Chromatography : Chromatography Rina Gokani Rachchh
S. J. Thakkar Pharmacy College, Rajkot Slide 2: 3/18/2009 © RGR 2 What is Chromatography? Derived from the Greek word Chroma meaning colour, chromatography provides a way to identify unknown compounds and separate mixtures Slide 3: 3/18/2009 © RGR 3 Introduction to Chromatography Definition
Chromatography is a separation technique based on the different interactions of compounds with two phases, a mobile phase and a stationary phase, as the compounds travel through a supporting medium.
mobile phase: a solvent that flows through the supporting medium
stationary phase: a layer or coating on the supporting medium that interacts with the analytes
supporting medium: a solid surface on which the stationary phase is bound or coated Slide 4: 3/18/2009 © RGR 4 1903 Tswett - plant pigments separated on chalk columns
1931 Lederer & Kuhn - LC of carotenoids
1938 TLC and ion exchange
1950 reverse phase LC
1954 Martin & Synge (Nobel Prize)
1959 Gel permeation
1965 instrumental LC (Waters) Milestones in Chromatography Slide 5: 3/18/2009 © RGR 5 Applications of Chromatography Forensics Research Pharmaceutical industry separating mixtures of compounds
identifying unknown compounds
establishing the purity or concentration of compounds
monitoring product formation in the pharmaceutical and biotechnology industries Types of Chromatography… : 3/18/2009 © RGR 6 Types of Chromatography… Paper HPLC Gas Thin layer Column Chromatographic methods classification : Chromatographic methods classification (1) Geometry of the system : 3/18/2009 © RGR 8 (1) Geometry of the system In column chromatography the stationary phase is contained in a tube called the column.
Planar chromatography. In this geometry the stationary phase is configured as a thin two-dimensional sheet.
(i) In paper chromatography a sheet or a narrow strip of paper serves as the stationary phase.
(ii) In thin-layer chromatography a thin film of a stationary phase of solid particles bound together for mechanical strength with a binder, such as calcium sulfate, is coated on a glass plate or plastic or metal sheet. (2) Mode of operation : 3/18/2009 © RGR 9 (2) Mode of operation Development chromatography. In terms of operation, in development chromatography the mobile phase flow is stopped before solutes reach the end of the bed of stationary phase. The mobile phase is called the developer, and the movement of the liquid along the bed is referred to as development.
Elution chromatography. This method, employed with columns, involves solute migration through the entire system and solute detection as it emerges from the column. The detector continuously monitors the amount of solute in the emerging
mobile-phase stream—the eluate—and transduces the signal, most often to a voltage, which is registered as a peak on a strip-chart recorder. Example of planar development chromatography – TLC or PC (3) Retention mechanism : 3/18/2009 © RGR 10 (3) Retention mechanism Classification in terms of the retention mechanism is approximate, because the retention actually is a mixture of mechanisms. The main interactions are:
hydrophobic (non-specific), dipole-dipole (polar), ionic, special. (4) Phases involved : 3/18/2009 © RGR 11 (4) Phases involved 1.) The primary division of chromatographic techniques is based on the type of mobile phase used in the system:
Type of Chromatography Type of Mobile Phase
Gas chromatography (GC) gas
Liquid chromatograph (LC) liquid Slide 12: 3/18/2009 © RGR 12 2.) Further divisions can be made based on the type of stationary phase used in the system:
Name of GC Method Type of Stationary Phase
Gas-solid chromatography solid, underivatized support
Gas-liquid chromatography liquid-coated support
Bonded-phase gas chromatography chemically-derivatized support Slide 13: 3/18/2009 © RGR 13 Liquid Chromatography
Name of LC Method Type of Stationary Phase
Adsorption chromatography solid, underivatized support
Partition chromatography liquid-coated or derivatized support
Ion-exchange chromatography support containing fixed charges
Size exclusion chromatography porous support
Affinity chromatography support with immobilized ligand 5) Principle of separation : 3/18/2009 © RGR 14 5) Principle of separation Adsorption chromatography.
