Chromatography Theory PDF

Basic Principles of Chromatography FDSC 624 1  Types of Differential Distribution Different chemical states of the same matter. - Distillation - Sublimation - Crystallization - Zone melting - Precipitation...

Basic Principles of Chromatography FDSC 624 1  Types of Differential Distribution Different chemical states of the same matter. - Distillation - Sublimation - Crystallization - Zone melting - Precipitation 2  Types of Differential Distribution Different chemical phases. - Absorption (partition) - Adsorption - Liquid-liquid extraction (LLE) - Solid-phase extraction (SPE) - Supercritical fluid extraction (SFE) - Solid-phase microextraction (SPME) 3  Types of Differential Distribution Different chemical environments or different locations. - Separation based on different rates of migration of analytes under the influence of a field. o Electric field: electrophoresis, field-flow fractionation o Gravitational field: centrifugation, filtration o Thermal field: Thermal diffusion o Membrane (semi-permeable): dialysis, osmosis, ultra-filtration 4 5 6 7 Chromatographic Technique 8 9 Absorption & Adsorption 10 11 12  Chromatography: Peak Resolution Poor resolution More separation Less band spread Chromatographic Definitions Chromatographic Relationships  Rate Theory H=Au1/3 + B/u + Cu H: plate height (as small as possible) A: Eddy Diffusion Term B: Molecular Diffusion Term C: Mass Transfer Term u: Velocity of Mobile Phase 16  Eddy Diffusion A=2λdp λ: packing factor dp: diameter of the particles packed in the column 17  Molecular Diffusion B=2ΨDM – The longer the molecule in column, the higher the diffusion Ψ: obstruction factor DM: diffusion coefficient for the solute in the mobile phase 18 Mass Transfer 8 𝑘𝑘 𝑑𝑑𝑓𝑓 2 𝐶𝐶 = 2 π (1 + 𝑘𝑘)2 𝐷𝐷𝑠𝑠 K: retention factor Ds: Diffusion coefficient of the analyte in the stationary phase df: average film thickness of liquid stationary phase 19  Chromatography - van Deemter Plot HETP: height equivalent to a theoretical plate 20 Column Efficiency - Kinetic variables  Summary of Separation Efficiency One cannot optimize any given operating conditions and column choices without compromising properties. Optimizing chromatographic resolution (small particle diameter, thin phase coating, long column lengths, and slow or optimum rate of mobile phase) will be at the cost of capacity (large column and thick phase coating) and speed (thin film coating, high velocity of mobile phase). Capacity will be at a cost of resolution and speed. 22  Quantitative Analysis Peak areas Peak height Calibration and standards Internal standard method  Liquid Chromatography Theory & Principle of Operation Using a pressurized polar SOLVENT to “carry” a sample through a tubular COLUMN filled with a non-polar packing; the organic species present in that sample can be properly separated for QUALITATIVE identification and QUANTITATIVE analysis with various detectors for optical or physical properties. 24  Liquid Chromatography (HPLC) MODES of chromatographic interactions for SEPARATION of MOLECULES based on polarity, charge, size & solubility. 25  Liquid Chromatography (HPLC): category Adsorption (Open Column) Partition (Normal Phase/ Reverse Phase) Ion-Exchange Size Exclusion (SEC) [Gel Permeation (GPC)] 26 Liquid Chromatography (HPLC) 27 Liquid Chromatography (HPLC) 28 Liquid Chromatography (HPLC) 29 Liquid Chromatography (HPLC) 30 Liquid Chromatography (HPLC) 31 Liquid Chromatography (HPLC) 32 Liquid Chromatography (HPLC) 33 Liquid Chromatography (HPLC) 34 Liquid Chromatography (HPLC) 35 Liquid Chromatography (HPLC) 36 Liquid Chromatography (HPLC) 37 Liquid Chromatography (HPLC) 38 Liquid Chromatography (HPLC) 39 Basic HPLC Theory ALL organic molecules will have a characteristic retention time on a specific column with a specific solvent & flow which can provide both qualitative & quantitative data. 40  Basic HPLC Theory (con’t) There are 4 basic HPLC analysis: Reverse Phase (RP) Normal Phase (NP) Ion-Exchange (IEC) Size Exclusion (SEC) 41  Basic HPLC Theory (con’t) The principle of REVERSED Phase Chromatography Separation in reverse phase HPLC depends upon the reversible ADSORPTION of organic molecules according to their hydrophobicity & polarity under conditions where the stationary phase (column) is more hydrophobic than the mobile phase (solvent). 42  Basic HPLC Theory (con’t) RP MATERIALS: Column = C-18 (ODS) LEAST Packing C-8 (Octyl) C-Ø (Phenyl) C-NH2 (Amino) C-C3CN (Cyanopropyl) MOST Solvents = Water MOST Acetonitrile Methanol Tetrahydrofuran LEAST 43 Basic HPLC Theory (con’t) 44 Basic HPLC Theory (con’t) 45 Basic HPLC Theory (con’t) 46 Basic HPLC Theory (con’t) 47 Basic HPLC Theory (con’t) 48 Basic HPLC Theory (con’t) 49 Basic HPLC Theory (con’t) 50 Basic HPLC Theory (con’t) 51 Basic HPLC Theory (con’t) 52  Basic HPLC Theory (con’t) The principle of NORMAL Phase Chromatography Separation in normal phase HPLC is limited to the primarily HYDROPHOBIC interactions between a HIGH polarity Column and a mixture of LOW polarity Solvents to selectively partition the sample molecules off the column. 