Chromatography Chromatography: a sample (or sample extract) being dissolved in a mobile phase (which may be a gas, a liquid or a supercritical

fluid). The mobile phase is then forced through an immobile, immiscible stationary phase.

The phases are chosen such that components of the sample have differing
solubilities in each phase. A component which is quite soluble in the stationary phase will take longer to travel through it than a component which is not very soluble in the stationary phase but very soluble in the mobile phase. As a result of these differences in mobilities, sample components will become separated from each other as they travel through the stationary phase.

Chromatography : separations method based on differences in partitioning behavior between a flowing mobile phase and a stationary phase to separate the components in a mixture. Chromatographic separation process based on

the difference in the surface interactions of the analyte and eluent (mobile
phase) molecules. A column (or other support for TLC) holds the stationary phase and the mobile phase carries the sample through it. Sample components that partition strongly into the stationary phase spend a greater amount of time in the column and are separated from components that stay predominantly in the mobile phase

and pass through the column faster.

As the components elute from the column they can be quantified by a detector and/or collected for further analysis.

Gas Chromatography and Liquid Chromatography Gas and liquid chromatography are methods to separate sample components from each other and from matrix components and the solvent.

This is accomplished by partitioning the compounds between a stationary

phase (the column packing or coating) and a moving phase (a gas in GC and a liquid in HPLC). Compounds with little partitioning into the stationary phase pass through the column more swiftly and elute first. Those that spend a lot of time in the stationary phase are retarded and elute later. In gas chromatography, boiling point or vapor pressure is the major factor in elution order, although polarity has a small but important effect. In

HPLC, polarity is the primary factor in elution order.

Since elution is based on physical properties, retention time or elution pattern is an attribute of a molecule and can be used to determine identity. A particular compound will have a characteristic retention time (time from injection to detection) when run on similar columns under similar conditions. Two different compounds may have very close or identical retention times on one column type, but when analyzed on a column of differing polarity usually have different retention times. This is the basis for identification based on chromatographic retention time.

GC and HPLC are also used in quantitation. The signal from the detector

is in the shape of a peak, and the height or area of the peak is proportional
to the amount injected.

Quantitation utilizes standard curves, where known amounts from standards of known concentration are injected into the instruments, and the response compared to a sample of unknown concentration.

CHROMATOGRAPHY THEORY

Chromatography theory consists of empirical relationships to

describe chromatographic columns and the separation of peaks in chromatograms. The underlying principle that controls chromatographic

separations is a dynamic behavior that depends upon partitioning and mass transport. Separation of solutes depends on the rates at which the species

are eluted.

These migration rates are determined by the

partition coefficient, K or the equilibrium constant of the distribution of the solutes between the mobile phase (elution solvent) and stationary phase (column packing).

Distribution of analytes between phases The distribution of analytes between phases can often be described quite simply. An analyte is in equilibrium between the two phases; Amobile Astationary

The equilibrium constant, K, is termed the partition coefficient; defined as the molar concentration of analyte in the stationary phase divided by the molar concentration of the analyte in the mobile phase.
K = [A] stationary [ A] mobile kVM Vs

K=

detector signal time

sample

mobile phase

A+B B A
packed column

B A B A

t0

t1

t2

t3

t4

Retention factor (capacity factor), k', is often used to describe the migration rate of an analyte on a column. The retention factor for analyte A is defined as;
k'A = t R - tM / tM

Retention time,( tR ) Retention time, tR is the time for the analyte to pass through the column or the time between sample injection and an analyte peak reaching a detector at the end of the column. Each analyte in a sample will have a different retention time. Dead Time (tM) The dead time is the time a non-retained compound spends in the mobile phase which is also the amount of time the non-retained compound spends in the column. The time taken for the mobile phase to pass through the column is therefore called tM.

Adjusted Retention Time (tR') The adjusted retention time is the time a compound spends in the stationary phase. The adjusted retention time is the difference between the dead time and the retention time for a compound.

t R and tM are easily obtained from a chromatogram. When an analytes retention factor is less than one, elution is so fast that accurate determination of the retention time is very difficult. High retention factors (greater than 20) mean that elution takes a very long time. Ideally, the retention factor for an analyte is between one and five. Selectivity Factor (alpha) The selectivity is a ratio of the capacity factors of two peaks which describes the separation of two species (A and B) on the column. The selectivity is always greater than one. If the selectivity equals one, the two compounds cannot be separated. The higher the selectivity, the more separation between two compounds or peaks. a = k 'B / k 'A When calculating the selectivity factor, species A elutes faster than species B.

