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High Performance Liquid Chromatography (HPLC) of Three Analgesics OVERVIEW/THEORY Real-life samples are often complex mixtures of many substances (e.g., sea water contains many salts and other impurities along with the water). When samples contain several components, the individual analytes often must be separated from each other prior to their detection so that their signals do not interfere with one another. High performance liquid chromatography (HPLC) is a powerful separation technique in which a liquid mobile phase (eluent) is passed through a solid stationary phase contained in a column. The analytes are separated (resolved) from each other as they are pumped through the column based upon their relative affinities for the stationary phase versus the mobile phase. After being separated on the column, the analytes pass directly to an in-line detector that generates a chromatogram showing the individual signal peaks versus time. The schematic diagram of a typical HPLC with UV-vis detector is shown below (Figure 1)

Figure 1. Schematic of an HPLC. a, solvent reservoir; b, high-pressure liquid pump; c, high-pressure injector valve; d, column; e, detector; f, chart recorder; g, waste reservoir.

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Figure 2: HPLC system. a, solvent reservoir; b, high-pressure liquid pump; c, high-pressure injector valve; d, column; e, detector; f, chart recorder; g, waste reservoir (not shown).

In this experiment, three analgesics are separated using “reversed-phase” HPLC in which a polar mobile phase is used with a nonpolar stationary phase. The column in this experiment contains a bonded C18 (octadecylsilane) nonpolar organic stationary phase. The mobile phase is a polar methanol/water mixture also containing some acetic acid. The three analytes are eluted in order of decreasing polarity, with the most polar analyte, acetaminophen, coming off first followed by acetanilide and then phenacetin. Since the molecules all have some aromaticity, signal is provided by a UV-vis absorbance detector with the resulting chromatogram showing signal (absorbance units; AU) versus time. The concentrations of the three analytes will be determined by comparing their peak areas from a diluted sample mixture to the corresponding peak areas from a diluted standard mixture you prepare according to the directions below. SAFETY Protective eyewear must be worn at all times. Notify the instructor if there is a problem with the instrument. For more general safety in the laboratory, please refer the appendix. METHOD

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Preparation of the standard mixture Using the analytical balance, accurately weigh between 0.1 to 0.2 grams acetaminophen on a small weighing boat and record the weight. Zero the balance, clean the spatula and weigh between 0.1 and 0.2 grams acetanilide into the same weighing boat and record the weight. Zero the balance again, clean the spatula, and weigh between 0.1 and 0.2 grams phenacetin into the same weighing boat. Carefully transfer this mixture to a 250-mL volumetric flask with the HPLC grade methanol wash bottle from the chemical cabinet (do not use the methanol meant for rinsing). If you already have a few mL’s of methanol in the flask before you transfer, this will aid in the dissolution of the solid. Add just enough methanol (about 20-25 mL) to dissolve the mixture. DO NOT CONTINUE TO THE NEXT STEP UNTIL THE SOLID HAS COMPLETELY DISSOLVED IN THE METHANOL. Add nanopure water while swirling the flask but stop before the mark. The methanol/water mix heats up slightly so let the flask cool to room temperature before bring the solution to the mark and mixing completely. The solution is too concentrated for analysis so dilute it by a factor of 25 and mark the diluted sample “diluted working standard.” Preparation of the unknown mixture Obtain the unknown from the instructor. This sample contains all three analytes that have been accurately weighed to four decimal places. Transfer the sample to a 250-mL volumetric flask with about 25-mL methanol and allow to fully dissolve before diluting with nanopure water. Fill the flask almost to the mark while swirling and then let it cool to room temperature before diluting to the mark and mixing. Dilute this sample also by a factor of 25 and label “diluted unknown.” Use of the HPLC * NOTE: the figures for the handheld controller for the HPLC is in the instruction only to give you an idea what to expect when you look at the controller screen. All the important information that you should be checking is in the TEXT of the instruction, not in the figures. Take the diluted standard and unknown to the instrument room. Pick up the handheld controller. Press “ESC” button until you see the screen that resembles below. If you have already done GC or GC/MS for this course, you should be familiar with the handheld controller. If you have not done those experiments, then, you should know one thing: the handheld controller does not use a touchscreen control. Instead, you should use the Function keys (F1, F2, F3, etc) near the screen.

Figure 3: the Analysis screen.

Note: this figure is shown as an example. You should see the screen that resembles this, but will not be identical.

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Make sure the Method listed on the screen says “466” (not “ISOCRAT” as shown above the Figure 3). If not, ask for assistance from the laboratory instructor. Press F7 to turn on the system (the pump and the detector). You should see the screen that resembles Figure 4.

