UV-Vis Analysis of a Suspected Date-Rape Sample

Figure 1. Structure of Ketamine

Image from: http://www.chemspider.com/Chemical-Structure.3689.html

1.0 Introduction

Ketamine is an analgesic and anaesthetic (prevents pain) which is managed in the medical industry. It has various forms such as powder, pills and liquid form which can be injected (Stevens, 2018). It is one of many Date-Rape drugs due to its ease of camouflage in drinks as it is colourless, odourless and tasteless (Negrusz, A., & Gaensslen, R.E. (2016).  Exposure to drugs of abuse such as Ketamine, can induce a wide range of sedative effects. This is caused by the blockage of N-methyl-D-aspartate (NMDA) receptors and so effects range from unconsciousness and disorientation, dependent on the dosage, to anterograde amnesia, which is the incapability to form memories after the occurrence that induced the amnesia, which may lead to the victim of sexual assault to not remember the event. This would make the victim less likely to report it (Narang, Malhotra, Singhal, Mathur, Chakraborty, Anil, . . . Pundir, 2017). This is because NMDA receptors (Bell, S. 2014, pp.219-220) are located in nerve cells, and they are involved in synaptic plasticity (Collingridge et al., 2017), and synaptic plasticity is a biological process which is involved with learning and the development of the memory (Zhang et al., 2018). The time it takes to become unconscious and dizzy is reduced when it is mixed with a depressant e.g. alcohol. In addition, long-term side effects of consuming Ketamine (if taken more than twice a week) are severe abdominal pain which is more commonly known as “K-Cramps” and damage to the kidneys and bladder. On the other hand, Ketamine is proven to be a good source for antidepressant purposes, (Zhong, Xiaomei, He, Hongbo, Zhang, Chunping, Wang, Zhijie, Jiang, Miaoling, Li, Qirong, . . . Huang, Xiong, 2016) as studies have shown it stabilizes patients much faster compared to other antidepressants (ScienceDaily, 2017).

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UV-Visible Spectroscopy (Biowave II) measures the numerical determination of different analytes by the amount of electromagnetic radiation a substance absorbs. Inside a UV-vis Spectroscopy machine, a visible and/or UV light source hits a mirror and is separated into its component wavelengths by a diffraction grating. The single wavelengths divide into two by a half-mirror – the sample beam and the reference beam. The sample beam passes through the sample cuvette containing the solution studied and then passes through a lens, which is then identified at the end by an electronic detector. The reference beam also passes through a (reference) cuvette, through a lens and then is detected. The intensities of each beam are compared (Www2.chemistry.msu.edu, 2006).

Electrons exist in pairs in orbitals at discrete energy levels. The energy level an electron is at is dependent on the amount of energy an electron has. If an electron has the minimum amount of energy it will be at ground state. Molecules can absorb radiation and move up to a higher energy state; they become excited and this process is called electronic excitation (Robinson, Frame, & Frame, Eileen M. Skelly, 2014). The electron transition requires so much energy that photons of ultraviolet and visible light are involved (Lewis, Evans, Evans, Wynne, & MyiLibrary, 2006). So, this analytical technique can be used to analyse colourless solutions and therefore can be used to determine the identification of drugs such as Ketamine.

The aim of the practical was to determine if Ketamine was present in the crime scene sample and to calculate the concentration of this Ketamine. This was done by comparing wave scans from a UV-Visible spectrometer to see if they had the same absorbance peaks as this indicates that they have the same energy requirements because they will absorb the same photons of light.

2.0 Experimental method

Five standard solutions were prepared to produce wave scans in a UV-Visible Spectrometer. These were: Ketamine, Aspirin, Paracetamol, Caffeine and 5% Ethanol. In addition, a crime scene sample was prepared by adding 1ml of the crime scene solution to a volumetric flask using a pipette. 5% ethanol was added to the conical flask until the meniscus sat on the line of the volumetric flask. Stopper the flask with a plastic stopper and shake the solution holding the flask with both hands.