Chromatography in which separation is based mainly on differences between the adsorption affinities of the sample components for the surface of an active solid. Slide 15: 3/18/2009 © RGR 15 Partition chromatography.
Chromatography in which separation is based mainly on differences between the solubility of the sample components in the stationary phase (gas chromatography), or on differences between the solubilities of the components in the mobile and stationary phases (liquid chromatography). Slide 16: 3/18/2009 © RGR 16 Ion-exchange
chromatography. Chromatography in which separation is based mainly on differences in the ion-exchange affinities of the sample components. Anions like SO3- or cations like N(CH3)3+ are covalently attached to stationary phase, usually a resin Slide 17: 3/18/2009 © RGR 17 Molecular exclusion chromatography.
A separation technique in which separation mainly according to the hydrodynamic volume of the molecules or particles takes place in a porous non-adsorbing material with pores of approximately the same size as the effective dimensions in solution of the molecules to be separated. Slide 18: 3/18/2009 © RGR 18 Affinity chromatography. The particular variant of chromatography in which the unique biological specificity of the analyte and ligand interaction is utilized for the separation. Slide 19: 3/18/2009 © RGR 19 The affinity chromatography After a wash step the adsorbed molecules are released and eluted by changing the conditions towards dissociation or by adding an excess of a substance that displaces the target molecule from the affinity ligand. Slide 20: 3/18/2009 © RGR 20 The development of an affinity chromatography method boils down to:
Finding a ligand specific enough to allow step elution.
Finding conditions for safe binding and release within the stability window of the target molecule and the ligand. Slide 21: 3/18/2009 © RGR 21 Theory of Chromatography
1.) Typical response obtained by chromatography (i.e., a chromatogram):
chromatogram - concentration versus elution time Wh Wb Where:
tR = retention time
tM = void time
Wb = baseline width of the peak in time units
Wh = half-height width of the peak in time units Inject Slide 22: 3/18/2009 © RGR 22 Note: The separation of solutes in chromatography depends on two factors:
(a) a difference in the retention of solutes (i.e., a difference in their time or volume of
(b) a sufficiently narrow width of the solute peaks (i.e, good efficiency for the separation
A similar plot can be made in terms of elution volume instead of elution time. If volumes are used, the volume of the mobile phase that it takes to elute a peak off of the column is referred to as the retention volume (VR) and the amount of mobile phase that it takes to elute a non-retained component is referred to as the void volume (VM). Peak width & peak position
determine separation of peaks Slide 23: 3/18/2009 © RGR 23 2.) Solute Retention:
A solute’s retention time or retention volume in chromatography is directly related to the strength of the solute’s interaction with the mobile and stationary phases.
Retention on a given column pertain to the particulars of that system:
- size of the column
- flow rate of the mobile phase
Capacity factor (k’): more universal measure of retention, determined from tR or VR.
k’ = (tR –tM)/tM
k’ = (VR –VM)/VM
capacity factor is useful for comparing results obtained on different systems since it is independent on column length and flow-rate. Slide 24: 3/18/2009 © RGR 24 The value of the capacity factor is useful in understanding the retention mechanisms for a solute, since the fundamental definition of k’ is:
k’ is directly related to the strength of the interaction between a solute with the stationary and mobile phases.
Moles Astationary phase and moles Amobile phase represents the amount of solute present in each phase at equilibrium.
