Chromatographic Separations Flashcards
What is the basis method of distillation?
Differences in volatility of compound
What is the basis of method of extraction?
Differences in solubility in two immisible liquids
What is the basis method of chromatography and electrophoresis
Samples that are multicomponent and complex
What is the definition of chromaotography
Chromatography is the separation method in which components in a mixture to be separated are distributed between two phases
What are two phases of chromatography and what is its definition
Two phases includes stationery phase (the one that stays in place inside the column or solid surface) and mobile phase (the solvent that moves through and carry with it the component mixture.
What are the chromatography system composed of?
Stationery phase, Mobile Phase, and Mixture to be separated
What are the types of chromatography?
Gas chromatography (GC), high performance liquid chromatography (HPLC), thin layer chromatography (TLC) and supercritical fluid chromatography (SFC)
What is the principle of chromatography?
Components of a mixture are carried through the stationery phase of a mobile phase. Separation occurs because of differing affinities of components with stationery phase and mobile phase
What are the relationships between retention and stationery phase?
If components are strongly retained by the stationery phase, it moves slowly with the flow of mobile phase. If it is weakly retained by sp, it moves quickly to mobile phase. Differences in mobility causes sample to separate easily
What is a chromatogram and what is its function?
Plotting the function of time and solute concentration through the peaks that were obtained, detectors were placed at the end of the column during elution.
What are the differences between peak positions and peak areas?
Peak position is used to identify components meanwhile peak areas are to determine amounts of each component or analyte
What is elution?
Process in which components are washed through a stationery phase by the movement of a mobile phase
What is ELUENT?
fluid used to eluate a substance (mobile phase)
What is ELUATE?
samples that have been separated/left the column
What is a SOLUTE?
Solid that dissolved in liquid
What is an ANALYTE?
The chemical substance that is determined in an analytical procedure (component)
What is retention time tR
Time for the analyte to pass through the column or the time from sample injection to detection
What happens to compound that are not retained by stationery phase?
It will elutes out of the column at time tM (void time) or dead time also labelled as t0
What is a tM
The time a non-retained compounds spends in the mobile phase or time taken for mobile phase to pass through the column
What is a tM
The time a non-retained compounds spends in the mobile phase or time taken for mobile phase to pass through the column
What is t’R and tS?
Time the compound spends in the stationery phase. t’R is the difference between tR and tM of a compound.
What is k’ ?
K’ is the retention or capacity factor. It is used to describe the migration rate of solutes on column.
What is the relationship between k’ and a separation and elution time?
Large K’ (>20-30) favours good separation, with increased elution time
What is the range of K’ that signifies good separation?
2
What is the selectivity factor? a-alpha
a-alpha is described as the separation of two species (A&B) on the column
What to know about a-alpha?
a-alpha must be greater than 1
if a-alpha=1, the two compounds cannot be separated
As a-alpha increases, the separation between 2 compounds/peaks increases
When calculating a-alpha, species A elutes faster than B
What are the factors that affect the efficiency of a separation?
Difference in retention time between peaks( farther apart better separation)
Peak widths ( narrow peaks are produced if separation is efficient)
What is Resolution? Rs
Degree of how well separation is between two peaks (peaks widths are taken into account)
What are the values and how does it signifies a good resolution?
if R=1.5 represents baseline resolution or complete separation between two neighbouring solutes (ideal)
if R=1.0 it is considered adequate for most separations
R must be more and equal than one to be good.
The higher the Rs, the better the separation
How are column efficiency expressed as?
Theoretical plates, N and plate height
What is theoretical plates, N?
It views column as divided into a number of adjacent imaginary segments. N indicates how good a column is for a separation.
What is the relationship between N and separation of two columns?
As the value of N increases, the better the column will separate the two compounds
What are the traits of columns with high N?
More efficient and have narrower peak at given tR than a column with lower N
What is the relationship between tR and N? What is the
As tR increases, N increases
How to improve Rs (Retention)
Lengthen the column to increase the N
What is plate height (H) or Height Equivalent to a Theoretical Plate (HETP)
It is the approximate length of a column required for one equilibration of solute between stationery and mobile phase
Why do zones/band broadens?
It is due to the loss of column efficiency
What is the relationship between rate of mass transfer processes and the band broads?
As the rate of mass-transfer processes occurring while a solute migrate through a column, the broad of band increases.
What are the mechanisms that lead to band broadening
Multiple paths / Eddy diffusion, A
Longitudinal diffusion, B
Mass transfer between phases / equilibration time between phases, C
How to minimise band broadening (to increase the efficiency)
Increase the rate of mass transfer
Decrease analyte interaction (interact with sp)
Increase the velocity of analyte
What is the purpose of the Van Deemter equation?
It is to tell how the column and the flow rate affect the H
What is A-term?
A term stands for multiple paths, it is directly proportional to the packing particle diameter, dp
A is small when using smaller particle size and uniform size.
What is B-term?
B-term is longitudinal diffusion. Diffusion is when analyte migrate from a more concentrated to a more dilute region. Longitudinal diffusion is when solute diffuse from the concentrated centre of zone to more diluted region
What is B-term effect on H?
Its effect on H is inversely proportional to v because solute spends less time in the column at high v and less diffusional broadening occurs.
What is the relationship between B term and Dm?
B is directly proportional to solute diffusion coefficient in the mobile phase Dm.
What is C-term?
C-term is mass transfer between phases. Finite time required for solute to equilibrate between mobile and stationery phase
What is the relationship between affinity towards stationery phase and velocity of mobile phase?
If velocity of mobile phase increases, the molecule has strong affinity to stationery phase. The molecule in mp will move ahead of the molecule in the stationery phase. Band of analyte is broadened, higher velocity worsens the broadening.
Why is the mass-transfer effect on H is directly proportional to v?
It is because solute residence time is longer at low v, the deviation from equilibrium is less and zone broadening or H is smaller.
What is df?
Stationery phase film thickness (most important factor)
What is Ds
Diffusion coefficient of the solute in the film, complex function of fs(k’) of the retention factor k’
What is the relationship between df and efficiency?
Decreasing df reduces H and increase the efficiency because solute can diffuse faster from the farthest depth of the stationery phase into the mobile phase
How to reduce zone broadening?
Smaller particle sizes
Narrower column
Thinner liquid stationary phase
Why optimization of flow rated is one of the important steps in chromatographic analysis?
For B/v, high flow rate will decrease the term and thus reduces band broadening
For Cv, low flow rate is required to give the molecules enough time to reach equilibrium
What can we conclude about the optimum flow rate?
high enough to reduce diffusion and low enough to give enough time for achieving equilibrium
