Peak Detection and Selection

Here we can find several stages where automation procedures should be used. These are already partially well known from standard LC analysis. However, as the number of analysed samples, the types of analysis and various other requirements are completely different to these standard applications, the usage of such procedures therefore needs to be discussed in some detail. 

1 SAMPLE PREPARATION AND INTRODUCTION ( INJECTION ) INTO THE CHROMATOGRAPHIC SYSTEM 

The injection of the sample into the LC-NMR system can be carried out by an autosampler or a manual injection valve. The only difference which has to be considered is the fact that the amount of sample required for the NMR system is larger than that required for UV detection. Therefore, it is often necessary to inject sample volumes which exceed 100 jjlL In such cases, it is mandatory to dissolve the sample in the starting solvent phase in order to avoid additional LC gradients created by the sample solvent being injected. 

The basic conditions of a chromatographic separation conducted for an LC-NMR experiment do not differ from those required for (routine) analytical 

The chromatographic equipment which is responsible for the separation includes the pump, and in many systems a column oven. The parameters which affect the separation are the flow rate, the solvent composition and the LC gradients. Many different software packages are available which allow completely unattended automatic chromatography. Such systems also include control of the sample injection process. 

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Even if HPLC is chosen as the main stability indicating method, CE will be useful as orthogonal technique, especially in cases where the reason of mass balance deviations is unclear. In addition, due to its increased peak capacity and selectivity in comparison to HPLC, the detection of possible enantiomers, stereoisomers, and position isomers, having the same molecular weight and equal/similar spectra as the drug substance may be separated by a secondary CE method. 

Whatever the application, MS-based analyses of FAC effluent will always be faced with the need to support a wide range of buffer components, ranging from variable ionic strength, surfactant levels to required cofactors. In select situations such as indicator analyses, online methods may be appropriate but it is clear that the insertion of an intermediate LC step offers sigmficantly improved performance. This changes the nature of the data analysis, from the detection of sigmoidal breakthrough curves to peak detection and differential analysis across multiple fractions. 

Figure 9. Identification data of the human plasma scan. The data were inerted into Melanie and two scan points were selected pi and Mr were calculatedfrom the position of a point on the gel and are shown in the first field. The secondfield contains a list of the matching proteins (SWISS-PROT accession number and SWISS-PROT name) and their score. Clicking on this field displays the whole list (an asterisk means that the protein was not found a third time in a 3x3 neighbourhood of the point). Clicking on the third field renders the ctrum attached to the scan point with DataExplorer, which allows verifying peak detection and gives information about the intensity of the peaks.

Linear dependence of current of additional peak 1 on concentration of Zr(IV) can be used for elaboration sensitive and selective determination of zirconium with detection limit of 1.7x10 mol/1. 

It is difficult to compare separation techniques in any general way. Comparison may be based on the traditional figures of merit, such as resolution Rs (including column efficiency N, selectivity, retention, and peak capacity), chromatographic speed, sample capacity, sensitivity, detection and column impedance, as well as breadth of application. Usually a tradeoff between these attributes is found. Berger [26] has compared GC, pSFC, cSFC, LC and CE on the basis... 

Ions derived from the N-terminus of the original peptide are termed a, b, or c (in other words, the charge is retained on the N-terminus), while those originating from the C-terminus are named x, y, or z. Numerical subscripts contain information on the number of amino acid residues present in a given ion. It must be remembered that we are interested only in charged species since the neutral ones are not detected and do not yield peaks in the spectrum. The main idea behind this system is shown in Fig. 6.5, and the structures of selected ion types are depicted in Fig. 6.6. 

Because of its advantages (high sensitivity and selectivity, low cost and miniaturization) amperometric detection has been frequently used in flow injection analysis (FIA) and RP-HPLC. However, it has been established that the peak area (detector response) considerably depends on the flow rate. A general approach has been proposed to predict the effect of flow rate on the peak area in FIA and RP-HPLC. The general form of the correlation describing the flow in a parallel plate cell with short rectangular electrodes is... 

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