5.1: : Identification of Drug Type by UV-Visible Spectroscopy

C1: Identification of Drug Type by UV-Visible Spectroscopy

Safety Notes: The compounds used in these experiments as "unknown" samples are drug compounds and care must be taken to avoid ingestion and skin contact.

The drug samples are suspected to be barbiturate. This is a quick method of determining the type of barbiturate present.

Apparatus

UV-Vis Spectrometer 5 ml pipettes 100 ml volumetric flasks 100 ml beakers Matched UV Silica Cells pH paper

Reagents

Drug samples – 10 mg dissolved in 1ml methanol and made up to 10 ml with water Distilled or deionized water 0.5 M sodium hydroxide 16% ammonium chloride 50% sulphuric acid

Procedure

1. Dilute drug solution 1:25 with water.
2. Take 2 ml of this dilute sample solution, and add 2 ml 0.5 M sodium hydroxide (pH=13).
3. Scan this solution against 0.5 M sodium hydroxide as the blank between 200 and 350 nm.
4. Determine the absorbance maximum wavelength ( max).
5. Take a further 2 ml of the dilute sample solution.
6. Add 1.5 ml 0.5M sodium hydroxide and 0.5 ml 16% ammonium chloride solution (pH=10).
7. Re-scan at this lower pH of 10. Note any change in max
8. Add 0.5ml 50% sulphuric acid to pH 13 sample to give pH <2.
9. Re-scan, noting any change in max.

Thinking about making valid measurements

To give evidence in court you have to show that the analytical method is producing valid results. Are you certain that any sample pre-treatment has not invalidated the analysis? How did you ensure the integrity of the components?

Table 2: Ultraviolet spectrometry of the weak acid fraction. Wavelength of absorbance maxima (nm).

Compound	pH 13	pH 10	pH <2
Chlorpropamide	232	-	232
N-methyl substituted barbiturates	246	246	-
5,5-substituted barbiturates	254	239	-
Paracetamol	257	-	245
Phenylbutazone	264	264	237
Thiol substituted barbiturates	303	-	285

Improving your practical technique

• The instrument should be allowed to warm up before use.
• A wavelength check should be carried out to check the instrument is correctly set up.
• Instrument needs to be calibrated using a set of standards of differing concentration for quantitative analysis.
• Cells must be kept scrupulously clean, in particular the region where the light beam passes through the cell.
• There can be subtle differences in the cell shape, therefore always put the cell the same way round when performing quantitative measurements.
• If practicable, use matched cells or the same cell throughout the series of analyses.
• Air bubbles in the cell cause serious problems. Samples may have to be degassed before analysis.
• Solid material must be removed from the sample before analysis.
• There is an absorbance value (above 1 is not recommended) above which the Beer Lambert law no longer applies.
• There is also an absorbance below which the absorbance values are unreliable.
• If used ,an auto-flow cell sampler need to be checked : Is liquid being drawn up at reasonable speed? Is liquid exiting cell chamber at suitable speed? Has waste container been emptied?
• It may be appropriate to use a double beam spectrophotometer and to have a cell containing solvent in the reference beam.
• Measurements can be easily repeated – there should be not significant differences between the readings (e.g. < ± 0.003 absorbance units). Significant differences should be investigated.
• Consider doing duplicate samples.
• Analyze a blank sample (i.e. solvent blank).
• In many cases, samples may need to be diluted so that the measured absorbance is between the lowest and highest standard on the calibration curve.
• You may need to perform a wavelength scan to determine the λ(max).
• Qualitative identification by UV-Vis is very crude, where practicable use a more specific technique such as infrared spectroscopy.