Instant Notes: Analytical Chemistry

types of HPLC column, conventional and microbore, and a comparative

summary is given in Table 2.

Microbore columns have three principal advantages over conventional

columns, i.e.:

● solvent consumption is about 80% less because of the much lower mobile

phase flow rate (10–100ml min-^1 )

● the low volume flow rate makes them ideal for interfacing with a mass

spectrometer (Topics F3 and F4)

● sensitivity is increased because solutes are more concentrated, which is

especially useful if sample size is limited, e.g. for clinical specimens.

However, in practice, they are not as robust as conventional columns and are

not necessary for many routine applications.

Columns are connected to the sample injection valve and the detector using

short lengths of very narrow bore (~0.15 mm internal diameter) stainless steel

or PEEK (polyether ether ketone) tubing to minimize dead-volume which

contributes to band spreading in the mobile phase by diffusion.

HPLC stationary phases are predominantly chemically-modified silicas,

unmodified silica or cross-linked co-polymers of styrene and divinyl benzene.

The surface of silica is polar and slightly acidic due to the presence of silanol (Si-

OH) groups. It can be chemically modified with reagents, such as chlorosilanes,

which react with the silanol groups replacing them with a range of other func-

tionalities (Fig. 4(a)). The resulting bonded phases, which are hydrolytically

stable through the formation of siloxane (SiæOæSiæC) bonds, have different

chromatographic characteristics and selectivities to unmodified silica.

Octadecyl silica(ODSor C18) is the most widely used of all the stationary

phases, being able to separate solutes of low, intermediate and high polarities.

Octyland shorter alkyl chains are considered to be more suitable for polar

solutes. Aminopropyl and cyanopropyl(nitrile) silicas are good replacements

for unmodified silica, which can give variable retention times due to traces of

water in the solvents. Polar, and especially basic solutes, tend to give tailing

D6 – HPLC: principles and instrumentation 159

Injection

port Waste

To

column

From

pump

Sample

loop

Injection

port Waste

To

column

From

pump

Sample

loop

(a) (b)

Fig. 3. Sample-injection valve and loop. (a) Sample-loading position; (b) sample-injection position.