HPLC Flashcards
Describe the HPLC system
Consist of a solvent connected to a intricite system of tubes.
- System includes (in order) a pump, Injector (introduces sample), HPLC coloumn, detector
Purpose of the solvent?
Used as a mobile phase
- Often have one aqueous and one organic
- Can have a single mixed solvent
- Can use isocratic or gradient elution
What are some general addatives to solvent in HPLC?
Addatives:
- Buffers for pH control
- Salts
- Ion-pair reagents e.g. ion pair reagent allow hydrophobic stationary phase to bind onto ionic compounds (hydrophilic)
Form MS:
- Need something to promote adducts
- Acids because MS require analyte ions to be able to detect
How many pumps are used in an ioscratic gradient?
Only one
What is a typical HPLC coloum?
Stainless steel that can withstand high pressures, with packing material inside. Packing material is held in place by a frit
What is a guard coloum?
Protects the more expensive analytical coloum from particulates. May contain packing material or just a filter
HPLC particles are usually…..
A. 80-150um
B. 45-80nm
C. 2.5-15um
D. 300-400um
C
What are some potential functional groups added to the particles?
C8, C18, C4, phenyl, amide, CN, PFP, HILIC
With examples to the phases how can resolution improved?
Via the mobile phase:
- Composition
- Flow rate
- Gradient/isocratic
- pH
- Temp
Column
- Phase
- Length - theoretical plates
- Particles and pore size - affect eddy diffusion
How can the mobile phase be manipulated to increase to optimisation of chromatography?
Isocratic - constant composition
Gradient - composition changes throughout the run
Gradient mobile phase is more attractive in when there are multiple analytes
What are some variables to the gradient?
Range- % organic at start and end
Time - shallow or steep gradient
Always important to ensure to reequilibriate the column before next sample
Non-polar compounds need what % of organic solvent to elute?
A. 20-30%
B. 30-60%
C. 0-30%
D. 60-100%
D
Low/medium polarity compounds need what % of organic solvent to elute?
A. 20-30%
B. 30-60%
C. 0-30%
D. 60-100%
B
Polar compounds need what % of organic solvent to elute?
A. 20-30%
B. 30-60%
C. 0-30%
D. 60-100%
C and may require specialised chromatography - HILIC
What is the eluotropic series?
Not all solvents are equal and can be classified according to polarity
More non-polar compounds will require a more non-polar sovlent for elution from the stationary phase.
A good starting point is a medium polarity and go up or down the series as required
Why does pH need to be optimised?
pH affects ionisable analytes as two different ions will exist in the same sample depending where the pH is relative to pKa of the analyte.
How does temperature affect the column?
AS temp increase the efficiency of the column as the resistance to mass transfer (C) decrease
So therefore more mass transfer will occur in the column
Note. Mass transfer is the time it takes to equilibriate between the phases by the analyte
What are some polar groups?
Hydroxyl, carboxyl, amino, phosphate, carbonyl, sulphydryl (R-CH2-SH)
Examples of polar analytes
Metanephrine, normetanephrine, tobramycine
Name some non-polar groups?
Methyl
Example of non-polar analyte
25-OH-VitD
Some parts are polar but majority is non-polar
With example why is it important to regard the analytes size?
For chromatography using pores.
- 99% of chromatographic surface is inside the pores
Mobile phase must be allowed into the pore in order for chromatographic retention of the analyte to take place
How is pore size adjusted to enhance chromatographic results?
Especially important for analysing larger molecules, e.g. peptides
If the analyte is too large then chromatography will simply not occur and analyte will elute.
How is column length adjusted to enhance chromatographic results?
Remember the more theoretical plates the better resolution.
- BUT increased column length results in higher back pressure and longer run times
- The run time can be decreased by increasing flow rate, but causes an increase to back pressure
The increased back pressure can severily impact the lifetime of the column
How can the the column efficiency be adjusted?
By decreasing the particle size the number of particle plates increase.
- Thus, increasing column efficiency
Smaller more regular particles decrease eddy diffusion- good to decrease peak broadening
What characteristics of the analytes are useful in choosing column type?
Ionic groups Polar groups Hydrophobicity Double bonds Size Benzene rings
What is the most common column type in clinical setting?
C18 column (very common)
- Hydrophobic phase good for retention of non-polar analytes, e.g. testosterone
- C18 has many variations with end-capping and other side chains which increase pH stability or analyte retention. E.g. Butyl side chains improve the peak shape
When is a C8 column more favourbale than C18?
- Good for analytes that are retained for too long on C18
e. g. 25-OH Vit D
When is a phenyl column applicable?
- Used for the seperation of aromatic compounds
- Seperate based on hydrophobic and pi-pi interactions
e. g. retinol or vit E
Describe one modification of the phenyl column.
Penta-fluoro phenyl column
Four different retention mechanism:
- Polar interactions
- Dipole-dipole
- pi-pi
- Hydrophobic
Beware - acetonitrile suppresses pi-pi interactions so avoid using with PFP column
How does a cyano column achieve seperation?
By hydrophobic interactions (propyl group), dipole interactions with CN group
- Can be used for normal or RP chromatography
- In past, this phase was not very robust
What is a hydrophobic interaction liquid chromatography (HILIC)?
Used for retention of very polar compounds that are not retained well in reverse phase chromatogrpaphy
- An acetonitrile-rich mobile phase is used in the inital condition; analytes are eluted using aqueous mobile phases
- Elution pattern is similar to normal phase chromatography but the organic/aqueous mobile phase combination used is similar to reverse phase
Why might amide columns be more appropriate than HILIC?
Provide wider pH and temp stability and suitbale for use with 100% aqueous mobile phases
