1. What is Chromatography?
Chromatography is a physico‑chemical process that belongs to fractionation methods same as distillation, crystallization or fractionated extraction.
It is believed that the separation method in its modern form originated at the turn of the century from the work of Tswett to whom we attribute the terms chromatography and chromatogram
The method was used for preparation and purification purposes until the development of sensitive detectors
The detector signal, which is registered in continuum, leads to a chromatogram that indicates the variation of the composition of the eluting phase with time.

2. Chromatographic separations
Sample is dissolved in a mobile phase (a gas, a
liquid or a supercritical fluid)
The mobile phase is 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 a varying degree.

4. The Chromatogram
It reveals, as a function of time, a parameter the depends on the instantaneous concentration of the solute as it exits the column
The components entering the detector will be shown as a series of peaks that would be more or less resolved from one another as they rise from the baseline
obtained in the absence of analyte.
If the detector signal varies linearly with the concentration of analyte, the same variation will occur for the area under the peak in the chromatogram.
A constituent is characterized by its retention time, tR,
Retention time is defined by the time taken between the moment of injection into the chromatograph and the peak maximum recorded on the chromatogram.

5. In an ideal case, the retention time tR is independent of the quantity injected.
A compound not retained will elute out of the column at time tM, called the void time or the dead time (sometimes designated by to ).
The separation is complete when as many peaks are seen returning to the baseline as there are components in the mixture.
In quantitative analysis, it suffices to separate only the components that need to be measured.
Identification by chromatography is arbitrary. For a better confirmation, another identification method has to go along with thechromatography
When tM = tR ; there would be no separation, why?
All components will move st the same rate through the colum

6. a. Retention time
b. Distribution of the
peak
c. Significance of the
three basic
parameters and
features of a
Gaussian
distribution
d. Example of a real
chromatogram

7. Definition of plate height H =

8. The Theoretical Plate Model
Many theories have been suggested to explain the mechanism of migration and separation of analytes in the column.
The oldest one, called the theoretical plate model, corresponds to an approach now considered obsolete but which nevertheless leads to relations and definitions that are universal in their use and are still employed today.
In this model, each analyte is considered to be moving progressively through the column in a sequence of distinct steps, although the process of chromatography is a dynamic and continuous phenomenon.
Each step corresponds to a new equilibrium of the entire column.
In liquid‑solid chromatography, for example, the elementary process is described as a cycle of adsorption/desorption.

9. Column Efficiency
As the analyte migrates through the column, it occupies an increasing area
This linear dispersion L, measured by the variance L
increases with the distance of migration.
When this distance is equal to L, the column length, the variance will be:  2L= HxL
where H is the same as the value for the height equivalent to one theoretical plate
Since N = L/H
N = L2/  2L
N here is called theoretical efficiency of the compound.
Thus, N = t2R/  2
The more appropriate equation for N is
N = t2R/w21/2

10. Dispersion of a solute in a column and its translation into a chromatogram
On the chromatogram, 
Represents the peak width
At 60.6% of the height
Isochronic image of the concentration
of an eluted compound at a particular instant.
Variation of the concentration at the outlet of the column as a function of time.

11. Effective plate number
If the performance of different columns has to be compared for a given compound, more realistic values are obtained by replacing the total retention times tR, by the adjusted retention times t’R
t’R does not take into account the void time tM spent by the compound in the mobile phase.
The mathematical relationships:

12. Retention parameters VM = tM x F
In chromatography, three volumes are usually considered.
Volume of the mobile phase in the column (dead volum
The volume of the mobile phase in the column (called the dead volume) VM corresponds to the accessible interstitial volume. It can be determined from the chromatogram provided a solute not retained by the stationary phase is used.
VM can be expressed as a function of tM and flow rate F:
VM = tM x F

13. . This volume does not appear on the chromatogram
2. Volume of the stationary phase, Vs
. This volume does not appear on the chromatogram
Vs can be determined by subtracting the volume of the mobile phase from the total volume inside the column.
3. Retention volume, VR
The retention volume VR of each analyte represents the volume of mobile phase necessary to cause its migration throughout the column.
On the chromatogram, the retention volume corresponds to the volume of mobile phase that has passed through the column from the time of injection to the peak maximum.
VR = tR x F

14. Retention factor k (historically called capacity factor, K’)
When a compound is injected onto a column, its total mass mT is divided in two quantities:
mM, the mass in the mobile phase and ms, the mass in the stationary phase.
The values of these quantities are dependent on MT and K but their ratio, the retention factor, is constant:
The factor k, which is independent of the flow rate and length of
the column, can vary with experimental conditions.
k is the most important parameter in chromatography for
determining the behavior of columns.
The value of k should not be too high otherwise the time of
analysis is unduly elongated.

15. Experimental determination of the retention factor, k
The mobile phase progressively transports the analyte towards the end of the column
It is assumed that the ratio of the retention volume VR to the dead volume VM is identical to the ratio which exists between the total mass of the compound and the mass dissolved in the dead volume. Consequently:

16. Using the equations: VM = tM x F
and VR = tR x F
Then,
tR = tM ( 1 + k)
Therefore, the value of k is accessible from the chromatogram and can be obtained using the following equation

17. Separation factor between two solutes
The separation factor, , allows the comparison of two adjacent solutes 1 and 2 present in the same chromatogram
Thus, the separation
factor is given
by the equation:

18. Resolution factor between two peaks
To quantify the separation between two peaks, the resolution factor R is used and can be obtained from the chromatogram