Set the UV-Visible spectrometer to scan mode. Press number 1 for applications, then number 3 to produce the scan. Physically input the start and end wavelength which is 200-350nm. Next press the green button indicating OK. For the first scan ethanol is used to blank the apparatus to zero. Pour the 5% ethanol solution into a cuvette until the line is reached. Place the cuvette with the arrow facing towards the light in the UV-Visible spectrometer, then press the blue button. Once the noise has stopped remove ethanol and place in one of the scan solutions until they’ve all produced absorbance wave scans. To print out the wave scan you press number 2 on the spectrometer.

A stock solution of ketamine 100µg/ml in ethanol was prepared. A serial dilution took place to produce the standards.

When measuring the absorbances, the spectrophotometer was set to a single wavelength of 220nm. Firstly, analyse the blank (ethanol) solution in a cuvette (use this cuvette for all the standards to reduce error to the calibration curve) to zero the apparatus by pressing the blue button. Then place standard 1 inside and press the green button. Repeat this until you have scanned all the standards. Record your results in a table. Using a clean cuvette, pipette the crime scene sample into the cuvette and scan this. Analyse this sample three times.

After you have collected all the absorbances, plot a standard curve of absorbance against ketamine concentration as shown in figure 1. From this you can calculate the concentration of ketamine in the crime scene sample by adding a line of best fit and rearranging the equation of the line to find x.

3.0 Results and Discussion

This graph (figure 2) shows a strong positive correlation; as the concentration of ketamine increases, the absorbance at 220nm increases.

During the part of measuring absorbances a different group used the UV-Visible spectrometer in the middle of standards 4 and 5. This meant using another cuvette to blank the apparatus and hence introduced error to the calibration curve so the R2 value was not as strong as it could have been.

R2 is the

              Figure 2.

Table 1

Samples

Absorbance at 220nm

Crime scene

0.525

Crime scene

0.519

Crime scene

0.528

The calculation for the concentration of Ketamine from the crime scene sample is:
x=y0.0196

Table 2

Absorbance at 220nm

Concentration of Ketamine µg/ml

 0.525

0.5250.0196
= 26.78571…

 0.519

0.5190.0196
= 26.47959…

0.528

0.5280.0196
= 26.93877…

The average concentration of Ketamine in the crime scene sample was 26.7… µg/ml (3.s.f).  This was correspondent to 13.36… mg/500ml.

Due to the 1 in 25 dilution the final concentration of Ketamine was 334mg/500ml (3s.f.)

The concentration is high and so results in visual hallucinations, delirium and anterograde amnesia.  

4.0 Conclusion

In the conlusion, the research indicated that the crime scene sample absorbance scan and the ketamine scan were matches as they both had absorbance at similar values – for ketamine the peaks were 223nm and 269nm and for crime scene sample it was 226nm and 269nm. On top of this there may have been caffeine in the sample because caffeine also has a peak at 223nm. The aims were met as the intention was to prove ketamine was in the crime scene sample, however the R2 value was not as strong as intended because of errors produced by interference with the apparatus. Next time the experiment would be done whilst no one else requires the apparatus to avoid it happening in the future.

References

Bell, S. (2014). Forensic chemistry (Second edition.; International ed.). Harlow, Essex: Pearson pp.219-220

Chemistry, R. S. (2015). Ketamine. Retrieved from ChemSpider: http://www.chemspider.com/Chemical-Structure.3689.html

Collingridge, G., Mellor, J., Apps, R. and Warburton, C. (2017). NMDA Receptors | Centre for Synaptic Plasticity | University of Bristol. [online] Bristol.ac.uk. Available at: http://www.bristol.ac.uk/synaptic/receptors/nmdar/ [Accessed 24 Oct. 2018].

Lewis, Evans, Evans, Wynne, & MyiLibrary. (2006). Chemistry (3rd ed., Palgrave foundations). Basingstoke [England] ; New York: Palgrave Macmillan.

