HPLC Techniques PDF

HPLC Chromatogr. Techniques 1 Contents  Overview, Basic terms  HPLC Columns  Mobile Phases  HPLC Equipment  HPLC Applications 2 HPLC system 4 HPLC System 5 HPLC system Pumps Elution mode 7 HPLC system Gradient s...

HPLC Chromatogr. Techniques 1 Contents  Overview, Basic terms  HPLC Columns  Mobile Phases  HPLC Equipment  HPLC Applications 2 HPLC system 4 HPLC System 5 HPLC system Pumps Elution mode 7 HPLC system Gradient systems Mobile phase reservoirs Quarternary pump (quarternary A B C D gradient, low pressure gradient) Low pressure Mixer ▪ Mixing of up to 4 different solvents ▪ Cheaper instrumentation (one degasser, Degasser one pump). ▪ Higher gradient delay volume High pressure Pump (compared with the binary pump) ▪ Gradient is mixed in the mixer and it is based on opening of the valves. Injection Column 8 HPLC system Gradient systems Mobile phase reservoirs Low pressure Binary pump (binary gradient, high A B pressure gradient) Degasser Degasser ▪ Mixing only of two solvents Pump B ▪ Expensive, sophisticated instrumentation (two degassers, two High pressure pumps, control unit for pump synchronization). Mixer ▪ Very small gradient delay. ▪ Superb gradient reproducibility. ▪ Gradient is achieved by control of flow Injection Column rate of the pump. 9 HPLC system INJECTION 10 HPLC system INJECTION HPLC system INJECTION HPLC system COLUMNS 14 HPLC system COLUMN HPLC system  HPLC System Column Column is the most important for chromatographic separation: Stationary phase selection depends on sample/analyte properties (solubility, polarity, etc.) Factors for the selection of appropriate column and sorbent: ▪ Separation efficiency, peak symmetry ▪ Column lifetime, stability of the bonded phase ▪ Reproducibility ▪ Time of analysis ▪ Selectivity Column length: Column diameter: ▪ Resolution ▪ Back pressure Decrease with ▪ Separation efficiency ▪ Mass sensitivity diameter ▪ Back pressure Increase with ▪ Separation efficiency ▪ Mobile phase length ▪ Column capacity consumption ▪ Mobile phase Increase with ▪ Analysis time consumption diameter 17 HPLC system Columns and Stationary Phases History of the HPLC particles development Year Particle size Number of plates (15 cm) Particle parameters: ▪ Size (diameter), ▪ Shape (regular, irregular) ▪ Type (porous, non-porous) Pore diameter ▪ Active surface area 18 HPLC system Columns and Stationary Phases Particle size and separation efficiency ▪ Smaller particle diameter allows to achieve more separation plates ▪ Back pressure significantly increase when small particles are used. HETP (µm) ▪ Flow rate significantly influences the column efficiency and analysis time. ▪ Columns with small particles provides optimal separation in the wide range of flow rate, which allows to use higher flow rates. Linear velocity (cm/s) 19 20 HPLC system Columns and Stationary Phases Types of particles Bonded Phase Silica ▪ Silica bead containing silanol (Si-OH) are bonded with hydrocarbon groups. ▪ The nature of the bonded phase determines the chromatographic behavior. Pellicular Packing ▪ An inert core provides physical support ▪ A thin layer of coating on the core provides functional groups for the separation of analytes Microporous ▪ Gel-type resin consisting of cross-linked polymers Macroporous ▪ Highly cross-linked (>50%) resin ▪ Stable from pH 1 to 14 ▪ Available in a variety of particle and pore sizes HPLC system F5…pentafluorophenyl phase Phenyl…butyl-phenyl phase Columns and Stationary Phases C4…butyl phase C8…octyl phase Stationary phases C18…octadecyl phase IONIC HYDROPHOBIC POLAR As colunas podem ser feitas de sílica ou SCX…strong cation exchange material polimérico, com superfícies SAX…strong anion exchange quimicamente modificadas para aumentar a Silica …bare silica phase polaridade. CN…cyanopropyl phase Exemplos comuns incluem: fases de sílica, NH2…amino phase amino, diol, ciano e zwiteriónicas, como a modificação especial de ácido amónio- sulfónico.  