they have less affinity for it. 6 3 09/10/2023 Thin-Layer Chromatography (TLC) Flow of the mobile phase → non selective for unsorbed solutes All eluted or non-sorbed components or solutes spend equal time in the mobile phase. Differences in time arise because the solutes spend different amounts of time on the sorbent as determined by the interactions of the chromatographic system Depending on its nature, the layer promotes: physical sorption of solutes to the surface active groups of the layer particles (adsorption) dissolvation of the solutes into the stationary liquid held on the layer (partition) attraction of opposite charge on the layer (ion exchange) retention or rejection on the basis of size and shape (size-exclusion) 7 Thin-Layer Chromatography (TLC) Usually liquid/solid chromatography: SP = alumina or silica gel coated onto glass, aluminium or rigid plastic film. MP = solvents (usually fairly non-polar). MP ascends a thin layer (~100 µm) of material spread over a flat surface. Microscale, rapid (10-20 min). 2-20 µg. Not good for preparative work (10 mg max). Silica pH range: ~2-8. Alumina is more basic. Also ‘reverse phase silica’: aliphatic hydrocarbons attached to silica. 8 4 09/10/2023 Thin-Layer Chromatography (TLC) Choice of MP for silica chromatography including TLC & column Vary polarity of MP to increase or decrease time polar solutes spend on SP → separation. Chromatographic strength of solvent: how fast it moves analytes. For silica: more polar → more strength. Polar surface = SP MP ~ non-polar O Si O O Si O OH O Si OH Van der Waals, ionic & dipolar interactions → adsorption of polar species O Adsorption solvent strength parameter: εo = measure of adsorption energy / unit area of solvent. Admits factors other than polarity. Can choose weak and strong solvent and mix to get required strength. Non linear effect! Solvents must be miscible. 9 Thin-Layer Chromatography (TLC) The solubility of the compound in each solvent provides a valuable preliminary clue as to the required solvent strength for the mobile phase (all solutes should be moderately soluble in the mobile phase). The solvent that moves the zones near the centre of the plate has the correct strength. If the resolution is not adequate with any of the single solvents, a mixture of solvents should be used. Examples of solvent systems for silica gel TLC: Ethyl acetate has proven to be a good solvent If Rf values are too high add 5-10 % of hexane to reduce polarity If Rf values are too low add 5-10 % of methanol A percentage of acetic acid or ammonia (or pyridine) can be added to maintain acidic or basic solutions non-ionized and to prevent tailing of zones 10 5 09/10/2023 Thin-Layer Chromatography (TLC) XB XA 11 Thin-Layer Chromatography (TLC) Two dimensional TLC Used for the examination of complex mixtures Following application on one corner of a square plate the plate is run in order to achieve maximum resolution Plate is removed from the chamber and dried Plate is rotated at 90° and re-run in another solvent (the line of the partially resolved components from the first run becomes the origin for the second development) 14 6 09/10/2023 Thin-Layer Chromatography (TLC) Multiple TLC (unidimensional multiple development) 15 Thin-Layer Chromatography (TLC) Multiple TLC (unidimensional multiple development) This process increases the resolution of components with Rf values below 0.5 After a single run, the plate is removed from the chamber and partially dried The plate is placed again in the same in the same solvent and run in the same direction final Rf = 1 – (1 – Rf)n Multiple development can also be carried out in different solvents (stepwise development) 16 7 09/10/2023 Thin-Layer Chromatography (TLC) Detection and visualisation Self absorption in UV (254 nm) Fluorescence (UV excitation) Iodine (1 % alcoholic solution sprayed or iodine crystals in a jar, after some time iodine sublimes and other methods can be used as well sensitivities 0.1-0.5 g can be achieved) Charring reagents (charring occurs after spraying with corrosive reagents and heating) Geiger for radioactive materials Rhodamine B (solution of 50 mg / 100 ml of ethanol produced violet spots on pink background for organic compounds) Spraying with water (lipophilic solutes such