Chromatography Flashcards
Basis of Chromatography
Partiton of sample compounds between a stationary phase and a mobile phase which flows over and/or through the stationary phase
Basis of Adsorption Chromatography
Solid stationary phase and liquid/gas mobil phase. Solutes are separated according to their different adsorption characteristics onto the stationary phase
Basis of Partition Chromatography
Thin film formed on surface of solid support by liquid stationary phase. Solutes equilibrate between mobile and stationary phase
Basis of Ion exchange Chromatography
- A resin is used to covalently attach anions or cations onto it which acts as the stationary phase, with an aqueous mobile phase.
- Solutes ions of the opposite charge are attracted to the resin by electrostatic forces
- Type of separation is difficult to achieve using other techniques as charge is easily manipulated by pH of the buffer used
Basis of Molecular or size exclusion Chromatogrpahy (Gel permeation/ filtration)
- Lacks any interactions between stationary and mobile phase.
- The liquid/gas mobile phase passes through a porous gel that separates them according to size.
- Gel consists of spherical beads containing pores of a specific size distribution. Small pores allow larger solute molecules to enter causing them to be retained but exclude larger ones.
- Therefore larger molecules pass through the column more quickly.
Define Normal Phase in HPLC
Polar stationary phase & non-polar mobile phase
Define Reversed Phase in HPLC
- Alkyl chain covalently bonded to a silica support
- hydrophobic stationary phase which has a stronger affinity for non polar compounds
- opposite (reverse) of normal phase chromatography
- polar mobile phase with solutes separated based on their molecular polarity. More polar compounds interact less with stationary phase and will be retained less
Define Simulated Moving Bed
Continuous Countercurrent Chromatography
- Solvent is pumped in opposite direction to solute diffusion
- Faster moving solutes moves faster than the moving bed
- Slower diffusing solute does not
- Therefore the solutes move in opposite direction allowing for continuous separation
Capillary Electrophoresis
- Separates ions based on their electrophoretic mobility with the use of an applied voltage (electroosmotic flow) - Gives flat profile which improves peak resolution and separation efficiency
Define the retention (or capacity) factor
- The time taken for a compond to travel through a column is known as the retention time.
- If a column is not retained at all it will still take time to travel through the column (dead time)
- The time a solute is interacting with the stationary phase can be estimated by correcting its retention time by that taken for an unretained species.
Retention Factor Equation
k = {time spent (or mass) in stationary phase} / {time spent (or mass) in mobile phases}
Partition Coefficient Equation
K = [stationary phase]/ [mobile phase]
Phase Ratio Definition
Beta = (Volume of Mobile Phase)/(Volume of Stationary Phase)
Relationship between retention factor and partition coefficient
k = K/Beta
Define Retention time
Time it takes for a compound to travel through the column
Define Dead Time
Time taken for an unretained compound to travel through the column
Equation relating retention factor and retention time
k = (tr - to)/to
tr = retention time to = dead time
Definition of Selectivity
alpha = k1/k2
Number of Theoretical Plates (N) equation
N = 5.54(tr/wh)^2
wh = peak width at half height
Use of Number of Theoretical Plates (N)
It is a measure of the column efficiency. The higher N the narrower the peak which is beneficial as it is more likely to give fully resolved (separated) peaks for each component
Definition of Height Equivalent to a Theoretical Plate (H)
A measure of column efficiency. The shorter each theoretical plate, the more plates are contained in any length of column.
Define Resolution (R = 1.5)
The higher the resolution the less the overlap between the two peaks. It takes into consideration both selectivity and the width of the peaks. (Baseline resolution occurs at R = 1.5, values less than this shows some co-elution)
Resoluton equation (width at half peak height)
R = 1.18( t_R2 - t_R1)/(w_h1 + w_h2)
Resoluton equation (width at peak base)
R = 2( t_R2 - t_R1)/(w_b1 + w_b2)
Equation relating Resolution, Retention factor and selectivity
R = sqrt(N)/4 x (alpha - 1)/alpha x k2/(k2 + 1)
Van Deemter Relationship for theoretical plate height
H = L/N
Four factors affecting the theoretical plate height
- Mobile phase velocity
- Multipath diffusion
- Diffusion of compound in mobile phase
- Transfer of compound between the mobile and stationary phases
Theoretcial Height equation for packed columns (HPLC)
H = A + B/u + (Cs + Cm)u
u = average linear mobile phase velocity A = Diffusion due to non-uniformity of packing (Constant) B = Longitudinal Diffusion coefficient in mobile phase (Constant) Cs = Mass transfer term in stationary phase Cm = mass transfer term in mobile phase
What does the A term mean in the equation: H = A + B/u + (Cs + Cm)u
It is the diffusion due to the non-uniformity of packing. It is affected by the nature of the column material and how well it is packed. It is generally proportional to particle size of the packing material and = 0 for capillary columns as there are no particles
What does the B/u term mean in the equation: H = A + B/u + (Cs + Cm)u
It is the Longitudinal Diffusion coefficient. This contributes to peak broadening as analytes diffuse to areas of lower concentrations infront or behind the moving band. It is negligible at higher velocities and is more important in gasses as diffusion ceofficients are much higher
What do the (Cm + Cs)u terms mean in the equation: H = A + B/u + (Cs + Cm)u
These are the mass transfer termsn (S = in Stationary phase, M = in mobile phase). These terms are based on diffusion but take place perpendicular to the flow rate. Therefore, the faster the mobile phase moves the less time there is for equilibrium between the phases. Therefore the mass transfer effect on peak broadening is directly related to the mobile phase velocity.