The most important separation modes in HPLC SP– solid POLAR: LSC or Adsorption Chromatography NORMAL PHASE (NPLC) REVERSED PHASE LIQUID CHROMATOGRAPHY (RPLC)(RPLC) SP– NONPOLAR large surface area of solid; porosity; and SP, in direct contact porous particles; small particles with the liquid MP often with a chemically modified surface Chromatogr. Techniques 23 Liquid Chromatographic Modes  Normal-phase or Liquid-solid (NP, LSC) › Separation based on adsorption/desorption of the analyte onto a polar surface (silica)  Reversed-phase (RPC) › Separation based on analytes’ partition coefficients between the mobile phase and the bonded stationary phase  Ion-exchange (IEC) › Separation based on ion-exchanging with the counter-ions and ionic interaction with the bonded ionic group  Size-exclusion (SEC or GPC) › Separation based on analyte’s molecular size and sieving action of the column packing Modes of High Performance Liquid Chromatography Types of Compounds Mode Stationary Mobile Phase Phase Neutrals Reversed Phase Non-polar modified Water/Organic Weak Acids chromatography silica (C18, C8, cyano, Modifiers Weak Bases amino, phenyl) Ionics, Bases, Acids Ion C-18, C-8 Water/Organic Pair Ion-Pair Reagent Compounds not Normal Silica, Amino, Organics soluble in water Phase Cyano, Diol Ionics Inorganic Ions Ion Anion or Cation Aqueous/Buffer Exchange Exchange Counter Ion Resin High Molecular Weight Size Polystyrene Gel Filtration- Compounds Exclusion Silica Aqueous Polymers Gel Permeation- Organic 25 HPLC system MAIN TYPES IN HPLC HPLC system REVERSE PHASE (RPLC) HPLC system - RPLC Reversed-phase (RPLC)  Reversed phase › Most popular since it can handle the broadest variety of sample types › Opposite of normal phase – stationary phase is non-polar and mobile phase is polar › C18 columns (e.g. octadecyl, -C18H37) › Eluents used – aqueous solution of organic solvents e.g. MeOH, ACN, THF, etc.  Applications › Amino acids, homologs, herbicides, etc.c.  Reversed Phase Liquid Chromatography ▪ Partition type of chromatography ▪ Main type of interaction is hydrophobic (van Der Waals interaction), but in real is the retention mechanism complex. ▪ Elution order: Strong Lewis acids (carboxylic acids) < Weak Lewis acids (alcohols, phenols) < Strong Lewis bases (amines) < Weak Lewis bases (ethers, aldehydes, ketones) < permanent dipoles (CHCl3) < induced dipoles (CCl4) < aliphatics ▪ Retention increases also with the number of carbon atoms in molecule: … Pentan < Hexan < Heptan… ▪ Branched-chain isomers are eluted earlier than linear form.  Reversed Phase Liquid Chromatography Stationary phases ▪ C18 modified silica is the most common stationary phase, providing high retention (other phases are C8, phenyl, CN, diol, NH2 – providing lower retention and alternative selectivity). ▪ Carbon load: Retention strenght for C18 could be estimated from „carbon load“ – more carbon means thicker stationary phase and consequently higher retention (for non-polar analytes, columns with lower carbon load could be recommended). ▪ Pore size (Å, Ångström) determines suitability of the phase for small or large molecules – small pore size providing better capacity, but it is not for large molecules. 1Å = 0.1 nm (1×10−10 meter) 32  Reversed Phase Liquid Chromatography Stationary phases ▪ Introduction of polar (hydrophilic) groups stabilise the stationary phase even 100% water mobile phase is used. ▪ Polar-encapped phase – Hydrophobic interaction silmilar to the traditional phase, stronger hydrogen bonding and silanol activity. ▪ Polar-embedded phase – Opposite behaviour, reduction of the hydrophobic intercation, reduced silanol activity. A. Common C18 phase B. C18 + polar-embedded group C. C18 + polar-encapping HPLC system - RPLC  Reversed Phase Liquid Chromatography Stationary phases ▪ Effect of chain lenght on retention. 1. Acetone 2. p-methoxyphenol 3. Phenol 4. m-cresol 5. 