HPLC - What is it?

Hewlett Packard HPLC sale: You know you have to use HPLC in your laboratory...you haven't done it yourself...so where do you start?.....

High Performance Liquid Chromatography (HPLC) is an analytical process utilizing special instruments designed to separate, quantify and analyze components of a chemical mixture. Samples of interest are introduced to a solvent flow path; carried through a column packed with specialized materials for component separation; and component data is obtained through the combination of a detection mechanism coupled with a data recording system. All this occurs under pressures which may reach or exceed 6,000 psi! The basic components of an HPLC system include solvent resevoir sample injector pump(s) analytical column detector(s) data recorder waste container (or fraction collector) Other important elements are an inlet solvent filter, post-pump inline filter, sample filter, precolumn filter, guard column, back-pressure regulator and/or solvent sparging system. The function of each of these components is briefly

Model 1050 HPLC System Including: 1050 Quaternary HPLC Pump 79852A, 1050 Variable Wavelength UV-VIS Detector 79853, 1050 21 Position Auto sampler 79855A, Computer and Monitor, ChemStation. Installation and Training Options are Available. Other Pump, Detector and Autosampler Options are available. HP 1050 autosampler HP 1050 quaternary pumps HP 1050 interface 35900-C HP UV 79853 detector Computer and new Chemstation analysis and control software HP 1046A fluorescent detectors available

$ Inquire at 763-712-8717 Other HPLC arrivals: HP 1090 series, with uv or pda Beckman system Gold HPLC Gilson (very affordable system) Hitachi components Shimadzu 10 series Waters 600E system with uv vis detector Kontron HPLC pumps Pharmacia FPLC Perceptive Biosystems BioCAD

described below. An HPLC system begins with the solvent reservoir, which contains the solvent used to carry the sample through the system. The solvent should be filtered with an inlet solvent filter to remove any particles that could potentially damage the system's sensitive components. Solvent is propelled through the system by the pump. This often includes internal pump seals, which slowly break down over time. As these seals break down and release particles into the flow path, an inline solvent filter prevents any post-pump component damage. The next component in the system is the sample injector, also known as the injection valve. This valve, equipped with a sample loop of the appropriate size for the analysis being performed, allows for the reproducible introduction of sample into the flow path. Because the sample often contains particulate matter, it is important to utilize either a sample filter or a precolumn filter to prevent valve and column damage. Some systems utilize an automatic injector or autosampler to accomplish this task. Following the injector, an analytical column allows the primary sample separation to occur. This is based on the differential attraction of the sample components for the solvent and the packing material within the column. However, a sacrificial guard column is often included just prior to the analytical column to chemically remove components of the sample that would otherwise foul the main column. Following the analytical column, the separated components pass through a detector flow cell before they pass into the waste reservoir. The sample components' presence in the flow cell prompts an electrical response from the detector, which is digitized and sent to a recorder. The recorder helps analyze and interpret the data. Recorders can be as simple as a pen recording what happens in the detector onto moving chart paper or as sophisticated as a computer with powerful software for hardware control and data interpretation. As a final system enhancement, a back pressure regulator is often installed immediately after the detector. This device solvent bubble

Water 600E systems

When molecules in a mixture are very similar, direct quantification becomes difficult. HPLC is a separating technology used to separate very similar molecules from a mixture and then quantify them accurately. It is a member of a group of technologies known as "CHROMATOGRAPHY". In chromatography, the components are separated on the basis of their chemical composition and structure. Any molecule present in the mixture will have its own unique composition and structure and therefore will have different interaction with its surrounding. These interactions make them all move uniquely on the support provided to it. This support is known as "stationary phase" and the energy to move is provided by a liquid or a gas "mobile phase". On the basis of the material being used as either phase, the chromatographic technique is titled accordingly. HPLC (High Performance Liquid Chromatography) is a mode of chromatography wherein the stationary phase is used in form of fine particles packed in a cylindrical column made up of inert material like stainless steel. Stationary phase adsorbs the analytes and holds them reversibly for particular time. All the molecules in the mixture will spend different time with different stationary phase and therefore come out at the end one by one. Adsorption being the base of separation, usage of fine particles offers more surface area for the molecules to interact with. The time that a molecule takes to travel from one end to other end of the stationary phase is known as the RETENTION TIME. Hence all molecules will display their own unique retention time for a particular stationary-mobile phase combination. Selection of stationary phase and mobile phase is done on the basis of the molecule of interest to get optimum retention time. As a result, HPLC acquires a high degree of versatility not found in other chromatographic systems and it has the ability to easily separate a wide variety of chemical mixtures. Silicagel is the most frequently used adsorbent. The adsorbent is packed in a column. The density of the packing directly affects the resolution. Hence the HPLC columns are densely packed with the adsorbent to achieve significant degree of resolution. But very high density of packing demands for the mobile phase to be introduced at very high pressure to generate effective flow. So the technique is also sometimes named as HIGH PRESSURE LIQUID CHROMATOGRAPHY. At the end of the column, a detector is kept. As the molecules come out from the end, the detector detects and gives signal. Till the molecules keeps traveling through the detector's path it keeps detecting it and gives a peak on CHROMATOGRAM. The peak area calculation quantifies the molecule of interest on the basis of standard values. Remarkable interference can be observed by minor

