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
