CHROMATOGRAPHIC THEORY

Prof. Derick Carboo

Chemistry Department University of Ghana Legon
E-mail: dcarboo@ug.edu.gh

Prof Derick Carboo

2/14/2010

Individual solutes interact with stationary phase to different degrees. The interactions may be adsorption, relative solubility, charge, or dipoledipole interaction, van der Waals etc. So they are retarded by the st. phase differently.
For example: A component which is quite soluble in the st. phase will take longer to travel through the column than one which is less soluble in the st. phase but more soluble in the mobile phase.
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Chromatographic theory:

As a result of these differences in mobilities, sample components will become separated from each other as they travel through the column.

The sample is transported through the column by continuous addition of mobile phase (Elution).

The average rate at which an analyte moves through the column is determined by the time it spends in the mobile phase. (It is

assumed that solute does not move in the st. phase)

So if it has a higher affinity for the st. phase it would be retarded even more.
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Chromatographic theory:

The Retention time, tr for each component is the time needed after injection of the mixture onto the column until it reaches the detector. The Void time, tm (or dead time) is the time taken by the (un-retained) mobile phase to travel through the column The adjusted retention time for a solute is the additional time required for the solute to travel the length of the column beyond the time required by the unretained mobile phase (solvent).

Adjusted retention time : tr = tr tm

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 Relative retention (also known as selectivity factor) : For any two components 1 and 2 the Relative retention is given by the ratio of the adjusted retention times: = tr / tr (where tr2 > tr1 , so > 1 )

is a measure of the separation between the

two components. The greater , the greater

the separation between the two solutes (on the chromatogram).

can also be related to the partition coefficients of the solutes: = Ka/Kb (the ratio must be greater than unity) If Ka > Kb , then solute A is more retained on the stationary phase than solute B.

Relative retention is fairly independent of flow rate and can therefore be used to help identify peaks when flow rate changes.

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2. Capacity factor
or partition ratio)

(also known as retention factor, capacity ratio

Is a measure of solute velocity through a column compared to mobile phase

In a chromatographic column, the solute is distributed between the stationary phase and the mobile phase. The fraction in the mobile phase moves at the same velocity as the mobile phase The fraction in the stationary phase is considered as having zero velocity.

For solute A, the capacity factor is given by : kA = time solute spends in stationary phase
time solute spends in mobile phase

Or

kA = tr tm/tm
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The longer a solute is retained by the column the greater is the capacity factor. Very short capacity factor (<1) means elution is so fast that determination of retention time cannot be done accurately. Large capacity factor (>20) means elution time too long (leading to band broadening) Ideal capacity factor 1 5. Capacity factors can be manipulated in GC by changes in Temp and column packing (packed column) , and In LC by changes in mobile phase composition and stationary phase.
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Efficiency is how well a chromatographic system can separate compounds. This depends on two factors: 1. Differences in the retention times of the solutes: the further apart the peaks the better the separation 2. How broad the peaks are: the broader the peaks the poorer the separation. So the Efficiency of the column also refers to the extent of band broadening that occurs when solute traverses the column.

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As a band of solute moves through the chromatographic column it tends to spread. This manifests itself in a broadening of the chromatographic peak. Typical injected volume is 5-20 L; Typical collected volume is 1000L.
Band Broadening is caused by:
1) 2) 3)

the non-even flows around and inside the porous particles, slow adsorption kinetics, longitudinal diffusion, and other factors.
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The longer the component is retained, the more broad its zone. Band broadening is, in general, dependent on the a) adsorbent particle size, b) adsorbent porosity, c) adsorbent pore size, d) column size, shape, e) and packing performance.

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In the ideal case, the chromatographic peak can be represented by a Gaussian curve with the standard deviation . The ratio of standard deviation to the peak retention time /tr is called the relative standard deviation, which is independent on the flow rate. The width of the curve is measured by :
(1) Peak width at half height, w1/2 = 2.35

(2) Peak width at base,

(3) Peak width at 0.67 height

w = 4
w0.67 = 2

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In chromatography resolution of two peaks is a function of the separation between the peaks. Its defined quantitatively by: Resolution = tr / wav = Vr / Wav

tr or Vr = separation between the peaks ; Wav = average width of the two peaks at base

Example: Given trA = 407s, width at base = 13s; trB = 424s, width at base = 16s Find the resolution.

Resolution = tr / wav = (424 404) / 0.5(13 + 16) = 1.17

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(s)
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Diffusion is the spontaneous movement of solute molecules from a region of high concentration to a region of low concentration Fick's first law of diffusion describes the number of moles crossing each square meter per second:

J(mol/m2 s) =D dc/dx where J is the flux ; D is diffusion coefficient; dc/dx is concentration gradient The () sign shows the decrease with distance.

