Awad Nasser Albalwi

Analysis of Food Additives by HPLC and UV-Vis

Aims: • To determine the concentration of some additives in various beverages and an unknownsample using High Performance Liquid Chromatography (HPLC).• To use ultraviolet absorbance to measure the food additives caffeine and benzoic acid in(non-diet) soft drinks, using Beer’s law and by solving simultaneous linear equations.• To compare and contrast these two analytical methods as applied to the determination ofartificial sweetners.format

Part A- HPLCProcedure- The Calibration standards were prepared in the ranges of 0-20,40,60,80&100 ppm for caffeine, saccharin and benzoic acid by pipetting the required amount of stock solution into 100ml volumetric flasks and making up to the mark with MilliQ water.

The Soft drink was diluted by a factor of 10 to ensure the concentration of the analytes in the can were within the calibration range.

The tea bag was weighed and the tea made up in a 250ml volumetric flask. And then it was diluted by factor 20.

the pH of the eluent was measured using pH electrodes. The unknown solutions were then collected and made up to the mark with MilliQ

waterin 100ml volumetric flask. The samples were than put through the HPLC starting with the most dilute standards and the peak areas for each solution were recorded.

Pure caffeine and benzoic acid were injected into the HPLC column last to determine which peak belonged to them. The caffeine peak was identified as a larger peak at 3min , Benzoic Acid peak at the duration time (6min).

The parameters for the HPLC were as follows:

Eluent25% MeOH/75% ,1ml Acetic acid buffered to pH 3

Flow rate 1.4ml/minDetector UV Detector – 254nmStationary phase Band clone10u –C18Column Length 150x3.9mmPressure 1494 psiLoop size 20µL

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B-UV-Vis PartProcedure- Standards in the range of 0,2,5,7.5&10ppm for benzoic acid and 0,4,10,15&20ppm for Caffeine. The zero solution was prepared in 100ml volumetric flasks and 0.01M HCl. The soft drink was diluted by a factor of 100. The tea was by a factor of 100 for analysis. the unknown solution was collected and made up to the mark with milliQ water.

Once all samples and unknown were collected and prepared a baseline of the blank was recorded The spectrum of each of the highest concentrated blanks was then recorded and the wavelength of peak absorption was then recorded(230nm for benzoic acid and 273nm for caffeine).

The absorption of all standards was then recorded at 230 and 273nm and the absorption of both caffeine and benzoic acid at both wavelengths was recorded. The unknown, diluted soft drink & tea solutions were then recorded and the absorptions at both wavelengths recorded.

The Calibration curves allowed the extinction coefficients for caffeine and benzoic acid to be calculated at both wavelengths, this then allowed the concentrations of caffeine and benzoic acid in the unknown, Soft drink and tea samples to be calculated by employing the simultaneous equations described in the lab manual.

Results: The following table shows the results obtained by both the HLPC and UV methods:Standard mg/l Saccharin (Peak

Area)Caffeine Peak Area Benzoic

Acid(Peak area)20 91184 418983 9564240 198222 831018 19561260 283020 1224027 28702780 491768 1686236 395409100 522328 2028593 476241

a

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sample HPLC UV

b

c

de

f g

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Unknown [X] mg/L [X] mg/L Caffeine 63±4 25mg/l 53±325mg/lBenzoic Acid 84±425mg/l 88±225mg/lSaccharin 50±2025mg/l

Soft drink [X] mg/l [X] mg/l

Caffeine 330±12mg/lOr 33±1 in 100mg 465±25mg/l

Benzoic Acid ------- 87±225mg/lSaccharin 231±46mg/l

Tea [X] mg/l Mg/l

Caffeine104±4mg/lOr 26±1mg/250ml 279 ±7mg/l

Discussion- Both methods were successfully used to determine the concentrations of caffeine, benzoic acid in various samples. Both methods gave results closely in case of unknown solution.

By using HPLC data , The calibration curves obtained by plotting the peak areas

against concentrations are very linear with R2 values being 0.999 for Caffeine and benzoic acid and 0.974 for saccharin. These linear results give very accurate results as indicated by relative errors of ±4 for caffeine, benzoic acid , but for saccharin was high in the unknown solution .

