Chemistry 3200 High Performance Liquid Chromatography: Quantitative Determination of Headache Tablets Liquid chromatography was developed by Tswett in early 1900 s and was shown to be a powerful separation method. The differential partitioning of solutes between the stationary phase packed in the column and the mobile phase flowing through the column is the basis of separation. It was recognized that if finely divided particles were used to pack the column the peak widths were narrower and sharper separations were obtained. Unfortunately, this also resulted in very slow flow rates and very long analysis times. Consequently, prior to around 1970, liquid chromatography was a slow separation method depending on gravity flow of the mobile phase through packed columns. Modern liquid chromatography, HPLC, was the result of advances in the theory of chromatography, the development of chemically bonded stationary phases, the use of packings of very small diameter (3-10 µm), and the development of pumps, valves, and flow-through detectors for handling fluids under high pressure. Very efficient columns only 3 cm in length packed with 3 µm totally porous particles which are capable of generating 5000-8000 theoretical plates are now in routine use with flow rates of up to 10 ml/min, although flow rates of 1 to 2 ml/min are more suitable for most separations. This experiment will demonstrate the separation of the compounds (acetylsalicylic acid, acetaminophen, and caffeine) commonly found in analgesics. The stationary phase consists of 5 µm silica beads with C 18 H 37 (octadecyl) groups bonded to the surface. The mobile phase is a solution composed of 0.2 % triethylamine, 0.2 % glacial acetic acid, 8.0 % acetonitrile, 88.0 % water and 3.6 % methanol water flowing at 2 ml/min. The mode of separation used here is called reversed phase chromatography, and it functions by partitioning the compounds between the highly lipophilic (oil loving) stationary phase and the more hydrophilic (water loving) mobile phase. In normal phase liquid chromatography (e.g. separations on silica gel), the more polar components show greater affinity for the stationary phase, and the less polar components favor the mobile phase. The other way to express this is to say that the more polar the solute, the greater is its partition coefficient, K; (K = C s /C m ). In reversed phase chromatography, the opposite behavior is observed; the more polar components favor the mobile phase (i.e. K is greater for the non-polar solutes). Another way to view these variations is: in normal phase chromatography, the polar phase is the solid substrate; in reverse phase chromatography, the polar phase is the solvent. All of the species used in this laboratory experiment absorb light in the UV region, so you will use UV detection (254 nm light). In principle, any detection technique which is sensitive to the components being separated can be used. Some modern instruments use more sophisticated detection schemes such as fluorescence, conductivity, mass spectroscopy, nuclear magnetic resonance (NMR) or electrochemical detectors.
The chromatograph you will be using can best be viewed as a series of components. The pump draws eluent from a reservoir and delivers it to the column. The injector is a multiport valve that allows samples of known volume to be reproducibly introduced to the column without interrupting the flow. It is positioned between the pump and the column. Naturally, the column itself is the key component wherein the separation takes place. The detector (UV in our case), positioned down-stream of the column, converts changes in light absorbance to electrical signals. The chart recorder displays the electrical signals received from the detector. The output corresponds to a record of component response versus time. One of the advantages of the HPLC technique is the ease and the reproducible nature of the injections. A sample is introduced into a loop of precisely measured size. Upon turning the injector handle, this loop is mechanically switched into the high-pressure line leading from pump to column. Thus, exactly the same size sample is injected in exactly the same way every time. Because of the reproducible nature of the injection and the reasonable longterm stability of the detector and signal amplifiers, the same instrument will give the same response for repeated injections of the same sample over a considerable period of time. Consequently, external standards may be used to establish a calibration curve that may be used for unknowns. However, each HPLC unit is slightly different (the detector response varies somewhat from one unit to another among other things) and a calibration curve obtained on one instrument cannot be used to run samples on another. The determination of the proper mobile phase for a particular analysis is the first job for the analyst. Once the method is established, samples can be analyzed quantitatively or qualitatively for the presence of particular components. Since this a very time consuming process, the HPLC units will be set up with the proper mobile phase for the samples to be run. The samples will be analyzed by the retention times and retention volumes. Data can be converted to capacity factors, k, which are a measure of the affinity of a particular species for the stationary phase under the particular mobile phase composition used. k = (tr - tm)/tm (1) The definitions of the terms in this equation are as follows: t r is the retention time of a component that was retained on the column; tm is the void volume of the column, which is the time it takes an unretained compound to travel from the injector through the column and to the detector. You will calculate the capacity factor, k, for each standard. Notice that the capacity factor is a unitless quantity. Analgesics are prepared in various formulations that often include one or more of the following compounds: acetylsalicylic acid (aspirin, hereafter abbreviated SA), acetaminophen (the active ingredient in Tylenol, hereafter abbreviated AC), and caffeine (a vasodilator that accelerates the delivery of the active ingredients, hereafter abbreviated CAF). Certain over-the-counter pain relievers contain all three of these ingredients, and this combination is marketed as a particularly effective remedy for headaches. Pharmaceutical manufacturers must maintain tight quality control of their products, and routinely use chromatographic methods for the analysis of medications. In this lab you will perform a quantitative determination of the CAF and SA content of an over-the-counter headache tablet using HPLC.