In general: Diffusion in liquid is 10,000 times slower than in gases. Macromolecules diffuse 10 to 100 times slower than small molecules.
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If the solute was injected as infinitely sharp layer with m moles per unit cross section area and spreads by diffusive mechanism as it travels, then the Gaussian profile of the band is described by :

m.e 4Dt where t is time, and x is distance along the column from the current center of the band
and c is the concentration mol/m3.

c =

-x2/4Dt

The standard deviation of the band is: = 2Dt

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Two approaches can be taken to explain the separation process:

The PLATE THEORY proposed by in 1941 by Martin and Synge. The theory is based on an

analogy with distillation and counter current extraction.

RATE THEORY proposed by van Deemter in 1956 accounts for the dynamics of a

separation.

Each has its own advantages and limitations.

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The Plate theory: Distillation is a technique used to separate liquids over their volatilities or boiling points. A mixture of the liquids is heated and the vapours are in equilibrium with the liquid. The more volatile or the one with the lower boiling point rises and is collected by cooling it in a condenser. We can identify simple distillation where the boiling points of the components are very different ( 30oC )so separation is achieved without a fractionating column. In case where the boiling points are closer fractional distillation is used. Here a column is placed in between the vessel and condenser to allow a longer path for the components to separate.
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In fractional distillation a mixture of two or more liquids having slightly different boiling points can be separated using a fractionating column. A typical fractionating column is the bubble cap column. This contains a number of shallow trays or plates capable of holding a thin layer of liquid. Each plate has an overflow, which allows excess liquid to flow to the plate below, and several bubble caps through which vapour rising upward can escape only after bubbling through the liquid. The vapour is condensed at the top of the column and part, called reflux, is allowed to flow back down the column. In the bubble cap column actual plates exist where vapour passes through the liquid phase causing the phases to mix; The height of a plate can often be directly measured as the plate height
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In packed distillation column the plates cant be observed but can be calculated; so they are called theoretical plates,

According to the PLATE THEORY the chromatographic column, is likened to a distillation column which 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".

Amob

Ast
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The analyte moves down the column by transfer of equilibrated mobile phase from one plate to the next. The more equilibrium points there are in the column the narrower the plate height. The smaller or narrower the plate height, the narrower the band width; hence the better separated the bands will be.

1. 2.

An efficient column has a small plate height An efficient column has more theoretical plates.

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The band has a gaussian profile with a standard deviation of

If a solute has travelled a distance of x at a linear flow rate of ux (m/s), then the time it has been on the column is: t = x/ux ; therefore 2 = 2Dx/ux Replacing 2D/ux with H : 2 = (2D/ux)x = H x

2 Dt

H = 2 /x

H is called the plate height and is the width of the band after the solute had travelled a certain distance. H is proportional to the variance of the band
H is the height equivalent of a theoretical plate (HETP).

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HETP is a quantity relating the width of a band to the distance travelled through the column. The smaller the plate height, the narrower the band width. So The ability of a column to separate the components of a mixture is improved by decreasing plate height. Therefore an efficient column has more theoretical plates. Different solutes passing through the same column will have different plate height because each diffuses differently in the column. ( D is different for different solutes)

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N can be calculated from the chromatogram:

N = L/H = 16L2/w2 N = 16tr2/w2 = 5.55tr2/w21/2

Generally plate height for GC are between 0.1 1.0mm HPLC plate height 10m Capillary electrophoresis plate height <1m

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The relationship between the number of plates N and resolution R is given by: R = N ( 1) k2 4 () (1+ kav )
= selectivity factor k2 = capacity factor for the more retained component kav = average capacity for both compounds It follows that :

R N L Because N is proportional to the length of column doubling the column length increases resolution by 2

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Band broadening outside the column.

The solute cannot be injected as infinitely thin zone. The band has some finite width even before entering the column. If the band is applied as a plug of width t, the contribution to the final variance is: 2injection = (t)2 / 12

The broadening in the detector holds the same relationship, because some finite time is required for the sample to pass trough. 2detector = (t)2 / 12
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Given :Elution rate = 1.35 ml/min; w1/2 for the collected band is 16.3 s. Volume of the sample applied is 0.30 ml. Detector volume is 0.20 ml. Find : 1. the variances introduced by injection and detection. 2. The width at half-height, which is caused by column only.

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SOLUTION. From w1/2 = 2.35 the observed total variance is: 2obs = (w1/2/2.35)2 = (16.3/2.35)2 = 48.11s2

The time of injection is: tinjection=(0.30 ml)/(1.35 ml/min) = 0.222 min =13.3s.
Similarly,

2injection = 14.78s2

tdetector= (0.20 ml)/(1.35 ml/min) = 8.89 s, and 2detector= 6.58 s2.

from 2obs = 2column + 2detector + 2injector column = 5.17 s.