formation until the solvent is completely through the detector. This is important because bubbles in a flow cell can interfere with the detection of sample components. Alternatively, an inert gas sparging system may be installed to force dissolved gasses out of the solvent being stored in the solvent reservoir. Each of the components described above requires fittings to couple it into a system. It is important to note that improper selection or installation of these fittings can lead to leaks or the formation of dead volume, both of which can result in poor HPLC performance. GMI can assist in component choice or complete system configuration to accomplish most lab and production requirements. We spend extra time in our reconditioning 'up front' so that you, our customer, receive a COMPLETE working system upon arrival to your laboratory. HPLC LINGO: ELUTION: This term describes the transport of a species through the stationary phase by the continuous flow (addition) of mobile phase. ELUANT: Mobile phase that carries the sample through the column. ELUATE or EFFLUENT: Mobile phase with separated components after they emerge from the column. ISOCRATIC ELUTION: A separation in which the mobile phase composition remains unaltered. The mobile phase may comprise of a single solvent or a pre-mixed mixture of solvents. GRADIENT ELUTION: HPLC is frequently used for the separation of mixtures that contain compounds with a wide range of polarities. In such situations, isocratic conditions may not provide an acceptable separation (i.e., it is not possible to obtain sufficient resolution or the separation takes an unacceptably long period of time). To solve these problems, the composition of the mobile phase is changed during the separation. Two or three solvents that differ in polarity are employed. After sample introduction,

changes in the stationary and mobile phase composition, purity and/or density. Therefore it is must to get standard grade reagents for HPLC. Such quality control requirements makes HPLC a little expensive technology as compared to other similar technologies. GMI staff have performed chromatography and can assist you with your technology selection. You never have to be embarrassed by your level of knowledge with these technologies since it is our goal to HELP you with intelligent and educated decisions in procuring equipment. We will never 'over sell' you or talk you into unneeded purchases unlike some 'dealers' with the sole goal of unloading their inventory.

GMI IS
on the lookout for your un-needed instrumentation especially your HP / Agilent HPLC's, Waters, and Perceptive Biosystems BioCADS

DISTRIBUTION CONSTANT: It describes the equilibrium involving the transfer of an analyte between the mobile and stationary phases. This constant, also called partition ratio or partition coefficient, is defined as K= Cs/Cm, a ratio of the analyte molar concentration in the stationary phase to that in the mobile phase. RETENTION TIME, t: The time taken by the analyte peak to reach the detector after sample introduction is called the retention time. A more accurate measure of the retention time of an analyte is obtained by subtracting from this value the time taken for an unretained solute to emerge from the column (i.e., the dead time, t0), resulting in the adjusted retention time, t. The retention time is the most important parameter for component identification under set experimental conditions. CAPACITY FACTOR (or Retention Factor) k : A measure of the retention volume (or time) of a particular component of the sample with a given combination of stationary phase and mobile phase. It is defined for species A as k A = (tA-t0)/t0, or, kA = tA/t0 where t0 is the retention time for an unretained compound and tA =

the ratio of these solvents is programmed to vary either continuously or in steps, resulting in enhanced separation efficiency. The terms binary gradient, ternary gradient, and quaternary gradient refer to the use of 2, 3, and 4 solvents, respectively, to make up the mobile phase composition in a gradient elution method. CHROMATOGRAM: When a detector that responds to solute concentration is placed at the column outlet, a plot of the generated signal versus time (or volume of mobile phase) is called a chromatogram. Such a plot, which usually comprises of one or more peaks, may be used for qualitative and quantitative purposes the location of a peak on the time (or volume) axis serves to identify the component, and the area under the peak provides a quantitative measure of that component. SENSITIVITY: Minimum limit of detection of a given species. Determined by the smallest ratio of peak height-to-baseline noise (signal-to-noise ratio) that allows accurate and reproducible determination of peak height or area. This varies with detection method, instrument used and species being detected. UNRETAINED COMPOUND: Component of a mixture that moves through a column at the same rate as the mobile phase, i.e., migration is not retarded by physical or chemical interaction with the stationary phase. VOID VOLUME: Total volume of mobile phase in a fully wet packed column - the space between the particles of the stationary phase (interstitial volume) plus the volume within the particles (pore volume). It is also defined as the volume of mobile phase required to carry an unretained component through a column. RESOLUTION: Measure of the degree of separation between two successively eluting components in a chromatographic run (two adjacent peaks in a chromatogram). The resolution between species A and B may be expressed as: R = 2 [tB tA]/ (WA + WB), where t and W correspond to the retention time and peak width

adjusted retention time of species A. ALPHA ( ) VALUE: A measure of the separation of any two components under a given set of conditions. It is defined for two components A and B as: = k A/k B (where k is the respective capacity factor). It is the ratio of the retention volumes of the two components; i.e., their relative retention. BAND (or Zone): Layer of sample or component moving through the stationary phase, carried by the mobile phase. Represented on the chromatogram as a peak when it emerges from the column. BAND-BROADENING (or Zone-broadening): Spreading of the sample or component band. Arises due to inefficiency of the column bed or inappropriate choice of mobile phase. COLUMN EFFICIENCY: Degree to which species flow through the column as "bands", without being spread; less band-broadening implies less likely overlap of peaks in the chromatogram. Efficiency is expressed numerically as Plate Count, N, or as a Height Equivalent to Theoretical Plate (HETP), H. It is a measure of the quality of a filled column. HETP, H = L/N number of plates N = 16(t/W)2 where L = column length, t = retention time, and W = peak width at baseline. The origin of these terms is from the treatment of the chromatographic column as made up of a number of discrete narrow bands called theoretical plates, similar to a distillation column.

at baseline, respectively. A resolution value of 1.5 implies a complete separation of the two species