3,5-xylenol 6. Anisole 7. p-phenylphenol Longer chain provides higher retention. Reversed Phase Liquid Chromatography Lipophilicity Polar compounds are eluted earlier than the non-polar. CH3 CH3 CH3 OH OH OH CH3 CH3 CH3 CH3 OH OH H3C OH H3C H3C CH3 CH3 CH3 CH3 CH3 Polarity 36 HPLC system NORMAL PHASE (NPLC) HPLC system - NPLC HPLC system - NPLC  Normal Phase Liquid Chromatography ▪ Adsorption chromatography, -OH on the surface of silica are active sites (or Al3+ an O2- in case when alumina is used). ▪ Types of interactions are dipole-induced dipole, dipole-dipole, hydrogen bonding, π-complex bonding. ▪ Adsorption strengths (k) increase in the following order: saturated hydrocarbons < olefins < aromatic ≈ halogenated compounds < suphides < ethers < nitro compounds < esters ≈ aldehydes ≈ ketones < alcohols ≈ amines < suphones < suphoxides < amides < carboxylic acids ▪ Only functional groups or double bond are used for separation, it is not possible to distinguish between molecules that are identical except the aliphatic moiety. ▪ The most polar functional group in the molecule determines its retention. ▪ The strength of interaction depends also on steric factors, isomers are suitable for separation by adsorption chromatography. Normal Phase Liquid Chromatography Polar compounds are eluted later Lipophilicity than the non-polar. CH3 CH3 signal 2 CH3 3 OH OH 1 OH void CH3 CH3 CH3 CH3 OH OH H3C OH H3C H3C CH3 time CH3 CH3 CH3 CH3 Polarity Comparison of normal and reversed phase liquid chromatography 42 Chromatogr. Techniques 43 Mobile phase  HPLC mobile phases are usually a mixture of one or more solvents with these characteristics › Desirable Physical properties  High purity, low cost, UV transparency, non-corrosive, low viscosity, low toxicity, non-flammable, sample solubility › Strength  Strength is related to polarity of solvent; Water is a strong solvent in normal phase but a weak one in reversed-phase  Solvent strengths under normal phase are characterized by Hildebrand’s scale (Eo) › Selectivity  Depends on dipole moment, induced dipole, H-bonding, and dispersive characteristics of the solvents Mobile phase  Viscosity › Low viscosity solvents decreases the pressure to achieve a given flow rate › Low pressure extends the lifetime of pumps and columns  Boiling point › Low b.p. facilitate solvent removal from collected fractions › Some pumps have difficulty in pumping liquids with b.p. 8 pontos por pico) para definir um pico são precisos pelo menos 3 pontos a “subir” e 3 a “descer”  armazenagem (identificação)  Tratamento de dados  acumulação / “smoothing” / integração / derivatização  determinação de tempos (tr) e intensidades (áreas / alturas) integração - definição do princípio e fim do pico picos mal definidos (falta de resolução e “tailing”) – correção da linha de base  tratamento à posteriori utilizando novos parâmetros (“threshold” / integração) – não melhora a separação!! 85 Separation Techniques I have two separation techniques in my lab, High Performance Liquid Chromatography and Gas Chromatography. Which should I use? 86  Comparison of HPLC and GC Sample Volatility Sample Polarity HPLC HPLC Separates both polar and No volatility requirement non polar compounds Sample must be soluble PAH - inorganic ions in mobile phase GC Sample must be volatile GC Samples are nonpolar and polar 87  Comparison of HPLC and GC Sample Thermal Lability Sample Molecular Weight HPLC HPLC Analysis can take place No theoretical upper limit at or below room temperature In practicality, solubility is limit. GC GC Sample must be able to survive high Typically < 500 amu temperature injection port and column 88  Comparison of HPLC and GC Sample Preparation Sample Size HPLC HPLC Sample must be filtered Sample size based upon column i.d. Sample should be in same solvent as mobile phase GC GC Solvent must be volatile Typically 1 - 5 L and generally lower boiling than analytes 89  Comparison of HPLC and GC Separation Mechanism Detectors HPLC HPLC Both stationary phase Most common UV-Vis and mobile phase take Wide range of non- part destructive detectors 3-dimensional detectors Sensitivity to fg (detector dependent) GC GC Most common FID, Mobile phase is a universal to organic sample carrier only compounds 90  Advantages of HPLC Rapid and precise quantitative analysis – Typical analysis time of 5-20 min, precision 60% of all existing compounds vs. 15% for GC – Can analyze samples with little or minimal preparation 91