Lab Report on High Performance Liquid Chromatography

The lab report below was submitted as part of the coursework for CM2142 Analytical Chemistry. Please do not plagiarise from it as plagiarism might land you into trouble with your university. Do note that my report is well-circulated online and many of my juniors have received soft copies of it. Hence, please exercise prudence while referring to it and, if necessary, cite this webpage.

1.      Aim
This experiment aims to determine the concentration of caffeine and tartrazine present in an unknown soft drink sample with reversed phase High Performance Liquid Chromatography (HPLC).
2.      Results and calculations
Preparation of sample
5.0 ml of sample was first diluted with 20.0 ml of deionised water to form 25.0 ml of sample solution. The solution was filtered before analysis with HPLC.

Preparation of individual standards
For 20ppm solution of caffeine:

1.00 mL of 500ppm standard caffeine solution was used to prepare 25 ml of 20ppm caffeine standard.
By likewise calculations, 2.50 mL of 50ppm standard tartrazine was used to prepare a standard solution with the requisite 5 ppm concentration.
Preparation of combined standards
Volume of caffeine needed for standard 1 = (10 x 25) ÷ 500 = 0.50 mL
Volume of tartrazine needed for standard 1 = (1 x 25) ÷ 50 = 0.50 mL
 [Caffeine]/ ppm Volume of Caffeine used (mL) [Tartrazine]/ ppm Volume of Tartrazine used (mL) Std 1 10 0.50 1 0.50 Std 2 20 1.00 3 1.50 Std 3 30 1.50 6 3.00 Std 4 50 2.50 10 5.00
Table 1: volume and concentration of caffeine and tartrazine used
Table 2: retention times and peak areas of caffeine and tartrazine after HPLC
From Graph I (in Supplementary Data), using the equation, y = 14293 x - 10548, where y is the peak area and x is the concentration of caffeine (ppm),  concentration of caffeine present in the soft drink, 5x = 5(400725.75+10548)/14293 ≈ 143.8 ppm
From Graph II (in Supplementary Data), using the equation, y = 10405 x +711.1, where y is the peak area and x is the concentration of tartrazine (ppm), the concentration of tartrazine present in the soft drink is 5x = 5(21943.84-711.1)/10405  = 10.2 ppm.
The x value obtained from the graph must be multiplied by five as the concentration of the compounds in the soft drink was diluted with deionised water (in 1:4 ratio) prior to injection. Retention times of the eluted caffeine and tartrazine were noted as well. The following results were obtained.
 Retention time (min) Concentration (ppm) Caffeine 3.23 143.8 Tartrazine 1.46 10.2
Table 3: Retention time and experimental concentration of the components in the unknown sample
3.       DISCUSSION
In this experiment, HPLC is used to determine the concentrations of caffeine and tartrazine in a soft drink sample. Although gas chromatography (GC) has a higher sensitivity and speed, it cannot be utilised in this case as both compounds are not sufficiently volatile and may degrade at high temperatures.
Figure 1: Setup of HPLC
As seen Fig 1, a sampling loop is often used in HPLC to inject miniscule volumes of samples into the column; this prevents column overloading and band broadening. The system used in the experiment makes use of a dual reciprocating plunger solvent delivery which is connected to a degasser, a manual injector with 5 μL sample loop. This ensures that only the required volume is introduced into the column and the excess is drained away.
Reversed-phase partition HPLC
In this experiment, octylsilane serves as the liquid stationary phase. To increase column lifespan and reduce column bleed, the liquid stationary phase is chemically bonded onto the silica particles (right figure). This bonded stationary phase is non-polar.

The mobile phase, on the other hand, is polar. It consists of an isocratic solvent made up of 60% methanol and 40% water. Because the solvent composition remains constant throughout the elution, this is therefore known as an isocratic elution.

In reverse-phase chromatography, the more polar component – which is tartrazine in this experiment – will elute first. There is greater interaction between the more polar tartrazine and polar solvent. Hence, tartrazine will prefer to remain dissolved in the polar solvent than adsorbed on the non-polar bonded stationary phase; it will be carried by the mobile phase and eluted first. This accounts for its shorter retention time. On the other hand, the less polar caffeine has a lower interaction with the mobile phase and a greater interaction with the non-polar stationary phase. This results in a greater retention time and a slower elution.
Normal-phase partition chromatography is not used in this experiment to separate the polar compounds. In normal-phase chromatography, the stationary phase is polar and a non-polar phase mobile solvent is used. The mobile phase used is usually organic solvent. As both caffeine and tartrazine are polar and water-soluble compounds, the retention time would be much longer if normal-phase chromatography is used. This may lead to band broadening and lowered efficiency.
Analytes present
In HPLC, separation is based on the interactions between the sample, stationary and mobile phases.

