Chromatography Flashcards
Principle of hplc
Injection of a Smakk volume of liquid sample into a column packed with Tiny particles called stationary phase
Individual components of the sample are moved down packed column with the mobile phase forced through the column by a hugh pressure pump.
The components are separated by one another by the column packing that involves various Chemical or physical interactions between molecules and the packing particles.
The separated components are detected at the exit of the column by a detector that measures their amount. An out out from thus detector is called a liquid chromatography
Types of HPLC
Normal phase
Reverse phase
Adsorption
Ion chromatography
Size exclusion chromatography
Normal phase chromatography
This is not common.
It is the traditional separation mode based on the adsorption/ desorption of analyte onto a polar stationary phase (typically silica or alumina )
Polar analytes are going to migrate slowly through the column due to strong interactions with the polar stationary phase
It is also particularly useful for the separation of non polar compounds and isomers
One disadvantage us the easy contamination of surfaces by sample components
Reverse phase chromatography
This is more common
Based on analytes partition coefficient between a polar mobile phase and non polar stationary phase
Non polar analytes will interact more strongly with hydrophobic groups for informing a liquid like layed around the solid silica support.
Sutible for analysis of polar , medium polarity and some non polar analytes
Ion exchange chromatography
Based on the exchange of ionic analytes with counter ions of ionic groups attached to the solid support
Typical exchange groups are anionic (so3-) or cationic (nh4+) groups bonded to polymeric or silica materials
Common applications are the separation of ions and biological components such as amino acids
Size exclusion chromatography
Separation mode based on analytes moleculer size
Larger molecules are excluded fro, the pores and migrate faster than the smaller Molecules that can penetrate the pores
Instrumentation of hplc
See notes
What affects HPLC resukts
SAMPLE PARAMETERS
conc
Nature of sample eg biological
Sample extraction
Volume of injection
COLUMN PARAMETERS
column material type
Column material particle size
Column length dimsgees etc
Temp and pressure
MOBILE PHASE
Slovent
Ratio of solvent
Flow rate or pressure
PH
DETECTOR
wavelength
Sensitivity
How does mp need to be prepared
Needs to contain a buffer
Degas solvent as Small gas bubbles in the mobile phase can lead to collection in pump heads and detectors and ruin the analysis
Should be filtered to remove any particulate matter which could cause wear to the pumping system
What does mobile phase have an influence on
Solute retention and serparation
What is solvent strength refer to
It’s ability to elute solutes from a column
Increased solvent strength = faster elution
What is solvent strength related to
It’s polarity
In rpc what is a string and weak solvent
Strong - non polar hexane
Weak - water
Role of ph in mobile phase
If you ionise a drug by changing the ph it’s going to spend more time in the polar phase.
In rpc low ph is used to suppress the ionisation of weakly acidic analytics leading to a higher retention
Used a buffer eg acetic acid
% ionised
Sheet
How does flow rate and column temp affect mp
Operating at a higher flow rate Will reupducd retention and analysis time
Hugger column temps reduce the viscosity of mp and has effect in retention
Purpose of pump
Ensure delivery of precise, reproducible , constant and pulse free flow of mobile phase
What type of way can the pump deliver solvent
Constant mobile phase compition (isocratic )
Or increasing mobile phase composition (gradient )
Isocratic elution
The same mobile phase is used throughout the entire elution of the sample , mobile phase solvent remains constant with time
This is best for simple separations
Often used in quality control applications that support and are in close proximity to the manufacturing process
Gradient elution
The strength of the mobile phase in increasing with elution
Best for analysis of complex samples
Often used in method development for unknown liquids
Gives better resolution and faster elution
Generation of gradient flow
LOW PRESSURE MIXING
The amount of each solvent (uo to four solvents ) is regulated by a proportioning valves . The mixed solvent then enter the hugh pressure pump and flows into the column
It is less expensive as it requires only one pump however gas bubbles are formed as the solvents are mixed at atmospheric pressure
HIGH PRESSURE MIXING
the delivery of multiple hugh pressure pumps is controlled individually with a programming deceive , and mixture is sent ti a hugh pressure mixing chamber.
Is expensive bc multiple required but a operate reliably without de gassing because the solvents are mixed at sufficndlty hugh pressure
What is a HPLC column
Stainless steel tube filled with small particle packing
Structure or silica particles surface
What is retention time
The time between sample injection and peak Maximum (tR)
Draw a graph with all measurements
Where is peak width usally measured
At the base (wb) or at the peak half height (w1/2)
What is vR
Retention volume. The volume of mobile phase needed to elute an analyte at a given flow rate.
VR= tRF
What is VM
Void volume of liquid mobile phase needed contained in the column
Vm= tM F
Peak volume
Also called band with is the volume of mobile phase containing the elute weak
Peak cpume = wbF
How to calculate assymetry value
How to calculate tailing factor
What is resolution
Resolution (Rs) is a measure of the degree of separation of two separate analytes
What does rs value need to be for baseline separation
1.5
Now to work out resolution
What Is a measure of column efficiency
Number of theoritical plates of plate number (N)
How to work out number of theoretical plates
HETP
Height of a theoritical plate = L/N
L= length of column
What is the main factor controlling HETP
Particle diameter of the packing
What number is h/ HETP usallly
2.5
What is capacity factor
Capacity factor k’ is a measure of the degree of retention of solute by the column
It is the time the solute spends in the stationary phase tR’ relative to the time it resides in the mobile phase (tM)
Capacity factor /retention factor eqn
What do you use to establish peak identity
Retention time
What do you use to measure conc
Band size
How do you overcome errors reuniting from calibration curve when trying to calculate concentrations
Use an internal standard
What is an internal standard
An exogenous compound that is added to both the calibrators and samples at the same time
What determines the choice of is
Never be found in sample
Well resolved
Structural similar to analyte
Stable available in pure form
How to work out conc
A series of solutions are prepared under varying concentrations of the compound under investigation plus a fuxed conc of internal standard.
Instead of dealing with area now , dealing with ratio of analyte response to is response
When you inject these solutions into the chromatogram obtained it will show a peak for the analyte sand is
From such traces yiu can calculate the peak area or height rations
Phr = huegh of sample peak / height if IS peak
Plot graph of peak height ration v conc of analyte
Get equation of lie and r2
Advantages of hplc
Rapid and précise quatatuve analysis
Amendable to diverse samples
Automated operates
Can be used to quantify
Hugh sensitivity
Limitations
No universal detector
Less seperation efficiency than capillary gc
More difficult for novices
