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Liquid Chromatographic Separation Methods

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    Liquid chromatographs are fairly simple pieces of equipment. There is a solvent reservoir that holds the mobile phase, a high pressure pump, an injection valve, a column, and a detector (Figure 47).


    Figure 47. Components of a liquid chromatograph.

    The pumps are usually reciprocating small-volume pumps that use inlet and outlet check valves (Figure 48). There is a small cylinder with a sapphire piston. The inlet and outlet valves essentially have a moveable ball in them. When the cylinder is full of fluid and pushing it into the chromatograph, notice how the ball in the inlet check valve will move down and block the opening to the solvent reservoir. Also notice how the ball in the outlet check valve will pop up, allowing flow into the column. When the cylinder is almost empty and the piston recycles to fill (this recycle step is very fast), the ball in the inlet check valve pops up and allows flow from the solvent reservoir to the cylinder. The ball in the outlet check valve moves down, blocking the hole, and prohibiting flow back from the column. The flow out of a reciprocating small volume pump such as this is pulsed (when the piston recycles to fill, flow is stopped). Many liquid chromatographs employ some form of a pulse-dampening device to reduce these pulses.


    Figure 48. Diagram of the pump head of a reciprocating small-volume pump.

    The injection valve is usually a six-port, two-way valve with a sample loop (Figure 49). In one position (the load position), the flow comes in from the pump through one port and goes out another port to the column. The other two ports connect the syringe adapter to a sample loop and a vent. A common sample loop size is 20 µL. Usually you inject about 100 µL of sample into the loop. The first 80 µL cleans the loop and goes out the vent. The last 20 µL are retained in the loop and ready for injection in the system.

    In the second position (the inject position), the flow from the pump now goes through the sample loop and then to the column. Moving the valve to this position allows the mobile phase to sweep the sample into the column for analysis.


    Figure 49. Flow diagram of a six-port injection valve.

    We have already discussed most of the important features of liquid chromatographic columns. The column lengths are usually 3, 15, or 25 cm depending on the particle size used. The internal diameter of conventional liquid chromatographic columns is 4.5 mm (about ¼ inch).

    Many methods have been used for detection in liquid chromatography. The most common detection method is to use ultraviolet absorption. Fluorescence spectroscopy is also used for certain classes of compounds. More recently people have figured out ways to adapt mass spectrometers to liquid chromatographs. The large volume of solvent in liquid chromatography is incompatible with conventional mass spectrometric methods, so these techniques are quite specialized. Also, micro columns with smaller diameters and less solvent are often used with liquid chromatographic-mass spectrometers. One advantage of mass spectrometry over most other detection methods is that the mass spectrum provides information that may allow you to identify the chemical structure of an eluting compound.

    This page titled Liquid Chromatographic Separation Methods is shared under a CC BY-NC-SA 4.0 license and was authored, remixed, and/or curated by Thomas Wenzel.