Equilibrium is achieved or approached at the center of a chromatographic peak. When k' is # 1.0, separation is poor
When k' is > 30, separation is slow
When k' is = 2-10, separation is optimum Slide 25: 3/18/2009 © RGR 25 A simple example relating k’ to the interactions of a solute in a column is illustrated for partition chromatography: A (mobile phase) A (stationary phase) » KD where: KD = equilibrium constant for the distribution of A between the mobile
phase and stationary phase Assuming local equilibrium at the center of the chromatographic peak: k’ = [A]stationary phase Volumestationary phase [A]mobile phase Volumemobile phase k’ = KD Volumestationary phase Volumemobile phase As KD increases, interaction of the solute with the stationary phase becomes more favorable and the solute’s retention (k’) increases Slide 26: 3/18/2009 © RGR 26 k’ = KD Volumestationary phase Volumemobile phase Separation between two solutes requires different KD’s for their interactions with the mobile and stationary phases
peak separation also represents different changes in free energy DG = -RT ln KD Slide 27: 3/18/2009 © RGR 27 3.) Efficiency:
Efficiency is related experimentally to a solute’s peak width.
- an efficient system will produce narrow peaks
- narrow peaks ? smaller difference in interactions in order to separate two solutes
Efficiency is related theoretically to the various kinetic processes that are involved in solute retention and transport in the column
- determine the width or standard deviation (s) of peaks Wh Estimate s from peak widths, assuming Gaussian shaped peak:
Wb = 4s
Wh = 2.354s Dependent on the amount of time that a solute spends in the column (k’ or tR) Slide 28: 3/18/2009 © RGR 28 Number of theoretical plates (N): compare efficiencies of a system for solutes that have different retention times
N = (tR/s)2
or for a Gaussian shaped peak
N = 16 (tR/Wb)2
N = 5.54 (tR/Wh)2
The larger the value of N is for a column, the better the column will be able to separate two compounds.
- the better the ability to resolve solutes that have small differences in retention
- N is independent of solute retention
- N is dependent on the length of the column Slide 29: 3/18/2009 © RGR 29 Plate height or height equivalent of a theoretical plate (H or HETP): compare efficiencies of
columns with different lengths:
H = L/N
where: L = column length
N = number of theoretical plates for the column
Note: H simply gives the length of the column that corresponds to one theoretical plate
H can be also used to relate various chromatographic parameters (e.g., flow rate, particle size,
etc.) to the kinetic processes that give rise to peak broadening:
Why Do Bands Spread?
a. Eddy diffusion
b. Mobile phase mass transfer
c. Stagnant mobile phase mass transfer
d. Stationary phase mass transfer
e. Longitudinal diffusion Slide 30: 3/18/2009 © RGR 30 Slide 31: 3/18/2009 © RGR 31 a.) Eddy diffusion – a process that leads to peak (band) broadening due to the presence of multiple flow paths through a packed column. As solute molecules travel through the column, some arrive at the end sooner then others simply due to the different path traveled around the support particles in the column that result in different travel distances. Longer path arrives at end of column after (1). Slide 32: 3/18/2009 © RGR 32 A solute in the center of the channel moves more quickly than solute at the edges, it will tend to reach the end of the channel first leading to band-broadening The degree of band-broadening due to eddy diffusion and mobile phase mass transfer depends mainly on:
1) the size of the packing material
2) the diffusion rate of the solute b.) Mobile phase mass transfer – a process of peak broadening caused by the presence of different flow profile within channels or between particles of the support in the column. Slide 33: 3/18/2009 © RGR 33 c.) Stagnant mobile phase mass transfer – band-broadening due to differences in the rate of diffusion of the solute molecules between the mobile phase outside the pores of the support (flowing mobile phase) to the mobile phase within the pores of the support (stagnant mobile phase). Since a solute does not travel down the column when it is in the stagnant mobile phase, it spends a longer time in the column than solute that remains in the flowing mobile phase. The degree of band-broadening due to stagnant mobile phase mass transfer depends on:
1) the size, shape and pore structure of the packing material
2) the diffusion and retention of the solute
3) the flow-rate of the solute through the column Slide 34: 3/18/2009 © RGR 34 d.) Stationary phase mass transfer – band-broadening due to the movement of solute between the stagnant phase and the stationary phase. Since different solute molecules spend different lengths of time in the stationary phase, they also spend different amounts of time on the column, giving rise to band-broadening. The degree of band-broadening due to stationary phase mass transfer depends on:
1) the retention and diffusion of the solute
2) the flow-rate of the solute through the column
3) the kinetics of interaction between the solute and the stationary phase Slide 35: 3/18/2009 © RGR 35 e.) Longitudinal diffusion – band-broadening due to the diffusion of the solute along the length of the column in the flowing mobile phase. The degree of band-broadening due to longitudinal diffusion depends on:
1) the diffusion of the solute
2) the flow-rate of the solute through
the column Slide 36: 3/18/2009 © RGR 36 Van Deemter equation: relates flow-rate or linear velocity to H:
H = A + B/m + Cm
m = linear velocity (flow-rate x Vm/L)
H = total plate height of the column
A = constant representing eddy diffusion & mobile phase mass transfer
B = constant representing longitudinal diffusion
C = constant representing stagnant mobile phase & stationary phase mass transfer
One use of plate height (H) is to relate these kinetic process to band broadening to a parameter of the chromatographic system (e.g., flow-rate).