19. The van Deemter equation in gas chromatography
When the characteristics of a separation were expressed previously, the speed of the mobile phase in the column did not appear.
However, the speed has to affect the progression of the solutes, hence their dispersion within the column, and must have an effect on the quality of the analysis.
These kinetic considerations are collected in a famous equation proposed by van Deemter.
The simplified form of this equation is given below

20. Van Deemter Equation
The three experimental parameters A, B and C are
related to column parameters and also to experimental
conditions.
If H is expressed in cm, A will be expressed in cm, B in
cm cm2/s and C in s (where velocity is measured in
cm/s). The function is a hyperbolic function that goes
through a minimum (Hmin) when

21. Van Deemter plot for gas phase chromatography
showing domains for A, B and C.

22. Van Deemter equation which has been expanded to cover both gas and liquid chromqtography
The equation shows that there is an optimum flow rate for each separation and that this does indeed correspond to the minimum on the curve represented by equation
The loss in efficiency that occurs when the velocity is increased represents what occurs when trying to rush the chromatographic separation by increasing the flow rate of the mobile phase.
However, it is hard to predict the loss in efficiency that occurs when the flow is too slow. How?

23. Term A, packing term (Eddy diffusion)
The term A is related to the flow profile of the mobile phase as it traverses the stationary phase.
The size of the stationary phase particles, their dimensional distribution, and the uniformity of the packing are responsible for a preferential path and add mainly to the improper exchange of solute between the two phases.
This phenomenon is the result of Eddy diffusion or turbulent diffusion, considered to be non‑important in liquid chromatography or absent by definition in capillary columns, and WCOT (wall coated open tubular) in gas phase chromatography

24. Term B, diffusion coefficient in the mobile phase
The term B is related to the longitudinal molecular diffusion in the column.
It is especially important when the mobile phase is a gas.
This term is a consequence of entropy, telling us that a system will tend towards the maximum degrees of freedom as demonstrated by a drop of ink that diffuses after falling into a glass of water.
Hence, if the flow rate is too slow, compounds being separated will mix faster than they will migrate.
This is why one must never interrupt the separation process, even momentarily.

25. Term C, mass transfer coefficient
The coefficient C is related to the resistance to mass transfer between the two phases
It becomes important when the flow rate is too high for equilibrium to be obtained.
Local turbulence within the mobile phase and concentration gradients slow down the equilibrium process (Cs = Cm).
The diffusion of solute between the phases is not instantaneous, hence the solute will be in a non‑equilibrium process.

26. Optimization of a chromatographic analysis
Because analytical chromatography is used inherently in quantitative analysis, it becomes crucial to precisely measure the areas of the peak.
Therefore, the substances to be determined must be well separated.
In order to achieve this, the analysis has to be optimized using all the resources of the instrumentation and, when possible, software that can simulate the results of temperature modifications, phases and other physical parameters.
This optimization process requires that the chromatographic process is well understood.

27. Classification of chromatographic techniques

28. Classification of chromatographic techniques
Chromatographic techniques can be classified into three categories depending on
the physical nature of the phases,
the process used,
or the physico‑chemical phenomenon, which is at the basis of the Nernst distribution coefficient K, also defined as:
We will take here the classification based on the
nature of the phase present

29. 1. Liquid‑solid chromatography
The mobile phase is a liquid and the stationary phase is a solid.
This category, which is widely used, can be subdivided depending on the retention phenomenon into:
Adsorption chromatography
Ion chromatography
Molecular exclusion chromatography

30. a. Adsorption chromatography
The separation of organic compounds on a thin layer of silica gel or alumina with solvent as a mobile phase
Solutes bond to the stationary phase because of physisorption or chemisorption interactions.
The physico‑chemical parameter involved is the coefficient of adsorption.

31. b. Ion chromatography
The mobile phase in this type of chromatograph; is a buffered solution and the stationary phase consists of spherical m diameter particles of a polymer
The surface of the particles is modified chemically in order to generate ionic sites.
These phases allow the exchange of their mobile counter ion, with ions of the same charge present in the sample.
This separation relies on the coefficient of ionic
distribution

32. c. Molecular exclusion chromatography
The stationary phase is a material containing pores, the dimensions of which are chosen to separate the solutes present in the sample based on their molecular size.
This can be considered as a molecular sieve allowing selective permeation.
This technique is known as gel filtration or gel permeation, depending on the nature of th mobile phase, which is either aqueous or organic.
The distribution coefficient in this technique is called the coefficient of diffusion.

33. 2. Liquid‑liquid chromatography (LLC)
Stationary phase is a liquid immobilized in the column.
It is important to distinguish between the inert support which only has a mechanical role and the stationary phase immobilized on the support
Impregnation of a porous material with a liquid phase was used earlier but had the problem of bleeding
In order to immobilize the stationary phase, it is preferable to fix it to a mechanical support using covalent bonds.
The stationary phase still acts as a liquid and the separation process is based on the partition of the analyte between the two phases at their interface.
The parameter involved in the separation mechanism is called the partition coefficient.

34. 3. Gas‑liquid chromatography (GLC)
The mobile phase is a gas and the stationary phase is a liquid.
The liquid can be immobilized by impregnation or bonded to a support,
The partition coefficient K is also involved
4. Gas‑solid chromatography (GSC)
Stationary phase is a porous solid (such as graphite or
silica gel) and the mobile phase is a gas.
This type demonstrates very high performance in the
analysis of gas mixtures or components that have a
very low boiling point.

35. 5. Supercritical fluid chromatography (SFC)
The mobile phase is a fluid in its
supercritical state, such as carbon dioxide at about 50 °C and at more than 150 bars (15 MPa).
The stationary phase can be a liquid or a solid.
This approach combines the advantages of the LLC and GLC techniques