Chromatography Flashcards
Definition of chromatography?
Chromatography is a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary (stationary phase) while the other (the mobile phase) moves in a definite direction
What are the intermolecular and inter ionic forces used in chromatography?
Ionic interactions
Van der Waals forces: dipole-dipole, dipole-induced dipole, induced dipole-induced dipole
Hydrogen bonding
Size exclusion - molecular sieving
How does chromatographic separation work?
Mixture of compounds is introduced as a narrow band at time t0 on top of the column into a continuously flowing mobile phase, components A and B are separated by differential distribution between the mobile phase and the stationary phase
How do you know which analyte has a higher affinity for the stationary phase?
The analyte that moves through the phase slower has the greater affinity for the stationary phase, the one that has the lower affinity for the stationary phase will be eluted quicker. Height of peak or peak area can be used for contraption, peak area will increase with increasing concentration
What is GC?
an cope only with substances that are volatile or can be evaporated intact at elevated temperatures from which volatile derivatives can be readily obtained (only about 20% of known organic compounds can be analysed by GC without prior treatment)
What is LC?
Main requirement: the sample must be dissolved in a solvent and apart from cross linked high molecular mass substances all organic and inorganic products satisfy this condition, as long as they can be dissolved in solution
How can something be made more suitable for GC?
Derivatisation, block functional groups and introduce new functionality so becomes more voltaic for GC
Retention?
For a single analyte A involved in transfer between mobile and stationary phases an equilibrium is reached as described with constant K
Why is one type of interaction needed in chromatography?
To be effective to maintain symmetrical Gaussian peaks need only one type of interaction, hence why columns are designed to utilise one type of interaction, if more than one type of interaction peaks will not be good
What is a linear isotherm?
If the distribution coefficient K is a constant independent of sample concentration since K = Cs/Cm a plot of Cs against Cm should be a straight line of slope K. This is called a linear isotherm as it is obtained at a single temperature, the peak resulting for an analyte obeying a linear relationship is symmetrical and Gaussian and can be described by the equation for the normal distribution - assume all molecules around each analyte interact in the same way
Broadening and peak splitting?
The narrower the peak is the higher it is, widening of the peak affects the height, this can occur due to diffusion. Peak splitting can take place due to more than one type of interaction
What does the chromatographic peak represent?
The chromatographic peak represents the frequency distribution of all molecules of a particular analyte as they move as a group through the system or as they are detected as they exit from the system
Where is the average molecule found in the distribution?
The average molecule is found at the centre of the distribution at the position of the peak maximum as it is this average position that is used to characterise the particular analyte
How is the distribution of a peak measured?
The distribution of a peak about its mean position is measured in terms of the peak variance (standard deviation squared) or standard deviation
Width at half height?
Wh = 2.354 standard deviation
Width at the base?
Wb = 4 standard deviation
What happens to the molecules when the distribution is Gaussian?
When the distribution is Gaussian, all analyte molecules migrate at the same time and the retention time is independent on sample size
Non linear isotherms?
Langmuir isotherm - peak tailing
Anti-Langmuir isotherm - peak fronting
Chemisorption isotherm
What is a Langmuir isotherm?
Peak shape tailing, the analyte molecules adsorb on the most active surface sites of stationary phase, therefore additional molecules cannot adsorb with the same strength, seen for reactions occurring on surface of the material
When is the Langmuir isotherms observed in adsorption systems?
Solute stationary phase interactions are strong and solute-solute interactions are relatively weak
A stationary phase having heterogeneous energy sites (free silanol groups Lewis acid sites on the surface of silica supports
What is an Anti-Langmuir isotherm?
Peak shape fronting, the analyte molecules which are the first to adsorb on the surface of stationary phase, facilitate the sorption of additional molecules
When is the Anti-Langmuir isotherms observed in adsorption systems?
Solute stationary phase interactions are relatively weak and solute-solute interactions are relatively strong
Column overload occurs as a result of application of an excessive amount of solute to the system
What is a Chemisoprtion isotherm?
Peak shape varying, chemical reaction between analyte and the surface of stationary phase leading to severe cases to total retention of the analyte by the stationary phase
What do non linear isotherms do to retention times?
Non linear isotherms result in retention times that vary with sample size
Other sources of peak asymmetry?
Unresolved analytes
Column voids in HPLC (this may lead to the response being split)
Measure of asymmetry?
As = b/a
where a and b are measured at a specified fraction of total peak height (10% of the peak height)
How to tell if the peak is asymmetric or symmetric?
Symmetrical response As = 1
Tailing peak As > 1
Fronting peak As < 1
What do values between 0.8 and 1.2 mean?
Symmetry factor values between 0.8 and 1.2 do not give great losses in separation efficiency or quantitative accuracy in measuring peak area
If the values are <0.8 or >1.2?