Procedure (Students will work in groups for this experiment) According to the label on the bottle, each tablet has the following content: acetaminophen- 250 mg, acetylsalicylic acid-250 mg, caffeine-65 mg, and filler-100 mg. Filler is inert material that should be water soluble. It is of no interest to us in this analysis, though pharmaceutical manufacturers must also control its content. You will measure the concentrations of CAF and SA in a single tablet by crushing the tablet and extracting the active ingredients in preparation for analysis by HPLC. You will also prepare a set of 4 standards containing both CAF and SA in order to make a quantitative determination of the mass of these compounds in the tablet. 1. Select one tablet for analysis and record its mass. Use a mortar and pestle to crush the tablet into a fine powder. 2. Weigh ~20 mg of this powder directly into a 100 ml beaker. Record the added mass. Add a few milliliters of methanol followed by less than 50 ml of mobile phase to the beaker to dissolve the powder. Be careful not to use more than 50 ml because you will deliver this solution to a 100 ml volumetric flask for final dilution. The mobile phase is used to dissolve the tablet so that the solvent does not alter the composition of the mobile phase in the column. 3. Carefully pour the solution from the beaker to a clean 100 ml volumetric flask. Rinse the beaker with ~5 ml of mobile phase 2 or 3 times, and pour these into the flask to make a quantitative transfer of the dissolved material to the volumetric flask. Be sure to mix the solution by agitating it before you have filled the flask. Pour a small volume of mobile phase into the beaker and use it to dilute the solution in the flask to the mark. Mix the solution again by inverting the stoppered flask, and label the flask SAMPLE. If the actual contents of the tablet are equal to the content stated on the label, the sample will contain CAF and SA in the following concentrations: CAF 1.955 mg in 100 ml solution 0.01955 mg/ml SA 7.519 mg in 100 ml solution 0.07519 mg/ml We use mg/ml as our unit of concentration because we will ultimately want to calculate the mass of each analyte in the tablet. 4. Next you will prepare standards that contain both CAF and SA in the approximate concentrations listed below. You do not need to use these exact concentrations, but you need to record precise mass values so that the actual concentrations are accurately known. You will notice two features of this set of standards. Firstly the concentrations vary with opposing trends for each analyte. This allows you to make an unambiguous qualitative determination of the retention time of each analyte, since one will have an increasing peak height as the standard number increases, and the other will have a decreasing peak height. Secondly the standard concentrations closely bracket the expected concentrations of the analytes. This is appropriate because we expect a very narrow variation in the concentration range of the sample.
5. Prepare separate stock solutions of SA and CAF as follows: SA: Label a 25 ml volumetric flask SA STOCK. Weigh ~16 mg of SA and deliver it into a 25 ml volumetric flask. Note the actual mass delivered to the flask in your notebook. Add ~12 ml of mobile phase and dissolve the SA. Dilute to the mark to give a stock solution containing ~0.64 mg/ml. Using the actual mass, calculate the exact concentration of the SA stock solution and note it in your notebook. CAF: Label a 25 ml volumetric flask CAF STOCK. Weigh ~6 mg of CAF and deliver it into a 25 ml volumetric flask. Note the actual mass delivered to the flask in your notebook. Add ~12 ml of mobile phase and dissolve the CAF. Dilute to the mark to give a stock solution containing ~0.24 mg/ml. Using the actual mass, calculate the exact concentration of the CAF stock solution and note it in your notebook. 6. Label four 10 ml volumetric flasks and add the following volumes of the stock solutions to each flask with a 2 ml graduated pipette: Standard label Volume of SA stock soln. Volume of CAF stock soln. #1 1.300 ml 0.600 ml #2 1.200 ml 0.850 ml #3 1.100 ml 1.000 ml #4 1.000 ml 1.250 ml Dilute each flask to the mark with mobile phase. Your instructor will demonstrate the proper operation of the chromatograph. The injection volume is fixed by the size of the injection loop (10 µl). The flow rate will be maintained at 2 ml/min. The column is an Alltech adsorbosphere C18 reversed phase column with dimensions of 150 x 4.6 mm. The UV detector is set at 254 nm, the detector range is set to 0.01, and the chart recorder is set to 3 cm/min. 7. Collect a chromatogram of each standard and the sample by injecting 10 ml of each solution onto the HPLC. The standards should give two peaks (SA and CAF) and the sample should give three (SA, CAF, and AC). Obtain a chromatogram for your unknown (tablet) in triplicate. Compare this to the chromatogram of the standard. Qualitatively determine the components of your unknown by comparing the retention volumes of each peak to the standards. Quantitatively determine the amount of each SA and CAF in your unknown.
Student Name: Chemistry 3200 High Performance Liquid Chromatography: Quantitative Determination of Headache Tablets Date: Lab Instructor: Section: Data Total tablet mass: Tablet powder mass: Standard solutions SA stock mass: mg CAF stock mass: mg SA stock concentration: mg/ml CAF stock concentration: mg/ml Concentrations of standard solutions Std # 1 Concentration of SA mg/ml Concentration of CAF mg/ml 2 3 4
Student Name: Chromatogram Standard solutions Std # 1 2 3 4 Sample SA CAF Retention time (t r ) Mass (mg) Retention time (t r ) Mass (mg) Sample SA CAF Retention time (t r ) Mass (mg) Retention time (t r ) Mass (mg) Trial 1 Trial 2 Trial 3 Average Calibration Curves attach the calibration plots 1. Use the peak areas (mass) and known concentrations of the standards to make calibration plots (peak area vs concentration). 2. Give the mathematical formula for the best line describing the calibration curve. (area or mass) SA = m SA C SA + b SA (area or mass) CAF = m CAF C CAF + b CAF Formula for calibration lines: SA: CAF: 3. Use these values and the peak areas from your unknowns to determine the concentrations of SA and CAF in the sample solution. Calculate the mass of CAF and SA in the pain reliever tablet that you analyzed. SA concentration in sample solution: mg/ml SA mass in tablet: mg CAF concentration in sample solution: mg/ml CAF mass in tablet: mg
Student Name: Calculation for unknown SA/CAF concentration in sample solution: Calculation for SA/CAF mass in original tablet:
Student Name: List the possible sources of error: