Page 229 - Academic Press Encyclopedia of Physical Science and Technology 3rd Analytical Chemistry

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Encyclopedia of Physical Science and Technology EN008B-382 June 30, 2001 18:58

696 Liquid Chromatography

FIGURE 25 Guide to selecting HPLC conditions.

The mobile phase viscosity should be less than 1.0 cP volves the gradual increase of the mobile-phase solvent
(see Table III) to minimize high column back-pressures. strength with time to increase the speed of peak elu-
Optimization of the sample separation with respect to res- tion. Usually the percent composition of a component
olution, speed, and capacity should be the goal. However, of the mobile phase is changed. However, addition of a
compromise of each of these factors to some degree is new stronger solvent can also be done. Gradient elution
necessary; if not, undue cost or labor is expended. Finally, should be used instead of an isocratic mobile phase if
the conditions of an HPLC method can often be decided T g /T g ≥ 0.25, where T g represents the difference in
simply by checking the scientific literature for similar pre- retention times between the first and last eluting peaks,
vious studies. and T g is gradient time. The optimum gradient time can
be predicted from the equation T g = 20 V M φ/F, where
V M = column dead volume, φ = change in volume
B. Programming an LC Variable
fraction of the strong solvent during the gradient, and
If the sample components have a widely varying degree F = flow rate. An average capacity factor of 5 and the
of retention, separation times can be excessively long for isocratic parameter for small molecules equal to 4 must
reasonable peak resolution. To improve the separation, be assumed. The shape of the gradient program can be lin-
programming of some variable during the course of the ear, concave, convex, or stepwise. In choosing a solvent
separation is necessary. The most common parameters are pair, the only requirements are the solvents be nonreac-
flow rate, solvent strength, temperature, and column type. tive and miscible. For adsorption LC, methylene chloride
Flow programming involves initially using a low flow is often added to hexane. For reversed-phase LC, ace-
rate to better resolve the early peaks and then increas- tonitrile is added to water. For ion-exchange LC, a buffer
ing the flow rate to elute well-retained components. It or salt solution is added. A gradient program can be op-
can be carried out step-wise or continuously if desired. timized by following these steps: (1) Run a linear pro-
Basically, improvement of front-end resolution at the ex- gram at about 2%-min of the strong solvent B to deter-
pense of back-end resolution is the result. Decreasing the mine if and where all the components elute, (2) choose
flow rate by 75% will yield a resolution improvement of the initial % solvent B composition to be half of the
about a factor of 1.4. The primary advantages of flow pro- %B where the first peak elutes, (3) optimize the pro-
gramming are its ease of implementation and no need for gram by starting at the front of the chromatogram, in-
column reequilibration between samples. serting sharper %B rates at the appropriate times, and (4)
Solvent strength programming or gradient elution is the fine-tune the program adding short %B holds to main-
most effective and popular programming method. It in- tain good peak resolution. Computer-assisted methods