Instant Notes: Analytical Chemistry

● Solvent reservoirswith in-line filters(2mm porosity or less) for each solvent

to remove dust and other particulate material. This reduces pump wear and

protects the column from becoming clogged which results in increased back-

pressures.

● A means of de-gassingthe solvents to remove dissolved air. Air interferes

with the detector response by forming bubbles in the flow-through cell as the

pressure reduces to atmospheric at the end of the column. De-gassing is

normally accomplished by bubbling helium through each solvent to displace

the air, or by passing them through a commercial permeable-membrane de-

gassing unit.

● A gradient formerto generate binary, ternary or quaternary mixtures of

solvents with a pre-programmed composition profile during a separation

(gradient elution).

Sample injection Liquid samples and solutions are injected directly into the pressurized flowing
mobile phase just ahead of the column using a stainless steel and Teflon valve
fitted with an internal or external sample loop(Fig. 3). The loop, generally of
between 0.5 and 20ml capacity, is first filled or partially filled with sample from
a syringe while the mobile phase flows directly to the column. By turning a
handle to rotate the body of the valve, the mobile phase is diverted through the
loop thus injecting the sample onto the top of the column without stopping the
flow. A disposable guard columnis sometimes positioned between the injector
and the analytical column to protect the latter from a buildup of particulate
matter and strongly retained matrix components from injected samples. It
consists of a short length of column tubing, or a cartridge, packed with the same
stationary phase as is in the analytical column.
Valve injection can easily be automated, controlled by computer software and
used with autosamplers. For quantitative analysis, filled-loop injection has a
relative precision of about 0.5%.

The column is where the separation process occurs and it is, therefore, the

central component of a high-performance liquid chromatograph. There are two

Column and
stationary phase

158 Section D – Separation techniques

Common inlet

Common outlet

Fig. 2. A typical twin-headed reciprocating pump. Reproduced from W.J. Lough & I.W.

Wainer, High Performance Liquid Chromatography, 1996, first published by Blackie Academic

& Professional.