Instrumentation Flashcards
describe preparative column chromatography
This involves a column packed with quite large stationary phase molecules (e.g. silica or alumina). The sample mixture is coated onto the top and the eluent is flushed down by the liquid mobile phase, taking the sample with it. Because of the large particle size efficiency is bad. Generally used for preparative work, to separate products, and not for analytical determinations.
High performance liquid chromatography (HPLC) is?
High performance liquid chromatography (HPLC) is an extension of liquid column chromatography where particle size is smaller and the eluent is pumped around the column under pressure rather than relying on gravity.
HPLC can be used for ________________
HPLC can be used for most forms of liquid chromatography i.e. Adsorption, partition, ion exchange, and gel permeation.
The column packing material may be ____________, ____________,and ___________________
Microspheres –particulate packing with thin surface area 🡪 low mass transfer and high efficiency. Low capacity since only surface is used for partitioning. Used in ion exchange chromatography.
Porous microsphere- these particles are completely permeable, but very small (3,5 and 10μM) to give a large surface area. Also have good efficiency due to low mass transfer, but have a higher capacity.
Bonded phases –where silica has different groups bonded to the surface. This gives a wide range of possible stationary phases e.g.
Silica gel and ODS silica gel are _________________
two of the most commonly used packing for normal and reverse phase chromatography respectively. The extent to which a compound is retained depends primarily on polarity (silica gel) and lipophilicity (reverse phase ODS silica gel).
Name two of the most commonly used packing for normal and reverse phase chromatography respectively
SILICA GEL and ODS SILICA GEL
Most drug molecules have both ______________ and ___________
lipophilic and polar groups.
The other factor which influences the degree of retention of a particular compound is__________________
the nature of the mobile phase.
Generally, the more polar the mobile phase the more ____________________
quickly it will elute a compound from silica gel column
Generally, the more lipophillic a mobile phase the more ___________________
quickly it will elute a compound from a reverse phase OSD column
In normal phase chromatography four commonly used solvents are:
hexane
DCM
isopropanol
methanol
In other of increasing Polarity/ strength
H>D>I>M
In reverse phase chromatography, four commonly used solvents are:
water
methanol
acetonitrile
tetrahydrofuran
In other of increasing strength
An ideal detector for HPLC should have;
Rapid and reproducible response to solutes
A linear response
Stability of operation
There are two major classes of detectors for HPLC these are;
Optical detectors
Electrochemical detectors
There are three important optical detectors;
explain each one
Refractivity index (RI) monitors :
UV/VIS Detector
- Fixed wavelength detectors
- Diode array spectrometers
Florescence detector
-
explain Refractivity index (RI) monitors
and state its advantage and disadvantage
Refractivity index (RI) monitors : These monitor the difference between the refractive index of a reference stream (the mobile phase solvent), compared to the eluent emerging from the column. Any solute in the elute, cases a change in RI.
Visible light is shone through a double cell containing both the reference and sample.
If the two are the same then the light is refracted to a certain extent into a photo-detector.
When the solute elutes from the column this alters the RI and this is picked up by the photo-detector.
If the two are the same then the light is refracted to a certain extent into a photo-detector.
Advantage
1. It is universal
Disadvantages – not very sensitive (need quite high concentrations of solute sensitive to about 1μg of compound); sensitive to temperature changes. Only really used if there is no other choice of detector.
explain Fixed wavelength detectors
and state its advantage and disadvantage
fixed wavelength detectors: fixed wavelength (at which you expect the solute to absorb) is selected and the absorption of that wavelength of light is monitors in the sample coming from the column.
Advantages – Response is linear providing the concentration is not too high; it is not temperature sensitive; it is much more sensitive than RI (sensitivity up to 0.01μg)
Disadvantages – can only use for species that absorb at a set know wavelength in the UV/Vis region. You can’t really analyse unknown samples.
explain Diode array spectrometers
These detectors enable you to measure absorbance of the sample coming from the column over all wavelengths simultaneously, thereby enabling you to analysis for more than one component (which absorb at different wavelengths).
Light of many wavelengths is passes through onto a holographic mirror which splits light up into all its component wavelengths and focuses them on many microdiodes, switching them all on. If some wavelengths are absorbed by the sample then not all of the wavelengths pass through and therefore some diodes switch off.
it measures absorbance at different wavelengths
explain Florescence detector
and state its advantage and disadvantage
Similar to the UV/Vis detector except that it detects the light fluorescing for naturally florescent solutes (when light of the correct wavelength is shone on it).
Advantages - This is more sensitive than both RI and UV/Vis and you can detect very low concentrations (at the ng level for highly fluorescence compounds).
Disadvantages – you can only use it if the sample naturally fluoresces (very few do). However you can derivatise molecules to make them fluoresce. Phenols, amine, amino acids can all be made to fluoresce by reacting them with the following molecule;
explain types of Electrochemical detectors
Conductivity detector
This is used in ion exchange chromatography where the sample components are ionic and the mobile phase has very low conductivity. It is useful for detecting anions and cations.
Redox electrochemical detector
These detectors are useful for solutes than can be oxidised or reduced.
The extent to which a compound is retained depends primarily on ———————– and ——————-
Polarity(silica gel) and Lipophilicity(reverse phase ODS silica gel)
