HPLC Determination of Caffeine, Lab report PDF

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Detailed report on practical for determination of caffeine via HPLC...

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Title: High Performance Liquid Chromatography (Part 8) Date: 22/2/16 Aim: The aim of this experiment was to learn the skills and techniques necessary for the optimisation of HPLC operating conditions. By varying the parameters flow rate, wavelength and mobile phase composition, the optimum conditions will be used for the analysis of caffeine and Theobromine in caffeine tablets and carbonated beverages. Materials and Methods: Chemical Caffeine Theobromine Methanol

CAS No. 58-08-2 83-67-0 67-56-1

Hazard Statements H302: Harmful if swallowed H301: Toxic if swallowed H225: Highly Flammable liquid and vapour H301: Toxic if swallowed H311: Toxic in contact with skin H331: Toxic if inhaled H370: Causes damage to organs

Instruments: 

Agilent Compact 1120 Isocratic HPLC system using EZChrom elite Compact, version3.30.



Cary UV-Vis 50 Spectrophotometer

Introduction: Chromatography is the analytical technique used for separating mixtures into their components due to their molecular composition and structure. (1) It involves using a stationary phase, which could be a solid or a liquid supported in a solid, and also a mobile phase which can be a liquid or gas. The mobile phase carries the sample being tested through a column containing the stationary phase. The different interaction and affinities between the stationary phase, mobile phase and the sample leads to the separation of components. High performance liquid chromatography, or HPLC is a form of column chromatography, but instead of using gravity, HPLC uses high pressures of up to 400 atmospheres to force the mobile phase through the column leading to separations in much faster times. HPLC is a widely used analytical technique due to its speed and efficiency in separating mixtures. Separations tend to be much better than that of normal column chromatography as smaller particles can be used in both phases. This gives a much greater surface area for interactions between the stationary phase and mobile phase.

Below is an image of the pathway used in HPLC. The mobile phase reservoir we used contained Methanol. The pump solvent manager then provides the high pressure as the sample is injected and passes with the mobile phase down through the HPLC column where the interaction between both phases separates the solution. The components then pass out, or elute, from the column and are detected with the results being passed through to a computer data station with the results being shown as peaks on a chromatogram.

Factors which are taken into account when using HPLC and also other forms of chromatography include the retention time, resolution between peaks and column efficiency. Retention time or Rt can be defined as the time taken for the components to flow through the column. Long retention times may lead to higher costs and wasted time while analysing solutions. Resolution can be defined as how well two elution peaks can be differentiated on a chromatogram. It is calculated using the difference in retention times between two peaks, divided by the combined widths of the elution peaks. These factors can be optimised in HPLC by altering the mobile phase composition, the flow rate and the wavelength to give the best separation. Mobile phase composition alters results due to changes in its polarity. For normal phase chromatography the stationary phase generally used is polar (e.g. Silica). For this experiment we used a mixture of methanol and water. Methanol is non polar whereas water is polar. The more water added to our methanol the more polar the mobile phase will become. As polar molecules will interact more with polar molecules, thus leading to better separation/ longer Retention times, this ratio for mobile phase composition is very important when determining the optimum HPLC parameters.

] Methanol structure

For this experiment caffeine and Theobromine were analysed. Caffeine (C 8H10N4O2) is naturally occurring in plants such as coffee, tea, and guarana and caca beans. It is used to paralyze and kill pests feeding on the plants. When it is purified it is a bitter, odourless white crystalline powder and is used in soft drinks to add a bitter taste and also caffeine is also acts as stimulant. Caffeine stimulates the central nervous system, increases heart rate and alters respiration. Caffeine has a melting point of 238°C and a molecular weight of 194.19g mol-1. Its structure can be seen below. Caffeine Structure

Theobromine (C7H8N4O2) is an alkaloid molecule in the group of methylxanthines similar to caffeine. Theobromine’s structure is also similar to that of caffeine with the molecule also having stimulant effects however the effects are approximately ten times weaker. Theobromine has diuretic, stimulant and relaxing effects. It can lower the blood pressure due to its ability to dilate blood vessels and can also relax the bronchi muscles of the lungs which allow it to be sometimes used as a cough medicine. (3) Theobromine has a melting point of 375°C and a molecular weight of 180.16402 g/mol. (4) Procedures: The experiment for the main part was carried out in accordance with laboratory manual CS351A (pgs 51-54) with one deviation. For part C, The analysis of caffeine in tablets and carbonated beverages, 2mls of the beverage was used instead of 5mls as stated in the manual. This was due to 5mls being too concentrated and its results would not fit within the standard curve. Results and Discussions

