(1) This document summarizes an experiment using High-Performance Liquid Chromatography (HPLC) to analyze a drug sample.
(2) The experiment used HPLC to separate and detect three components in the drug sample - Metformin hydrochloride, Povidone, and magnesium stearate.
(3) The results showed Metformin hydrochloride eluted first due to its high polarity, followed by Povidone and then magnesium stearate, with differences in their retention times and absorbance peaks due to their varying interactions with the non-polar stationary phase.
(1) This document summarizes an experiment using High-Performance Liquid Chromatography (HPLC) to analyze a drug sample.
(2) The experiment used HPLC to separate and detect three components in the drug sample - Metformin hydrochloride, Povidone, and magnesium stearate.
(3) The results showed Metformin hydrochloride eluted first due to its high polarity, followed by Povidone and then magnesium stearate, with differences in their retention times and absorbance peaks due to their varying interactions with the non-polar stationary phase.
(1) This document summarizes an experiment using High-Performance Liquid Chromatography (HPLC) to analyze a drug sample.
(2) The experiment used HPLC to separate and detect three components in the drug sample - Metformin hydrochloride, Povidone, and magnesium stearate.
(3) The results showed Metformin hydrochloride eluted first due to its high polarity, followed by Povidone and then magnesium stearate, with differences in their retention times and absorbance peaks due to their varying interactions with the non-polar stationary phase.
School of Chemical, Biological, and Materials Engineering and Sciences, Mapua University, Intramuros, 1002 Metro Manila, Philippines
dhmanasala@mymail.mapua.edu.ph1
I. Introduction Further discussing the process
that occurs during HPLC, the In the study of Biology, more presence of a high pressure, typically specifically, but not limited to, in the around 400 atm, forces the solvent to field of applied laboratory medicine, pass through a column which is filled samples are actively collected and by adsorbent. In technical terms, the treated. Following this is completing immense pressure allows the mobile specific methods and procedures in phase to proceed to the order to determine distinct aforementioned column known as the components found in the mixture. One stationary phase [ CITATION Jimst \l of the more common methods used in 13321 ]. experiments similar to this is a method called “High-Performance Being an advanced and highly Liquid Chromatography” or HPLC sensitive, HPLC machines are able to [ CITATION Hig20 \l 13321 ] which is read and detect various magnitudes of basically a more developed technique interaction between the components in derived from column the mixture and the adsorbent present. Chromatography. The HPLC machine has the ability of doing so by considering the Briefly differentiating the two differences in chemical and physical techniques mentioned, column properties. Additionally, the concept chromatography heavily relies on of polarities is also heavily applied. gravity which allows the solvent to [ CITATION Ver16 \l 13321 ] drip down in the column. On the other hand, HPLC utilizes a high-pressure Before proceeding with the pump instead of gravity which makes discussion of polarities and property the process quicker relative to column differences, it is important the two chromatography. major types of HPLC namely “normal phase” and “reverse phase”. The two types can be distinguished in the technique alongside the function of an polarities of their mobile and HPLC machine. (2) Successfully stationary phases. For the normal- complete an HPLC analysis on the phase HPLC, the mobile phase is drug sample (3) to determine the considered to be non-polar and is then effects of external factors and followed by a polar stationary phase. parameters (temperature, humidity, The reverse-phase simply has these mobile and, stationary phase two polarities reversed [ CITATION composition) Lak19 \l 13321 ]. Among the two types, II. Methodology this experiment utilizes the reverse- phase type. This is generally the more This experiment was executed common type being that is it relatively through Labster which is an online more ideal towards the retention of virtual laboratory simulator. organic analytes [ CITATION Mat13 \l 13321 Prior to proceeding with the ]. actual experiment, the HPLC machine For the components in the was calibrated. This is completed by mixture found in the sample, each forcing incremental pressure through