HPLC is a popular method of analysis because it is easy to learn and use and is not limited by the volatility or stability of the sample compound.
In isocratic HPLC the analyte is forced through a column of the stationary phase (usually a tube packed with small round particles with a certain surface chemistry) by pumping a liquid (mobile phase) at high pressure through the column. The sample to be analyzed is introduced in a small volume to the stream of mobile phase and is retarded by specific chemical or physical interactions with the stationary phase as it traverses the length of the column. The amount of retardation depends on the nature of the analyte, stationary phase and mobile phase composition. The time at which a specific analyte elutes (comes out of the end of the column) is called the retention time and is considered a reasonably unique identifying characteristic of a given analyte. The use of pressure increases the linear velocity (speed) giving the components less time to diffuse within the column, leading to improved resolution in the resulting chromatogram. Common solvents used include any miscible combinations of water or various organic liquids (the most common are methanol and acetonitrile). Water may contain buffers or salts to assist in the separation of the analyte components, or compounds such as Trifluoroacetic acid which acts as an ion pairing agent.
A further refinement to HPLC has been to vary the mobile phase composition during the analysis, this is known as gradient elution. A normal gradient for reverse phase chromatography might start at 5% methanol and progress linearly to 50% methanol over 25 minutes, depending on how hydrophobic the analyte is. The gradient separates the analyte mixtures as a function of the affinity of the analyte for the current mobile phase composition relative to the stationary phase. This partitioning process is similar to that which occurs during a liquid-liquid extraction but is continuous, not step-wise. In this example, using a water/methanol gradient, the more hydrophobic components will elute (come off the column) under conditions of relatively high methanol; whereas the more hydrophilic compounds will elute under conditions of relatively low methanol. The choice of solvents, additives and gradient depend on the nature of the stationary phase and the analyte. Often a series of tests are performed on the analyte and a number of generic runs may be processed in order to find the optimum HPLC method for the analyte - the method which gives the best separation of peaks.
These new “HPLC” instruments could develop up to 6,000psi (400 bar) of pressure, and included improved detectors and columns. HPLC really began to take hold in the mid to late 1970’s. With continued advances in performance, the name was changed to High Performance Liquid Chromatography (HPLC). High Performance Liquid Chromatography (HPLC) is now one of the most powerful tools in analytical chemistry, with the ability to separate, identify and quantitate the compounds that are present in any sample that can be dissolved in a liquid. Today, trace concentrations of compounds, as low as “parts per trillion” (ppt), are easily obtained. HPLC can be applied to just about any sample, such as pharmaceuticals, food, nutraceuticals, cosmetics, environmental matrices
Applications for HPLC
Preparative HPLC refers to the process of isolation and purification of compounds. Important is the degree of solute purity and the throughput, which is the amount of compound produced per unit time. This differs from analytical HPLC, where the focus is to obtain information about the sample compound. The information that can be obtained includes identification, quantification, and resolution of a compound.
Chemical Separations can be accomplished using HPLC by utilizing the fact that certain compounds have different migration rates given a particular column and mobile phase. Thus, the chromatographer can separate compounds (more on chiral separations) from each other using HPLC; the extent or degree of separation is mostly determined by the choice of stationary phase and mobile phase.
Purification refers to the process of separating or extracting the target compound from other (possibly structurally related) compounds or contaminants. Each compound should have a characteristic peak under certain chromatographic conditions. Depending on what needs to be separated and how closely related the samples are, the chromatographer may choose the conditions, such as the proper mobile phase, to allow adequate separation in order to collect or extract the desired compound as it elutes from the stationary phase. The migration of the compounds and contaminants through the column need to differ enough so that the pure desired compound can be collected or extracted without incurring any other undesired compound.
--HPLC of Proteins and Polynucleotides
Identification of compounds by HPLC is a crucial part of any HPLC assay. In order to identify any compound by HPLC a detector must first be selected. Once the detector is selected and is set to optimal detection settings, a separation assay must be developed. The parameters of this assay should be such that a clean peak of the known sample is observed from the chromatograph. The identifying peak should have a reasonable retention time and should be well separated from extraneous peaks at the detection levels which the assay will be performed. To alter the retention time of a compound, several parameters can be manipulated. The first is the choice of column, another is the choice of mobile phase, and last is the choice in flow rate. All of these topics are reviewed in detail in this document.
Identifying a compound by HPLC is accomplished by researching the literature and by trial and error. A sample of a known compound must be utilized in order to assure identification of the unknown compound. Identification of compounds can be assured by combining two or more detection methods.
Quantification of compounds by HPLC is the process of determining the unknown concentration of a compound in a known solution. It involves injecting a series of known concentrations of the standard compound solution onto the HPLC for detection. The chromatograph of these known concentrations will give a series of peaks that correlate to the concentration of the compound injected.