High-Performance Liquid Chromatography (HPLC)

High-Performance Liquid Chromatography (HPLC)  HPLC is a type of chromatography that employs a liquid mobile phase and a very finely divided stationary phase.  To obtain satisfactory flow rates, the liquid must be pressurized to several hundred or more pounds per square inch.  HPLC is a process that was developed in the late 1960's.  Major technical advances that developed at that time included  Solid packing particles were reduced in size from 150-200 µm to 5-10 µm.  Smaller particles increased the resolution of separation, allowing smaller, shorter columns. High-Performance Liquid Chromatography (HPLC)  HPLC is a type of chromatography that employs a liquid mobile phase and a very finely divided stationary phase.  To obtain satisfactory flow rates, the liquid must be pressurized to several hundred or more pounds per square inch. Instrumentation  Liquid phase samples (mixtures) are injected onto the column usually using a syringe and specially devised injection valve.  The sample is swept onto the chromatographic column by the flowing mobile phase and chromatographic separation occurs as the mixture travels down the column.  Normal HPLC detectors detect the elution of a compound from the end of the column based on some physical characteristic such as ultraviolet light absorption, ability to fluoresce, or the difference in index of refraction between the analyte and the mobile phase itself. Mobile phase Pump Injector Pre-Column Column Detector Reservoir Data Typical apparatus for HPLC Mobile phase reservoir, filtering  One or more glass, plastic or stainless steel solvent reservoirs are used to hold mobile phase  Mobile phase solutions are usually pre-filtered before use to remove dust and particulates.  Most of the manufacturers supply these bottles with the special caps, Teflon tubing and filters to connect to the pump inlet and to the purge gas (helium) used to remove dissolved air.  Purging is a process in which dissolved gases are swept out of a solvent by bubbles of an inert, insoluble gas.  Mobile phase is usually degassed via a sparger  Helium purging and storage of the solvent under helium was found not to be sufficient for degassing of aqueous solvents.  It is useful to apply a vacuum for 5-10 min. and then keep the solvent under a helium atmosphere.  As the solute capacity, or k', for the compound decreases, the compound begins to migrate through the stationary phase.  Each of the other compounds in the sample subsequently migrate as their k' values decrease.  Compared with isocratic elution, resolution and separation are improved, and bandwidths are nearly equal Mobile phase selection  Solvent strength (o ) is a quantitative representation of solvent strength.  In reversed phase LC there is an inverse relationship between solvent strength and chromatographic elution power.  For example, water is a strong solvent but has little elution power in RP-LC and must, therefore, be mixed with a less polar organic modifier to elute a solute with a low k’ value.  The series of weak, medium strength and strong solvents thus derived is referred to as the eluotropic series.  If the solvent competes strongly for the active sites, leaving less time for sample molecules to be adsorbed and hence t hey are rapidly eluted. This type of solvent is comparatively strong  The eluotropic series gives the chromatographer a method of influencing the k’ values of sample components.  Decreasing the solvent strength, k’ values may be increased.  To increase chromatographic resolution per unit time during the course of a chromatographic run, the solvent strength can be increased in a stepwise or continuous manner causing k’ values to change. This is known as solvent programming. Solvent characteristics of mixed solvent. Solvent Selectivity Group Solvent strength weighting factor( Si) Reverse Phase* Normal Phase Methanol II 2.6 5.1 Acetonitrile VI 3.2 5.8 4.5 4.0 --- 0 10.2 VIII 4.1 Tetrahydrofuran III Water Chloroform Methylene chloride V 3.1 Methyl t-butylether I ca.2.5 Ethyl ether Hexane I 2.8 --- 0 * Approximate value for some other common solvents are acetone(3.4), dioxane(3.5), ethanol(3.6), and isopropanol(4.2). ST = i Siqi ST = total solvent strength of the mixture Si = solvent strength weighting factor qi= volume fraction of solvent in the mixture Example) methanol/water ( 60 : 40 v/v %) ST = i Siqi = SCH3OHqCH3OH + SH2OqH2O ST = (2.6)(0.6) + (0)(0.4) = 1.56 Iso-eluotropic solvents Binary system: acetonitrile/water 1.56 = (3.2)(qCH3CN) + (0)(qH2O) (qCH3CN) = 0.49 = 49 % Components of HPLC instrumentation 1. Stationary Phase  The stationary phase in HPLC refers to the solid support contained within the column over which the mobile phase continuously flows.  The sample solution is injected into the mobile phase of the assay through the injector port.  As the sample solution flows with the mobile phase through the stationary phase, the components of that solution will migrate according to the non-covalent interactions of the compounds with the stationary phase.  The chemical interactions of the stationary phase and the sample with the mobile phase, determines the degree of migration and separation of the components contained in the sample.  Some of the more common stationary phases include: Liquid-Liquid, Liquid-Solid (Adsorption), Size Exclusion, Normal Phase, Reverse Phase, Ion Exchange, and Affinity. Liquid-Solid  Operates on the basis of polarity.  