have their specific interactions with the column beginning with 0.1 the beads inside the column which is mL/min for 10 minutes. After such, a affected by the polarity of the volume of the subject solvent was stationary phase and the analyte itself. utilized to flush out interferents that Ultimately, this yields different rates may be contaminate the whole of elution for the distinct components experiment. while flowing out the column. After calibration, the sample In displaying the signal peaks was then prepared accordingly. Using caused by the elution rates, a a mortal and pestle, the drug sample chromatogram is produced. The peaks was powderized to aid in dissolving it are caused by the detected retention in water. times which indicates which With the needed preparations component eluted first up to the last completed, the process of HPLC was [ CITATION Chr \l 13321 ] commenced starting by injecting the The success of this experiment prepared drug solution into sample is then guided by these set of injector for the HPLC machine to be objectives: (1) to understand HPLC as able to process the sample following a chromatographic separation constant conditions. A chromatogram of the signal detect will be yielded Being the component with the accordingly. lowest value for retention time, Metformin hydrochloride was For the next half of the detected first being the peak is found experiment which focuses on the at around the 5-minute mark. effects of external factors such as Followed by Povidone which was temperature, the components in the detected by the 9th minute and then mobile and stationary phase were finally at the 16-minute mark is swapped with acetonitrile and C-18 magnesium stearate. The differences respectively. However, prior to doing in retention time and absorbance are so was the washing and re- affected by the degree of interactions conditioning of the machine by between the non-polar silica beads allowing new variables to be flushed found in the column. through. In dissecting and discussing III. Results and Discussion each peak that is found on the graph After the completion of the above, Metformin hydrochloride HPLC processes, the following (C4H12ClN5), which is highly polar, chromatogram were produced and yielded the fastest relative retention analyzed. time [ CITATION WFa11 \l 13321 ] caused by the low degree of interaction between the silica beads which is non-polar, allowing it to be detected quicker as soon as it was injected. The strong absorbance peak of this component is explained by the occurrence that it exited the stationary phase immediately. Following this component is Figure 1. Absorbance VS Retention Povidone (C6H9NO)n as shown by the (at 25 Co) middle, small-sized peak in the graph. Regarding the position of this peak on Observable from Figure 1 are the middle of the graph, it indicates three peaks with distinct degrees of that its retention time is not the fastest absorbance and value for retention (in nor the slowest relative to the drug minutes). components. This is due fact that this compound is soluble in non-polar as Figure2.Chromatogram well as polar solvents [ CITATION JRS20 \l comparison (Normal VS Extreme 13321 ] Additionally, it is observable conditions) that it has the weakest absorbance For the next half of the peak out of the three. These experiment, a comparative analysis observations are made possible by the was done. Between the two fact that Povidone is an organic chromatograms shown in Figure 2, the substance that contains a hydrophobic left chromatogram shows the readings or non-polar group and a hydrophilic and detections subjected under normal or polar group causing a variance in conditions (25 Co and 40% humidity). the interactions in the mobile and The chromatogram on the right was stationary phase. oppositely conditioned being it was Lastly for this graph, it is subjected to relatively more intense observed that the largest value for conditions (40 Co and 95% humidity). retention time was detected and The motive for this section of identified as Magnesium stearate the experiment is to identify how such (Mg(C18H35O2)2). This phenomenon is external factors such as humidity and caused by the structure of the temperature affect the readings for compound which makes it non-polar absorbance and retention times for or hydrophobic [ CITATION Mag \l 13321 ]. certain compounds in a sample. With that, it tends to actively interact with the non-polar silica in the Initially, it can be observed stationary phase. The increase in between the two chromatograms, that degree of