Chromatography Flashcards
What is quality control
Checking that the right ingredient is present in the qaunity
What is QA
Checking SOP such that the quality of the product can be assured
What is the BP
a book that contains info of on the acceptable parameters for the quality of formulated drugs
What is Chromatography
It is a process which a chemical mixture is carried around by a liquid or gas over
What is Chromatography
It is a process which a chemical mixture is carried around by a liquid or gas over a stationary phase
What is the stationary phase and what is it comprised of
A solid or liquid material this is coated onto thin layer if inert material.
What is the mobile phase and what is it comprised of
A gas of liquid that is allowed to flow over and through the stationary phase
What is Colum chromatography
It is when the stationary phase is held in a tube and the mobile phase is forced through the column under pressure or gravity
What are the two types of column chromatography
Liquid
Gas
What are the 4 different types of liquid chromatography
-Absorption:
> S phase is an adsorbent solid- charcoal or silica
-Partition
> S phase is a liquid coated solid
-Ion exchange
> S phase has ionic bonded phase
-Gel permeation
> S phase is porousWhat is the S phase of Absorption chromatography made of?
Made of a polar groups
> SiO2, Al2O3
>Molecules are attracted by Dipole-dipole force, Hydrogen bonding or weak Van der waals forces.
What is the S -phase of partition chromatography made of
Made of an inert solid, coated with a liquid SiO/H2O
What is a reverse phase chromatography
This when the stationary phase is non-polar and the mobile phase phase is polar
Why does separation occur in partition chromatography
Due to the difference in solubility of the solutes
>The solute is partitioned between the two phases, the amount of time spent in each is dependant on solubility.
What is the S phase of ion exchange chromatography
coating of solid of resin ions (cations and anions) is covalently bonded to it.
> Ions of opposing charges bind electrostatically to it.
How dose Ion exchange chromatography work
When eluent containing ions is eluted through the column, the electrostatically bound ions are released as others are preferentially bound.
>the smaller and more highly charged an ion is, the stronger it will be retained in the column
How do Gel permeation chromatography work
-Works without equilibrium between the solutes and stationary phase
-The stationary phase is passed through a porous gel
> Depending on size the molecules will, the larger molecules elute first
Preparative of liquid column chromatography (driven by gravity)
This is when the column is packed with large stationary molecules such as Silica or alumina.
Sample mixture is coated unto the top and Eluent is flushed down by the liquid mobile phase
Describe HPLC
It is an extension of liquid column chromatography
> particles are Small and passed through column by an eluent pumped under pressure of by gravity
What type of stationary phase can be used in HPLC
It can use all sorts >Adsorption >Partitions > Ion exchange > gel permeation
What are the options for the HPLC packing material
- Microspheres
- Particulate with thin SA - Porous microspheres
- Bonded phase: When silica has different groups bonded to it.
Which is the most common material used as packing material in reverse phase chromatography
ODS silica
Which factors affect retention time
The nature of the mobile phase
> More polar phase means more quickly eluted in Silica
> More lipophilic = quicker elution in ODS
What are the ideal factors of a detectors for HPLC
- Rapid and reproducible response to solutes
- A linear response
- Stability of operation
What are the 2 classes of detectors
Optical
Electrochemical
Name the types of optical detectors and the Advantages vs disadvantages
- Refractivity index: Monitor difference between a reference stream and the eluent.
> Advantage: it is universal
Disadvantage: It is not very sensitive
-Sensitive to temperature change
- UV/VIS
a) fixed wavelength: A fixed wavelength (at which you expect the solute to absorb) is selected
>Advantages: Response is linear providing the concentration is not too high
>Disadvantage: can only use for species that absorb at a set know wavelength in the UV/Vis region. You can’t really analyse unknown samples.
b)Diode array spectrometer: measures absorbance of the sample coming from the column over all wavelengths simultaneously
>How it works:
>Results in a 3D graph - Detects light fluorescing from natural fluorescent solutes (fluoresces in the current wavelength of light )
What are the two types of electrochemical detectors
Conductivity: Used in ion exchange chromatography.