Figure 1: Structure of Caffeine                                               Figure 2: Structure of Tartrazine
Caffeine has the chemical name of 1,3,7-trimethylxanthine and a molecular formula of C8H 10N4O2[1]. It is a psychoactive stimulant that is usually added into soft drinks; as an additive, it contributes a bitter taste as well as induces a stimulating effect. Caffeine is less polar than tartrazine due to the absence of ionic groups. Therefore, caffeine is eluted later than tartrazine and has a longer retention time.
Caffeine, being colourless, can be detected within the UV absorption range of 210nm and 275nm. Thus in this experiment, a wavelength of 220nm was used to measure the elution of caffeine using the photodiode array detector conducting a UV/VIS scan.
Tartrazine has a molecular formula of C16H9N4Na3O9S2[2]. It is a synthetic lemon yellow food colouring that is widely used in the food industry. From figure 3, tartrazine has more polar groups such as the negatively charged sulphonic acid groups as well as the hydroxyl group. Hence, it will interact more with the polar mobile phase than the non-polar stationary phase resulting in a shorter retention time relative to caffeine and will be eluted first.

 Figure 3: Colour chart at different wavelenghts of absorbance
Tartrazine is yellow. This is because it absorbs the complementary colour of violet. From figure 4, its absorbance should occur at wavelengths in the visible light range approximately from 380 nm to 430nm.  In this experiment, the absorbance of tartrazine was measured at 425nm to determine the concentration and identify tartrazine in the unknown sample.
Caffeine absorbs in the UV range of around 220 nm. Because it absorbs in this range, it is colourless to the naked human eye.
Analysis of Data
As seen from Graphs I and II, the R2 value of the calibration curves is almost 1. This suggests that there is a strong linear relationship between the area under the peaks and the concentration of the components. The concentrations of the components in the unknown sample can therefore be accurately extrapolated from these calibration graphs.
Column efficiency is also one important factor in chromatography which is affected by the plate height, H and the number of theoretical plates, N. the 2 components are related in the following equations:
In liquid chromatography, the minimum in plate height occurs at a very low linear velocity of the mobile phase as compared to gas chromatography. Thus, in order to maintain a suitably reasonable flow rate, the sample can be injected at high pressures.
Experimental techniques
The sample and standards must be prepared carefully to minimise the errors in their concentrations. Deviations from the concentrations may affect the recorded peak areas and subsequently, the calibration curves of peak area against concentration. This may implicate the eventual results obtained.
The 5 μL sampling loop used ensures that only the required volume is introduced into the column. This reduces human errors and allows for consistent and accurate introduction of small samples into the column.
The sample is degassed prior to introduction into the column. Degassing removes any dissolved gases present in the sample. Otherwise, these gases may cause band broadening as well as reduce the efficiency of the column.
The syringe is rinsed with the sample at least 3 times before filling with the sample for injection. This reduces chances of contamination with may affect the concentration and the composition of the standards.
In most soft drinks, there’ll be some UV absorbance from the matrix of these drinks. This does cause a small systematic error, but this error is negligible compared to the levels of caffeine and tartrazine in the sample.
 Figure 5: Structure of aspartame
In this analysis, a non-diet soft drink is probably used for analysis[3]. This is because the sugar substitute found in diet drinks, aspartame, also absorbs UV radiation due to its conjugated system. Aspartame therefore interferes in the analysis. Drinks containing caramelised sugar are avoided too, as this colorant absorbs in the UV region of interest.
Suggestions for improving accuracy of results
To ensure the reliability of the results, the peak area of each standard was measured twice. This allows the average peak area to be calculated and reduces random errors. Greater accuracy may be ensured by taking more readings and using their average values to plot the calibration curves.
In this experiment, the concentration of caffeine and tartrazine in the sample is derived from calibration curves. These results may be double-checked by recording the absorbance of the sample and calculating its concentration with Beer-Lambert’s law. According to this law, A = ε c l, where A is the absorbance, ε is the molar absorptivity, c is the concentration of the absorbing species and l is the path length of the sample[4]. Once the absorbances of the samples are known, their concentrations can be measured.
Safety Precautions
According to safety data, caffeine is a skin irritant. Upon exposure, wash away with water.
The HPLC eluent, with 60% methanol and 40% water, is a flammable mixture and an irritant. Any residue should be placed in the waste container and spillages should be washed away with plenty of water.
4.       CONCLUSION
As seen from table 4, the unknown sample contains caffeine with a concentration of 143.8 ppm with retention time of 3.23 min and tartrazine with a concentration of 10.2 ppm and retention time of 1.46 min.
5.       REFERENCES
[1] Medscape Reference. Structure and properties of caffeine. Article retrieved on 25 Mar 2012:  <http://emedicine.medscape.com/article/821863-overview>
[2] About.com – Chemistry. Tartrazine chemical structure. Article retrieved on 25 Mar 2012:  <http://chemistry.about.com/od/factsstructures/ig/Chemical-Structures---T/Tartrazine.htm>
[3] The University of Rhodes Island, UV spectroscopy of caffeine. Article retrieved on 26 Mar 2012: <http://www.chm.uri.edu/sgeldart/chm_414/414%20 Ultraviolet%20Spectroscopy.pdf>
[4] Sheffield Hallam University. Beer Lamber Law. Article retrieved on 27 Mar 2012:                              < http://teaching.shu.ac.uk/hwb/chemistry/tutorials/molspec/beers1.htm>

1.
2.       SUPPLEMENTARY DATA
i.         Calibration graph of caffeine
ii.       Calibration graph of tartrazine
iii.      Datasheet

Graph I: calibration curve of caffeine
Graph II: calibration curve of tartrazine