interaction yields a the retention time and degree of consequence of an increase is the time absorbance for Povidone and of retention inside the column. magnesium stearate are similar with the possible presence of miniscule discrepancies, which can be securely disregarded. However, for the first peak, which is the identified metformin hydrochloride, it can be observed that there seems to be a small yet quantifiable discrepancy between the degree of absorbance at normal conditions compared to the extreme chromatograms in terms of retention one. time and absorbance. The success in the completion of the technique Note that the degree of allowed the further understanding of absorbance or the area under the curve the mechanisms that takes place in an corresponds to the concentration of HPLC machine together with the that specific components in the polaristic effects and interactions sample, the slight decrease in the between molecules. “height” of the peak indicates that there is a loss in the concentration at For the next comparative half an extreme condition [ CITATION Int \l of the experiment, it is determined 13321 ]. This occurrence can be that the change in the environment or attributed to the possibility of the conditions affects the compound compound degrading. The increase in possibly through thermal degradation temperature causes the breakdown of as seen in the decrease in the components found in the concentration for metformin compound possibly forming other hydrochloride [ CITATION Mic18 \l 13321 ] products [ CITATION Typ \l 13321 ]. Additionally, for the References comparative analysis of the two given chromatograms, it indicated that the components present in the sample are able to function even with the changes in environment while noting the small degradation possible for metformin hydrochloride. CONCLUSION After the completion of the entirety of the experiment, the objectives were effectively sufficed. The theory and application behind chromatography, more specifically HPLC, were demonstrated and applied by identifying the components present and analyzing the Chromatography. (n.d.). Retrieved from Columbia University: http://www.columbia.edu/cu/chemistry/ugrad/hssp/EXP_8.html Clark, J. (2020, August). HIGH PERFORMANCE LIQUID CHROMATOGRAPHY - HPLC. Retrieved from Chem Guide: https://www.chemguide.co.uk/analysis/chromatography/hplc.html Faulkner, W. (2011). Analysis of Metformin on a Perfluorophenyl. Retrieved from Thermo Fisher Scientific: https://assets.thermofisher.com/TFS-Assets/CMD/Application- Notes/ANCCSMETFORMIN_0611.pdf High Performance Liquid Chromatography. (2020, August 16). Retrieved from Chemistry LibreTexts: https://chem.libretexts.org/Courses/University_of_California_Davis/UCD_Chem_115_Lab_Man ual/Lab_2%3A_High_Performance_Liquid_Chromatography Integral of the plasma drug concentration-time curve. (n.d.). Retrieved from Pharmacokinetics: https://sepia2.unil.ch/pharmacology/parameters/areaunderthecurve/#:~:text=The%20area %20under%20the%20plasma,body%20and%20the%20dose%20administered. Judge, M. (2018, June 4). Factors That Affect RF Values in Thin Layer Chromatography. Retrieved from Sciencing: https://sciencing.com/factors-values-thin-layer-chromatography-8561359.html Koester, V. (2016, June 16). What is HPLC? Retrieved from ChemistryViews: https://www.chemistryviews.org/details/education/9464911/What_is_HPLC.html Lakna. (2019, September 25). Difference Between Normal Phase and Reverse Phase Chromatography. Retrieved from PEDIAA: https://pediaa.com/difference-between-normal-phase-and-reverse- phase-chromatography/#:~:text=The%20main%20difference%20between%20normal,and%20a %20polar%20mobile%20phase. Magnesium Stearate. (n.d.). Retrieved from chemicalland21: http://www.chemicalland21.com/industrialchem/organic/MAGNESIUM%20STEARATE.htm S., J. (2020, January 1). Will povidone iodine dissolve a polar molecule? Retrieved from Wyzant: https://www.wyzant.com/resources/answers/737801/will-povidone-iodine-dissolve-a-polar- molecule#:~:text=Povidone%20is%20polyvinylpyrrolidone%20and%20is,water%20and%20non %2Dpolar%20solvents.&text=It%20is%20soluble%20in%20water,isopropanol%2C%20which %20are%2 Scott, M. (2013, February 16). Why is reversed phase high-performance liquid chromatography (HPLC) preferred? Retrieved from Research Gate: https://www.researchgate.net/post/Why-is-reversed- phase-high-performance-liquid-chromatography-HPLC-preferred Types of Reaction. (n.d.). Retrieved from Bitesize : https://www.bbc.co.uk/bitesize/guides/zqd2mp3/revision/5
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