> Samples are ionic and mobile phase had low conductivity
Redox electrochemical: These detectors are useful for solutes than can be oxidised or reduced.
What are the structural factors that effect the rate of elusion in HPLC in neutral compounds?
The balance between polarity and lipophilicity
Altering the pH of the mobile phase has no influence on this
What are the structural factors that effect the rate of elusion in HPLC in ionisable compounds?
pH - Usually in reverse phase where the mobile phase is aqueous
>drugs are ionisable, and the degree of ionisation is dependant on the pH
What is liquid chromatography-mass spec (LCMS)
It has 2 separate analytical techniques : chromatography and mass spec
What is gas chromatography
mobile phase; Gas
stationary phase:
>Liquid adsorbed to a solid (GLC)
>organic species bonded to a solid surface
Separation is based on volatility
What are the 2 factors that effect the desperation of GLS
> The volatility of the component at column temperature
>The polarity (solubility) of the component in the stationary phase
What are the 2 different types of GC columns
Packed column:
Capillary column: Longer (up to 500m) and thinner
> Require small sample size so has very sharp peaks
Types of stationary phases of GC and their properties
All based on silica polymers
> Methyl silicate: Non polar separation
> Cyanopropyl phenyl methyl silicate: food for moderate polarity eg Aromatic compounds
> Triflurosilicone: Good for polar separation eg Alcohol
Good properties of a GC detectos
> Must respond rapidly
>Response must be linear (steep slope)
How can the sensitivity of the detector be measured
- Measure the slope on a detector response versus amount of sample graph
- Look at the limit of detection, i.e. the smallest peak one can actually say with confidence is a peak and not just background noise
For a 95% confidence of a peak being an actual component the limit of detection (L.O.D) is equal to the mean background + 2 standard
An ideal GC detector is:
Sensitive Linear response temp ranges to at least 400 C Quick response High reliability Does not destroy the sample Respond similarly to all solutes
What is a flame ionisation detector and how does it work?
A detector used in GC
The column gas is mixed with air and H2(g) then burnt using a plasma to get to very high temperatures (>3000oC).
compounds are ionised (+ve) and detected by the cathode
Electrons flow to anode
Advantages of Flame ionisation detector
- Sensitive
- Quick response time (<1 millisecond)
- Peaks are proportional to amount of solute
disadvantages of Flame ionisation detector
- Can only detect combustible materials (mainly organic – carbon containing)
- Destroys the sample
What are chromatograms
A detector that responds to solute concentration which give different peaks which represent a separated component of mixtures
What is retention time
The time it takes a solute to reach the detector after injection
What determines a good chromatographic separation
- Resolution
- Efficiency
What is resolution
It is the measure of how well a peak separated and is needed for quantitative calculations.
R= 2(tR2-tR1)/ (wb1+wb2)
wb = with of base of each peak measured
The greater the value of R, the better the resolution of the two compounds
What is an alternative calculation for the resolution if the base with is unclear
1.18 * (tR2 - tR1)/ wh1+ wh2
What are the ways to improve resolution
Increase the column length but this affects efficacy
- Alter mobile and stationary phase
- Anything that increases efficacy and leads to narrow peaks
How is efficacy calculates
n= 5.54*(tR)^2/ L x (Wh)^2
Wh = width at half height of the peak
What are the factors that affect efficacy in a column
The length of the column
> The molecules of a single compound, despite having the same properties, take different lengths of time to travel through a column.
- The multiple pathway affect
- Non equilibrium mass transfer
- Sample size
- Diffusion
- Time on column
- The multiple pathway affect
Packaging of column is not uniform so solutes can take different pathways and so travel at different speeds
- Non equilibrium mass transfer
Some molecules may travel further into the stationary phase or be too far away for the surface
- Sample size
if you increase the solute concentration too much, the stationary phase may become saturated
- Diffusion
Diffusion occurs mostly in the mobile phase and is grater in gas chromatography, compare to liquid chromatography.
- Time on column
longer a compound is on the column the more opportunity the molecules have had to diffuse
Overall factors required for high efficiency (narrow bands)
Small sample size Small particle size Thin layer of stationary phase Optimum mobile phase velocity Low dead volume