Description of chromatographic columns The resolution of chromatographic columns is described by the theoretical plate height, H, or the number of theoretical plates, N. H and N provide useful measures to compare the performance of different columns for a given analyte.

The efficiency is related to the number of compounds that can separated by the column. The efficiency is also expressed as the number of theoretical plates (N, unitless) or as the height equivalent to a theoretical plate (HETP, generally in millimeters).
The efficiency increases as the height equivalent to a theoretical plate decreases, thus more compounds can be separated by the column. The efficiency increases as the number of theoretical plates increases, thus the column's ability to separate two closely eluting peaks increases

The Theoretical Plate Model of Chromatography The plate model supposes that the chromatographic column is contains a large number of separate layers, called theoretical plates. Separate equilibrations of the sample between the stationary and mobile phase occur in these "plates". The analyte moves down the column by transfer of equilibrated mobile phase from one plate to the next.

It is important to remember that the plates do not really exist; they are a figment of the imagination that helps us understand the processes at work in the column.They also serve as a way of measuring column efficiency, either by stating the number of theoretical plates in a column, N (the more plates the

better), or by stating the plate height; the Height Equivalent to a Theoretical Plate
(the smaller the better). If the length of the column is L, then the HETP or H is H=L/N

The number of theoretical plates that a real column possesses can be found by examining a chromatographic peak after elution; N = 16 (tR / W)2 ; where W is the width of the peak at its base or

where w1/2 is the peak width at half-height. As can be seen from this equation, columns behave as if they have different numbers of plates for different solutes in a mixture. Chromatographic Resolution Although the selectivity factor, a, describes the separation of band centres, it does not take into account peak widths. Another measure of how well species have been separated is provided by measurement of the resolution. The resolution (Rs) between two peaks in a chromatogram is given by: where delta Z is the separation between peaks A and B; and Wa and Wb are the widths at the base of peaks A and B, respectively.

Acceptable resolution is on the order of Rs = 1.0, and baseline resolution between two peaks (as shown in the figure) requires an Rs > 1.5. It is useful to relate the resolution to the number of plates in the column, the selectivity factor and the retention factors of the two solutes;

To obtain high resolution, the three terms must be maximised. An increase in N, the number of theoretical plates, by lengthening the column leads to an increase in retention time and increased band broadening - which may not be desirable. Instead, to increase the number of plates, the height equivalent to a theoretical plate can be reduced by reducing the size of the stationary phase particles.

It is often found that by controlling the capacity factor, k', separations can be greatly improved. This can be achieved by changing the temperature (in Gas Chromatography) or the composition of the mobile phase (in Liquid Chromatography). The selectivity factor, a, can also be manipulated to improve separations. When a is close to unity, optimising k' and increasing N is not sufficient to give good separation in a reasonable time. In these cases, k' is optimised first, and then a is increased by one of the following procedures: A. B. C. D. Changing mobile phase composition Changing column temperature Changing composition of stationary phase Using special chemical effects (such as incorporating a species which complexes with one of the solutes into the stationary phase)

Optimization of Column Performance

Can be achieved by:

increasing the difference in migration rates (retention time) of components reducing peak width (zone broadening)
Original Chromatogram

detector signal

Increase difference in migration rates

Reduce zone broadening

Time

The Rate Theory of Chromatography A more realistic description of the processes at work inside a column takes account of the time taken for the solute to equilibrate between the stationary and mobile phase (unlike the plate model, which assumes that equilibration is infinitely fast). The resulting band shape of a chromatographic peak is

therefore affected by the rate of elution. It is also affected by the different

paths available to solute molecules as they travel between particles of stationary phase. If we consider the various mechanisms which contribute to band broadening, we arrive at the Van Deemter equation for plate height;

HETP = A + B / u + C u where u is the flow rate (cm/s) or the average velocity of the mobile phase, A is Eddy diffusion term, B is a longitudinal diffusion term, and C is

a mass transfer term. A, B, and C are factors which contribute to band or

zone broadening

A - Eddy diffusion : Each molecule takes a different path through column The mobile phase moves through the column which is packed with stationary phase. Solute molecules will take different paths through the stationary phase at random. This will cause broadening of the solute band, because different paths are of different lengths.

