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Experiment Developed by Adam Schellinger 12-04, Revised by R. Machado 8-05 & 8-08, P. Buhlmann 7-06, 5-07; J. Katzenmeyer 5-08

Chapter IX Experiment #6 Quantification of 5 Pharmaceutical Solutes Using Gradient Elution Reversed Phase Liquid Chromatography Goals 1. To identify five pharmaceutical solutes chromatographically by varying their concentration in several mixtures and confirming the elution order based on polarity (i.e., pKa and / or functional groups), molecular weight and size. 2. To understand why gradient elution solves “the general elution problem.” 3. To determine the identity and quantify known solutes in an unknown mixture. Introduction In this experiment, you will use high performance liquid chromatography (HPLC) to separate a pharmaceutical mixture that consists mainly of antihistamines. Chromatography is a powerful technique commonly used in many fields to purify and / or quantify particular substances within a mixture. By far, gas chromatography (GC) is the most powerful and simplest technique for separating volatile organic compounds. Unfortunately, over 85% of all known organic substances are relatively nonvolatile and must be analyzed using liquid chromatography (LC) (1). There are two main techniques for performing LC: normal phase liquid chromatography (NPLC) and reversed phase liquid chromatography (RPLC). Overall, the most common mode of liquid chromatography is RPLC, where the retention and separation of the solutes is governed by their polarity, molecular weight, and size. In RPLC, the mobile phase is more polar than the stationary phase; in NPLC, the opposite is true. The mechanism of retention in RPLC is governed by the partitioning of solutes from the mobile phase into the stationary phase. Thus, solutes that are more polar will be less retained by the RPLC stationary phase. As the polarity of the solute decreases, the solute is eluted at a longer time due to a greater extent of partitioning into the stationary phase (1). Many solute mixtures that must be separated with RPLC contain some analytes that are weakly retained and other analytes that are highly retained by the stationary phase. Changing the eluent strength (i.e., polarity) of the mobile phase is the most common way to control the retention of each analyte. For example, increasing the volume fraction of the organic modifier in an RPLC eluent increases the eluent strength (i.e., decreases the polarity). In an isocratic separation, the eluent composition of the mobile phase is held constant as a function of time. Imagine that the sample mixture of interest contains solutes weakly, moderately, and highly retained by the stationary phase. To perform an adequate separation, we require the weakly retained solutes to be retained above some lower limit and the highly retained solutes must be eluted within a reasonable time. Using isocratic elution only provides two options for optimizing the separation. Increasing the eluent strength of the mobile phase will lower the retention time of the highly retained components but may decrease the retention of the weakly retained components below the required limit. Alternatively, decreasing the eluent strength would allow adequate IX-1

Experiment Developed by Adam Schellinger 12-04, Revised by R. Machado 8-05 & 8-08, P. Buhlmann 7-06, 5-07; J. Katzenmeyer 5-08

retention of the weakly retained components, but now the highly retained components do not elute in a reasonable time. This dilemma is commonly referred to as the “general elution problem.” The most common way to overcome the “general elution problem” in LC is to perform gradient elution, which is comparable to a temperature program in GC. In gradient elution, one changes the eluent strength as a function of time. Thus, starting with a weak eluent strength will allow for adequate retention of the weakly retained solutes; ending with a strong eluent strength will reduce the retention of highly retained solutes to a reasonable value. Gradient elution also improves the peak capacity, which is defined as the number of separable peaks within a defined separation space. In this experiment, you will separate a mixture of 1 antidepressant (amitriptyline) and 4 antihistamine solutes (methapyrilene, pheniramine, promethazine and triprolidine) using gradient elution RPLC. The mobile phase consists of methanol (the nonpolar organic modifier) and water with 10 mM perchloric acid (HClO4) which serves as a low capacity buffer system to maintain a constant degree of ionization (i.e., polarity) for the basic solutes and to improve retention time reproducibility and peak shape. As these pharmaceutical solutes will possess some charge, the pKa values of each solute will be required to understand their elution order in the chosen mobile phase. The stationary phase used in this experiment is silica based; silica is the most popular stationary phase support for LC. Since the bare silica particles are polar, the nonpolar stationary phase required for RPLC is obtained through the bonding of octyldecyl chains onto the surface of the silica. A diagram is shown in Figure 1.