Figure 4: System On and System Off

Note: this figure is shown as an example. You should see the screen that resembles this, but will not be identical.

Use the arrow keys to move from “System Off” to “System On” if necessary. Once you have “System On” selected, press “Enter” key. This should start the HPLC pump as well as turn on the lamp in the UV detector. You should hear the pump running. Wait about 5 minutes to allow the instrument to equilibrate. In your notebook, record the pump flow rate (mL/min), the approximate pump pressure (bar), and the wavelength (nm) of the UV-vis detector. Each values should be approximately the following: Flow rate: Pressure: Wavelength: At this point, you are ready to make your first injection of the diluted standard mixture. From here on, you must follow the instruction for each sample you run. First, make sure the injector is in the “Load” position (Figure 5).

Figure 5: the injector. From the left, the injector without the syringe, the injector with the syringe in “Load” position, and the injector with syringe in “Inject” position. Rinse the 1-mL syringe several times with the diluted standard solution and then fill it completely. Insert the syringe at the center of the injector all the way in. Inject almost the entire contents of the syringe into the instrument, but be sure to leave a little left in the syringe in case an air bubble is there. What you have done is fill a small 20-µL loop many times, with the excess draining into the waste bottle. Leave the syringe in the injector once you have loaded the sample.

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You are now ready to begin the analysis. Turn the injector to “Inject” position. The run should start at this point and the integrator should start moving. Once the run has started (the integrator has started), turn the injector back to “Load” position for the next injection. After less than three minutes, the three analytes will be eluted as three separate peaks on the chromatogram. The run should stop in 3 minutes. The integrator should print out the result below the collected chromatogram. The chromatogram obtained should resemble Figure 6.

Figure 6: sample chromatogram

Your goal is to get three successive trials that the area % of all of the three peaks agree with each other within about 1%. After running sufficient trials of the standard mixture, change the computer heading to read "unknown" and run the unknown exactly as the standards. When you are finished, turn off the instrument by selecting the “System Off” as you turned on the system (see Figure 3 above). After turning off the system, the pump should stop and the pressure should go to zero. Make a table in your notebook showing the peak areas for the three analytes for both the standard and unknown. You will also turn in all the integrator chart paper. Remember, the unknown and standard must both be run on the same day and lab period, ideally within 60 minutes of each other. If too much time passes between the injections of the standard and the

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unknowns, then results can be skewed because the response of the instrument changes over time (this is known as “signal drift”). CALCULATIONS To determine the amount of each analyte in your unknown, set up simple proportion between its average area % in the unknown chromatogram versus its average signal in the standard chromatogram. What you are essentially doing is creating a proportionality constant that cancels itself out because the known and unknown were diluted and measured identically. Report the weight of each of component in the original sample to four decimal places.

Instrumental Analysis Laboratory Safety Rules A. Instructions: Carry out all manipulations in accordance with instructions and the

safety rules and procedures given herein. B. Eye Protection: All students and staff working in the laboratory must wear safety

glasses at all times. If a student needs to be reminded more than three times to wear goggles, she/he will

be dismissed from lab for the remainder of the day, and will not be given an opportunity to make up the work.

C. Apparel: The clothes you wear in lab are an important part of your “safety

equipment,” and should offer protection from splashes/spills. Closed toed shoes (sneakers are fine), Full-length pants or a full-length skirt, and A shirt that completely covers your torso (i.e. at minimum, a t-shirt).

In other words, you must NOT wear shorts to lab. You must NOT wear flip-flops, sandals, or crocs. You must NOT wear tank tops, halter tops, spaghetti-strap tops, or low cut jeans to lab. Exposed abdomens, hips, and backs are not safe in the lab.

D. Gloves: Gloves are an important part of personal protection. Gloves will be available

at all times in the laboratory. Your instructor will require their use when appropriate. E. Food: Food, drinks, and gum are not allowed in lab. None at all, not even water

bottles. F. Sanitation Issues: Be sure to wash your hands before leaving lab, before you eat

anything outside of lab, and before you answer your cell phone. G. Music: Individual headphones are not allowed. Your may choose to play music for

the entire class. H. Other: All students are explicitly prohibited from:

1. conducting any unauthorized experiments. 2. removing chemicals or apparatus from the laboratory for any reason. 3. working in the lab alone, or at other than regularly scheduled lab periods. 4. smoking in the laboratory or within 20 feet of any doorway. 5. impeding movement in aisles or through doorways with bags, skateboards, etc.