2501 High Performance Liquid Chromatography

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1 2501 High Performance Liquid Chromatography High Performance Liquid Chromatography Scheme Chp25:: 1 High Performance Liquid Chromatography

2 Components of HPLC High Performance Liquid Chromatography Scheme The basic liquid chromatograph consists of six basic units. The mobile phase supply system, the pump and programmer, the sample valve, the column, the detector and finally a means of presenting and processing the results. A block diagram of the basic liquid chromatograph is shown. Chp26: 2 High Performance Liquid Chromatography

HPLC Instrumentation High Performance Liquid Chromatography (HPLC) is an analytical technique for the separation and determination of organic and inorganic solutes in any samples especially

3 HPLC Instrumentation High Performance Liquid Chromatography (HPLC) is an analytical technique for the separation and determination of organic and inorganic solutes in any samples especially biological, pharmaceutical, food, environmental, industrial, etc. In a liquid chromatographic process a liquid permeates through a porous solid stationary phase and elutes the solutes into a flow-through detector. The stationary phase is usually in the form of small-diameter (5-10 mm) uniform particles, packed into a cylindrical column. The typical column is constructed from a rigid material (such as stainless steel or plastic) and is generally 5-30 cm long and the internal diameter is in the range of 1-9 mm. 3 High Performance Liquid Chromatography

Principles Chromatography is the term used to describe a separation technique in which a mobile phase carrying a mixture is caused to move in contact with a selectively absorbent stationary phase.

4 Principles Chromatography is the term used to describe a separation technique in which a mobile phase carrying a mixture is caused to move in contact with a selectively absorbent stationary phase. Different components of the sample are carried forward at different rates by the moving liquid phase, due to their differing interactions with the stationary and mobile phases. There are a number of different kinds of chromatography, which differ in the mobile and the stationary phase used. HPLC: In HPLC, the mobile phase is a solvent. This solvent is pumped under high pressure through a column. The Stationary Phase is a finely divided solid held inside the column. HPLC Vs. GC: Comparison of HPLC over Gas Chromatography Less volatile and larger samples can be used with HPLC. It was discovered that better separation of the components of the mixture occurs if the particles in the stationary phase are very small. However, it was also found that if very small particles were used in the column, then the liquid passed very slowly through the column. Therefore, a pump is used to force the liquid through the column. This is not necessary in GC but a shorter column can be used in HPLC because the separation is more efficient. 4 High Performance Liquid Chromatography

5 Mobile Phase; Solvent Delivery The mobile phase is either a single solvent or a blend of two or more having the appropriate eluting power for the sample components. It ranges from a nonpolar liquid to aqueous buffers mixed with an organic solvent. The solvent delivery system comprises a means of degassing, filtering and blending up to four solvents which are then delivered to the top of the column under pressure by a constant flow pump. Liquids samples or solutions are introduced into the flowing mobile phase at the top of the column through a constant or variable volume loop and valve injector that is loaded with a syringe. Column are straight lengths of stainless steel tubing tightly packed with a micro particulate stationary phase. The column packing are chemically modified silicas, unmodified silica or polymeric resins or gels. Solute are detected in the mobile phase as they are eluted from the end of the column. The detector generates an electrical signal that can be amplified and presented in the form of a chromatogram of solute concentration as a function of time. A dedicated micro computer is an integral part of a modern high performance liquid chromatograph. Software packages facilitate the control and monitoring of instrumental parameters, and the display and processing of data. 5 High Performance Liquid Chromatography