Part A: Quality Control Point Quality Control was run with 50:50, methanol to water, mobile phase composition and at a wavelength of 254nm. The flow rate was 1.0mL/min. Retention factor (K) and Efficiency (N) are both calculated using the following formulas. These figures help in determining how efficient and consistent the machines data is. K= (tR-t0) / t0 N= 5.54 (tR / half the peak base width) 2 TR= Retention Time T0= Dead Time

Retention Factor (k) for Quality Control Run 1

Run2

Run3

Average

0.5

0.48

0.49

0.49

Standard Deviation 0.01

% RSD 2.040816

The retention factor was out of range on the Shewart Chart; however the data correlated with the figures from previous weeks. This was perhaps due to a change in the system. Efficiency values below also varied greatly. This may be down to experimental error calculating base widths due to the tailings present on the peaks. (Graph 1 at end) Efficiency values (N) for Quality Control Run 1

Run2

Run3

Average

1465.61

3297.63

1237.96

2000.4

Standard Deviation 1129.186

% RSD 56.448

Part B: Optimization of Operating Conditions To Calculate and compare optimum performance for each HPLC parameter, resolution was calculated. Rs=2(tr2-tr1) / (Wb1+Wb2) Tr= Retention time Wb= Width of peak base Resolution was used to decide which variable of each parameter gave the best separation between the caffeine and Theobromine. A resolution factor of 1.5 is usually the minimum value which is accepted as a good separation between peaks

Mobile Phase Composition To Optimise Mobile phase composition the ration of Methanol to Water was varied. These were run at a wavelength of 254nm and a flow rate of 1.0mL/min.

Resolution of different mobile phase ratios Mobile T2(min) T1(min) Phase 50/50 2.277 1.747 50/50 2.277 1.747 70/30 1.747 1.590 70/30 1.590 1.750 35/65 3.477 2.077 35/65 3.507 2.087

Wb1(min)

Wb2(min)

R

0.2 0.25 0.18 0.21 0.2 0.33

0.27 0.33 0.36 0.24 0.35 0.22

2.255319 1.827586 0.581481 -0.711111 5.090909 5.163636

Both The 50:50 compositions and the 65:35 compositions of the mobile phases had resolutions over 1.5. The 65:35 compositions had a higher resolution; however this ratio was not used as the optimum composition due to this ratio having a longer retention time, which slows down the overall analysis time. The 50:50 ratios were chosen due to it having a satisfactory resolution and lower retention times which made the overall run time faster.

Resolutions at varying flow rates Flow Rate T2 (mL/min) (min) 0.4 5.68 0.7 3.233 1 2.183 1.3 1.68

T1 (min) 4.367 2.49 1.717 1.32

Wb1 (min) 0.5 0.2 0.25 0.19

Wb2 (min) 0.5 0.25 0.325 0.31

R 2.626 3.302222 1.62087 1.44

Resolution for the flow rate was high at flow rates of both 0.4mL/min and 0.7mL/min, well over the necessary 1.5 resolution value. However the flow rate of 1mL/min has a resolution of over 1.5 and a faster run time for both analytes to elute. This led to the 1mL/ rate being chosen as the optimum flow rate. 1.3mL/min did not have a resolution high enough to be considered.

Resolutions art varying wavelengths Wavelength T2 T1 (nm) (min) (min) 254 2.183 1.717 265 2.18 1.717 280 2.21 1.717

Wb1 (min) 0.25 0.23 0.23

Wb2 (min) 0.325 0.32 0.33

R 1.62087 1.683636 1.760714

The results received at each wavelength were very similar. Each wavelength had retention times which were extremely close for both at all wavelengths. This led to all wavelengths having resolutions in the 1.6 to 1.7 range with the wavelength of 280nm having the greatest resolution. This figure was confirmed via the UV scan analysis report. The scan ranged from 200.1nm to 399.9 nm and both Theobromine and caffeine had an optimum absorbance at 280 nm, which led to this wavelength being chosen as the optimum

Internal Standards for Caffeine and Theobromine Caffeine Concentration (ppm) 2 10 15 20 40

Caffeine Peak area 1

Caffeine Peak area 2

Caffeine average

Theobromine Peak area

[Caf Peak] [TB Peak]

4261294 7784362 9840841 14406202 27459422

4299851 7848574 9821236 14392666 27628968

4280572.5 7816468 9831038.5 14399434 27544195

7596410 8794921 9316788 9120704 9065209

0.563 0.889 1.055 1.579 3.038

The above concentrations were chosen to complete an internal standardisation procedure. For each standard the concentration of canine was increased while the concentration of Theobromine was kept constant. The resulting peak areas were used to create a plot so that unknown concentrations can be discovered. Due to the varying concentrations of caffeine, by studying the chromatograms, it was clear that the Theobromine eluted first and caffeine eluted second. This was clear due to the rising peak height as the value of caffeine concentration rose. The peak area of caffeine relative to that of Theobromine was plotted against the concentration of caffeine to create a calibration curve.

