Introduction to Chromatography

Introduction Chromatography permit the scientist to separate closely related components of complex mixtures. In all chromatographic separations the sample is transported in a mobile phase, which may be a gas, a liquid, or a supercritical fluid. This mobile phase is then forced through an immiscible stationary phase, which is fixed in place in a column or on a solid surface. The two phases are chosen so that the components of the sample distribute themselves between the mobile and stationary phase to varying degrees.

Classification of Chromatographic Methods Chromatographic methods can be categorized in two ways. The first classification is based upon the physical means by which the stationary and mobile phases are brought into contact. In column chromatography planar chromatography

Classification of Chromatographic Methods Three general categories of chromatography: (1) liquid chromatography, (2) gas chromatography, (3) supercritical-fluid chromatography.

Elution Chromatography on Columns Elution involves washing a species through a column by continuous addition of fresh solvent. The sample is introduced at the head of a column, whereupon the components of the sample distribute themselves between the two phases. Introduction of additional mobile phase (the eluent) forces the solvent containing a part of the sample down the column, where further partition between the mobile phase and fresh portions of the stationary phase occurs.

Chromatograms If a detector that responds to solute concentration is placed at the end of the column and its signal is plotted as function of time (or of volume of the added mobile phase), a series of peaks is obtained. Such a plot, called a chromatogram, is useful for both qualitative and quantitative analysis. The positions of peaks on the time axis may serve to identify the components of the sample; the areas under the peaks provide a quantitative measure of the amount of each component.

MIGRATION RATES OF SOLUTES The effectiveness of a chromatographic column in separating two solutes depends in part upon the relative rates at which the two species are eluted. These rates are determined by the magnitude of the equilibrium constants for the reactions by which the solutes distribute themselves between the mobile and stationary phases.

Increase in band separation Decrease in band spread

A mobile A stationary K=C S /C M K Distribution coefficient C S Molar concentration of solute in stationary phase C M Molar concentration of solute in mobile phase Distribution Coefficient

v = L/t R v Linear rate of solute migration L Length of column packing t R Solute retention time Retention Time u = L/t M u Linear rate of movement of mobile phase molecules t M Dead time v = u × (fraction of time solute spends in mobile phase

Time Detector Signal trtr tMtM Typical chromatogram for a two component mixture. The small peak on the left is represent a species that is not retained on the column and so reaches the detector immediately after elusion is started. Thus its retention time t m is approximately equal to the time required for a molecule of mobile phase to pass through the column.

The Relationship Between Retention Time and Distribution Coefficient

Relative Migration Rates: The selectivity Factor The selectivity factor  of a column for the two species A and B is defined as  = K B /K A where K B is the distribution constant for species B and K A is the distribution constant for species A.  is always greater than unity. A relationship between the selectivity factor and retention factors:  = k` B /k` A Where k` B and k` A are the retention factors. An expression for the determination of  from an experimental chromatogram:

Definition of Plate Height

Time Detector Signal trtr tMtM w Determination of standard deviation  from a chromatographic peak: w = 4 

H = A + B/u + ( C S + C M ) u Plate height Multiple flow paths Longitudinal diffusion Mass transfer between phases

The Multipath Term(A) Zone broadening arises in part from the multitude of pathways by which a molecule (or ion) can find its way through a packed column. The length of these pathways may differ significantly; thus, the residence time in the column for molecules of the same species is also variable. Solute molecules then reach the end of the column over a time interval, which leads to a broadened band. This effect which is called eddy diffusion, is directly proportional to the diameter of the particles making up the column packing.

Typical pathways of several molecules during elution.

The Longitudinal Diffusion Term (B/u) Longitudinal diffusion in column chromatography is a band broadening process in which solutes diffuse from the concentrated center of a zone to the more dilute regions ahead of and behind the zone center. The longitudinal diffusion term is directly proportional to the mobile-phase diffusion coefficient D M. The contribution of longitudinal diffusion is seen to be inversely proportional to the mobile phase velocity.

Mass-transfer Coefficients (C s and C M ) The need for the two mass-transfer coefficients C s and C M arises because the equilibrium between the mobile and the stationary phase is established so slowly that a chromatographic column always operates under nonequilibrium conditions. Effect of Mobile-Phase Velocity Fig provides the quality of the fit of the van Deemter equation. The upper curve was obtained from a numerical fit of the van Deemter equation to the data. The lower plots in the figure also show the contribution of the longitudinal diffusion, and the masstransfer effects.

Linear flow rate cm/s H mm A van Deemter plot for a packed liquid chromatographic column. The points on the upper curve are experimental. The contribution of various rate terms are shown by the lower curves. A: multipath effect; B/u, longitudinal diffusion; Cu, mass transfer for both phases. H = A + B/u + ( C S + C M ) u

Methods for Reducing Zone Broadening Two important controllable variables that affect column efficiency are the diameter of the particles making up the packing and the diameter of the column. To take advantage of the effect of column diameter, narrower and narrower columns have been used in recent years. With gaseous mobile phases, the rate of longitudinal diffusion can be reduced appreciable by lowering the temperature and thus the diffusion coefficient D M. The consequence is significantly smaller plate heights at low temperatures.

Linear flow rate cm/s H mm mm mm mm mm mm Effect of particle size on plate height. The numbers to the right are particle diameters.

Methods for Reducing Zone Broadening Two important controllable variables that affect column efficiency are the diameter of the particles making up the packing and the diameter of the column. To take advantage of the effect of column diameter, narrower and narrower columns have been used in recent years. With gaseous mobile phases, the rate of longitudinal diffusion can be reduced appreciable by lowering the temperature and thus the diffusion coefficient D M. The consequence is significantly smaller plate heights at low temperatures.

Column Resolution The resolution R s of a column provides a quantitative measure of its ability to separate two analytes. Column resolution is defines as It is evident from Fig that a resolution of 1.5 gives an essentially complete separation of the two components, whereas a resolution of 0.75 does not. At a resolution of 1.0, zone A contains about 4% B and zone B contains a similar amount of A. At a resolution for 1.5, the overlap is about 0.3%. The resolution for a given stationary phase can be improved by lengthening the column, thus increasing the number of plates.

0 0 RS = 0.75 RS = 1.0 w A /2 A B w B /2 0 RS = 1.5 (t R ) A (t R ) B ZZ tMtM Separation at three resolutions. Here R S = 2  Z/(w A + w B )

Retention factor k B ’ R S /Q or (t R ) B /Q’ Effect of retention factor k B ’ on resolution R S and elution time (t R ) B. It is assumed that Q and Q’ remain constant with variation in k B ’

(a) 70% Methanol 30% water (b) 60% Methanol 40% water (c) 50% Methanol 50% water (d) 40% Methanol 60% water Inject Effect of solvent variation on chromatograms. Analytes: (1) 9,10-anthraquinone; (2) 2-methyl 9,10-anthraquinone; (3) 2-ethyl-9,10-anthraquinone; (4) 1,4-dimthyl 9,10-anthraquinone; (5) 2-t- butyl-9.10-anthraquinone.

Time Signal Illustration of general elution problem in chromatography