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Aims and Objectives
Qualitative Analysis Overview
Peak Identification and Assignment
Spectral Peak Identification
5.1. Peak Purity
5.2. Spectral Characterization
6 Quantitative Analysis Overview
7 Chromatographic Requirements
8 Peak Integration Events
8.1. Integration Events
9 Peak Height or Peak Area
10 Principles of Quantitative Analysis
11 Area%/Height% (Normalization)
12 External Standard Quantitation
13 Calibration Curve
13.1. Statistical Information
14 External Standard Multi-Level Calibration
14.1. Calibration Curve Information
15 External Standard Multi-Level Calibration Curve - Typical Calculation
16 Internal Standard Analysis
17 Multi-Level Calibration Curve
1. Aims and Objectives
To define Quantitative HPLC and explain the information that can be derived from this type of HPLC analysis
To examine the use of Peak Height or Area in quantitative calculations and to investigate
Integration of chromatographic peaks
To explain the use of Calibration and Calibration curves in Quantitative HPLC
To outline the principles of Single and Multi-level calibration in Quantitative analysis
To investigate practical uses of External Standard, Internal Standard and Normalization methods of Quantitative analysis
To outline example calculations for the various Quantitative methods
At the end of this Section you should be able to:
To define Qualitative HPLC and explain the information that can be derived from this type of
To define and explain the principles of peak identification and analyte characterization from a practical perspective
2. Qualitative Analysis Overview
The aim of qualitative analysis is to answer the question ‘What’ is in the sample?
Figure 1: Options for qualitative HPLC analysis.
Two discrete situations exist for qualitative analysis:
the sample components are known and peaks within the chromatogram need to be assigned to the known components the sample is a complete unknown which you are attempting to characterize
In the former case, it might be possible to inject standards of the pure compound, and assign the peaks in the chromatogram based on the retention time of the standard. Having a selective detector, such as diode-array UV or Fluorescence detector, which assists in identification by producing spectra or a specific response, can assist in peak assignment.
In the second case, it may be necessary to employ detectors that can be used to aid in identification, such as mass spectrometers. It may also be necessary to collect the eluent fraction containing the peak of interest for off-line characterisation using Infra-red or Nuclear Magnetic Resonance
3. Peak Identification and Assignment
Perhaps the most straightforward way to assign peaks within the chromatogram of a sample solution is to inject standard solutions under identical analytical conditions. By comparing the retention factor (k) and response of the peak in the chromatogram of the standard solution, with the sample chromatogram, peaks may be tentatively assigned.
It is important that the concentration of the standard solution is matched to the sample solution as closely as possible. This avoids peak mis-assignment due to peak shape effects.
Figure 2: Using reference standards for peak identification.
For more rigorous peak assignment it is important that the analysis is also carried out under orthogonal (different) conditions. The analysis is carried out in the reversed phase using a different column and mobile phase, and the retention factor and response compared…