as steroids show as while opaque spots) 17 Column Chromatography 19 8 09/10/2023 Column Chromatography Usually a preparative technique rather than analytical Usually silica used (range pH:2-8), particle diameter 40-60 m Use alumina if basic solution is required. Vary polarity of MP to increase or decrease time polar solutes spend on SP → separation. Chromatographic strength of solvent: how fast it moves analytes. For silica: more polar → more strength. Polar surface = SP MP ~ non-polar O Si O O Si O OH O Si OH Van der Waals, ionic & dipolar interactions → adsorption of polar species O Adsorption solvent strength parameter: εo = measure of adsorption energy / unit area of solvent. Admits factors other than polarity. Can choose weak and strong solvent and mix to get required strength. Non linear effect! Solvents must be miscible. 20 Column Chromatography Dry pack Voids form as silica packs more tightly after introduction of solvent Avoid! Mobile phase added continuously Mixture of components Separation into components Sand/Na2SO4 Silica Fractions Slurry pack Acid washed sand Glass wool Put silica in conical flask, add just enough solvent to make a very thick slurry. Pour into column as evenly as possible. Keep column “wet” when stored 21 9 09/10/2023 Column Chromatography stationary phase polarity INCREASING POLARITY Carbowax (polyethylene glycol) C18 (hydrocarbon coated silica) – reverse phase Paper Cellulose Starch Calcium sulphate Silica Florosil (magnesium silicate) Magnesium oxide Alumina (aluminium oxide) Activated carbon 22 Column Chromatography INCREASING POLARITY Mobile phase polarities Helium Nitrogen Petroleum ether (pentanes) Ligroin (hexanes) Cyclohexane Carbon tetrachloride Toluene Chloroform Dichloromethane t-butyl methyl ether Diethyl ether Ethyl acetate Acetone 2-propanol Pyridine Ethanol Methanol Water Acetic acid 23 10 09/10/2023 Column Chromatography Elution sequence by functional group on silica or alumina (polar) TLC & Column Chromatography INCREASING FUNCTIONAL GROUP POLARITY FAST Alkane hydrocarbons Alkenes (olefins) Aromatic hydrocarbons Ethers Esters Ketones Aldehydes Amines Alcohols Phenols Carboxylic acids SLOW 25 Outline Lecture 1 What is Analytical Science Errors and units What is chromatography? Terminology Classification History Theoretical plate model Rate theory Kinetic theory Normal phase chromatography (column, TLC) Reverse phase chromatography (HPLC) Gas chromatography Other chromatographic techniques Lecture 2 Lecture 3 Lecture 4 26 11 09/10/2023 HPLC High performance/pressure liquid chromatography Components are dissolved in a liquid (solvent) and then forced to flow (via the mobile phase) through a column (stationary phase) under high pressure. SP packed in a stainless steel column. To ‘push’ MP through the column, pressure must be v. high (typically between 800 -4000 psi although modern UHPLC systems operate up to 15000 psi). Separation achieved in the column. 27 HPLC Flow rates: Analytical: 0-10 mL/min, 3-10 µm packing Preparative: 45-180 mL/min, larger diameter packing Sample: injected into sample loop then carried into column MP: Isocratic elution: single or mixture of solvents remains constant Gradient: two or more solvents, ratios vary during analysis HPLC includes: Adsorption, partition, ion exchange, affinity, etc. Most common is reversed phase (75 % of analysis) 28 12 09/10/2023 HPLC Modes of Separation Normal Phase: Classic form of liquid chromatography using polar stationary phases and non-polar mobile phases The analyte is retained by the interaction of its polar functional groups with the polar groups on the surface of the packing Analytes elute from the column starting with the least polar compound followed by other compounds in order of their increasing polarity Reversed Phase: The stationary phase is non-polar and the mobile phase is polar The analytes are attracted to the surface by their non-polar functional groups The most polar analyte elutes from the RP column first followed by other analytes in order of decreasing polarity Ion-exchange Affinity chromatography 29 Samples HPLC applications: Separation of small organic molecules (MW < 500) e.g. pesticides, small drug molecules, steroids, Separation of ions (RP with phase modifiers, ion exchange) Separation