The width due to column broadening alone is: w1/2 = 2.35column = 2.35 x 5.17 s = 12.1 s.
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The Plate Theory assumes that diffusion is the only source of the band broadening. The rate theory realizes that band broadening is a kinetic effect occasioned by the finite rate at which mass transfer occurs during migration of solute down the column and that this effect also depends on the length of possible passages between the mobile phase and stationary phase and is therefore proportional to the flow rate of the eluent. The theory therefore attempts to investigate the dependence of the plate height on the linear flow rate.
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The Van Deemter Equation.

J.J. Van Deemter proposed in 1956 the equation, which summarizes the on-column effects that contribute to the plate height. The equation takes into account three components:
1.

2.
3.

multiple path of an analyte through the column packing; molecular diffusion; effect of mass transfer between phases.
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H A + B/ux + C.ux
H = plate height A = multiple paths term (or eddy diffusion) B = longitudinal diffusion term C = equilibration time (or mass transfer) term Ux = linear flow rate

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The most significant result is that we can find an optimum eluent flow rate where the column efficiency will be best.
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The velocity of mobile phase in the column may vary significantly across the column diameter, depending on the particle shape, porosity, and

the whole bed structure.

For packed columns: A, B, C 0 For open-tubular columns: A = 0 For capillary electrophoresis: A = C = 0

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Band broadening due to differing flow velocities can be written in form: A = Hpath = 2 dp A is theoretical plate height (HETP) arising from the variation in the zone flow velocity; dp is average particle diameter; is the constant (very close to 1), describing the particle size distribution; The narrower the distribution, the smaller is .
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The term A may be reduced (efficiency increased) by (1) reducing the particle diameter (which will lead to the increasing of the column back pressure) and (2) by narrowing the size distribution.

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The B term arises as a result of dispersion or

mixing of the molecules due to diffusion.

The longitudinal diffusion (along the column long axis) leads to the band broadening of the chromatographic band.
As it was shown before, the variance resulting from diffusion is: = 2 Dt 2 = 2Dt = 2D x/ux = (2D/ux)x = Hx Plate height due to diffusion:

Hdiffusion = 2/x = 2D/ux B/ux

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The plate height due to diffusion is inversely proportional to flow rate. The faster the linear flow rate , the less time is spent in the column and the less diffusion occurs . Therefore : The higher the eluent velocity, the lower the effect on the band broadening. Longitudinal diffusion is a common source of band broadening in GC but is of little significance in LC because molecular diffusion in the liquid phase is about five orders of magnitude lower than that in the gas phase.
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The term Cux arises from the mass transfer term which refers to the finite time required for solute to equilibrate between the mobile and the stationary phases. Plate height due to finite equilibration time of the mass transfer is: Hmass transfer = Cux = (Cs + Cm)ux
where Cs describe the rate of mass transfer through stationary phase, and Cm describes mass transfer through mobile phase.

Two mass transfer coefficients Cs and Cm are needed because the equilibrium between the mobile phase and the stationary phase is established so slowly that the chromatographic column always operate under non-equilibrium conditions.
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Therefore :
analyte molecules at the front of a band are swept ahead before they have time to equilibrate with stationary phase and be retained. Equilibrium is not reached at the trailing edge of a band, and molecules are left behind in the stationary phase by the fastmoving mobile phase The slower the linear flow rate the more complete the equilibration (or mass transfer) and less zone broadening occurs The mass transfer equations are different for LC and GC.

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where

2k ' d Cs 2 3k '1 Ds

k' is the capacity factor, d is the thickness of stationary phase, r is column radius. Ds and Dm are the diffusion coefficients in stationary and mobile phases.

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Therefore: 1. Decreasing the stationary phase thickness d

reduces plate height and increases

2.

efficiency, because solute diffuse faster across the stationary phase. Decreasing the column radius r reduces

plate height and increases efficiency by

reducing the distance trough which the solute must diffuse to reach the stationary phase.
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Mass transfer for the modern types of packing materials combines two effects: adsorption kinetics; mass transfer (mainly due to diffusion) inside the particles. Modern packing materials for HPLC are thefore spherical, totally porous, rigid particles with average diameter ~5 m and pore diameter ~100. Ratio of the particle to the pore diameter is 500/1. There is no pressure propelled flow inside the particle, and molecules can move there only by diffusion.
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Adsorption kinetics is almost negligible compare to the diffusion inside the particles, and band spreading of the peak may be written in form:

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The Van Deemter equation can be further expanded to:

H = 2dp + 2GDm/ + (dp or dc)2 /Dm + Rd2f /Ds
H is plate height is particle shape (with regard to the packing) dp is particle diameter G, , and R are constants Dm is the diffusion coefficient of the mobile phase dc is the capillary diameter df is the film thickness Ds is the diffusion coefficient of the stationary phase.

Where:

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