Describe the Van Deemter plot - Is there an optimum velocity
Yes there is an optimum velocity. The equation is H = A + B/u + (Cs + Cm)u
- Multipath diffusion (A) is relatively constant
- Mobile phase mass transfer (Cm x u) increases with velocity
- longitudinal diffusion (B/u) has a lesser effect as velocity increases
- Generally speaking higher velocities are used than the optimum in the interest of speed
What is the effect of decreasing the particle size on the van deemter plot?
- It decreases the diffusion path lengths so solutes can travel in and out of the particle faster
- Therefore particles spend less time in the particle where peak diffusion can occur
- Therefore smaller particles limit the effect of flow rate on peak dispersion
- However they do result in much higer back pressure
Define reduced plate height
Dimensionless plate height
h = H/dp
H = Theoretical Plate Height dp = mean particle diameter
Define reduced mobile phase velocity
Dimensionless measure of the mobile velocity:
v = u x dp/Dm
u = mobile phase velocity dp = mean particle diameter Dm = Diffusion coefficient of solute in mobile phase
Gas Chromatography Detectors (There are 2)
Thermal Conductivity Detector
- No Calibration
- Ratio of peak areas for concentration
- Low sensitivity
- Large dead volume
- Not suitable for capillary work
Flame Ionisation Detector (FID)
- Good sensitivity
- Excellent linearity
- Insensitive to organic gases
- Low dead volume
HPLC Detectors (5)
- UV (Uses Beer’s Law)
- Refractive Index
- Fluorescence
- Electrochemical
- Mass Spectrometry
4 principle methods of obtaining quantative information from chromatograms
- Normalising peak ares
- Does not give absolute amount just molar fractions - Internal Standard Method (inclusion of an reference standard in each sample)
- External Standard Method (prior experiments performed to generate a calibration curve)
- Standard Addition (Multiple samples taken with varying amounts and made up to the same volume. The [standard] varies linearly so [sample] can be calculated from plot
Define Flash Chromatography
A form of preparative chromatography:
- Uses smaller silica gel particles
- Overcomes the increased pressure drop by using pressurised gas to drive the solvent flow
Column Overloading
When a retention factor obtained at low [sample] changes by more than 10% as [sample] is increased
Can be done by increasing [sample] (concentration overload) or injection Volume (Volume overload)
Volume overload vs Concentration overload
Good Solubility = Concentration overload
- Column efficiency has little effect on this method and selectivity tends to be the dominant factor
- [Sample] increased beyond linear region resulting in asymmetric bands)
Poor Solubility - Volume Overload
- Heavily influenced by stationary-particle size and column diameter
- [Sample] remains in linear region and volume is increased until throughput optimised
Scale Up Factor Equation
(Dp^2 x Lp)/(Da^2 x La)
D = Column Diameter L = Column Length p = preparative experiment a = analytical
Flow rate Scale up equation
Fp = Fa x (Dp/Da)^2
Internal standard response factor (Rf)
Rf = AxCis/AisCx
A = Peak Area C = Concentration is = Internal Standard x = Sample
- Use of internal standard procedure is recommended for accurate quantative work as it eliminates the need for accuracte injections as a reference is included in each sample
Relative Retention Time
Retention time of sample relative to that of a standard compound:
RRT = Standard RT/ Sample RT
The use of RRT reduces the effects of variables that can affect the retention time (e.g. flow rate, Temp, etc)
Sample RT = retention time measured from the point of injection of the compound of interest, likewise Standard RT from the point of injection of the internal standard
Reversed Phase Chromatography preparation
- alkyl chain covalently bonded to solid support
- Alkyl bonded phases are silica based and are prepared by reacting hydroxyl groups on the surface of the silica with organic silyl esters or chlorides.
- Any remaining unreacted silanol groups are blocked by subsequent methylation (e.g. trimethylsilazane)