6 Putting the Pressure One of the biggest differences between HPLC and classical LC is, of course, pressure. High pressure not only profoundly affects run speed making quicker, more routine, and automated analysis practicable. High pressure increases the number of theoretical plates available. A variety of pump types are routinely used in HPLC. Syringe pumps, developed in the 1960s, use a motorized piston to push fluid from a reservoir into the column. The accuracy of solvent delivery of syringe pumps is generally better than that of other pumping systems. Single-piston pumps, including the fast fill variety (developed in the 1970s), have also achieved a measure of popularity. Fast-fill pumps use pistons made of sapphire or ruby; the single piston is driven through a housing into a Teflon seal using a motor-driven cam; it returns via spring action. Diaphragm pumps use a movable membrane to deliver fast-cycling flow that is useful for microbore column analyses. Single-piston pumps and others generally require pulse dampers to prevent uneven solvent flow, especially in more sensitive systems. For all systems, gradients can be formed using multi-pump systems to deliver the individual solvent components into mixing chambers that can equilibrate and then deliver the solvent at either high or low pressures. 6 High Performance Liquid Chromatography

7 Columns; The stationary phase HPLC columns are packed with very fine particles (usually a few microns in diameter). The fine particles are required to attain the low dispersion that give the high plate counts expected of modern HPLC. Plate counts in excess of 25,000 plates per column are possible but these are very rarely found with real samples because of the dispersion associated with injection valves, detectors, data acquisition systems and the dispersion due to the higher molecular weight of real samples as opposed to the common test samples. Packing small particles into the column is a difficult technical problem but even with good packing a great amount of care must be given to the column end fittings and the inlet and outlet connection to keep dispersion to a minimum. Some state of the art systems are now 'chip' based and may use no particles at all. LC columns, in general, achieve their separation by exploiting the different intermolecular forces between the solute and the stationary phase and those between the solute and the mobile phase. The column will retain those substances that interact more strongly with the stationary phase than those that interact more strongly with the mobile phase. Normal chromatography uses a polar stationary phase for all forms of partition chromatography. The use of a nonpolar stationary phase is known as reversed-phase (RP) partition chromatography. Horváth was one of the most powerful promoters of the use of RP-HPLC and its applications for a wide variety of separation tasks. In fact, the power of RP-HPLC for dealing with biological molecules has been so great that it has grown from an LC curiosity to become the most popular separation mode in HPLC. Typically, an octadecylsilica chemically bonded phase is attached to a highly purified and sized silica gel. The RP mobile phase consists of polar solvents such as methanol or acetonitrile. The addition of varying percentages of water increases polarity and provides a way to control retention and selectivity. 7 High Performance Liquid Chromatography

8 Detector HPLC detectors use the same detection principals as those used in LC techniques with extra care being given to the small solute elution volumes that result from the combination of high column efficiencies with small volumes. In order to give an accurate chromatographic profile the detector sampling (cell) volume must be a small fraction of the solute elution volume. If the detector volume were larger then the elution volume then you would have peaks that appeared with flat tops as the whole peak would be resident in the detector at the same time. This means that as column volumes decrease and system efficiencies increase the volume of the detector cell volume must also decrease. This is of course at odds for the requirement for detector to maintain high sensitivity as this is usually dependant on having a larger cell volume. Again, this requires the very careful design of modern detectors. 8 High Performance Liquid Chromatography

9 Characteristics of an Ideal Detector The ideal detector has the following characteristics good sensitivity, good stability, reproducibility, linear response over a few orders of magnitude, short response time, ease of operation, and is non-destructive in nature. UV/Vis Absorbance Detectors: Most modern UV/Vis detectors consist of a scanning spectrophotometer with grating optics. The independent or combined use of a Deuterium source (UV range, nm) with a Tungsten source (visible range, nm) provides a simple means of detecting absorbing species as they emerge from the column PDA Detectors: The most powerful UV/Vis absorbance detectors in use today are photodiode-array (PDA) based instruments that permit very rapid collection of data over a selected spectral range. Thus, spectral data for each chromatographic peak can be collected and stored. This stored data may then be compared with the spectrum of a pure standard from a library - a spectral analysis study of peak purity. Refractive Index (RI) Detector: RI detectors have the significant advantage of responding to nearly all solutes. The difference in the refractive index of the reference mobile phase versus the column effluent results in the detection of separated components as peaks on the chromatogram. The detection limits are usually lower than those observed with absorbance detectors. Electrochemical Detector: Detection based on amperometry is the most common electro-analytical method used in HPLC. Although these have not yet been exploited to the same extent as optical detectors, they offer the advantage of wide applicability in addition to sensitivity. Conductivity Detector: This instrument provides universal, reproducible, high-sensitivity detection of all charged species. This detector may be used with an HPLC system for the simple and reliable quantification of anions, cations, metals, organic acids, and surfactants down to the ppb level. The addition of a chemical suppressor between the column and conductivity detector serves to reduce the eluant conductivity, allowing the use of gradient elution and the determination of ppb levels with minimum baseline drift. 9 High Performance Liquid Chromatography