53  Basic HPLC Theory (con’t) NP MATERIALS: Column = Silica (SiO2) LEAST packing Alumina (Al2O3) Titania (TiO2) Zirconia (ZrO2) MOST Solvents = Chloroform MOST THF, DMF Toluene, Ethyl Acetate Hexane LEAST 54  Basic HPLC Theory (con’t) The principle of ION-EXCHANGE Chromatography Separation in Ion-Exchange HPLC is a function of the reversible adsorption of CHARGED solute molecules in the mobile phase to the stationary Ion-Exchange groups of OPPOSITE charge. 55  Basic HPLC Theory (con’t) IEC MATERIALS: Column = Sulfonates [R-SO3H] ACIDIC packing Amines [R-N(R3)] BASIC Solvents = WATER [with various Acidic (Acetate, Phosphate) or Basic (Quaternary Amine) BUFFERS] 56 Basic HPLC Theory (con’t) 57 Basic HPLC Theory (con’t) 58 Basic HPLC Theory (con’t) 59 Basic HPLC Theory (con’t) 60 Basic HPLC Theory (con’t) 61 Basic HPLC Theory (con’t) 62 Basic HPLC Theory (con’t) 63  Basic HPLC Theory (con’t) The principle of GEL (Filtration) PERMEATION Chromatography Separation in Gel-Permeation LC is based on the differences in SIZES from biological and synthetic polymers as they pass through a column packed with a “GEL” of very specific pore sizes that will “delay” the movement of certain size molecules. 64  Basic HPLC Theory (con’t) GPC MATERIALS: Column = Styrene-DivinylBenzene Resins Packing with Specific Molecular Weight ranges based on the “Average” PORE SIZE Distribution Solvents = Water / THF / DMSO / DMF for Bio-Polymers Toluene / Xylenes / Chloroform for Petrochemicals 65 Basic HPLC Theory (con’t) SDVB Beads are available from only TWO companies in the World… one in Japan, the other in Germany. 66 Basic HPLC Theory (con’t) 67 Basic HPLC Theory (con’t) 68 Basic HPLC Theory (con’t) 69 Basic HPLC Theory (con’t) 70 Basic HPLC Theory (con’t) 71  Basic CHROMATOGRAPH Design ALL Analytical Chromatographs have several Common design components: SOLVENT / PUMP (Mobile Phase) / INJECTION PORT / VALVE / COLUMN (Stationary Phase) / DETECTOR [up to FOUR] / OUTPUT (Analog & Digital) 72 HPLC System Design 73  HPLC System Design PUMP: Reciprocating, cam-driven, single piston PUMPS that usually employ a simple pulse dampener to even out the solvent flow are common. 74  HPLC System Design INJECTION VALVE: High pressure, fluoropolymer material that accommodates a variety of sample loops, liquid syringes & auto- sampler ports. 75  HPLC System Design TUBING & FITTINGS: Conventional stainless steel for high-pressure & solvent-resistance for GPC / SEC work & NPC; or Fluoropolymer (PEEK) with simple finger tight fittings for reduced IONIC contamination for bio- assays & RPC. 76  Columns & Fittings Packed columns with 3-10 micron silica-based stationary phases to provide maximum data quality in minimum time using metal or polymer high-pressure fittings & seals. 77  HPLC System Design COLUMN: * Normal or Reverse-Phase * Gel Permeation, Ion-Exchange ANALYTICAL = 3.0 to 4.6 mm DIAM, 75 to 300mm length, 3-10 micron. PREPARATIVE = 10 to 50 mm DIAM, 150 to 7500 mm length, 5-25 micron. 78  Normal Phase Columns Alumina and Silica use hydro-phobic solvent so sample adheres to packing in the column. Useful for low polarity and petrochemical and polymer type samples, certain tests for molecular weight determinations. 79  Reverse Phase Columns C-18 and other polar bonded phase columns. Opposite of normal phase where column packing has a long chain hydrophobic coating and high polarity solvents are used to separate more polar samples. Useful for high polarity and hydrophilic type samples. Most popular form of HPLC! 80  Solvent Selection NORMAL Phase: hexane, methylene chloride, THF, DMF, DMSO -=> NOTE: Attacks PEEKK Polymer, must use stainless steel contact surface. REVERSE Phase: 90% done with water, methanol & acetonitrile; some methods add small amounts APROTIC solvents (THF). 81  HPLC System Design DETECTOR: UV/Vis (fixed & variable wavelengths) Refractive Index Fluorescence Evaporative Light Scattering Electro-Chemical 82  LC Detectors UV-Vis (195-800 nm): Useful for most general purpose analyses Detects most organics Fixed, variable wavelength & PDA systems. 83  LC Detectors Differential Refractive Index: Looks at difference between a static reference cell and a sample flow cell. Specialized for sugars and carbohydrates and polymer (molecular weight) determinations. 84  LC Detectors Differential Refractive Index detector: Sugar separation 85  LC Detectors Filter Fluorescence: Use excitation & emission filters allow drugs, natural products, vitamins, and other specialized chemicals to be determined at PPT level. Also used for Poly- Aromatic (PAH, PNA) analyses for Environmental work 86  HPLC: Important Features Solvent Pump Rate – Micro-LC (0.01 - 2.0 ml/min) – Analytical (0.1 - 10.0 ml/min) – Semi-Prep (0.5 - 50 ml/min) System Pressure Rating – Normal Phase = 200-1000 psi – Reverse Phase = 1000-3000 psi – Most systems have a max. pressure of 5000 psi 87  Chromatographic Controls Higher Solvent Flow = Closer Peaks More Polar Solvent = Faster Separation Higher Pressure = Closer Peaks Less Flow, Polarity, Press. = Better Resolution Controlled Flow = Closer Peaks with minimum loss of resolution 88