This relationship is used to predict what the resulting effect would be of varying this parameter on the overall efficiency of the chromatographic system. Number of theoretical plates(N) (N) = 5.54 (tR/Wh)2 peak width (Wh) H = L/N Slide 37: 3/18/2009 © RGR 37 m optimum Plot of van Deemter equation shows how H changes with the linear velocity (flow-rate) of the mobile phase Optimum linear velocity (mopt) - where H has a minimum value and the point of maximum column efficiency:
mopt = rB/C mopt is easy to achieve for gas chromatography, but is usually too small for liquid chromatography requiring flow-rates higher than optimal to separate compounds Slide 38: 3/18/2009 © RGR 38 4.) Measures of Solute Separation:
separation factor (a) – parameter used to describe how well two solutes are separated by a chromatographic system:
a = k’2/k’1 k’ = (tR –tM)/tM
k’1 = the capacity factor of the first solute
k’2 = the capacity factor of the second solute,
with k’2 $ k’1
A value of a $1.1 is usually indicative of a good separation Does not consider the effect of column efficiency or peak widths, only retention. Slide 39: 3/18/2009 © RGR 39 resolution (RS) – resolution between two peaks is a second measure of how well two peaks are separated:
tr1, Wb1 = retention time and baseline width for the first eluting peak
tr2, Wb2 = retention time and baseline width for the second eluting peak tr2 – tr1 (Wb2 + Wb1)/2 Rs is preferred over a since both retention (tr) and column efficiency (Wb) are considered in defining peak separation. Rs $ 1.5 represents baseline resolution, or complete separation of two neighboring solutes ? ideal case.
Rs $ 1.0 considered adequate for most separations. Example : 3/18/2009 © RGR 40 Example What is the resolution of two Gaussian peaks of identical width (3.27 s) and height eluting at 67.3 s and 74.9 s, respectively?
ANS: Resolution = 2.32 Example : 3/18/2009 © RGR 41 Example Substance A and B were found to have Retention time of 16.40 and 17.63 min, respectively, on a 30.0cm column. An un retained species passed through the column in 1.30 min. the peak widths (at base) for A and B were 1.11 and 1.21 min, respectively. Calculate:
The column resolution
The average number of plates in the column
The plate height
The length of column required for resolution of 1.5.
The time required to elute substance B on the longer column
The plate height required for a resolution of 1.5 on the original 30 cm column and in the original time VARIABLE AFFECT COLUMN PERFORMANCE : 3/18/2009 © RGR 42 VARIABLE AFFECT COLUMN PERFORMANCE Retention factor (k’), selectivity factor (a) and
theoretical plates (N), HETP (H)
Varation in N
Variation in H
Variation in Retention factor
Variation in selectivity factor Application of chromatography : 3/18/2009 © RGR 43 Application of chromatography Qualitative analysis
- Analysis based on peak height
- Analysis based on peak area
Calibration and standards
The internal standard method
The area normalization method