Narang, Malhotra, Singhal, Mathur, Chakraborty, Anil, . . . Pundir. (2017). Point of care with micro fluidic paper based device integrated with nano zeolite–graphene oxide nanoflakes for electrochemical sensing of ketamine. Biosensors and Bioelectronics, 88, 249-257.

Negrusz, A., & Gaensslen, R.E. (2016). Sexual Offenses, Adult: Drug-Facilitated Sexual Assault. In Encyclopedia of Forensic and Legal Medicine (pp. 267-271).

Robinson, J. W., Frame, E. M., & II, G. M. (2014). Undergraduate instrumental analysis. Florida: CRC Press.

ScienceDaily. (2017). Study answers why ketamine helps depression, offers target for safer therapy. [online]                                                                                                           Available                                      at: https://www.sciencedaily.com/releases/2017/06/170621165928.htm [Accessed 23 Oct. 2018].

Stevens, O. (2018). DrugScience – Ketamine. [online] Drugscience.org.uk. Available at: http://www.drugscience.org.uk/drugs/dissociatives/ketamine [Accessed 23 Oct. 2018].

Zhang, H., Zhang, C., Vincent, J., Zala, D., Benstaali, C., Sainlos, M., Grillo-Bosch, D., Daburon, S., Coussen, F., Cho, Y., David, D., Saudou, F., Humeau, Y., Choquet, D., Xu, H., Perez, S., Cornil, A., Detraux, B., Prokin, I., Cui, Y., Degos, B., Berry, H., d’Exaerde, A., Venance, L., Mongillo, G., Rumpel, S., Loewenstein, Y., Castillo, A., Rossoni, S., Niven, J., Park, H., Kang, H., Jo, J., Chung, E., Kim, S., Filippo, M., Portaccio, E., Mancini, A., Calabresi, P., Yates, D., Dell’Acqua, M., Woolfrey, K., Bray, N., Villanueva, M., Whalley, K. and Huber, K. (2018). Synaptic plasticity – Latest research and news | Nature. [online] Nature.com. Available at: https://www.nature.com/subjects/synaptic-plasticity [Accessed 24 Oct. 2018].

Zhong, Xiaomei, He, Hongbo, Zhang, Chunping, Wang, Zhijie, Jiang, Miaoling, Li, Qirong, . . . Huang, Xiong. (2016). Mood and neuropsychological effects of different doses of ketamine in electroconvulsive therapy for treatment-resistant depression. Journal of Affective Disorders, 201, 124-130.

Www2.chemistry.msu.edu. (2006). UV-Visible Spectroscopy. [online] Available at: https://www2.chemistry.msu.edu/faculty/reusch/virttxtjml/spectrpy/uv-vis/uvspec.htm [Accessed 25 Oct. 2018].

Appendix

Table 3

Standard

Volume of Ketamine 100µg/ml stock solution (ml)

Volume of ethanol solution (ml)

Final volume (ml)

Concentration of Ketamine 50µg/ml

Standard 5

2.5

2.5

5

Standard

Volume of Ketamine 50µg/ml stock solution (ml)

Volume of ethanol solution (ml)

Final volume (ml)

Concentration of Ketamine 25µg/ml

Standard 4

2.5

2.5

5

Standard

Volume of Ketamine 25µg/ml stock solution (ml)

Volume of ethanol solution (ml)

Final volume (ml)

Concentration of Ketamine 12.5µg/ml

Standard 3

2.5

2.5

5

Standard

Volume of Ketamine 12.5µg/ml stock solution (ml)

Volume of ethanol solution (ml)

Final volume (ml)

Concentration of Ketamine 6.25µg/ml

Standard 2

2.5

2.5

5

Standard

Volume of Ketamine 6.25µg/ml stock solution (ml)

Volume of ethanol solution (ml)

Final volume (ml)

Concentration of Ketamine 3.12µg/ml

Standard 1

2.5

2.5

5

Standard

Volume of Ketamine 3.12µg/ml stock solution (ml)

Volume of ethanol solution (ml)

Final volume (ml)

Concentration of Ketamine 0µg/ml

Blank

0

5

5