10 Applications Medicinals: The 1970s ushered in great demands for the biomedical and pharmaceutical researcher. The potential for therapeutic compound development was increasing, particularly because of new recombinant DNA techniques in biotechnology demonstrated in The pressure was mounting for increased quality and efficiency in diagnostics, drug development, and pharmacological analysis. Throughout the decade, successful and efficient HPLC separations were demonstrated for all varieties of pharmaceutical and clinically relevant molecules, from anticoagulants and antibiotics to hemoglobin and serotonin. With the advent of computer-controlled HPLC in 1979, these accomplishments led to the broad acceptance of such systems. Because researchers can readily use HPLC at both the analytical and preparative scale, its effects are felt in all facets of drug research and development, from early identification to large-scale processing purification. In addition, much attention was paid to HPLC sample preparation throughout the 1980s, particularly regarding biological fluids. Introduction of direct injection techniques, which use mobile phase or stationary phase adjustments to remove binding proteins from the analyte(s) of interest, and advancements in sampling automation, widened possibilities for disease marker and pharmacological applications. Biotech: An important motivation for the general transfer from GC and traditional LC techniques to HPLC was the need for improved analysis of large biomolecules, especially as pertaining to the biotechnology revolution. In the mid-1970s, researchers demonstrated initial HPLC separations for proteins by using both ion-exchange and size-exclusion modes; others demonstrated such separations for peptides using ion pair (trifluoroacetic acid) reversed-phase chromatography. 10 High Performance Liquid Chromatography

11 Summary 11 High Performance Liquid Chromatography

12 ...the future The extensive impact of HPLC cannot possibly be conveyed in this introduction course. It offers compelling advantages to any situation in which efficient separations of nonvolatile samples are of use. For instance, forensic analysis is certainly one field with areas ripe for HPLC exploitation. In fact, illicit drugs, like their legal counterparts, have been successfully identified for years with HPLC techniques. In addition, advancements in polynucleotide separations provide possible applications for DNA crime analysis. HPLC has appeared in a limited capacity in crime labs, but it has not yet seen widespread adoption as compared to traditional GC-MS methods, largely because it is not cost effective. For fuel scientists, HPLC has become the method of choice, replacing open-column LC for hydrocarbon compositional analysis of fossil fuels, such as petroleum distillates; such analysis provides information on selecting optimal feedstock processing parameters. HPLC modes have also risen to prominence in polymer and materials chemistry, as a means of performing in-depth structural analysis. Overall, the decreasing run times that are possible on modern LC columns have allowed synthetic and mechanistic chemists to use them in the rapid monitoring of chemical reactions. The list can go on and on. As with any technique that has widespread application, particularly in multibillion-dollar industries, there is continual interest in, and vast resources are invested in, optimizing its technical and methodological parameters. For example, scientists are intensively pursuing theoretical retention studies of myriad species peptides, ionizable compounds, and so forth. Scores of researchers are also developing new stationary phases, as well as improving the coupling between extraction, separation, and detection steps. These and other endeavors can only lead to a higher level of performance and to expanding amounts of information. 12 High Performance Liquid Chromatography

Chemistry Instrumental Analysis Lecture 31. Chem 4631

Chemistry Instrumental Analysis Lecture 31. Chem 4631 Chemistry 4631 Instrumental Analysis Lecture 31 High Performance Liquid Chromatography (HPLC) High Performance Liquid Chromatography (HPLC) High Performance Liquid Chromatography (HPLC) Solvent Delivery