The concentration of caffeine in soft drink was determined to be 33±1 mg/100ml. It can be useful for measuring the accuracy ,if this result was comparing with that on the label of soft drink. Unfortunately, the label data of soft drink was not provide by the administrator.

The concentration of tea in the tea solution was calculated to be 26±1mg/250ml.

Caffeine & benzoic Acid -From Mcdevitt

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In the UV prac the first thing that was required to be done was to measure the wavelength of maximum absorption for caffeine and benzoic acid. This was done by measuring absorbance of the highest concentration standard of each and measuring the spectra from 210-300nm and then looking at the wavelength that corresponding to the maximum absorption. The absorption of unknown ,soft drink & tea solutions was then measured at both wavelengths (230 & 273nm). The concentration of caffeine and benzoic acid was then able to be determined by using beers law and solving the appropriate equations provided in the lab manual.

The concentrations of caffeine and benzoic acid in the unknown solutions were calculated to be 53±3 mg/L and 88±2 mg/L respectively. The results are more closer to those HPLC results (Table.1). On other hand , The concentration of caffeine in soft drink was much greater than it from HPLC. The reason for this might be the absorbance is for the caffeine and other contaminations such as saccharin.

The calculated concentration of caffeine in the soft drink was 465±25mg/l by UV/V. This value is much larger than that obtained via HPLC (table.1). It is interesting that the result coming from HPLC indicated that there is no area peak for benzoic acid while the UV/Vis gives result in present it in soft drink . It is necessary to make sure that there is no benzoic acid in soft drink by checking the label data of the soft drink or by measure the benzoic acid by using other method such as GC/MS. If the investigation of present of benzoic acid in soft drink is positive , that means there is systematic error in UV/Vis.

In the UV method beers law appears to hold for all analytes as can be seen by the very linear plots of absorbance vs concentration for caffeine and benzoic acid at the two wavelengths. However the graph for the absorbance of benzoic acid at 273nm has an R2

value of 0.9955 which is less than that for the other 3 graphs (graph ,e).

In order to overcome the problem of beers law not holding at higher concentrations samples with high concentrations of analyte -such as tea sample - can be diluted so their absorbance behaviour will obey beers law, the concentration of this diluted solution can be calculated and then by taking into account the dilution factor the concentration in the original sample can be calculated. It is also crucial to ensure the concentration of the analyte solution is within the calibration range to ensure accurate calculations for the concentration of the analyte are made.

conclusion :Both methods used have their advantages and disadvantages over the other. HPLC

is best used when there are numerous components present as it will separate the components out.

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The Questions :1. In HPLC, why is peak asymmetry a problem? Describe 3 possible causes of asymmetry.

1) Peak asymmetry is a problem in HPLC as it leads to non reproducible results which means the usefulness of HPLC as an analytical tool is greatly diminished. Possible causes of peak asymmetry include:

a) Overloading the column. Overloading can cause peak fronting which will produce a band shape that contains a gradual rise and then an abrupt fall2. Overloading is caused by having to much solute in the column. As the concentration of solute is increased it will become more soluble in the mobile phase and as the solute is eluted little solute will trail behind the peak resulting in an asymmetric band shape3.b) Peak tailing can occur when some sites in the stationary phase bind the solute more

strongly than others. Peak tailing results in a peak with a sharp rise in concentration at the beginning and a long tail of gradually decreasing concentration after the peak4

.

c) Peak tailing may also arise due to degradation of the column.

2. For both methods, comment on the sensitivity of each analyte and give options forimproving the sensitivity.

The sensitivity of a particular method for the analytes to be investigated is one of the major factors influencing which method to use. The sensitivity of a measurement gives a measurement of the instrument response for a change in concentration, for a calibration curve the sensitivity can be determined from the slope of the calibration line. The sensitivities achieved in this experiment for both methods are shown in the table below.

HPLC UV

Sensitivity (Peak area

units/ppm) Sensitivity (Abs units/ppm)Saccharin 5568 Benzoic Acid 273nm 0.0079Benzoic Acid 4803 Benzoic acid 230nm 0.0923Caffeine 20482 Caffeine 273nm 0.02486 Caffeine 230nm 0.0262

Both methods have different sensitivities towards the different analytes. The HPLC method was significantly more sensitive to caffeine than either benzoic acid or saccharin, whereas the UV method was most sensitive towards benzoic acid at the wavelength of its peak absorbance . The difference in sensitivities towards each analyte is due to the differing instrument response towards the presence of each species, as each method has a different type of detector each method will respond differently to different analytes.

