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Unsurprisingly, all 3 sodas tested contained roughly the same amount of the preservative benzoate. However, the Coke products contained more caffeine. The calculated values for all components correlated well to reported values by the manufacturers. HPLC is often used to purify peptide molecules.

In this example, transmembrane peptide complexes were prepared, and then stabilized by oxidative crosslinking the proteins with disulfide bonds.


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The proteins were then dissolved in formic acid, and purified using reversed phase HPLC. The sample was then eluted using a linear gradient of two solvents, and the purity confirmed with mass spectrometry. HPLC can also be used to identify organic compounds synthesized in the lab. In the Miller-Urey experiment, the abiotic synthesis of organic compounds on primordial earth was studied. Primordial gases, such as methane and ammonia, were introduced to a flask containing water, simulating early oceans.

Electrical discharge was then applied, imitating lightning on primordial earth. The water was then analyzed using HPLC coupled with mass spectrometry, and compared to known amino acid standards. You should now understand the basics of running the instrument, and analyzing the resultant data. The HPLC chromatograms are able to quantify each of the 3 components for all the samples based upon the calibration curves of the standards Figure 3.

From this set of experiments, it was determined that a oz can of these diet sodas contained the following amounts of each component:. Diet Coke: Coke Zero: Diet Pepsi: Not surprisingly, all 3 had roughly the same amount of benzoate, as it is just a preservative. The Coke products had a bit more caffeine, and the Coke Zero had much less aspartame than the other two sodas, as it also includes citric acid for some flavoring. The following numbers are the actual amounts of caffeine and aspartame in a oz can of the 3 diet sodas The caffeine content was obtained from the Coca-Cola and Pepsi websites.


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  • The aspartame content was obtained from both LiveStrong. Diet Coke: 46 mg caffeine; Sample Calculations Table 2 :. Concentration of caffeine in STD 1: The component solution for caffeine had 0. STD 1 had 1 mL of this solution diluted to STD 2 had 2 mL of this solution diluted to The results from the three calibration graphs Figure 4 yielded the following equations:. Since the sample was diluted by a factor of 2, the Diet Coke had Figure 3. The HPLC chromatograms of the 5 standards and the 3 samples. Figure 4. The calibration curves for each of the 3 components.

    Table 2. The data tables for the HPLC trials used for generating the calibration curves. HPLC is a widely-used technique in the separation and detection for many applications. It is ideal for non-volatile compounds, as gas chromatography GC requires that the samples are in their gas phase. Non-volatile compounds include sugars, vitamins, drugs, and metabolites. Also, it is non-destructive, which allows each component to be collected for further analysis such as mass spectrometry.

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    The mobile phases are practically unlimited, which allows changes to the polarity of pH to achieve better resolution. The use of gradient mobile phases allows for these changes during the actual trials. There has been concern over the possible health issues that may be associated with the artificial sweetener aspartame. Current product labeling does not show the amount of these components inside of the diet beverages.

    This method allows for quantifying these amounts, along with the caffeine and benzoate. Other applications include determining the amounts of pesticides in water; determining the amount of acetaminophen or ibuprofen in pain reliever tablets; determining whether there are performance-enhancing drugs present in the bloodstream of athletes; or simply determining the presence of drugs in a crime lab. While the concentrations of these samples, and often the identity of the components, can be readily determined, the one limitation is that several samples could have close to identical retention times, resulting in co-eluting.

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    Overview Source: Dr. Paul Bower - Purdue University High-performance liquid chromatography HPLC is an important analytical method commonly used to separate and quantify components of liquid samples. Principles During an HPLC experiment, a high-pressure pump takes the mobile phase from a reservoir through an injector. Procedure 1. Making the Mobile Phase Prepare the mobile phase by adding mL of acetonitrile to approximately 1. Carefully add 2. Dilute the solution to a total volume of 2. Add very slowly once the pH reaches 4. This should take around 50 drops to accomplish.

    It is important to degas the mobile phase to avoid having a bubble, which could either cause a void in the stationary phase at the inlet of the column or work its way into the detector cell, causing instability with the UV absorbance. Creating the Component Solutions The three components that need to be made are caffeine 0.

    Add 0.

    Practical High-Performance Liquid Chromatography, 4th Edition

    Place this solution in a refrigerator to avoid decomposition during storage. Making the 7 Standard Solutions The three components all have differing distribution coefficients, which affects how each interacts with both of the phases. Following the chart in Table 1 , pipet the proper amount of each component into a mL volumetric flask.

    Dilute each of the stock solutions to the mL mark on the volumetric flasks with mobile phase.

    Pour each standard solution into labeled small vials in a sample rack. This is high enough to allow all peaks to elute within 5 min and slow enough to allow for nice resolution. Verify that the minimum and maximum pressure and the flow rate are set to the correct values on the front panel of the solvent delivery system the pump. Minimum pressure setting: psi this is to shut off the pump, if a leak occurs. Maximum pressure setting: 4, psi this is to protect the pump from breaking, if a clog forms. Press "zero" on the detector's front panel in order to set the blank the blank is the pure mobile phase.

    Start with the 3 single-component samples, which allows for identifying the peak of each component of interest. Verify that the data collection program is set to collect data for s, which allows enough time for all 3 peaks to elute through the detector. When ready to start the trial, rotate the injector handle to the inject position which injects the sample into the mobile phase and click "Start Trial" on the computer data collection program immediately.

    For standards , only one of the three sequential peaks appear on the screen during the run Figure 1. Save the data under a suitable file name e. Analytical HPLC. Preparative HPLC. Separation of Enantiomers. Special Possibilities. ReversedPhase Chromatography. Chromatography with Chemically Bonded Phases. How to Perform the Instrument Test. Theoretical Principles. Permissions Request permission to reuse content from this site.

    Practical High-Performance Liquid Chromatography

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