This needs to be investigated, (restalish separation because cannot be quantified), because this could indicate:
On column degradation of analyte
Poor column packing
Unfavourable interaction between analyte and stationary phase
Poor injector design and set up
Why is band broadening and peak asymmetry bad?
Band broadening and peak asymmetry are detrimental to efficient separation of components of a mixture
What is desired from chromatography?
We desire to obtain sharp, symmetrical responses to allow analytes to be separated fully and to improve their detectability
How can the efficiency of the system be measured?
The efficiency of the system (not just the column) ie the ability to separate analytes fully may be measured by the sharpness and symmetry of the responses that are obtained
What are the two theories the efficiency of separation ca be considered in terms of?
Plate theory
Rate theory
What is plate theory?
The chromatographic column is considered to consist of a series of narrow discrete sections called theoretical plates, at each plate equilibration of analyte between the mobile and stationary phases occurs, the movement of analyte and mobile phase is considered as a series of transfers from one plate to another
How does speed affect plate theory?
Fast process is considered least effective
How does the number of plates affect efficiency?
The efficiency of a column increases as the number of theoretical plates (n) increases. The number of theoretical plays is used as a quantitative measure of efficiency. Higher number of plates increase efficiency, can control parameters in terms of mobile and stationary phase
What does ban broadening suggest?
Band broadening suggests a low performance of system, with efficient system can have more peak to analyse within same retention time - time saving
What is the number of theoretical plates defined as?
The number of theoretical plates (n) is defined as the square of the retention of the analyte divided by the peak broadening.
Why will n normally be inflated?
The measured retention time (tr) includes the dead time (tm) that part of the retention time that is constant for all analytes and will thus give an inflated value for n - this is particularly significant for early eluting analytes for which tm represents a greater proportion of the measured retention time, tr may thus be replaced by (tr-tm) in the equation where N is known as the number of effective theoretical plates
How does error in the measurement of column efficient increases?
The error in measurement of column efficient increases as the peak asymmetry increases, a number of approaches including various approximations, have been developed to allows the efficiency to be assessed from real chromatograms (asymmetrical or non Gaussian)
How is the most accurate value of plate number and effective plate number found?
The most accurate values of plate number and effective plate number are derived from computer analysis of the peak shape involved but the formulae provided enable a reasonable assessment to be made
What do plate number (n) and effective plate number (N) depend on?
The length of the column (L)
L = nh L = NH
Formula for plate height?
h = L/n
L - the length of the column
n - the number of theoretical plates
h - the plate height
Formula for effective plate height?
H = L/N
H - effective plate height
L - the length of the column
N - the number of effective plates
What is the main limitation to the plate model?
The main limitation to the plate model is that it does not relate the band broadening process to experimental parameters (eg flow rate of mobile phases, particle size and thickness of stationary phase etc)
Why are N and n useful parameters?
N and n however with certain limitations are useful parameters for characterising column efficiency and this capability is not affected by deficiencies in the plate model
What are the assumptions rate theory makes in its derivation and explanation of band broadening?
Band broadening due to eddy diffusion
Band broadening due to molecular (longitudinal) diffusion
Band broadening due to mass transfer
What is band broadening due to eddy diffusion?
The flow velocity through a packed column varies widely with radial position. Some molecules will find a relatively unobstructed way through the column and thus travel more quickly other will not and therefore travel more slowly. These different flow velocities cause zone dispersion and this effect is known as eddy diffusion
What is band broadening due to molecular (longitudinal) diffusion?
Molecular diffusion (in the axial direction) results from random molecular motion of analyse molecules in the mobile phase which leads to spreading of the zone. Band broadening resulting from molecular diffusion increases with the amount of time the solute (analyte) spends in the column
What is band broadening due to mass transfer?
Resistance to mass transfer in both the stationary and mobile phases prevents the establishing of an instantaneous equilibrium, if the blunt velocity is high and the analyte has a strong affinity for the stationary phase then the analyte in the mobile phase will move ahead of the analyte in the stationary phase giving band broadening
How is the overall band broadening expressed mathematically?
Van Deemter equation
H = A + B/u + Cu
H - column dispersivity
u - the average linear mobile phase velocity
A - contribution to zone broadening by eddy diffusion
B - contribution to zone broadening by molecular diffusion
C (Cm + Cs) - contribution to zone broadening by resistance to mass transfer in both the stationary (Cs) and mobile phases (Cm)
How do the parameters assume different amounts of importance?
For different forms of chromatography the different parameters assume greater or lesser importance
How do the parameters assume different amounts of importance in GC?
In GC molecular diffusion in the mobile phase and mass transfer effects in the stationary phase are important
How do the parameters assume different amounts of importance in LC?
In LC molecular diffusion can often be ignored but mass transfer in both phases is important