The sensitivity of a measurement was determined by the type of detector used.In the HPLC method a UV detector was employed. UV detectors are good for

gradient elutions with non absorbing solvents2. The most common way to improve the sensitivity of a measurement is to use a better detector, other detectors that may be employed with HPLC that have good sensitivity towards the analytes include electrochemical detectors and evaporative light scattering detectors2, however the sensitivity of each detector will be dependant on the analytes to be investigated.

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3)In HPLC, if a peak was observed in a soft drink that corresponded to saccharin eventhough it was not listed in the contents on the container, how could you go about

confirming the presence or otherwise of this compound?

3) The presence of saccharin in the can could be confirmed by a number of different methods. One of the most effective methods would be the use of mass spectrometry to identify the presence of sacchrain. The mass spectrum of saccharin could be viewed on a database, the soft drink could then be analyzed by having a mass spectrometer attached to the HPLC much like in GCMS and the mass spectrum of the peak compared to the database. If a peak at m/z=183 is observed this would correspond to the molecular ion of saccharin and indicate saccharin is most likely present.

The presence of saccharin could also be confirmed by injecting pure saccharin into the HPLC under different chromatographic conditions and comparing the retention times of the pure saccharin and the peak thought to correspond to saccharin as it is unlikely two components will have the same retention times under two lots of conditions.

4. For the tea sample the concentration of caffeine may be expressed as mg caffeine/L drink.Note that your extraction of caffeine from the solid food was not exhaustive; rather it was performed in such a way as to yield a typical serving. Thus your result (recovery) for mg caffeine/g solid food may be low. How could you check the completeness of recovery of caffeine from the tea leaves?

4) The remaining caffeine in the tea bag could be extracted via the Swiss water process. During this process the tea bags are soaked in hot water releasing the caffeine, the water is then passed through a carbon filter which traps the caffeine molecules. The caffeine can then be isolated and the amount obtained is weighed and compared to the amount extracted by just making the tea. This would give an indication for the completeness of recovery of the caffeine. The excess caffeine could also be extracted via an extraction process using methylene chloride 3.

6. Discuss the impact of saccharin and other compounds found in soft drinks on the determination of caffeine and benzoic acid by UV/Vis. Could you improve themethod to account for these additional compounds? How?

HPLC is very sensitive to other analytes that may be present. During HPLC all analytes are separated out in the column, however during the UV-Vis measurements other analytes are not separated out, these impurities may also absorb light at the two wavelengths used and thus give an absorbance reading that is higher than the actual reading if just caffeine and benzoic acid was present. This effect was demonstrated by measuring the spectra of the unknown solution with saccharin present, the spectra showed saccharin absorbed some UV radiation at 230 and 273nm, this resulted in the absorbance at these two wavelengths being higher than those corresponding to the unknown solution containing no saccharin. The experiment could however be modified to take into account the presence of saccharin in the unknown. The absorption of all species (caffeine, benzoic acid and

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saccharin) over a range of concentrations could be measured and from the calibration graphs the extinction coefficients could be determined. By noting that the total absorption is the sum of all the absorptions a least squares analysis can be used to minimize the square of the difference between the calculated absorbance and a guessed absorbance which is obtained by guessing a value of the concentration of the analytes, this can be done efficiently using the solver function in excel2. The calculated concentrations that result in the “guessed” absorbance being as close as possible to the measured absorbance such that the square of their difference is minimized will give the concentration of the unknowns. If not accounted for the presence of additional analytes that absorb over the wavelengths in question can lead to significant errors resulting in higher than actual concentrations being calculated. This is a significant disadvantage of the use of UV spectroscopy over HPLC methods.

References:

1 Mcdevitt, V.L, Rodriquez, A, Williams, K.R, 1998, Analysis of Soft Drinks: UV Spectrophotometry, Liquid Chromatography and Capillary Electrophoresis, Journal of Chemical Education, Vol 75, No5, pg625-628

2 Harris, D.C, Quantitative Chemical Analysis , W.H, Freeman and Company.

3http://departments.oxy.edu/tops/Caffeine/CAFFEINE-T.pdf