Aims and Objectives of the Experiment
The aim of this experiment is to study the separation of an extract of a non-prescription analgesic tablet containing acetylsalicylic acid (aspirin) and caffeine. The separation is achieved through a well-known process called liquid chromatography. The quantities of the aforementioned substances also need to be determined in the experiment (“High Performance Liquid Chromatography,” n.d.).
Theory and Principles
Chromatography is a technique used for the separation of the components, or solutes, in a mixture. The separation is based on the amounts of the relevant solute in the moving, or fluid, stream. There are two phases in chromatography, the mobile phase, and the stationary phase. The mobile phase is a liquid or a gas, while the stationary phase is usually a solid but it may be a liquid as well (Giddings & Keller, 2016).
Separations using chromatography are accomplished by employing a “forced transport” of the mobile phase, which holds the analyte mixture, through a porous medium. The mobile phase provides the transport to the analyte, while the stationary phase is immobile which is the porous medium. The mixture of components is usually referred to as analytes, which is dispersed in the mobile phase at a molecular level. Liquid chromatography is one in which the mobile phase is a liquid. Similarly, if the mobile phase is a gas, the process would be known as gas chromatography (Kazakevich & LoBrutto, 2007).
High Performance Liquid Chromatography (also known as HPLC) was first introduced by Prof. C. Horvath, when he decided to use small glass beads with porous layer on their surface in order to better facilitate the mass transfer. These beads were used as packings in columns but they showed resistance to the flow of liquid. As a result, the professor built an instrument to allow a continuous flow of liquid through the column, and this is where high performance liquid chromatography originated (Kazakevich & LoBrutto, 2007).
In HPLC, the mobile phase or the solvent is held in a reservoir, from where it is pumped into the system at a specified flow rate. The sample is introduced into the mobile phase using an injector, which is then introduced into the packed column. A detector is used in order to analyze the mobile phase, or solvent, that comes out of the column to evaluate the separation (Waters, n.d.).
Instrument Used
The instrument used in this experiment is a Hewlett Packard HPLC system with a Rheodyne injector, with 20µl loop, a UV absorbance detector, and a filtration unit, with a 0.45 µm filter. The instrument is shown in figure 1.
The HPLC system from Hewlett Packard consists of a pump, which pumps the solvent after adding the analytes into the column; a detector, which detects the exiting solvent from the column for the extent of separation; an injector, which injects the analytes into the solution at a molecular level; a computer which controls the entire HPLC system and is connected to the detector as well; and the software installed in the computer which provides the interface for the control of the HPLC system from the computer and to provide the results of the experiment which may be saved for printing at a later time or printed right away (Agilent Technologies, 1999).
Figure 1: Hewlett Packard HPLC System
Advantages and Disadvantages of the Technique
High Performance Liquid Chromatography is being used as one of the dominant techniques in the domain of separation technologies. There have been a number of innovations in HPLC, such as ultra-high-pressure liquid chromatography (UHPLC), liquid chromatography-mass spectrometry (LC-MS), two-dimensional liquid chromatography (2D-LC), and many others, which have taken HPLC to a level of higher performance in a diverse range of applications. HPLC is applicable to a diverse range of analytes, or sample types. It offers an opportunity for a precise analysis, along with a highly reproducible quantitative analysis. HPLC is an operation which is flexible in its approach and is highly customizable according to the application. The automated operation also offers an added advantage over other techniques. HPLC offers a high level of separation and an ability to detect even the most sensitive separations.
With all these advantages, benefits and returns that HPLC has to offer, it offers a few limitations as well. The HPLC technique lacks a detector which is ideal and universal. Another limitation, if considered, is that it offers a lesser separation efficiency as compared to capillary gas chromatography. It might be considered as a relatively difficult technique for new users. This technique has few other limitations as compared to other separation techniques, however, the benefits that it offers makes it an arguable choice (Dong, 2013).
Results and Calculations
1- Aspirin and Caffeine in analgesic tablets (Anadin)
(Aspirin):
Weight of whole tablet = 435.2 mg
Weight of tablet used = 20.1mg
y = 0.933x + 1.8633
When, y = 343.192
x = (343.192 + 1.8633)/ 0.933
x = 369.834 mg
The aspirin amount (SA) in sample S is 369.834 mg.
Aspirin in whole tablet = (SA/25) * (Weight of whole tablet/Weight of tablet used)
= (369.834 /25) * (435.2 /20.1)
= 14.793*21.65
= 320.29 mg
y = 6.5054 x + 4.4359
When, y = 2382.73
x = (2382.73 +4.4359)/ 6.5054
x = 366.951mg
The aspirin amount (SA) in sample S is 366.951mg.
Aspirin in whole tablet = (SA/25) *(Weight of whole tablet/Weight of tablet used)
= (366.951/25) *(435.2 /20.1)
= 14.678*21.65
= 317.631 mg
(Caffeine):
Weight of whole tablet = 435.2 mg
Weight of tablet used = 20.1 mg
Table2: the raw data of caffeine
y = 2.5937x + 1.4234
When, y = 46.304
x = (46.304 + 1.4234)/ 2.5937
x = 18.401 mg
The caffeine amount (Sc) in sample S is 18.401 mg.