Compounds that possess functional groups capable of strong hydrogen bonding will adhere more tightly to the stationary phase than less polar compounds. Liquid-Liquid  operates on the same basis as liquid-solid.  It is better suited for samples of medium polarity that are soluble in weakly polar to polar organic solvents.  The separation of non-electrolytes is achieved by matching the polarities of the sample and the stationary phase and using a mobile phase which possesses a markedly different polarity. Normal Phase  Use a polar stationary phase and a less polar mobile phase.  Thus hydrophobic compounds elute more quickly than do hydrophilic compounds. Reverse Phase  The stationary phase consists of silica based packings with n-alkyl chains covalently bound. For example, C8 signifies an octyl chain and C18 an octadecyl ligand in the matrix.  Thus hydrophilic compounds elute more quickly than do hydrophobic compounds. Normal-phase chromatography Polar stationary phase Reversed-phase chromatography nonpolar stationary phase More polar solvent has higher eulent strength Less polar solvent has higher eulent strength Types of HPLC depend on 1. Molecular weight of solute 2. Water solubility of the solute 3. Polarity of the solute 4. Ionic/non-ionic character of the solute Normal and reverse phase types cover almost 90% of all chromatographic applications. Selecting the Separation Mode Solute dissolve in water or organic solvents ? Guide to HPLC mode selection Affinity  Operates by using immobilized bio-chemicals that have a specific affinity to the compound of interest. The compounds are then eluted by changing the mobile phase conditions. Ion-Exchange  Operates on the basis of selective exchange of ions in the sample with counter ions in the stationary phase.  It is performed with columns containing charge-bearing functional groups attached to a polymer matrix. The functional ions are permanently bonded to the column and each has a counter ion attached.  The sample is retained by replacing the counter ions of the stationary phase with its own ions.  The sample is eluted from the column by changing the properties of the mobile phase that the mobile phase will now displace the sample ions from the stationary phase, (i.e. changing the pH). Ion-exchange chromatography 1. The column is packed with ... a. A polystyrene ion-exchange resin of beaded particles <10 µm available b. A silica-based ion-exchange resin (particles). 2. Samples are run through the columns packed with these particles and ion exchange would occur. For example,...  In the top diagram above, the resin would be exhaustively washed with acid to "load" the cation exchange sites (sulfonic acid groups in the example above) with protons (and elute any bound cations).  In the lower diagram, a sodium-containing sample is then loaded on the column. The sodium competes and displaces the protons and binds to the resin. The protons are then eluted.  The amount of sodium bound can be determined by titrated the eluted acid.  High concentrations of one species shift equilibrium, favoring binding of the higher concentration species and elution of the other. Size-Exclusion Chromatography  Operates on the basis of the molecular size of compounds being analyzed. Columns are packed with porous particles with different pore diameters. The particles usually are made of ... 1. Polystyrene that is cross-linked with divinylbenzene (semirigid) to withstand the high pressures. a. Organic solvents are commonly used (e.g., acetone, tetrahydrofuran, etc.) since water will not wet polystyrene. b. Slow flow rates are common. 2. Glass or silica. a. Silica allows high flow rates and high pressures. b. The pH must be kept below ~7.5 or the silica will dissolve. 3. Soft gels (cross-linked dextrans, polysaccharides or similar polymers). 1) Sephadex (dextran) 2) Sepharose (agarose) 3) BioGel P (polyacrylamide) a. Pressures must be kept low (<150 psi) to prevent crushing the gels. b. These are most useful for some water-soluble species (e.g., proteins, enzymes, etc.) Size-Exclusion cont’d---- The larger compounds will be excluded from the interior of the bead and thus will elute first.  However, there are some size-exclusion that are weakly anionic and slightly hydrophobic which give rise to non-ideal size-exclusion behavior. 2. Pumping system. a. The pumping system must... 1) Generate pressures up to ~6,000 pounds per square inch. 2) Give a pulse-free output (via a pulse damper). 3) Give flow rates of ~0.1 - 10 mL/minute. 4) Give flow reproducibility of 0.5% or better. 5) Resist corrosion by a variety of solvents. Write briefly the following pumping types: reciprocating, constant pressure and syringe type? 3. SAMPLE INJECTOR  Auto-samplers and manual sample injectors are available  Auto-samplers can store and sequentially inject multiple samples are popular in quality control applications and for high-throughput screening. Scale Sample Volume Micro 2 µL to 500 µL Analytical 1.0 µL to 5.0 mL Preparative 100 µL to 10 mL HPLC Sample Injection Loop  The sample is injected into the loop while the loop is switched out of the HPLC flow path.  After the loop is filled with sample it is switched back into the flow path and the sample is swept onto the head of the HPLC column for later elution. 