Practical implementation of the analytical test

All devices and columns used, are purchased from ‘Waters’.

1          Pump

The pump used, is a constant flow pump. This pump uses two alternating pistons and the fluid chambers in positioned in series. The flow rate can be set at different values (ml/min). For the analyzes performed here, a flow rate of 2 ml/min is chosen. The higher the flow rate, the faster the test is done, but the higher pressure is needed to pump the mobile phase through the stationary phase.

2          Column

This has already been discussed previously. All the used columns (Hypersil BDS, SunFire and XBridge) are comprised of stainless steel, filled with a non-polar stationary phase C18. The dimensions and particle size are given below:

1. Hypersil BDS C18        100 x 4.6 mm                       3 µm
2. SunFire C18                  100 x 4.6 mm                       3.5 µm
3. XBridge C18                  100 x 4.6 mm                       3.5 µm

After use, the column is rinsed with a mixture of 50% acetonitril/methanol and kept at 100% methanol.

3          Injectionsystem, detector and recorder

This has already been discussed previously. An amount of 20 µl of the sample, is injected each time. For the detection, a mass detector is used in combination with a single wave length UV detector. The size of the signal that is detected and integrated or recorded on the chromatogram, is dependant of a set value. This is called an attenuation which can vary between 1 and 10. The greater this value, the more the recorded signal is reduced. After this has happened, the integrator will automatically create a list in which the retention time of each peak registered is printed for four different methods: the two buffers and two solvents previously mentioned.

4          Eluent

The type of solvent/eluent used, has an influence on the separation. The elution[1] can be done with the use of a single solvent or a mixture of solvents (isocratic) or changing the concentration of the different solvents (gradient).

Elution in general

When an isocratic elution is used, the composition of the mobile phase is constant during the entire analysis. The ideal composition of the different solvents is one at which all products are eluted within an acceptable period. For components that are fast eluting, it is possible that the separation is insufficient, whereas slow eluting components can cause peak widening. A solution for this problem is the use of a gradient elution (see figure), which means that there is a transition of a weak solvent, to a stronger solvent, which may lead to a shorter method time, better separation and higher sensitivity. One disadvantage is that after every analysis, the column needs to be brought back to the initial conditions. Another disadvantage is the presence of a dwell volume. This is the device dependent volume that the mobile phase needs to pass, from mixing, until reaching the column. This dwell volume, in combination with the volume of the column, influences the retention times. A smaller column, such as the analytical column, has a bigger effect on the retention times, than the bigger columns of the preparative size. This means that fast eluting components can elute, even before the gradient elution has started.

Gradient Elution

1. The first period is the conditioning of the column, which takes about five minutes until the pressure is constant. During this period, the column will be brought in the conditions that is necessary for the separation of the monster. The appropriate condition for the actual, preparative separation can be chosen from the results of a smaller analytical test, with a sample of the monster.
2. The second period, is the period at which the monster is sucked by the syringe and positioned for injected on the column. This takes about two and a half minutes.
3. The last period, is the period of the actual separation. During this period, the first six minutes are crucial because the product is then injected and distributed over the column, and this is the period at which the pressure reaches its maximum. After the six minutes, separation and detection takes place and a report is made.

The detector and packing influence the choice of the solvent:

• in an apolar stationary phase, a polar solvent is needed
• the eluent should not interfere with the detection

The eluent used consists of a mixture of two different buffers (acetonitril and ammoniumcetate).

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[1] Elution: the process of extracting one material from another by washing with a solvent