Fig. 1. Surface derivatization of the silica substrate (i.e. particle) with an alkyl C18 chain to produce octyldecylsilane (ODS) silica particles. Figure adapted from ref. (1). This experiment will focus on the quantification of each pharmaceutical solute in an unknown mixture and a real world antihistamine sample (ActifedÒ). Knowing the abundance of a particular solute is extremely useful in the pharmaceutical industry to verify the purity of a substance or to determine the yield of a synthesis. 9.2 Theory Resolution The most common goal in RPLC is to obtain adequate separation of each solute in a reasonable analysis time; adequate separation allows one to isolate a specific component from a mixture for the purposes of identification and / or quantification. Resolution is a measure of the completeness of a separation and is quantitatively measured with the general equation: 2 × (t R,2 - t R,1) (1) Rs = 1.7(w1/ 2,2 + w1/ 2,1) where tR,1 and tR,2 represent the retention times of the least and most retained peaks, respectively, measured at the peak centroid (i.e., center of mass) and wb,1 and wb,2 represent the baseline widths of the IX-2

Experiment Developed by Adam Schellinger 12-04, Revised by R. Machado 8-05 & 8-08, P. Buhlmann 7-06, 5-07; J. Katzenmeyer 5-08

peaks (equal to four times the standard deviation of the peak, assuming a Gaussian peak shape). Baseline (i.e., complete) separation of two peaks is obtained when Rs ³ 1.5. The resolution has no units; thus, the retention time and peak width must be measured in the same units (i.e., time, volume or distance). Refer to Figure 2 for an illustration of separation based on various values of resolution.

Fig. 2. Resolution measures the degree to which two components in a mixture are separated. Rs is calculated using eq. 1. At low Rs, peaks are barely separated and Rs ³ 0.7 is required to distinguish between two peaks. Column Efficiency Another way to measure the separating power of a column is to measure the column efficiency (N), which is typically referred to as the number of theoretical plates. Column efficiency is measured with the following equation: 2

æ t ö (2) N = 5.54 × çç R ÷÷ w 1 / 2 ø è where tR is the retention time at the peak centroid and w1/2 is the half-width of the peak (i.e., peak width measured at the half-height of the peak). W1/2 values are the measurements actually being reported in your chromatograms. Both of these values are obtained directly from the data system in this experiment.

Peak Capacity A common concern in chromatography centers around the concept of peak capacity (nc), which is defined as the number of peaks that can fill a given separation space assuming that the peaks are optimally spaced. One important observation when discussing peak capacity is that each observed peak is not necessarily a single component. Also, a component in the sample is not restricted to eluting from the column within the chosen separation space. Therefore, the probability of observing single component peaks in any separation is often very low (less than 20%) (2), which is why the choice of the appropriate chromatographic conditions (i.e., stationary phase, mobile phase, eluent composition, temperature, elution mode, etc.) is pertinent to obtaining complete separation of every component. In isocratic elution, the peak capacity is determined with the following equation:

nc =

N × ln(k ' n +1) 4× R s IX-3

(3)

Experiment Developed by Adam Schellinger 12-04, Revised by R. Machado 8-05 & 8-08, P. Buhlmann 7-06, 5-07; J. Katzenmeyer 5-08

where k’n represents the retention factor (k’) of the last eluting component (n). The retention factor is calculated using the following equation: t -t (4) k' = R o to where to is the dead time of the column (we measure this value using the unretained solute of uracil). In gradient elution, the peak capacity is determined with a similar equation as in isocratic elution:

N (5) × k 'n 4 ×R s assuming that the peak width is constant in gradient elution. Based on the form of equations 3 and 5, it should be clear that, theoretically, gradient elution will always provide a higher peak capacity compared to isocratic elution, assuming that the value of k’n is comparable in each elution mode. nc =

9.3 Components of the Agilent 1100 HPLC Detection System: Agilent 1100 Series Variable Wavelength Detector (VWD) One of the ways to detect solutes from a mixture of components is to use ultraviolet (UV) detection. Solutes with a chromophore (i.e., UV absorbing functional group such as an aromatic ring) will absorb light at a particular wavelength according to the pathlength (b), their concentration (c), and the molar absorptivity (e) using Beer’s Law: (6) A(t) =e × b × c(t ) The detector records the absorbance as a function of time. Therefore, taking the integral of the absorbance as a function of time over the region in which a peak is being detected will provide one with the area of the eluting peak according to the equation: (7) Area = ò A(t ) × dt = e × b × ò c (t ) × dt To determine the moles of an absorbing species, which is related to the area of the peak, one can use the equation: moles = V × ò c (t ) × dt (8) The injector on this instrument has a fixed and reproducible injection volume. Therefore, the volume of sample injected (V) is a constant, and peaks from different injections are directly comparable. The peak height also serves as a measure of the number of moles of solute (assuming that the peak width and shape of a solute is constant for different injections). However, this experiment will make use of the peak area as a measure of the number of moles of solute present in each sample to account for any errors in the manual injection process that will affect the peak shape and / or width (1). The detector used in this experiment is the HP 1100 series VWD, which is capable of detection in the wavelength range of 190-600 nm using illumination from a deuterium lamp. The wavelength used in this experiment will be 230 nm, as the pharmaceutical solutes will absorb at this wavelength and the eluent will not (at least not significantly). Further specifications are available in the Agilent 1100 Variable Wavelength Detector Reference Manual (6). Instrument Control and Data System: Agilent Chemstation Software

