4.1.Test conditionsU.K.
4.1.1.Selection of optimum operating conditionsU.K.
4.1.1.1.Packed columnU.K.
In the selection of the test conditions, the following variables should be taken into account:
the length and diameter of the column;
the nature and amount of the stationary phase;
the temperature of the column;
the carrier gas flow;
the resolution required;
the size of the test portion, selected in such a way that the assembly of the detector and electrometer gives a linear response;
the duration of analysis.
In general, the values given in Table 1 and Table 2 will lead to the desired results, i.e. at least 2 000 theoretical Iplates per metre of column length for methyl stearate and its elution within about 15 minutes.
Where the apparatus allows it, the injector should be at a temperature of about 200 °C and the detector at a temperature equal to or higher than that of the column.
As a rule, the ratio of the flow-rate of the hydrogen supplied to the flame-ionization detector to that of the carrier gas varies from 1:2 to 1:1 depending on the diameter of the column. The flow of oxygen is about 5 to 10 times that of the hydrogen.
| Table 1 | |
| Internal diameter of columnmm | Carrier gas flowml/min |
|---|---|
| 2 | 15 to 25 |
| 3 | 20 to 40 |
| 4 | 40 to 60 |
| Table 2 | |
| Concentration of stationary phase% (m/m) | Column temperature°C |
|---|---|
| 5 | 175 |
| 10 | 180 |
| 15 | 185 |
| 20 | 185 |
4.1.1.2.Capillary columnU.K.
The properties of efficiency and permeability of capillary columns mean that the separation between constituents and the duration of the analysis are largely dependent on the flow-rate of the carrier-gas in the column. It will therefore be necessary to optimize the operating conditions by acting on this parameter (or more simply on the headloss of the column), according to whether one wishes to improve the separations or to make a rapid analysis.
4.1.2.Determination of the number of theoretical plates (efficiency) and resolution (See Figure 1)U.K.
Carry out the analysis of a mixture of methyl stearate and methyl oleate in about equivalent proportions (for example, methyl esters from cocoa butter).
Choose the temperature of the column and the carrier gas flow so that the maximum of the methyl stearate peak is recorded about 15 minutes after the solvent peak. Use a sufficient quantity of the mixture of methyl esters that the methyl stearate peak occupies about three-quarters of the full scale.
Calculate the number of theoretical plates, n (efficiency), using the formula:
and the resolution, R, using the formula:
where:
is the retention distance, in millimetres, from the start of the chromatogram to the maximum of the peak for methyl stearate;
are the widths, in millimetres, of the peaks for methyl stearate and methyl oleate respectively, measured between the points of intersection of the tangents at the points of inflection of the curve with the base-line;
is the distance, in millimetres, between the peak maxima for methyl stearate and methyl oleate[F1;]
Textual Amendments
[F2and the resolution index, lr, using the formula
Textual Amendments
where:
=
the height of the smallest peak, measured from the base line;
=
the height of the lowest point of the valley between the two adjacent peaks, measured from the base line.]
The operating conditions to be selected are those which will afford at least 2 000 theoretical plates per metre of column length for methyl stearate and a resolution of at least 1,25.