1IntroductionFor this experiment we will be using capillary electrophoresis (CE). CE is an analytical lab technique that allows for the separation of different components within a compound by applying a voltage potential and exploiting their differences in electrophoretic mobility, µcp. Electrophoretic mobility describes an analyte’s ability to move in an electric field and is dependent upon its atomic radius, ionic charge, viscosity, and the strength of the applied electric field. In the event that two species are of the same atomic size, the one with the larger charge willhave a higher migration rate. In the case where two atoms contain the same charge, the atom withthe smaller radius will have the higher migration rate. Larger applied voltages are also associatedwith higher migration rates. Species that contain no charge, i.e. neutral species, do not react to applied electric fields and are thus immobile, meaning CE is not useful for analyzing such compounds. In order to properly understand capillary electrophoresis one must first examine the instrumentation behind the technique. An example of a typical instrumental set-up for CE is depicted below in Figure 11. Most CE instruments usually consist of: a high voltage power supply connected to the anode and cathode, some sort of introduction system (sample trays), a capillary to connect the source and destination vials, a detection method, and an output device such as a computer1. In addition, a temperature control device can also be included as part of the instrumentation. This is to ensure there are no fluctuations in temperature that could cause avariance in viscosity, a property that we want to keep constant for each analyte.2Figure 1. Block diagram of a typical set-up for Capillary Electrophoresis Instrument1.Each side of the capillary tube is placed inside of an aqueous buffer that contains the electrolytic species of study. Prior to beginning the experiment, it is important to make sure the capillary tube is rinsed out using the buffer that is going to house the species being studied to avoid any cross contamination. Capillary tubes are usually made of fused silica and are sometimes coated with polyimide when high heat resistance is necessary2. Capillary tubes are also very useful due to their thin dimensions, which creates a high surface to volume ratio and eliminates overheating by high voltages1. The high voltage power supply is connected to both an anode and a cathode in order to create an electric field that can be used to induce migration of thesample through the capillary. In regards to detection, the cathodic end of capillary tubes usually contain a small opening that allows for researchers to shine UV-VIS light through and measure absorbance in order to determine sample concentration. In addition, a mass spectrum can also be constructed to determine mass to charge ratios of the ionic samples being studied via a photomultiplier tube that is connected at the cathodic end1. Once the species being analyzed reaches the detector, its components can then be identified based on their respective retention times and quantified via constructed calibration curves3.3Whenever a species moves under an applied voltage, the force that the ion experiences is equal to the magnitude of the net charge multiplied by the strength of the electrical field. The migration velocity, V, in a given electric field is governed by the following relationship3:V (cm s-1) = distance¿detector(Ld )¿migration time(t m)The electric field strength, E, is dependent upon the magnitude of the applied potential inaddition to the length over which the potential is applied and is represented via the following relationship3:E (V cm-1) = applied voltageTotal Column Length(L)Finally, electrophoretic mobility, µcp, i.e. the analytes mobility in the electric field, is given by the equation3: µcp (cm2 V-1s-1) = migration velocity(V )field strength(E)For this lab we are using capillary electrophoresis in order to determine the composition of commercially available analgesics (painkillers). Specifically, we are analyzing analgesics in order to find their commonly used active ingredients: caffeine, acetylsalicylic acid, salicylic acid,and acetaminophen. In addition, we will be quantifying the amount of acetaminophen within each analgesic. This will be achieved by constructing a calibration curve using five solutions of acetaminophen with molarities ranging from 0.5 mM to 50 mM. We will be using 50 mM of caffeine, salicylic acid, acetaminophen, and 1 mM of acetylsalicylic acid. We hypothesize that the manufacture Care One gave an accurate concentration of the acetaminophen found in their migraine relief and pain relief pills. We believe aspirin will not contain acetaminophen, as the manufacture did not include acetaminophen in the list of active4ingredients. We would expect aspirin and acetylsalicylic acid to have a similar elution times, since aspirin is predominantly acetylsalicylic acid. We would expect salicylic acid to have a similar retention time to acetylsalicylic acid, as it is a derivative of acetylsalicylic acid. MethodsFor this experiment we use a Beckman P/ACE MDQ Capillary Electrophoresis with a UV detector and 32KARAT software. The capillary column length to the UV detector (Ld) is 50 cm, the total capillary column length is 55 cm, the applied potential is 20 kV and the UV-Vis detector is set to 254 nanometers. We used caffeine 50 mM, salicylic acid 50 mM, and 1mM acetylsalicylic acid as qualitative standards. We also created a calibration curve with acetaminophen with concentration: 1 mM,7 mM, 20 mM, 35 mM, and 50mM. Are three unknowns were Care One low dose aspirin (baby aspirin), Care One migraine relief, and Care One pain relief. Six baby aspirin pills, one migraine relief pill, and one pain relief pill were each weighed and then crushed. The tablet were then dissolved in ~50 ml of Millipore water, then theydissolve tablets were transferred to 45 ml centrifuge tubes. The samples were centrifuged at an intermediate speed to remove insoluble materials. The supernatant was transferred to a 1 liter volumetric flask and diluted to a total volume of 1 liter with Millipore water. The prepared samples were transferred to CE vials and loaded into the sample tray. The tray started with salicylic acid (A1), then caffeine, acetylsalicylic acid, aspirin, migraine relief, and then pain relief. The next column of the tray include the acetaminophen