TB Peak Area/Caffiene Peak Area against Concentration of Caffiene(ppm) 3.5 3 f(x) = 0.07 x + 0.25 R² = 0.98

2.5 2 1.5 1 0.5 0 0

5

10

15

20

25

30

35

40

45

The above calibration curve gives the equation of the line. This allows the peak area from analysing both the Lucozade sample ad the caffeine tablets concentration to be found. The peak areas from the chromatogram from both analytes can be used and subbed in as the Y value so that the X value of caffeine concentration can be found.

Lucozade Caffeine peak area / Theobromine peak area = 119945350 / 11827914 = 10.14 Y = 10.14 Y=0.0674x + 0.2512 => 10.14 = 0.0674(x) + 0.2512 X = 146.718ppm Caffeine tablets Caffeine peak area / Theobromine peak area = 443219671 / 1189495 = 372.61 Y = 372.61 Y=0.0674x + 0.2512 => 372.61 = 0.0674(x) + 0.2512 X = 5524.64ppm Both the caffeine tablets and the Lucozade had concentrations of caffeine well in excess of the parameters of the standard graph. However the equation of the line aloud for a concentration to be found Personal Reflection: Overall this lab worked well and skills were gained in the functioning of a HPLC chromatogram and the computer that accompanies it. Chromatographs were interpreted and overall results for caffeine and Theobromine levels in the carbonated beverages were high as expected. If I was to repeat the experiment however I would improve certain areas. The experiment was long and our time management could have been improved. Also the organisation of data received with each chromatograph could have been improved which would lead to interpretation of results and the writing up of a report easier. Conclusion: To conclude, for this experiment, all aims were met. Both skills and knowledge on the applications and correct techniques needed for HPLC were gained throughout the lab. By analysing each parameter it was concluded that the optimum conditions for analysing both Theobromine and caffeine were a flow rate of 1mL/min, a wavelength of 280nm and a mobile phase ratio of methanol to water of 50:50. By using these values it was possible to calculate the concentration of caffeine in both caffeine tablets and Lucozade. Caffeine tablets contained a much higher concentration (5524.64ppm) of caffeine than the Lucozade (146.718ppm). After some research the level of caffeine which we analysed was similar to that stated on the label (46mg per 380ml bottle =>121ppm). This met the aim of the experiment of using internal standardised solutions to determine an unknown concentration of caffeine. Questions:

1. Gradient separations are used to separate samples containing many compounds. Gradient separations are useful because the mobile phase can be varied throughout the analysis so that a suitable polarity is found, which will lead to optimum separation. For normal phase HPLC, when a polar stationary phase is being used, increasing the mobile phase’s polarity (increasing the water content) will lead to better separation but longer Retention times. The correct balance has to be found. Both Isocratic and gradient separations have their own advantages and disadvantages. Some advantages of gradient separations over isocratic separation include improved resolution, increased sensitivity, shorter analysis time, and the ability to separate complex samples due to greater control over the mobile phase. However is that it can be time consuming to set up and more difficult to control. Isocratic separations on the other hand are always equilibrated and much simpler to use. They can have longer run times due to highly polar mobile phases being retained in the stationary phases for long periods of time. 2. By increasing column length, separation efficiency is increased. Separation efficiency is improved due to more surface area throughout the column leading to greater interactions between the stationary phase and the mobile phase improving separation. The increase in column length will increase retention time also; however this could be reduced by altering the flow rate. 3. For reversed phase HPLC the mobile phase solvent is polar and the stationary phase in nonpolar. This is due to the addition of hydrocarbon chains. Theobromine is less polar than caffeine. This causes caffeine to elute first from the column due to it having a low affinity to the non-polar stationary phase. The Theobromine will elute after due to it having a higher affinity for the stationary phase and more interactions occurring throughout the column.

References: (1) Laboratory Info : accessed on 19/02/2016 at 17:21 Online: http://laboratoryinfo.com/hplc/ (2) Pub Chem : accessed on 21/02/2016 at 21:06 Online: Synonyms

https://pubchem.ncbi.nlm.nih.gov/compound/caffeine#section=Depositor-Supplied-

(3) Phytochemicals : accessed on 20/02/2016 at 23:11 Online: http://www.phytochemicals.info/phytochemicals/theobromine.php (4) Pub Chem : accessed on 21/02/2016 at 22:48 Online: https://pubchem.ncbi.nlm.nih.gov/compound/theobromine#section=Chemical-and-PhysicalProperties (5) Caffeine informer : accessed on 23/02/2016 at 19:23 Online: http://www.caffeineinformer.com/caffeine-content/lucozade

Appendices: Chromatogram for Caffeine Tablet

Chromatogram For Lucozade Sample

UV scan analysis report for optimum wavelengths for Theobromine and caffeine...