MAAC: Chromatography 3 Flashcards
Describe the process of gas chromatography
The technique involves a sample mixture (gas or liquid) being injected (0.1 to 1μL) through a rubber septum into a heating chamber at the top of the column.
The heating chamber is set at approx 500C higher than the b.p of the mixture and causes rapid vaporisation which is then carried onto the column.
The top of the column is set lower than the heating block to encourage concentration via condensation at the top of the column.
The vaporised mixture is carried through the column by a continuous flow of inert gas, which acts as the MP, e.g. He, H2 or N2
The different components, depending upon the extent of their interaction with the SP, migrate at different rates through the column.
A detector at the end of the column monitors the effluent from the column and as each component emerges a peak is produced.
The output from the detector is generally displayed by a computer
The resulting trace of detector response against time is the chromatogram.
What is the importance of the fan in GC?
- Ensures consitant temperature throughout column
- Reduces waiting time between smaple analysis making it quick and it is also efficently done.
- Temp after analysis will be high therefore need to reduce to start next
Describe what packed GC columns are like and made off
- Packed columns have an external surface of STAINLESS STEEL, GLASS or COPPER
- Diameters are in the range of 1.6 to 9.5 mm.
- Length is usually < 3 metres
- Sample loading capacity: approx. 1 - 2 µg per component
- COLUMN PACKINGS or STATIONARY PHASES:-
- The columns are packed with solid particles (gas solid chromatography, GSC) or A solid support coated with a liquid either by adsorption or chemically bonded to it (gas liquid chromatography ,GLC)
Describe what capillary GC columns are like
- Capillary columns have a very much smaller internal diameter than packed columns, but are very much longer.
- Capillary or open tubular columns are of three basic types:-
- wall-coated open tubular (WCOT)
- support-coated open tubular(SCOT).
- Porous-layer open tubular (PLOT)
- WCOT columns are simply capillary tubes coated with a thin layer of the stationary phase.
- In SCOT columns the inner surface of the capillary is lined with a thin film of a support material, such as diatomaceous earth. This type of column holds several times as much stationary phase as does a wall-coated column and thus has a greater sample capacity.
- PLOT columns are SCOTs without the liquid.
- Capillary columns are made of FUSED SILICA WITH POLYAMIDE COATING ON OUTSIDE TO GIVE FLEXIBILITY.
- Diameters are in the range: 0.15 to 0.5 mm
- Length: 12 to 50 metres
- This increased length significantlyincreases the number of theoretical plates (N) in capillary columns compared to packed columns
- N> 100,000 plates in capillary GC
- Thus potential RESOLVING POWER (seperation capability) in capillary GC is much greater than in packed GC
What is used to calculate the polarity of the GC SP phase?
How is this detemirined
This is a classification system used to quantify the polarity of a GC stationary phase.
McReynold’s constant for a given stationary phase is determined by the retention of: benzene, n-butanol, pentan-2-one, nitropropane and pyridine on a particular phase.
The higher the McReynold’s constant the more polar the stationary phase.
What is the least polar SP?
A Very polar SP?
What is the realtionship with OV xx and polarity
- Squalene
- Carbowax
- The higher the number (xx) the more polar
Give examples of SPs and their upper temp limits
- Squalene: C30H62, upper temp limit 125 Degree C
- Apolane 87: C87H176, Upper temp 260. Non-polar but more polar than Squalene
- Polysilicones: e.g. dimethyl, diphenyl and cyanopropyl are general purpose liquid phases that are also useful in open tubular columns and for the separation of low-volatility samples. They are commonly used in different proportions to create stationary phases of varying polarityPolysilicones: e.g.
Example of non-polar, intermediate polar and polar bonded SP phases
Non-polar: Methy polysiloxane
Intermediate: Methyl 50% phenyl polysiloxane
Polar: Polyethylene Glycol
To avoid overloading the capillary column when injecting sample, what is used?
What can overloading lead too?
Overloading is when too much sample is injected into the collumn and this leads to the peaks from analysis being non-symmetrical. This can show as peak fronting- when the first half of the peak is non-symetrical. OVerloading can affect the seperation of 2 closely eluting peaks therefore can overlap resulting in erroes and intrgration and a shift in retention time
To avoid overloading a the sample is injected through a process called sample splitting. A split valve needs to be integrates into the GC hardware. This stops the whole sample entering the column and allows only a proportion of the sample to be injected into the column
describe injecting onto capilary column with split valve
In a split injection, 0.1-2.0 µL of sample , is introduced through a septum into a vaporisation region.
A glass or quartz liner protects the sample from the hot, catalytically active metal surface of the chamber and a plug of glass wool helps to ensure complete volatilisation.
Once it has evaporated, the sample mixes with the carrier gas stream.
Most of the sample is then vented out through the split exit; typically only 0.1-10% of the sample is carried on to the column.
A needle valve upstream of the splitter and a backpressure regulator at the split exit control the split ratio.
A buffer volume prevents condensation within the split line and a portion of the carrier gas is used to purge septum contaminants.
Describe GC detectors and disadvantages
- Popular because it is nearly universal for all organic compounds
- sensitivity is in the range of 10-10 to 10-12 g/sec
- Linear over at least six orders of magnitude
- It is a destructive detector but you could still collect the analytes by splitting the effluent stream before the FID and sending a portion of the sample to a non-destructive detector.
- Does not produce a signal for water
- The major disadvantages of this technique are that it requires three gases (carrier, air, and hydrogen) to operate and it is insensitive to inorganic compounds.
Describe how GC FID work
The sample stream is burned in a jet of hydrogen and air.
Ions are produced in the 2000C flame.
These ions are sent to a collector electrode through the application of a potential from the jet to the electrode.
The ions produce a current ranging from 10-12 to 10-5 amps, which is then amplified and recorded as the analyte signal.
What is an alternative detection method?
MASS SPEC
GC-MSD is a HYPHENATED technique
The combination of the two methods results in 3-d data providing both qualitative and quantitative information
- Good for distuingishing between samples of analytes with the same weight
- Most sensitive and useful
When might GC not be so useful. What needs to be done in this case and why?
Polar analytes/ drugs not analysed well therefore chemical derivations needed. This basically involves change to structure
- To increase volatility and decrease polarity of analytes
- To decrease thermal degradation of analytes
- To increase detector response
- To improve separation and decrease band broadening
- To improve extraction efficiency
Disadvantages for derviations?
There are some disadvantages:
The derivatizing agent may be difficult to remove and interfere with the analysis
It may cause unintended chemical changes in an analyte
It will increase the analysis time
It might cause extra expense
Examples of silylation reagents
MSTFA
Describe the meaurement of seperation
