Theory And Principles Of Liquid Chromatography Biology Essay

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High performance Liquid chromatography is a method of analysis which is mostly used in pharmaceutical analysis since it is able to carry out analysis without having to vaporize the analyte as in the case of gas chromatography (Braun, 1982). In this experiment, the main aim is to carry out analysis on samples of tablets that were suspected to be counterfeit. The amount of the aspirin and caffeine will be determined in these tablets and compared to the suspected sample of drug.

Theory and Principles of Liquid Chromatography

Chromatography is a technique that has been in use for quite a couple of years as a method of chemical analysis. It was initially used in the separation of coloured mixtures. However, in the modern world, this process has been developed and new techniques that are widely used in the separation and analysis techniques are in use in the modern method (Dolan, Kirkland and Snyder, 2009).

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Chromatographic techniques involves passing a sample mixture that is contained in solvent usually known as mobile phase which can either be a gas or a liquid through a an insoluble matrix or liquid that is stagnant but immiscible with the mobile phase usually known as stationary phase. The separation is based on the rate at which the sample distributes itself through the mobile phase.

There is various distributions mechanism in which the distribution of a sample may occur (Hill, 2000). Some of the examples include:

Adsorption

Partition

Ion Exchange

Size exclusion

Chromatographic techniques involves a wide range of methods involving simple methods with simple instruments such as paper chromatography to the more complicated and sophisticated instruments such as gas chromatography and high pressure liquid chromatography. Other chromatographic techniques include column chromatography, thin layer chromatography (TLC) among others (Robinson, 1988).

In this experiment high performance liquid chromatography was used. UV detector was used in the detection of the samples being eluted.

HPLC is especially used in the analysis due to its accuracy and precision in the analysis. It is also fast and many samples could be analysed at a record time for comparison.

Experimental procedure

Preparation of standards

The standard solutions of the three relevant solutions were prepared as follows:

0.4829g of aspirin, 0.0801g of caffeine and about 0.0534g phenacetin were measured and put in a beaker. 30ml of methanol was added in each beaker and stirred until they dissolved completely. The solution was then transferred to 50ml flask and the volumes made up to the mark with distilled water.

5ml of each solution was then diluted with 50ml of the mobile phase.

Preparation of the analgesic sample

The analgesic sample was prepared by accurately weighing about 0.05g phenacetin into a small beaker and the two suspected tablets. 30ml of methanol was then added and the tablets dissolved completely. The solutions were then transferred to the 50ml flask and the volume made to the mark with distilled water. 5ml of the sample was then diluted to 50ml using the mobile phase.

The chromatograms were obtained of each solution along with the peak area of each substance. The data was collected and analyzed.

HPLC Conditions

Reversed phase chromatography was used with the following conditions

Column: C18, non polar stationary phase, with internal diameter of 4.6mm and 150ml length

Mobile phase used was composed of 49.5% methanol, 49.5%water and 1% acetic acid

The flow rate was 1ml/min

Detector wavelength was 244mm

Injection volume was 10microlitre

Temperature was 250C

Result and Discussion

The elution order of the sample was caffeine, followed by aspirin and finally phenacetin. From the retention times of the standards, the samples retention time were used to give the identity of the peak. This was done and the results tabulated as shown below

Caffeine

Retention time

Peak area

standard 1

peak 1

2.724

757.0206

standard 2

peak 1

2.724

756.6738

standard 3

peak 1

2.724

756.6738

average

2.724

756.7894

Aspirin

Retention time

Peak area

standard 1

peak 2

4.08

4774.564

standard 2

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peak 2

4.08

4774.186

standard 3

peak 2

4.08

4774.186

average

4.08

4774.312

phenacetin

Retention time

Peak area

standard 1

peak 3

5.244

2545.532

standard 2

peak 3

5.243

2553.361

standard 3

peak 3

5.243

2553.361

average

5.243333333

2550.751

.

From the comparison of the retention time of the standards and that of the samples, the samples were identified as follows:

caffeine

Retention time

Peak area

sample 1

peak 1

2.723

267.3866

sample 2

peak 1

2.724

265.6188

sample 3

peak 1

2.725

266.7253

average

2.724

266.5769

aspirin

Retention time

Peak area

sample 1

peak 2

4.076

5748.23

sample 2

peak 2

4.078

5744.321

sample 3

peak 2

4.079

5750.743

Average

4.077666667

5747.765

phenacetin

Retention time

Peak area

sample 1

peak 3

5.24

2631.996

sample 2

peak 3

5.242

2629.23

sample 3

peak 3

5.244

2637.262

average

5.242

2632.83

Calculation of R value

The relative response factor (R) value was calculated using the following formula

R=

Standard solutions

From the equation, the R value for caffeine will be:

R=

Similar calculation for aspirin will be

R=

Sample solution

In the samples, rearranging the formulas and using the response factor indicated for each of the standard, the amount of each substance was obtained using the following formula:

Mass of caffeine in the sample will be =

% of caffeine =12.7975/

Mass of aspirin will also be =

The tolerance level for caffeine is between 90 and110% of the stated amount while that of aspirin is between 95 and 105%. Therefore, the range for caffeine in the tablet samples was supposed to be between 11.51775mg/tablet and 14.07725mg/tablet while that of aspirin was 250.5033mg/tablet and 276.8721mg/tablet.

Conclusion

The main aim of the experiment was to determine whether the samples of analgesic drug contained caffeine and aspirin as they claimed. This was precisely done and the result tabulated.

HPLC was especially used due to the major advantages that accrue to HPLC. This among others includes its stability to analyze samples without having to volatize them as is the cases in GC where it only analyzes samples that are do not decompose when they are volatized (Neue, 1997).

The separation is also very fast and efficient. The results are also repetitive and reproducible. The column can be continuously be monitored in case of any problem, it can be well identified before the analysis proceeds.

The types of detectors that are used in HPLC are of variety. If one detector does not give the desired results, another detector can be used and hence increasing the efficiency of the instrument.

The samples as they are being analyzed do not complicated sample pretreatment and the only treatment that is required is just to make the necessary concentration. In addition as these samples are being analyzed, multiple components can be analyzed with a single analysis. This gives the convenience in the use of HPLC in this analysis as the caffeine and aspirin could be analyzed in only one step.

HPLC conditions can be varied according to the samples being analyzed. Their also exists various columns for this. In addition, the stationary phase as well as the mobile phase can be varied according to the samples being analyzed.

Work cited

Braun, R.D. (1982), Introduction to chemical analysis; McGraw-Hill, New York

Hill, G. (2000); Chemistry in context (5th ed.), John Holman, United Kingdom, Britain

Robinson W.R., (1988), General Chemistry, Lexington; DC Health and Co

W.Dolan, J. Kirkland and L. Snyder; (2009) Introduction to the modern Liquid chromatography, Wiley publishers, New York

U. Neue; (1997); HPLC Columns, Theory and Practice, Wiley Publishers, New York.