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Boost your confidence with CHN microanalysis.

Using CHN microanalysis alongside other analytical techniques can provide precise and accurate data on a sample's composition and purity.

Sample characterization and purity determination requires a combination of analytical techniques to generate the package of data to fully characterize a sample. This article examines and demonstrates the role of CHN (carbon, hydrogen and nitrogen) elemental analysis and how it complements other established analytical techniques, including nuclear magnetic resonance (NMR) and mass spectrometry (MS), in helping laboratory scientists determine a sample's composition and purity. Furthermore, the article will demonstrate how CHN microanalysis can be used to determine sample purity by eliminating "blind spots" sometimes found with other analytical techniques enabling reliable calculation of compound stoichiometries as is required in life science research and development.

Determining Sample Composition and Purity

Research compounds typically produced in synthetic and medicinal chemistry laboratories often require final purification steps using flash chromatography or preparative HPLC. For flash chromatography, the samples are passed over a silica bed and eluted at an appropriate rate to afford separation of the product from by-products (often structurally related compounds) and impurities. The composition of the mobile phase is adjusted to allow this separation whilst ensuring that the product itself is retained on the column for the minimum time possible. The product is collected as a solution in the mobile phase, the solvent evaporated off and the product dried. Under certain mobile phase conditions, the silica gel may become sparingly soluble and trace levels elute with the product. This inorganic contaminant is 'invisible' to standard NMR and MS techniques, which could lead to a falsely elevated reaction yield. Yet, even with very low levels of inorganic impurity (silica gel) in a sample there will be a significant reduction in the percentage carbon, hydrogen and nitrogen levels as determined by CHN elemental analysis.


Similarly, whilst the presence of low levels of residual solvent in a 'dried' sample of 4-bromo-2,6-bis(benylthio)methylpyridine was readily identified by CHN elemental analysis as significant deviations from theoretical percentage values, the solvent has little or no effect on 1H NMR or MS data as shown in Figure 1.

In this example, CHN elemental analysis of 4-bromo-2,6-bis(benylthio)methylpyridine should give theoretical carbon, hydrogen and nitrogen values of 58.60%, 4.68% and 3.25%, respectively. Yet, analysis of the sample gave the values of C: 57.51%, H: 4.57% and N: 3.24%. This deviation of 0.4% from theoretical values as measured by CHN microanalysis indicates that the sample is not completely pure. Figure 2 shows how, by the inclusion of 0.5 moles of residual methanol in the calculations, the identity of the parent sample can be confirmed and the presence of trace levels of residual solvent confirmed and identified.

The principles of sample purification employed in reverse phase HPLC methods are similar to those discussed previously in that the sample is introduced onto a silica-based solid support and separation from impurities with subsequent elution from the column being determined by judicious modification of an aqueous/organic solvent mix of mobile phase. It is common practice to add modifiers such as trifluoroacetic acid (TFA) to the mobile phase to sharpen peaks and improve resolution. A large proportion of drug candidates, however, often have multiple basic ionization sites that are readily protonated and the presence of TFA may cause association to these sites at ratios not easily determined. TFA has a molecular weight of 114.03 and even at subunity ratios this will have a significant impact on the mass of sample taken whose mass itself may only be 300-400. This problem is even more significant in the case of peptides and proteins; they contain multiple basic nitrogen atoms within the molecule, each of which is susceptible to protonation and association. Using the TFA salt (C [F.sub.3] C [O.sub.2]H) of the diuretic amiloride ([C.sub.6][H.sub.9][N.sub.7]OF) as an example, Figure 3 demonstrates how %C, H and N changes with stoichiometry and how simple calculations and graphical representation can allow accurate determination of sample composition for dosage calculations or interpretation of biological activity.


How CHN Elemental Analysis Works

In CHN elemental analysis, samples are weighed (1-2 mg) into a tin capsule that is supported within a nickel sleeve. The prepared samples are then placed into an autosampler that is purged with helium--chosen for its chemically inert characteristics relative to the tube packing reagents, and its very high co-efficient of thermal conductivity. The tin capsule containing the sample is introduced via a ladle into a combustion tube held at 975 [degrees]C in a pure oxygen environment causing the tin capsule and sample to undergo flash combustion with an exothermic reaction at 1800 [degrees]C. These conditions, along with an option of variable combustion time create conditions such that even the most thermally resistant sample will oxidize. The products of this combustion process pass over a series of specialized reagents held in the combustion tube, which are ordered such that complete oxidation of the products is assured with conversion of the sample's elemental carbon, hydrogen and nitrogen to carbon dioxide, water, nitrogen gas and nitrogen oxides. These combustion products are subsequently flushed through a reduction tube packed with copper, held at 620 [degrees]C, where nitrogen oxides are converted to molecular nitrogen and residual oxygen is removed. In Exeter Analytical Inc.'s CE440 analyser (Figure 4) the combustion and reduction tubes (the combustion train, Figure 5) are orientated in a horizontal geometry to prevent build-up of sample ash.

To ensure total homogeneity, the mixture of combustion gases is pulsed into a mixing volume enabling a faster formation of a homogeneous mixture. Using a pressure transducer, the pressure in the mixing volume is measured until a pre-set pressure has been reached. The combustion products are then sealed in the mixing volume for a defined period of time after which a known volume of the combustion product mixture is released. This known volume of combustion mixture then passes through a series of traps where [H.sub.2]O and [CO.sub.2] are completely absorbed, with high precision thermal conductivity detector filaments located before and after each absorption trap. The difference between the output of each set of detectors before and after absorption can be seen to be proportional to the trapped component to allow determination of the carbon and hydrogen content, with the remaining gas containing only helium and nitrogen. Comparing this against a helium reference produces the nitrogen concentration.



Using CHN elemental analysis alongside analytical techniques such as NMR and MS, with an understanding of the synthetic pathways, generates with a much higher degree of certainty the quality of compounds being produced. This in turn affords a greater degree of confidence in the decision-making processes based around a product's performance further down the R&D path.

CHN elemental analysis is fast, easy to use, and can provide precise and accurate data on a sample's composition and purity. The versatility of the technique ensures that it is amenable to a wide range of life science sample types, including crystals, amorphous solids, peptides, proteins and, when used in conjunction with appropriate sample handling techniques, can even accommodate volatile samples such as colloids and gels.

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Date:Mar 1, 2012
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