What is Chromatography?

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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.

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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.

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1. Diagram showing the separation of a mixture of
   components A and B by column elution
   chromatography.
2. The output of the signal detector at the various
   stages of elution shown in (a).

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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

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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

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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.

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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

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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.

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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.

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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.

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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

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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.

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• 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

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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
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Definition of plate height H
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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.

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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 5.54 t2R/w21/2

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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.
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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 tR
• tR does not take into account the void time tM
  spent by the compound in the mobile phase.
• The mathematical relationships

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Retention parameters

• 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

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• 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

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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.

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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

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• 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

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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
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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

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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

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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

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

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• 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?

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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

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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.

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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.

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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.