Limits of Detection and Smoothing Procedure Validation

The limits of detection (LODs) of the RDP for nonpolarized IR spectra have been examined for n-component systems starting with optimization of the standard smoothing conditions for IR curve interpretation. For this purpose the complicated IR spectrum of a five-component mixture (for example, glycyl-L-methionyl-glycine, L-tyrosine, L-phenylalanine, L-phenylalanyl-L-phenylalanine, and DL-isoleucine) 

The IR spectrum of sample 1 is subtracted from the IR curve of pure glycyl-L-methionyl-glycine until disappearance of the peak at 3295 cm- to yield a final reduced IR spectrum. 

The L-tyrosine IR spectrum is subtracted from the latter curve at the maximum at 3205 cm-.  

A subsequent subtraction, where the subtracted spectrum corresponds to L-phenylalanine, results in an IR curve of the reduced IR spectrum. In this case the subtraction is aimed at disappearance of the 1624 cm- peak, typical for L-phenylalanine. However, the complex character of the spectral curve calls for monitoring of a second peak, also typical for a simple amino acid. For L-phenylalanine, the 844 cm band is suitable. 

Finally, the last spectrum is subtracted from the IR curve for individual L-phenylalanyl-L-phenylalanine until total elimination of the peak at 1495 cm-, which corresponds to pure DL-isoleucine in solid state, is reached. The comparison between the first and last curve demonstrates a qualitative correlation. It is noteworthy that the order of subtraction of different components in the mixture leads to identical final results. 

---