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Experiment Developed by Adam Schellinger 12-04, Revised by R. Machado 8-05 & 8-08, P. Buhlmann 7-06, 5-07; J. Katzenmeyer 5-08

In this experiment, Chemstation software is used to control the instrument and collect and process chromatographic data. Further details related to using the Chemstation software are found below in the experimental section. 9.4 Experimental Equipment List Check out the HPLC experiment kit from the stockroom. The kit will contain micropipettes (if you do not know how to use these please ask your TA BEFORE using them), 10.00 mL volumetric flasks, test tubes with cork stoppers, disposable 1 mL plastic syringes with 0.22 µm nylon filter tips, and an OTC antihistamine solution. The TA will provide the following stock solutions: • Amitriptyline (10 mg/mL) • Methapyrilene (10 mg/mL) • Pheniramine (15 mg/mL) • Promethazine (10 mg/mL) • Triprolidine (10 mg/mL) • Hexanophenone (Internal Standard) (5 mg/mL) • Uracil (Dead Time Marker) (1 mg/mL)

Warm Up the Instrument 1. Turn on the power to the computer and Agilent 1100 series vacuum degasser, quaternary pump, and VWD. 2. Load the Chemstation software by double clicking on the Instrument 1 Online button or by going to Start, Instrument 1 Online. 3. Load the Method named Chem2111 by going to “Method” and then “Load Method.” The path for this method is C:\HPCHEM\1\Methods\Chem2111\ 4. Make sure the in-line filter and column are properly attached to the instrument (ask your TA for help). 5. Check the eluent in all channels. The eluent should be 10/90 MeOH / H2O with 5 mM HClO4 in channel A, and MeOH with 5 mM HClO4 in channel B; channels C and D should contain pure MeOH. The eluent reservoirs should contain at least 750 mL of each eluent. 6. Start the instrument by clicking the on button in the Chemstation software or by going to RunControl and clicking “System On”. 7. Under the “Run Control” menu go to “Sample Information.” Type in a name for your group in the “Operator” box. Make sure “Prefix/Counter” is selected under the Data File area. Type in “Chem2111” for the Subdirectory and the date (example 092304 for 09-23-04) for the prefix . . . the counter should automatically be set to “00” after clicking in that area. Type in an appropriate name for the sample. EACH TIME YOU ARE INJECTING A NEW SAMPLE YOU SHOULD CHANGE THE SAMPLE NAME. Make a note of these values in your lab notebook in the event you need to retrieve your data. Please note that all data is stored under the location: C:\HPCHEM\1\DATA. Ask the TA for further instructions / help. 8. Start the method with no injected sample to flush out the column and finalize the instrument warmup procedure (suggested sample name: NO SAMPLE). A method is started by switching the injection valve from the LOAD to the INJECT position. Do not click “Run Method.” IX-5

Experiment Developed by Adam Schellinger 12-04, Revised by R. Machado 8-05 & 8-08, P. Buhlmann 7-06, 5-07; J. Katzenmeyer 5-08

Preparation of Known Sample Mixtures 1. Prepare the following solutions using the 10 – 100 µL Eppendorf pipette and the 10.00 mL volumetric flasks. Volume Added ( µL) Solute Solution Solution Solution 1 2 3 40 50 60 Amitriptyline 50 60 70 Triprolidine Promethazine 60 70 80 70 80 40 Pheniramine 80 40 50 Methapyriline 50 50 50 Uracil 90 90 90 Hexanophenone Table 1. Preparation of 5 known mixtures of pharmaceutical solutes

