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Appendix III Chromatographic Separation Techniques
(Ph. Eur. method 2.2.46)
Chromatographic paration techniques are multi-stage paration methods in which the components of a sample are distributed between 2 phas, one of which is stationary, while the other is mobile. The stationary pha may be a solid or a liquid supported on a solid or a gel. The stationary pha may be packed in a column, spread as a layer, or distributed as a film, etc. The mobile pha may be gaous or liquid or supercritical fluid. The paration may be bad on adsorption, mass distribution (partition), ion exchange, etc., or may be bad on differences in the physico-chemical properties of the molecules such as size, mass, volume, etc.
This chapter contains definitions and calculations of common parameters and generally applicable requirements for system suitability. Principles of paration, apparatus and methods are given in the following general methods:
— paper chromatography (2.2.26);
—
thin-layer chromatography (2.2.27);
— gas chromatography (2.2.28);
— liquid chromatography (2.2.29);
— size-exclusion chromatography (2.2.30);
— supercritical fluid chromatography (2.2.45).
DEFINITIONS
The system suitability and acceptance criteria in monographs have been t using parameters as defined below. With some equipment, certain parameters, such as the signal-to-noi ratio and resolution, can be calculated using software provided by the manufacturer. It is the responsibility of the ur to ensure that the calculation methods ud in the software are equivalent to the requirements of the European Pharmacopoeia and to make any necessary corrections if this is not the ca.
CHROMATOGRAM
A graphical or other reprentation of detector respon, effluent concentration or other quantity ud as a measure of effluent concentration, versus time or volume. Idealid chromatograms are reprented as a quence of Gaussian peaks on a baline (Figure
2.2.46.-1).
PEAK
The portion of a chromatogram recording the detector respon when a single component (or 2 or more unresolved components) is eluted from the column.
The peak may be defined by the peak area, or the peak height (h) and the peak width at half-height (w h), or the peak height (h) and the peak width between the points of inflection (w i). In Gaussian peaks (Figure 2.2.46.-1) there is the following relationship:
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RETENTION TIME (T
R
Time required for elution of a component (Figure 2.2.46.-1, baline scale being in minutes). RETENTION VOLUME (V
)
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Volume of the mobile pha required for elution of a component. It may be calculated from the retention time and the flow rate (F) in millilitres per minute using the following equation:
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HOLD-UP TIME (T
M
Time required for elution of an unretained component (Figure 2.2.46.-1, baline scale being小动物的外形特点
in minutes). In size-exclusion chromatography, the symbol t0(e below) is ud.
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HOLD-UP VOLUME (V
M
常吃土豆有7大好处Volume of the mobile pha required for elution of an unretained component. It may be calculated from the hold-up time and the flow rate (F) in millilitres per minute using the following equation:
In size-exclusion chromatography, the symbol V0 (e below) is ud.
RETENTION FACTOR (K)
The retention factor (also known as mass distribution ratio (D m) or capacity factor (k′)) is defined as:
K C=distribution constant (also known as equilibrium distribution coefficient);
V S=volume of the stationary pha;
V M=volume of the mobile pha.
The retention factor of a component may be determined from the chromatogram using the following equation:
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TOTAL MOBILE PHASE TIME (T
T
敬佩的人In size-exclusion chromatography, retention time of a component who molecules are smaller than the smallest gel pores (Figure 2.2.46.-2).
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TOTAL MOBILE PHASE VOLUME (V
T
In size-exclusion chromatography, retention volume of a component who molecules are smaller than the smallest gel pores. It may be calculated from the total mobile pha time and the flow rate (F) in millilitres per minute using the following equation:
RETENTION TIME OF AN UNRETAINED COMPOUND (T
)
In size-exclusion chromatography, retention time of a component who molecules are larger than the largest gel pores (Figure 2.2.46.-2).
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RETENTION VOLUME OF AN UNRETAINED COMPOUND (V
In size-exclusion chromatography, retention volume of a component who molecules are larger than the largest gel pores. It may be calculated from the retention time of an unretained compound and the flow rate (F) in millilitres per minute using the following equation:
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DISTRIBUTION CONSTANT (K
In size-exclusion chromatography, the elution characteristics of a component in a particular column may be given by the distribution constant (also referred to as distribution coefficient), which is calculated using the following equation:
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RETARDATION FACTOR (R
F
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The retardation factor (also known as retention factor (R f)), ud in planar chromatography, is the ratio of the distance from the point of application to the centre of the spot and the distance travelled by the solvent front from the point of application (Figure 2.2.46.-3).
b=migration distance of the component;
a=migration distance of the solvent front.