A = 2dp
A is directly proportional to the packing particle diameter, dp

is a function of packing uniformity and column geometry

In unpacked columns, A is zero

At low mobile-phase velocities, the rate at which each molecule moves down
the column tends to approach that of the average. Thus the molecules are not significantly dispersed by the multipath nature of the packing.

At high velocities, sufficient time is not available for diffusion averaging to

occur and band broadening due to the different path lengths is observed.

Thus, A depends on the stationary particles (want small) and their packing
(want uniform)

B - Longitudinal diffusion The concentration of analyte is less at the edges of the band than at the center. Analyte diffuses out from the center to the edges. This causes band broadening. If the velocity of the mobile phase is high then the analyte spends less time on the column, which decreases the effects of longitudinal diffusion. B/ = 2DM/ The longitudinal diffusion effect on H is inversely proportional to because the solute residence time is shorter at high and the extent of diffusion is less B is directly proportional to the solute diffusion coefficient in the mobile phase, DM DM is smaller in liquids than in gases, so B/ is less pronounced in LC than in GC

The obstructive factor, shows that longitudinal diffusion is hindered by the packing. is lower for a packed column (ie = 0.6) than an unpacked (capillary) column (ie = 1)

The longitudinal diffusion is the usual diffusion due to a concentration gradient, with analyte in a band moving through a column diffusing from the center of the band forward and backward.

Mobile phase

Forward and backward diffusion in mobile phase

Analyte band Solute molecules diffuse to regions of lower solute concentration in front of & behind zone; inversely proportional to mobile phase flow rate

C - Resistance to mass transfer The analyte takes a certain amount of time to equilibrate between the stationary and mobile phase. If the velocity of the mobile phase is high, and the analyte has a strong affinity for the stationary phase, then the analyte in the mobile phase will move ahead of the analyte in the stationary phase. The band of analyte is broadened. The higher the velocity of mobile phase, the worse the broadening becomes.
Mass-Transfer Coefficients (CS & CM)

The equilibrium between the mobile phase and stationary phase is established so slowly that a chromatographic column always operates under non-

Mobile phase

equilibrium conditions.
Stationary phase (SP) Analyte attracted to SP

So analyte molecules at the front of a band are swept ahead without equilibrating with the stationary phase and those at the trailing edge are left behind for a longer time in the stationary phase.

C = Cs + Cm Cs is a mass transfer term for the analyte in or on the stationary phase, and Cm is a mass transfer term for the analyte in the mobile phase; Cs = (fS(k)df2/DS)u df = film thickness of stationary phase (most important factor) CM = (fM(k)dp2/DM) dR = diameter of support particle

The mass-transfer effect on H is directly proportional to because the solute residence time is longer at low , the deviation from equilibrium is less, and zone broadening or H is smaller. Cs is less if the liquid stationary phase film thickness, df, is smaller , or the solute diffusion coefficient in the stationary phase, DS is larger. CM is less if dp is smaller (hence greater surface area), or the solute diffusion coefficient in the mobile phase, DM is larger Therefore, the mass transfer terms depend on the diffusion coefficients for the analyte in the stationary and mobile phases, and can be thought of as interactions that delay the analyte and lead to band broadening. The stationary phase mass transfer term, Cs, also depends on the thickness of the stationary phase, and the mobile phase mass transfer term, Cm, also depends on the particle size of the packing material due to its effect on eddy diffusion.

Van Deemter plots

A plot of plate height vs. average linear velocity of mobile phase.

Such plots are of considerable use in determining the optimum mobile phase flow rate.

Plot of H (and components) vs. flow rate

H, cm Cu A B/u u, cm/s

Note that there is an optimum flow rate to obtain the minimum theoretical plate height.

Zone Broadening can be reduced by using: smaller particles, narrower columns, lower temperature in GC,

thinner liquid stationary phases