of medium molecules (MW 500-5000) e.g. small peptides Generally poor for separation of large molecules (proteins, biomolecules) – used in conjugation with other methods Separation of chiral molecules (chiral SPs) 31 13 09/10/2023 HPLC Stationary Phase Silica: Porous silica particles are the most common substrate material used for HPLC column packing. Silica-based columns: can withstand high pressures are compatible with most organic and aqueous mobilephase solvents come in a wide range of bonded phases. Silica-based columns are often used for separations of low molecular weight analytes using mobile phase solvents and samples with a pH range of 2 to 7.5. Some new-generation silica materials, with extended pH ranges, are also available. 32 Stationary Phase Stationary Phase Functionality C18 (ODS) -Si(CH3)2C18H37 C8 -Si(CH3)2C8H17 C4 -Si(CH3)2C4H9 tC2 -SiC2H5 Aminopropyl -Si(CH3)2NH2 Cyanopropyl -Si(CH3)2CN Diol -Si(CH3)2OCH2CH(OH)CH2OH Phenyl -Si(CH3)nC6H5 Retention time for non-polar species Chain length: CN Phenyl NH2 C4 C8 C18 33 14 09/10/2023 Stationary Phase 34 Stationary Phase Particle Size Particle size for HPLC column packing refers to the average diameter of the packing particles. Particle size affects the back-pressure of the column and the separation efficiency. Column back-pressure and column efficiency are inversely proportional to the square of the particle diameter. This means that as the particle size decreases, the column backpressure and efficiency increase. 35 15 09/10/2023 Stationary Phase Particle Size A column with 3 μm packing produces almost twice the separation efficiency of a comparable 5 μm column, but a three-fold higher back-pressure with the same mobile phase and at the same flow rate. Small-particle (3 μm and 4 μm) columns are ideal for complex mixtures with similar components. Fast, high-resolution separations can be achieved with small particles packed in short (10-50 mm length) columns. Larger particle (5 μm and 7 μm) columns are typically used for routine analyses where analytes have greater structural differences. Large 10 μm packings have only moderate column efficiencies. Large particles (15-20 μm) are used for preparative-scale separations. 37 Stationary Phase Pore Size The pore size of a packing material represents the average size of the pores within each particle The size of the analyte should be considered when choosing the appropriate pore size for the packing material. The molecular weight of an analyte can be used to estimate the size of the molecule A pore size of 100 Å or less should be used for analytes below 3,000 MW A pore size of 100 Å -130 Å is recommended for samples in the range of 3,000 MW - 10,000 MW For samples >10,000MW, including peptides and proteins, a 300 Å material provides the best efficiency and peak shape. 38 16 09/10/2023 Mobile Phase Solvents Normal phase Reverse phase Heptane 1-chlorobutane chloroform methylene chloride isopropyl ether ethyl acetate acetonitrile methanol buffers water methanol acetonitrile acetone ethanol 2-propanol THF 39 Mobile Phase Elution Isocratic Mobile phase composition is kept constant throughout the analysis Gradient Mobile phase composition changes during the analysis 40 17 09/10/2023 Mobile Phase Why gradient elution? Gradient elution allows mixtures with wider ranges of hydrophobicity to be separated in one analysis Isocratic (47% B) – c.p. resolution 1.07, 75 min run time 0 10 20 30 40 50 60 70 Linear gradient elution – c.p. resolution 1.37, 15 min run time 0 5 10 41 Mobile Phase Effect of pH Reverse Phase : non-polar 42 18 09/10/2023 HPLC Detectors: Solute property not exhibited by MP UV/VIS (0.1-1 ng) Fixed Variable (VWD) Photodiode array (DAD) FTIR (1mg) Bulk property that changes with eluted solute Direct solute detection Refractive Index (100-1000 ng) Mass Spectrometry (1-1000 pg) Conductivity (500-1000 ng) ELSD (100-1000 pg) Electrochemical (10-1000 pg) Amperometric Coulobmetric Fluorescence (1-10 pg) Requires fluorophore 44 Recap How would you predict the order of elution of compounds with different polarity on a column or TLC plate? How would you optimise the resolution on a TLC plate/column? Schematic of an HPLC instrument. HPLC applications. Typical stationary phases, mobile phases in HPLC 46 19