(iii) Discuss the principles behind two different detection techniques that can be used with GLC instrumentation, and indicate the sort of analytes for which each is suitable.
Would expect them to choose FID and ECD but may choose MS. Should be able to describe principles of use incorporating a diagram, know that FID is non-specific and ECD relies on electronegative atoms and that ECD is more sensitive. Good students might mention derivatization with electronegative species to improve sensitivities. (70% of marks)
b) The results below were obtained during an analysis of the drug fluoxetine (C17H18F3NO), by gas liquid chromatography, of a suspected counterfeit pharmaceutical preparation. A 1 mg sample of the preparation was taken and dissolved in 10 mL of water, 100 ul of nisoxetine was added as internal standard (I.S.) and the whole made alkaline by addition of 1.0 mL of 0.100 mol l-1 sodium hydroxide. 1.0 mL of n-hexane-2-propanol was added and the mixture shaken vigorously. After allowing the layers to separate, the n-hexane-2 propanol layer was removed with a pipette, transferred to a glass tube and evaporated to dryness under a stream of nitrogen. The solid residue was re-dissolved in 100 uL of hexane and 3 replicate 1uL samples were injected into the chromatograph.
Assume RMMs C 12.0, H 1.0, F 19, N 14, O 16.0)
Students should first compute the peak area ratio and construct a graph of concentration vs peak area ratio
Peak area IS
Peak area ratio
10 umol L-1 Fluoxetine
20 umol L-1 Fluoxetine
30 umol L-1 Fluoxetine
40 umol L-1 Fluoxetine
extract sample 1
extract sample 2
extract sample 3
Assuming complete extraction, calculate:
(i) the concentration of Fluoxetine (in umol L-1) in the extract injected;
Students should read the concentration from the graph approx 20 umol/L.
(ii) the concentration of Fluoxetine (in ug L-1) in the extract injected;
RMM is 309 therefore 20 x 309 = 6180 ug /L or 6.18 mg/L
(iii) the % mass by weight of Fluoxetine in the original 1 mg sample.
(30% of marks)
The extract was dissolved in 100 uL therefore mass of drug in 100 uL is 0.1 / 1000. 6.18 x 01/1000 = 0.0.000618 mg / 1 mg x 100 = % by weight of 0.06%
Q3 The following data were obtained from a reverse- phase HPLC experiment using a 4.6mm i.d. column packed with 5m ODS with an isocratic mobile phase composed of 70%/30% water/acetonitrile. Samples were extracted from urine using a Molecularly Imprinted Polymer (MIP) solid phase extraction cartridge.
Retention time (min)
Peak width (s)
i) Discuss each of the terms underlined above. (30% of marks)
Students should be able to discuss these factors and not just describe them. This would require them to compare and contrast with other examples of e.g. phases, gradient elution; to state the strengths and weaknesses of MIP's compared to other SPE techniques and to discuss the features of particle size and hydrocarbon stationary phases.
ii) Calculate the capacity factor (k') for each of the compounds 1 to 3. (15% of marks)
k' = tr - tm
therefore 1=4.2; 2= 4.9 and 3 = 16.6
iii) Calculate the efficiencies (in numbers of theoretical plates) for
each of the compounds 1 to 3. (15% of marks)
N = 16 tr 2
Tricky bit here is to realise that retention time and peak width are in different units. The simplest solution is to convert both to seconds.
Then: 1= 4691 2 = 4494 and 3 = 3906
iv) Determine the selectivity for, and resolution between, compounds 1 and 2. Is this adequate resolution (20% of marks)
a = t'r2/t'r1 = 0.94
Resolution = 2(t2 - t1)
w1 + w2
again we must convert to seconds, then Rs =2 comment Rs should be > 1.5 i.e. this is adequate resolution
v) How could the above experiment be modified to reduce the retention time of compound 3? (20% of marks)
By using gradient elution. Possibly by making the mobile phase more non polar but this would compromise Rs of 1 and 2.
Q4 Answer ALL parts (a), (b) AND (c).
(a) Outline the theory of infra red spectroscopy and use this to explain why molecules such as CO2 are infra red inactive whereas H2C=O is infra red active.
(40% of marks)
Students are expected to provide a basic overview of origins of IR spectroscopy, starting with the point that it is a form of electromagnetic radiation that excites electrons in bonds making them vibrate.
Key point is that molecules must be polar in order to be infra red active. Therefore, although C=O bonds are polar, in CO2 the dipoles cancel and so the molecule is infra red inactive. In contrast, formaldehyde is polar and so would display an infra red spectrum.
(b) Using appropriate example, discuss the relative advantages of the different methods of sample preparation available for infra red spectroscopy.
(30% of marks)
The key issue here is that IR can be used for solids, liquids and gases (although the students are unlikely to encounter the latter at this stage).
For solids we have a choice - dissolve in a solvent, produce a mull, press as a KBr disc. Expect a brief discussion of advantages and disadvantage of each of these. More able students may mention that we can use a technique such as ATR-IR, or diffuse reflectance, to record the spectrum of un-diluted/un-dissolved solids
(c) Using the drug Warfarin (Figure 1) as an example, discuss the kind of structural data that infra red spectroscopy can provide.
(30% of marks)
An IR spectrum can be broken down into two general regions, functional group and fingerprint. The first of these provides an indication of the principal functionalities present in the species (each absorbs in a slightly different region depending on environment). In Warfarin should observe an OH stretch, C=O (carbonyl) and C=O, C-O (Ester), unsaturated sp2 carbon and saturated sp3 carbon. The fingerprint region is used mainly for confirmatory purposes.
A key point that one would expect to see is that IR is not the technique of choice for determining the identity of unknown compounds (due to its complexity), other techniques such as NMR are far superior. IR is chiefly a confirmatory technique.
Q5. After administration, ibuprofen (RMM = 180 ) undergoes extensive metabolism. An important pathway the formation of an O-glucuronide, following aliphatic hydroxylation is shown below.
(a) Describe how liquid chromatography - mass spectrometry - mass spectrometry (LC/MS/MS) might be employed to detect ibuprofen and each of the metabolites shown above in plasma. Your answer should include descriptions of both suitable instrumentation and the experiments to be performed.
(50% of marks)
(b) Outline the procedure you would adopt to develop a quantitative assay for the O-glucuronide metabolite of ibuprofen shown above.
(35% of marks)
(c) It has been suggested that Ibuprofen can be metabolised to a sulphate ester conjugate by a similar route. How would you use LC/MS/MS to confirm this? (15% of marks)
5A Descriptions of electrospray ionisation and its combination with a triple quadrupole mass spectrometer.
Experiments to be performed product ion scan of ibuprofen to look for characteristic product ions and assign them to parts of the molecule.
Product ion scans of components giving protonated molecular species at m/z197 (the hydroylated metabolite) and m/z 373 (the O-glucuronide) and relate to the product ion scan of ibuprofen.
Description of quantitation using MRM, including calibration etc ought to be aware that m/z 373-197 should be an important transition in the product ion scan of this particular metabolite.
Precursors ion scans of m/z 80 and 97 can be used to detect sulphate ester conjugates under negative ion electrospray MS/MS or might point out that these would be characteristic product ions in product ion scans.