Caffeine in whole tablet = (Sc/25) *(Weight of whole tablet/Weight of tablet used)
= (18.401 /25) * (435.2 /20.1) = 0.736*21.65
= 15.935 mg
y = 15.313x + 11.415
When, y = 280.416
x = (280.416+11.415)/ 15.313
x = 19.057 mg
It means caffeine amount (Sc) in sample S is 19.057 mg.
Caffeine in whole tablet = (Sc/25) *(Weight of whole tablet/Weight of tablet used)
= (19.057/25) * (435.2 /20.1)
= 0.762*21.65
= 16.503 mg
The number of theoretical plates:
Where N = Number of Theoretical Plates, tR = retention time of peak, W = the peak width at a given peak height, and a is a constant depending on method used.
In the calculation, the peak width at 1/2 height is used, so a is equal to 5.54.
Peak 2:
N w1/2 = 5.54(1.651/ 0.1198)2
1052.18 Plates
Peak 5:
N w1/2 = 5.54(4.179/ 0.1250)2
6192.050 Plates
Peak 6:
N w1/2 = 5.54(4.561/ 0.1346)2
6361.210 Plates
Capacity Factor (Retention Factor or Relative Retention)
Where k = capacity factor, tR = the elution time of retained component, and t0 = the elution time of the un-retained sample
Peak 2:
k = (1.651-1.317)/ 1.317
= 0.2536
Peak 3:
k = (4.179-1.317)/ 1.317
= 2.173
Peak 4:
k = (4.561-1.317)/ 1.317
= 2.463
Discussion
The amounts of aspirin and caffeine in Anadin tablet have been determined in the experiment. Two methods were used for the purpose; namely the graph between the weight of the tablet and the peak height, and the graph between the weight of the tablet and the peak area. The amount of aspirin using peak height was found to be 320.29 mg, while the amount in case of the peak area was found to be 317.631 mg. The commercial value suggests that there should be 325 mg of aspirin in the tablet. It may be observed that the experimental values are pretty close to the commercial value, thereby endorsing the authenticity of the experimental values. The small differences may have been induced due to errors during the experiment which may include buffer concentrations, inaccuracy in weighing samples, and any deviation in pH.
The amount of caffeine was found to be 15.935 mg using the peak height, while it was found to be 16.503 mg using the peak area. The commercial value suggests an amount of 15 mg of caffeine in the tablet. Therefore, it would be safe to say that the experimental values were near accurate and the slight differences may be accounted for the errors in the experiment. It may be observed in both aspirin and caffeine that the use of the values from peak height resulted in more accurate values as compared to peak area, however trivial the difference. This may suggest that the peak height method may be more accurate as compared to the peak area, however, all of the other circumstances should be checked before accepting this inference. It was made sure that the peak height and peak area for the unknown were kept in the range of calibration curves by using over 20 mg of the weight of the tablet.
The efficiency of the column was checked by performing tests to assess the performance of the column. The number of theoretical plates and the capacity factor for the column were determined to do so. The larger the number of theoretical plates, the more the efficiency of the column. The number of theoretical plates were found to be 1052.180, 6192.050, and 6361.210 showing the high efficiency of the column. The capacity factor of the column is a measure of the degree to which the component is retained in the column as compared to the component which is not retained (lcresources, n.d.). The highest capacity factor of the column was found to be 2.463 on peak 4.
Conclusion
The experiment was simulated to perform high performance liquid chromatography to determine the amount of aspirin and caffeine in Anadin tablet. The weight of the tablet was plotted against peak height and peak area, in both cases i.e. aspirin and caffeine. The amounts of aspirin and caffeine determined through the experiment were found very close to the actual commercial values meaning that the experimental values were accurate. The minor differences may be due to the errors during the experiment. The column was also checked for performance by measuring the efficiency of the column by measuring the number of theoretical plates and the capacity factor. The number of theoretical plates were found to be high signifying the high efficiency of the column. Overall, it may be said that the experiment was successful and showed close to accurate results.
References
Agilent Technologies. (1999). Agilent 1100 Series HPLC Value System: User’s Guide. Waldbronn, Germany: Hewlett Packard. Retrieved from https://www.agilent.com/cs/library/usermanuals/Public/G1380-90000.pdf
Dong, M. W. (2013). The Essence of Modern HPLC: Advantages, Limitations, Fundamentals, and Opportunities. LCGC North America, 31(6), 472–479. Retrieved from http://www.chromatographyonline.com/essence-modern-hplc-advantages-limitations-fundamentals-and-opportunities
Giddings, J. C., & Keller, R. A. (2016). Chromatography. In Encyclopaedia Brittanica. Britannica.com. Retrieved from https://www.britannica.com/science/chromatography
High Performance Liquid Chromatography. (n.d.), 1–9.
Kazakevich, Y., & LoBrutto, R. (2007). HPLC For Pharmaceutical Scientists. Hoboken, New Jersey: John Wiley & Sons, Inc.
lcresources. (n.d.). Definition: Capacity Factor. Retrieved January 28, 2017, from http://www.lcresources.com/resources/TSWiz/hs210.htm
Waters. (n.d.). How Does High Performance Liquid Chromatography Work? Retrieved January 27, 2017, from http://www.waters.com/waters/en_US/How-Does-High-Performance-Liquid-Chromatography-Work%3F/nav.htm?cid=10049055&lset=1&locale=en_US&changedCountry=Y