Solution 4 70 80 40 50 60 50 90

Solution 5 80 40 50 60 70 50 90

2. Dilute each solution to the mark by adding Eluent A. Mix thoroughly (invert at least 6 times). 3. Filter each sample into a clean, labeled 1mL Eppendorf tube . To do this, fill the syringe with the solution, then put the nylon tip filter on the syringe, and push the solution through the filter into the tube. Only use these filtered samples when you are injecting mixtures into the HPLC column! Preparation of the Unknown 1. The unknown sample provided by the TA. Dilute the sample (approximately 1/20) in Eluent A and add the dead time marker and internal standard to the sample (Why?). Filter the sample into a labeled 1 mL Eppendorf tube with a 1 mL disposable plastic syringe and 0.22 µm nylon filter. Chromatography (Blank Run) 1. Suggested sample name: BLANK 2. Rinse the 50 µL syringe three times with the channel A (i.e., 10/90 MeOH / H2O with 5 mM HClO4) eluent. 3. Draw 50 µL of eluent into the syringe and remove any air bubbles – check with the TA for the proper technique. 4. With the injector valve in the LOAD position, inject the eluent into the sample loop. Essentially, this step both flushes and loads the 20 µL sample loop. 5. Start the method (see step 9 of “Warm Up the Instrument”) 6. Wait for the method to end and then switch the injector valve back to the LOAD position. Stop the method (see step 10 of “Warm Up the Instrument”) 7. If you see peaks in the blank run, repeat steps 2 through 5, and inform your TA.

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Experiment Developed by Adam Schellinger 12-04, Revised by R. Machado 8-05 & 8-08, P. Buhlmann 7-06, 5-07; J. Katzenmeyer 5-08

Shut Down 1. Load the Method FLUSH and start the method (no injection). Wait ten minutes until the method is done. 2. Turn the HPLC off using the button in Chemstation or by going to RunControl, System Off. 3. Dispose of all standards and unknowns in the bottle marked “HPLC WASTE.” 9.5 Calculations 1. Identify the components in the known mixtures by comparing the changes in the ratio of the analyte peak area to the peak area of the internal standard (hexanophenone). The concentration of hexanophenone should be constant in all of your solutions. Therefore, taking the ratio of peak areas should account for any variation in injection volume or error from the detector that may exist from run to run. (Hint: hexanophenone should elute last and be a large peak). 2. Create plots of the peak area ratio (Asolute / Ahexanophenone) versus the concentration of each solute. Perform a linear regression analysis to obtain the slope and intercept of each curve and their standard deviation along with the correlation coefficient (R2) of the linear regression. Present the results of the linear regression analysis in graphical form. 3. Quantify the amount of each solute present in the unknown mixture. Calculate the appropriate concentration (mg/mL) and the expected error in that value. Tabulate your results. 4. Identify the antihistamine in the unknown sample and calculate its weight percent in the pill (1 pill = 150 mg). 5. For a known mixture you separated using gradient elution, calculate the plate count (N) of each solute and calculate the resolution of each adjacent pair of solutes. Using Rs = 1.5 and the average plate count of the solutes analyzed (excluding uracil), calculate the peak capacity available for the separation in gradient elution. Calculate the peak capacity available in an isocratic separation assuming the same plate count, resolution, and k’n value. Tabulate these results.

Questions (these should be answered in the discussion section of your lab report when you talk about your results) 1. What is the elution order of the solutes used in this study? Rationalize this elution order based on the polarity and molecular weight of each solute. Are there any discrepancies between the elution order you would expect in RPLC? Please explain.

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Experiment Developed by Adam Schellinger 12-04, Revised by R. Machado 8-05 & 8-08, P. Buhlmann 7-06, 5-07; J. Katzenmeyer 5-08

2. How linear are your calibration curves (i.e. can you place confidence in them to accurately predict the concentration of the same solute in other mixtures)? Please describe any errors that are associated with the calibration curves and how these errors might be reduced. 3. What components were present in your unknown mixture? What components were present in your real world mixture? Are the concentrations of the solutes in the unknown mixture and the real world mixture within the range of concentrations in your calibration curves? 4. What are some of the limitations of gradient elution? What are some of the advantages of gradient elution? (Hint: think in terms of reproducibility and sample throughput). 5. What are some of the limitations of isocratic elution? What are some of the advantages of isocratic elution? (Hint: think in terms of reproducibility and sample throughput). 6. Based on the peak widths obtained in gradient elution, estimate the number of peaks that could theoretically be baseline separated within 5 minutes.

9.6 Lab Report Your lab report should contain the following information: 1. Two chromatograms (the first labeled “calibration,” the second “unknown” ) 2. ...