Guide Selective Sample Handling and Detection in High-Performance Liquid Chromatography

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This reduces the adsorption peak from tailing, giving narrower peaks. Each group uses a set of the 7 vials containing different concentrations of the standard solutions Table 1. The first 3 are used to identify each peak, and the last 4 are for creating a calibration chart for each component. Standards 1—3 are also used for the calibration chart. Table 1. Volumes of stock standards used to prepare the 7 provided working standards total volume of each standard is 50 mL.

The three components that need to be made are caffeine 0.

These concentrations, once diluted in the same fashion, put the standards at the levels found in the soda samples. The three components all have differing distribution coefficients, which affects how each interacts with both of the phases. The larger the distribution coefficient, the more time the component spends in the stationary phase, resulting in longer retention times in reaching the detector.

Figure 1. The chromatogram of the 3 components. From left to right, they are caffeine, aspartame, and benzoate. This sufficiently gets rid of any gases in the samples. Figure 2. High-performance liquid chromatography, or HPLC, is a highly versatile technique that separates components of a liquid mixture based on their different interactions with a stationary phase. HPLC is an adaptation of column chromatography. In column chromatography, a column is packed with micro-scale beads called the stationary phase. The stationary phase beads are functionalized with chemical groups that induce an interaction between the bead and the components of a mixture located in the liquid, or mobile phase.

As the mixture flows through the column, the components interact with the stationary phase differently. In HPLC, column chromatography is performed at a higher flow rate, and therefore higher pressure, than classical column chromatography. This enables the use of smaller stationary phase beads with a greater surface area to volume ratio, which greatly increases the interaction of the stationary phase and components in the mobile phase.

This video will introduce the basics of the operation of HPLC by demonstrating the separation of components of various diet sodas. There are two types of HPLC used in the laboratory: analytical, and preparative. In analytical HPLC, the instrument is used to identify components of a small volume, and the analyzed sample is then discarded as waste. In preparative HPLC, the instrument is used to purify a mixture and a desired amount of each component is collected in fractions. The HPLC instrumentation consists of a series of simple components. First, the mobile phase, held in solvent reservoirs, is pumped through the system by one or more pumps at a constant flow rate.

The sample is injected into the mobile phase stream by the sample injector. The sample, diluted by the mobile phase, is then delivered to the HPLC column, where the components of the sample are separated. The components are then analyzed by the detector, and either saved in fractions for later use, or transferred to a waste bottle.

Nature of Samples

The HPLC column is the key component to the system. It is composed of a metal or plastic cylinder, packed with micro-scale beads of stationary phase, or chromatography resin. The sample mixture flows through the packed particle bed at a constant flow rate and each component interacts with the stationary phase as it flows by. The compounds interact with the stationary phase differently, and therefore travels down the length of the column to the detector at a different rate.

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HPLC - Detectores

The time required for a component to exit the column, or elute, is called the retention time. The result is a plot of retention time vs. The retention time is used to identify the component. The peak size, specifically the area under the peak, is used to quantify the amount of the compound in the initial solution.

Gas Chromatography

The choice of stationary phase depends on the properties of the components in the sample mixture. The most commonly used stationary phase is silica beads, as they are an inert nonpolar material that forms micro-scale beads, and achieves sufficient packing density. The most common type of HPLC is reversed-phase chromatography, which utilizes a hydrophobic stationary phase, typically silica beads with C18 chains bonded to the beads' surface.

The components are eluted in order of decreasing polarity. The mobile phase used in reversed-phase chromatography is typically a mixture of water and an organic solvent, such as acetonitrile. Depending on the sample, the mobile phase can remain a constant ratio of water and organic solvent, known as isocratic mode. The mobile phase ratio can also be changed linearly or stepwise during the separation, to create a mobile phase gradient. A gradient elution can prevent peak broadening of the less polar components, thereby improving the separation and shortening the elution time.


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In this experiment, HPLC will be used to separate and quantify three common components of diet soda. First, to prepare the mobile phase, add mL of acetonitrile to 1. Then carefully add 2. Dilute the solution to a total volume of 2 L. The resulting solution should have a pH between 2. Adjust the pH to 4. Filter the mobile phase through a 0. It is important to degas the solution, as bubbles can cause voids in the stationary phase, or work their way to the detector cell and cause instability in measurements.

Prepare three component solutions of caffeine, benzoate, and aspartame, which are three typical components of diet sodas. These component solutions are then used to prepare the standard solutions that will be utilized to determine the unknowns.

Analytical methods

Prepare mL of the caffeine and benzoate solutions. Prepare mL of the aspartame component solution. Store the solution in the refrigerator when not in use to avoid decomposition. Next, prepare 7 standard solutions, each with different concentrations of caffeine, benzoate, and aspartame. Pipet the proper amount of each component into a volumetric flask, and dilute to the mL mark with mobile phase. The first 3 solutions each contain one component, to enable peak identification.

The other 4 solutions contain a range of concentrations of all 3 components, in order to correlate peak height to concentration. Pour each standard solution into a labeled vial in a sample rack. Store the sample rack with samples and the remaining solutions in the refrigerator. First, set up the mobile phase and waste containers. Ensure that the waste lines are fed into a waste container, and are not recycling back into the mobile phase. Ensure that the inlet mobile phase line is fed into the mobile phase container. Verify that the flow rate of the mobile phase is set to 0.

This flow rate will enable all components to elute within 5 min, but is slow enough to ensure resolution of individual peaks.

Selective Sample Handling and Detection in High Performance Liquid Chromatography: Pt. B

Next, verify the minimum and maximum pressures on the solvent delivery system. These settings shut the pump off in case of a leak or clog, respectively. Press "zero" on the detectors front panel, to set the blank. Then fill the syringe with that solution. Begin with the 3 single-component samples in order to identify the peak of each component.

Next, manually inject the solution, by placing the injector handle in the load position. Verify that the data collection program is set to collect data for s, which allows for enough time for all 3 peaks to elute through the detector. When ready to begin the trial, rotate the injector handle to the inject position, in order to inject the sample into the mobile phase. It is using a mobile liquid or gaseous phase that is adsorbed onto the surface of a stationary solid phase. The equilibration between the mobile and stationary phase accounts for the separation of different solutes.

In this mode a polar sorbent i. The limited stability and reproducibility, as well as the bad peak shape which is frequently inherent with the analysis of neutral basic compounds explains its limited popularity. In most applications adsorption LC can be replaced by normal-phase NP partition chromatography. Nowadays, adsorption chromatography has been replaced by partition chromatography. This form of chromatography is based on a thin film formed on the surface of a solid support by a liquid stationary phase. The solutes equilibrate between the mobile phase and the stationary liquid.

Partition chromatography can be performed in the NP and in the reversed-phase RP mode. The most popular sorbents are the amino- and the cyanopropyl sorbent. Both sorbents can be used for the separation of neutral polar compounds. The cyanopropyl sorbent can be used in combination with both polar and non-polar eluents.

The use of NP-LC can be advantageous in case of fluorescence detection. Adsorption chromatography LSC. In this case a non-polar sorbent is combined with polar eluents.

Column Oven

The positive features of RP-LC are a fast equilibration, excellent stability, good reproducibility and peak symmetry, as well as a large application area. For neutral compounds, in principle, RP-LC is the method of choice. The most popular sorbents are the cyanopropyl, octyl and octadecyl phases.