4. Columns  Columns are usually stainless steel tubes.  Lengths are typically 10 - 30 cm.  Inside diameters of 4 - 40 mm are typical; newer high-performance columns are 1 - 4.6 mm.  Particulate packing size is typically 5 - 10 µm. HPLC column with replaceable guard column to collect irreversibly adsorbed impurities. Titanium frits distribute the liquid evenly over the diameter of the column. Columns that are secondary to the separating column or stationary phase. They are: Guard, Derivatizing, Capillary, Fast, and Preparatory Columns. A. Guard Columns  This serves as a protective factor that prolongs the life and usefulness of the separation column.  They are dependable columns designed to filter or remove: 1) Particles that clog the separation column; 2) Compounds and ions that could ultimately cause "baseline drift", decreased resolution, decreased sensitivity, and create false peaks; 3) Compounds that may cause precipitation upon contact with the stationary or mobile phase; and 4) Compounds that might co-elute and cause extraneous peaks and interfere with detection and/or quantification.  These columns must be changed on a regular basis in order to optimize their protective function. Size of the packing varies with the type of protection needed. B. Derivatizing Columns  Pre- or post-primary column derivatization can be an important aspect of the sample analysis.  Reducing or altering the parent compound to a chemically related daughter molecule or fragment elicits potentially tangible data which may complement other results or prior analysis.  This is one reason why HPLC was advantageous over gas chromatography. Because GC requires volatile, thermally stabile, or nonpolar analytes, derivatization was usually required for those samples which did not contain these properties.  Acetylation, silylation, or concentrated acid hydrolysis are a few derivatization techniques. Stationary Phases (Adsorbents) HPLC separations are based on the surface interactions, and depends on the types of the adsorption sites (surface chemistry). Modern HPLC adsorbents are the small rigid porous particles with high surface area. Main adsorbent parameters are:  Particle size: 3 to 10 m  Pore size: 70 to 300 m  Surface area: 50 to 250 m2/g Depending on the type of the ligand attached to the surface, the adsorbent could be: - Normal phase (-OH, -NH2), or - Reversed-phase (C8, C18, Phenyl), - Anion (NH4+), or - Cation (-COO-) exchangers. Plate height as a function of flow rate for stationary-phase particle sizes of 10, 5, and 3m. The Stationary Phase Schematic structure of silica gel. Aggregate of spherical particles (50% porosity, 150 m2/g surface area) and Sponge like structure (70% porosity, 300m2/g surface area) are available. Bonded stationary phase Silica surface dimethyl silane R= C8, C18, …… The octadecyl (C18) stationary phase is by far the most common in HPLC. It is frequently designated ODS, for octadecylsilane. Common polar phases Common nonpolar phases R = (CH2)3NH2 amino R = (CH2)17CH3 octadecyl R = (CH2)3C≡N cyano R = (CH2)7CH3 octyl R = (CH2)3C6H5 phenyl R = (CH2)2OCH2(OH)CH2OH diol Molecular model of octadecyl siloxane Bulky isobutyl groups protect siloxane bonds from hydrolysis at low pH. Siloxane (Si-OSiR) bond hydrolyze below pH2 PH 2-8 DETECTORS  The type of detector utilized depends on the characteristics of the analyte of interest.  Today, optical detectors are used most frequently in liquid chromatographic systems.  These detectors pass a beam of light through the flowing column effluent as it passes through a low volume ( ~ 10 ml) flow cell. The variations in light intensity caused by UV absorption, fluorescence emission, or change in refractive index (depending on the type of detector used) from the sample components passing through the cell, are monitored as changes in the output voltage. These voltage changes are recorded on a strip chart recorder and frequently are fed into an integrator or computer to provide retention time and peak area data.  The most commonly used detector is the ultraviolet absorption detector. A variable wavelength detector of this type, capable of monitoring from 190 - 460 nm. Detection Systems  Generally the most expensive part of the instrument  Depends on analyte properties, and required sensitivity (and money) A) Ultraviolet/Visible absorption (may extend to IR)  Most commonly used, LOD = 1pg, 0.1-1mg for IR Three major types:  The simplest (and cheapest) uses a mercury source & can only detect 254nm and 280nm  Tungsten and Deuterium sources used, together with interference filters  The best (and most expensive) uses a diode array to monitor the spectrum of the eluent over a wide range of wavelengths B) Fluorescence  LOD = 10fg  Monitors emission at specific wavelengths, in response to excitation with a specific wavelength Detection Systems cont’d---C) Refractive Index  LOD = 10ng  Monitors changes in the refractive index of the solvent, caused by the presence of analytes  Universal, but non-specific, not very sensitive D) Electrochemical  LOD = 100fg  May be potentiometric, conductometric or amperometric  Most common is conductivity (espcially for ion exchange and capillary electrophoresis) E) Conductivity  Simple, cheap, and robust  Need to solve the problem of detecting analyte ion in presence of large quantities of other ions (eluent)