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Chromatographic scince series - Cazes J.

Cazes J. Chromatographic scince series - Marcel Dekker, 1996. - 1098 p.
ISBN 0-8247-9454-0
Download (direct link): сhromatography1996.pdf
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Advantage
Disadvantage
Spot elution technique
Simple
Identification of unknown components By IR By NMR
By mass spectrometry Quantition can be performed by several methods, such as Colorimetry spectrophotometry Spectrofluorimetry HPLC
Gas chromatography
Simultaneous use of appropriate blank is required
Simultaneous use of appropriate reference meterial is required Losses caused by irreversible adsorption Nearly quantitative extraction is required on accurate location of the spots Chemical reaction can be optimized The measured property of the derivatives can be stabilized
In situ densitometry
Independence of the chromatographic separation from the detector in time and space The chromatogram can be preserved after measurement Total amount of the sample can be spotted The chromatogram is complete, every component can be detected The sensitivity of the detection is higher by about 2 orders of magnitude Reflectance spectra of the spots can be taken permitting certain identification of unknown components Selectivity of the separation can be increased by changing the detection wavelength Using transmittance, fluorescence modes both the sensitivity and selectivity can be significantly increased
Simultaneous running of appropriate reference standard is required Errors originating from the incomplete distribution of spraying Variation of layer structure along the plate
Disturbing effect of zones caused by solvent demixing Variation of results from plate to plate Instability of the formed derivatives on the plate
V. METHOD VALIDATION
The primary goal of every analytical investigation is to obtain qualitative and quantitative information about the sample being tested. Quantitation includes the entire process of analysis, from sampling to interpretation of the final results. Because quantitative analytical results cannot be better than those of the weakest step in the entire process, each step should be considered separately either in method validation when developing methods or when interpreting results. Method validation is, therefore, a summary of several validation steps involving exact proof and evaluation of the suitability, correctness, and precision of each chromatographic and nonchromatographic step and of the instrumental components.
840 Szepesi and Nyiredy
Table 4 Comparison of Peak Height vs. Peak Area Measurements and of Various Quantitation Techniques
Peak height vs. peak area measurements
Measurement Advantages Disadvantages
Peak height measurement Simple Errors originating:
Peak distortion
Effective Plate overloading
Sensitivity to:
Ri changes
Base line fluctuation
Peak area measurement Higher precision less sensi Linearity is valid only a
tive to: Rf changes narrow concentration
Base line fluctuation range
Ease in computerization
Comparison of internal standard, external standard, and area normalization methods
Property Internal standard External standard Area normalization
Advantages Not sensitive to loss dur Simple Simplest
ing complex sample Fast
preparation Fast No additional peak
Easy to calculate
More precise sample No additional peak No standard required
application
Disadvantages Error addition Insufficient accuracy Reliability of assay results
and precision of originating from:
Additional peak on the sample application Nonlinearity of concentra
chromatogram tion vs. detector response
Difficulties due to find curve
ing suitable internal Detector responses are func
standard tion of chemical structures
of analytes
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