Lecture 2 Flashcards
What characteristic does GC use to separate?
volatility
What is the mobile and stationary phase in GC?
Stationary; very high-boiling liquid polymer
which is immobilised on the inner wall of a narrow tube (the support)
Mobile; a gas, typically N2, H2 or He
Describe the process of GC
- a small volume of sample is injected into the start of the column
- the analytes in the sample are carried along the column by the gaseous mobile phase, interacting with the stationary phase
(strongly or weakly, depending on the chemical structure of the analyte and the chemical structure of the
stationary phase), and separating as they travel. - At the end of the column, the analytes are detected somehow – the instrument then outputs a chromatogram.
Give the 2 classifications of GC columns
- packed
- capillary
What is a packed GC column?
- Column length is typically a few metres (in a tightly wound circle).
- Column internal diameter is on the order of millimetres.
- The column is packed with small solid particles, which are coated in the stationary phase.
The mobile phase (gas) moves through the gaps between these particles. - The resolution achievable is relatively poor.
- Relatively robust and cheap
What is a capillary GC column?
- Column length is typically tens of metres (in a tightly wound circle).
- Column internal diameter is on the order of micrometres.
- The capillary (i.e. a column, but very narrow) walls are coated with a thin film (1-5µm) of
the stationary phase. The mobile phase moves through the middle of the capillary. - The resolution achievable is relatively good.
- Relatively fragile and expensive
Give 2 examples of a stationary phase
- polydimethylsiloxane (PDMS)
- polyethyleneglycol (PEG)
What is PDMS?
- polydimethylsiloxane
- It can be prepared
with a variety of molecular weights – n is not always the same number. - When used as the stationary phase, this
molecule is of low polarity, due to all of the methyl groups.
What is PEG?
- polyethyleneglycol
- It can also be prepared with a variety of molecular weights – for example, PEG-400 is commonly used. (400 refers to average molecular weight,
corresponding to n=8 or 9, on average). - When used as the
stationary phase, this molecule is of medium polarity, due to the OH groups.
In which scenarios can GC be applied?
- compounds are naturally volatile; such as gases, and liquids with low boiling points.
- compounds are not very volatile at room temperature, but become more volatile when heated up; such as liquids
with higher boiling points, and some solids. - compound is not naturally volatile, but can be chemically converted (“derivatised”) into volatile compounds.
What factors influence the degree of preference of analyte molecules for one phase or the other?
- an analyte with high affinity for the stationary phase (arising from a strong interaction between the molecules of the analyte and the molecules of the stationary phase), will be retained strongly by the
column, and elute more slowly. - An analyte which is volatile (low boiling point) will naturally spend more time in the mobile phase, so it will be retained more weakly by the column, and elute more quickly.
How can temperature influence retention time and separation?
- Since the GC column is mounted in an oven, we can control the temperature of the column. By heating it up,
we can increase volatility of analytes, which makes the volatility more significant. This is very
useful to speed up the elution of analytes which have either a very high affinity for the stationary phase, or a very high boiling point, or both. - But if temperature is too high then everything will elute quickly- gives poor separation
How do you get the best separation results?
In general, the best separation results are achieved when there is a good match between the intermolecular forces of the stationary phase and the analytes to be separated. If this is the case, then the elution order of the analytes should reflect their boiling points.
- A good match in intermolecular forces leads to stronger interactions between the analytes and the stationary phase which slows the analytes down which separates the analytes more effectively.
How does temperature influence elution?
- The temperature can typically be set from ambient up to 400 degrees C.
- High column temperatures increase the speed of elution, and therefore reduce the retention times of the species
- but this can also reduce the resolution of a separation
How does increasing temp to reduce retention time change resolution of chromatogram?
- reducing the retention times of the species means that they all elute more quickly, therefore the peaks are closer to one another; this has the effect of reducing the resolution of a separation
- high column temperatures also increase the random thermal motion that the analytes experience,
leading to broader peaks and a further reduction in resolution
How can issues associated with increasing temp in GC be solved?
Applying a time-dependent change of temperature to the oven, while the separation is taking place, is referred
to as programmed temperature elution. This is commonly used in GC to improve separations of analytes with
significantly different bps – especially in complex mixtures.
Name the 3 detection methods for GC
- Flame ionisation detection (FID)
- Thermal conductivity detection (TCD)
- Mass spec (GC-MS)
Give the strengths of FID
- reliable
- cheap
- robust
- works well for any hydrocarbon compound
How does FID work?
- In this method, the effluent gas (i.e. the mobile phase of the GC column) is combusted in a hydrogen flame.
- This generates a supply of CHO+ ions and electrons
- causes a measurable current to flow in the FID, which is reported as a signal to an interfaced computer.
How can detection be used to calculate amount of analyte in sample
- The magnitude of the current is proportional to the number of ions formed.
- This means that, as the amount of analyte exiting the column increases, the FID response increases.
- In this way, the magnitude of the FID response (the area of the peak in the chromatogram) is proportional to the amount of the analyte in the sample.
How does FID vary between molecule?
- for each different molecule, the FID response is proportional to the amount that was
injected, but the constant of proportionality (often termed the “response factor”) can differ significantly from
one analyte to another. - - For this reason, the analytical method must be standardised to get reliable quantitative
results.
How does TCD work?
- As an analyte molecule exits the column, the thermal conductivity of the effluent gas changes, since it is now a mixture of the carrier gas and analyte.
- In this way, monitoring changes in the thermal conductivity of the effluent gas as a function of time, produces a chromatogram.
- The change in thermal conductivity is proportional to the amount of analyte, so the method can be made quantitative (with appropriate standardisation).
What characteristic does TCD use?
- This method takes advantage of the differing thermal conductivities of molecules.
- The thermal conductivity of the effluent gas is monitored, and the baseline signal is set by the thermal conductivity of the carrier gas.
Give a disadvantage of GC-MS
- more expensive than others
How does GC-MS work?
It involves interfacing the GC with a Mass Spectrometer, so
that the effluent gas exits the column and enters the Mass Spec instrument.
Give advantages of GC-MS
- yields molecular mass data and structural information about the analyte molecules (from the MS instrument),
- analyte retention times (from the GC).
- If the identities of the analyte molecules are not already known
with absolute certainty, this extra information can be very valuable.
