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Case study 8: Analysis and interpretation

Answer the following questions and prepare a short report/presentation summarising your findings.

Click here to download a pdf document containing the drawings of the TLC plates, spectra and chromatograms.

Q1: What samples will the analyst require to carry out the investigation? 

Q2: What other materials will be required to carry out the various tests (for example, how might the analyst identify a spot seen on a TLC plate)?

Q3: Which techniques could be used for the analysis of the sugars?

Q4: What are the main advantages and disadvantages of TLC for sugar analysis?

The results from a TLC analysis of sugars are shown in TLC plate 1.

TLC Plate 1: Drawing of TLC plate from the analysis of sugars present in counterfeit and genuine samples

Q5: What does the TLC plate show?

The preliminary TLC results are investigated further using HPLC.

The HPLC system can provide quantitative information on sugars and qualitative information on oligosaccharides. The results from the HPLC analysis are shown in the following chromatograms:

Chromatogram 1: HPLC of mixed standard of glucose and maltose

Chromatogram 2: HPLC of counterfeit sample

Chromatogram 3: HPLC of genuine sample

Q6: What do the chromatograms show?

The quantitative information on sugars obtained from HPLC is not very precise. Enzymatic methods offer more reliable data than other procedures but are more labour intensive. Confirmation of the sugars identified by TLC and HPLC requires an enzymatic method of analysis. This provides more reliable quantitative information.

The content of glucose in a sample can be measured by phosphorylation in the presence of the enzyme hexokinase (HK), equation 1. This is then followed by an indicator reaction – a reaction that enables the amount of glucose-6-phosphate produced in the reaction indicated in equation 1 to be estimated.

Equation 1:

A second enzyme, glucose-6-phosphate dehydrogenase (G6PDH), in the presence of nicotinamide adenosine diphosphate (NADP⁺) yields 6-phosphogluconolactone and the NADP⁺ is reduced to NADPH (equation 2). The amount of NADPH produced is stoichiometric and proportional to the amount of glucose present. The increase in the concentration of NADPH is measured by monitoring absorbance at 340 nm. This gives a figure for the glucose content of the sample.

Equation 2:

To estimate the amount of maltose present the enzyme α-1-4-glucosidase is used. The enzyme hydrolyses the maltose to give two glucose units (equation 3). The amount of glucose is then measured using the above procedure.

Equation 3:

The assay will give a total glucose figure, i.e. the glucose produced as a result of the hydrolysis of the maltose and the glucose present in the sample. The maltose content is determined by subtracting the glucose concentration determined by the hexokinase experiment from the total glucose.

The results of the enzyme analysis are shown in Table 1.

Q7: What do these results show?

Q8: What techniques could be used to determine the colours present in the samples?

The colours in the various samples were concentrated using a cartridge clean up and extraction system. The extracts from the samples were examined using thin layer chromatography.

The samples were orange-yellow in colour and the genuine sample lists sunset yellow as an ingredient.

The samples were therefore examined for the presence of a variety of orange, yellow and red colours: sunset yellow, tartrazine, quinoline yellow, orange RN, carmoisine, red 2G, and amaranth. The results are shown in TLC plate 2.

TLC Plate 2: Drawing of TLC plate from the analysis of colourants extracted from genuine and counterfeit samples

In all TLC work, the identity of the analyte is assessed by comparing the R_f value for the analyte with the R_f values for standard compounds run on the same plate. The R_f value is the distance travelled by the analyte (or standard) relative to the distance travelled by the solvent front:

Q9: What do the results shown on the TLC plate suggest about the colours in the counterfeit sample?

The UV visible spectra of the samples and two standards (tartrazine and sunset yellow) where also recorded. The results are shown below.

Visible Spectrum 1: Counterfeit sample

Visible Spectrum 2: Tartrazine

Visible Spectrum 3: Genuine sample

Visible Spectrum 4: Sunset yellow

Q10: What do the spectra show?

Analysis of contaminants

Five aspects of contamination were investigated:

volatile organic compounds

metals

pesticides

nitrates, nitrites, sulfate

microbiology.

Volatile organic compounds: The samples were examined by headspace gas chromatography for the presence of volatile organic solvent residues and for other volatile components. A chromatogram for the standard used to determine volatile organic compounds is shown in chromatogram 4.

Chromatogram 4: Representative gas chromatogram of mixed Volatile Organic Compounds (VOC) standard obtained using a purge and trap injector and a mass spectrometry detector

The detection limits for the determination of the compounds shown above in beverage samples are typically in the range 1 to 5 mg L^-1. No solvent residues were detected in either the genuine or counterfeit samples.

Differences in the flavour components were observed but without extensive data from other samples the significance of these differences could not be determined (note that the results from the analysis of the flavour components are not presented in the case study).

Metals: The determination of metal content was carried out using graphite furnace atomic absorption spectroscopy (GFAAS). Using GFAAS the detection limit for lead is approximately 5 mg L^-1 and for cadmium it is approximately 0.5 mg L^-1. Multi-element analysis is possible using inductively coupled plasma – optical emission spectroscopy (ICP-OES). Using this technique the Ca, Mg, P, Fe, Cu, Mn and Zn content can be determined.

Using GFAAS to analyse the samples it was found that lead and cadmium were not present at levels higher than the detection limits. From the multi-element ICP-OES some differences were observed between the counterfeit sample and a genuine sample. However, differences were also observed between the genuine samples, thus no significant conclusions could be drawn from these results.

Pesticides: To screen for pesticides, the pesticide residues need to be extracted from the samples before being identified and quantified by gas chromatography with an electron capture detector or mass spectrometry detection. Following solvent extraction it is normally necessary to concentrate the sample using a cartridge clean-up system. The laboratory would have to be sure there were no interferences from other extractable organic compounds, and that components of the matrix did not reduce the efficiency of the extraction.

Chromatogram 5 for the standard used for the pesticide screen is shown below.

Chromatogram 5: Gas chromatogram of pesticide standards obtained from gas chromatography with electron capture detector used to screen for pesticides

No significant levels of pesticide were found in either the counterfeit or genuine beverages.

Nitrate, nitrite and sulfate: Inorganic ions such as nitrate, nitrite and sulfate are commonly analysed by ion chromatography. This is a technique similar to HPLC.

Chromatogram 6 shows the results obtained for a standard containing a range of ions.

Chromatogram 6: Ion chromatograph of standard

No significant levels of nitrite were found in any of the samples. The suspect sample appeared to contain significantly more nitrate (34 mg nitrate per litre of sample) than the genuine sample (12 mg nitrate per litre of sample). However this amount is below the level currently permitted for drinking water in the EU regulations (50 mg nitrate per litre of water).

Microbiological contamination: The microbiological laboratory would determine the total viable count, coliforms, yeasts and moulds, osmophilic organisms, and lactobacilli.

The results showed a low number of viable bacteria in the counterfeit sample that were not present in the genuine samples. These included some lactobacilli and some osmophilic organisms.

The levels found were not thought to represent a problem, particularly at the low pH of the products (pH 3.2 for the genuine product, pH 3.1 for the counterfeit product).

Q11: What conclusions about the counterfeit product can be drawn from the results of the contaminants study?

Conclusions

Q12: What conclusions can be drawn from the full analysis?