mobile phase, mobile, phase, HPLC, high performance liquid chromatography, high, performance, liquid, chromatography, solvent, solute, isocratic elution, column, carrier, stationary phase, sample, gradient, polytyptic, separation, compounds, composition, retention time, retention, time, bandwidth, resolution, capacity, factor, capacity factor, migration, migrate, Knox, analytical, chemistry, analytical chemistry, linear-solvent strength (LSS) gradient, linear, strength, university of kentucky, college of pharmacy
The mobile phase in HPLC refers to the solvent being continuously applied to the column, or stationary phase. The mobile phase acts as a carrier for the sample solution. A sample solution is injected into the mobile phase of an assay through the injector port. As a sample solution flows through a column with the mobile phase, the components of that solution migrate according to the non-covalent interactions of the compound with the column. The chemical interactions of the mobile phase and sample, with the column, determine the degree of migration and separation of components contained in the sample. For example, those samples which have stronger interactions with the mobile phase than with the stationary phase will elute from the column faster, and thus have a shorter retention time, while the reverse is also true. The mobile phase can be altered in order to manipulate the interactions of the sample and the stationary phase. There are several types of mobile phases, these include: Isocratic, gradient, and polytyptic.
In isocratic elution compounds are eluted using constant mobile phase composition. The separation of compounds can be described using several equations:
(Snyder,
1983)
All compounds begin migration through the column at onset.
However, each migrates at a different rate, resulting in faster
or slower elution rate. This type of elution is both simple and
inexpensive, but resolution of some compounds is questionable and
elution may not be obtained in a reasonable amount of time (Snyder,
1983).
In gradient elution different compounds are eluted by
increasing the strength of the organic solvent. The sample is
injected while a weaker mobile phase is being applied to the
system. The strength of the mobile phase is later increased in
increments by raising the organic solvent fraction, which
subsequently results in elution of retained components. This is
usually done in a stepwise or linear fashion. There are several
equations that describe gradient elution:
(Snyder,
1983)At the onset of sample introduction, the compounds are
initially retained at the inlet of the column. As the solute
capacity, or k', for the compound decreases, the compound begins
to migrate through the stationary phase. Each of the other
compounds in the sample subsequently migrate as their k' values
decrease. Compared with isocratic elution, resolution and
separation are improved, and bandwidths are nearly equal: 
(Snyder,
1983)
The Knox equation describes column efficiency or plate
number N in relation to certain experimental conditions, such as
column length, column diameter, temperature, flow-rate, molecular
weight, etc. (Equation).
Plate number N is equal to plate height value H divided by
particle diameter (dp). Plate height value H is in turn equal to
column length L divided by N. Two of the Knox coefficients, B and
C, depend on k' and size of the compound. In the equations above,
k' in the isocratic equations is replaced with average k' in the
gradient equations. In fact, this is the only difference in the
bandwidth and resolution equations between the two. Thus,
separation and height of the peak are dictated by the exact same
conditions for both isocratic and gradient elution (Snyder,
1983).
>From the equation for capacity factor in gradient elution, it
can be seen that average k' value depends on flow-rate, gradient
time, and column dead volume. This differs in isocratic elution
where k' is not dependent on time of separation, flow- rate, or
column dimensions.
A special feature in gradient elution is linear-solvent strength (LSS) gradients. These give approximately equal values of average k' for samples eluting at different times during separation. This is the reason why gradient elution can yield constant bandwidths for different compounds and equal resolution for pairs of compounds which have similar alpha or separation factor values.
Polytyptic Mobile Phase, sometimes referred to as
mixed-mode chromatography, is a versatile method in which several
types of chromatographic techniques, or modes, can be employed
using the same column. These columns contain rigid macroporous
hydrophobic resins covalently bonded to a hydrophilic organic
layer. SEC, IEC, hydrophobic or affinity chromatography are some
of the methods that may be utilized. By changing the the mobile
phase, the mode of separation is thereby changed which allows the
chromatographer to achieve the desired selectivity in the
separations.
Synder,
L.R.; Stadalius, M.A.; Quarry, M.A. Analytical Chemistry,
1983, Vol. 55, pp. 1412-30.
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mobile phase, mobile, phase, HPLC, high performance liquid chromatography, high, performance, liquid, chromatography, solvent, solute, isocratic elution, column, carrier, stationary phase, sample, gradient, polytyptic, separation, compounds, composition, retention time, retention, time, bandwidth, resolution, capacity, factor, capacity factor, migration, migrate, Knox, analytical, chemistry, analytical chemistry, linear-solvent strength (LSS) gradient, linear, strength, university of kentucky, college of pharmacy
