Gas chromatography Flashcards
LO
- Describe how the GC separation occurs and what can be done to improve it
- Describe all of the principal components of a GC instrument
o Injectors, column formats, detectors etc
o Know what combination to use for an analysis - Knows the strengths and limitations of GC and when to use it as a preferred technique over other methods
- Give some practical examples of GC in operation
o Carry out typical mathematical calculations in GC analyses
Reading list
What is Gas chromatography?
- “a technique for the separation of mixtures of thermally stable, volatile/ semi-volatile solutes in the gas phase”
* Separation technique- takes place due to chemical affinity for the stationary phase and molecular boiling point (BP) for distribution to gas phase - **Thermally stable- **high temperatures, solute degradation
* Volatile/ semi-volatile solutes- volatiles solutes readily move into gas phase
What is the stationary phase (SP) and how may it be present?
o The stationary phase in GC is a solid or a liquid coated onto a solid support
o Therefore, there are two types of GC- **gas-solid and gas-liquid chromatography
o In GC- the stationary phase may be packed into a ‘column’ (column chromatography) or coated onto the inner walls of a very narrow capillary (open tubular (OT) chromatography** e.g., capillary GC)
What is the mobile phase?
o This phase constantly moves over the stationary phase, and both are in equilibrium
o In GC, the mobile phase is termed the **carrier gas **
o The mobile phase is also sometimes called the **eluent gas **
o The process of pass liquid or gas through the column to remove bound compound is called **elution **
Whats the distribution ratio?
And the equation associated with it?
o The distribution ratio, D, for a solute is defined as the ratio of its analytical concentration in two immiscible solvents
o When D is high for a solute, has a high affinity for that solvent
o Differences in D for each solute in a mixture, gives rise to
separation
What is the GC principle?
Samples (which must be volatile and thermally stable at the operating temperature) are introduced via the injection port at the top of the GC column.
A continuous flow of gas elutes the compounds in order of increasing distribution ratio, D, from where they pass through a detector connected to a recording system”
Draw a schematic of a GC capillary column, labelling the key features and what the purpose of the main componenets are
- GC capillary column
- Brown and yellow is part of column housing, light brown is liquid coated onto wall
- Made of Glass or silica which is brittle
- Outer coating of polyamide adds stability
- Long columns between 30-100m
- Mechanical stability allows to twist or bend onto roll without breaking it
- Liquid coated on at different thicknesses depending on what we want to separate
- Analyte (A) in gas phase
- Stationary phase is a liquid/ solid
- If another compound B is added which has poor distribution ratio, it has a lower retention time and comes out first
Schematic:
• Container with the carrier gas/ eluent gas within
• Connected to a molecular sieve to remove any unwatedmoleucles such as hydrocarbons, water vapour and oxygen
• The column is coiled within a container in order to maintain a constant temperature in all of the column
• There is an inlet port for the sample to be injected which is a higher temperature than the column to ensure all samples are volatilised
• Sample can be read via flame ionisation detection where hydrogen and oxygen react to form a flame which the sample passes onto. The sample is ionised and the electrodes wither side detect the ions and electrons and produce a gas chromatogram which has been amplified. This shows the retention time and the area under the curve shows the concentration of the sample. This can then be compared against a known standard
• The column is usually made of glass or silica (something which can withstand high temperatures) and has a polyimide coating
What does separation depend on?
- Polarity of the SP
- Temperature (T)
- Flow rate (F) of carrier gas
- Amount of sample injected
- The length of the column
How does polarity of the SP affect separation?
o Higher D for the stationary phase –> higher retention or Dow value of solute
o Large differences in D for solute mix –> increased separation
How does temperature affect separation?
o Increase T –> increased proportion of solute in gas phase –> decreased retention
o Large differences in BP for solution mix –> increased separation
o If solutes have large range of BP then temp can be used to increase the degree of separation
How does flow rate of the carrier gas affect separation?
o Increase F –> decreased retention –> decreased efficiency (N) –> decreased separation power
How does the amount of sample injected affect separation?
oOverloading of optimum sample: SP ratio –> decreased N –> decreased separation power
How does the length of the column affect separation?
Longer columns give better separations, albeit longer runtimes, GC columns can be very long
The chromatogram and how different formulae are used
A typical gas chromatogram
- Gas chromatogram, peaks are sharp and well separated, separation due to heated gas phase so interaction with stationary phase being faster due to increased solute energy
- Increases mass transfer into stationery and gas phases
- Multistep gradient
- Optimised separation (NOT optimal)
How are the areas under the peak calculated?
Manual integration
What is GC used for in biomedicine and pharmaceutical science?
- QC in biomedicine drug manufacture (e.g., starting products; all stages of production and finished product)
- Quantification of API in biomedical preparations
- Pharmacognosy
- Stability studies
- Pharmacokinetic studies (metabolism studies, therapeutic monitoring of drug levels)
- Drug application studies (e.g., transdermal application)
- Checking suitability of packaging materials
- Detecting counterfeit drugs
- Biological system/ process characterisation via determination of biomolecules
o “omics”: e.g., proteomics, metabolomics, phenomics - Toxicology studies: analysis of tissues and excreta
- Manufacturing residues
- Flavour
What is GC used for in forensic science?
- Arson residues, accelerants
- Bulk explosives and explosives residues
- Chemical warfare agents
- Toxicology (overdose, drug abuse, poisoning, suspicious death)
- Doping in sports
- Drink driving
- Workplace drug testing
- Possession and trafficking of controlled substances
- Paint
- Environmental
- Food authenticity
What are classical GC components and the order in which they are used in?
A. Mobile phase (carrier gas) reservoir –>
B. Sample introduction device –>
C. GC separation column –>
D. Detector –>
E. Data logger/ computer
Draw a schematic of a GC instrument
Which aspects of the GC instrument need temperature control?
The Injector, detector and column
Each are independently controlled
What are the types of carrier gases present in GC?
Carrier gas/ mobile phase
Carrier gases: Gas purifiers
mobile phases can be unreactive gases such as; nitrogen, hydrogen, helium and aron
Tell me the the following about the mobile phase:
* how it is stored
* rate which it is supplied at
* how the rate is controlled
* the types of carrier gases
o Stored in a** compressed cylinder **
o Supplied at 10-40 psi, flow at 2-120 ml/min
o Fine control by needle valve or mass flow cylinder **
o Types of carrier gases: N2, He, H2 **(gases don’t need to be heated as out natural, they are inert and don’t react with solutes, all have pros and cons generally being cost vs efficiency)
In GC, does the carrier gas take part in the distribution processes?
NO (i.e., the analyte has no ‘affinity’ for any gas,just for the gas phase itself based on its BP)
What is used to control the movement along the column?
- It is mainly gas temperature that is used to control the rate of movement along a column
How can gas viscosity and density contribute to carrier gases rates?
- How, gas viscosity and density can contribute
**High density gas –> good efficiency –> faster analysis times **
- Large gas molecule means the larger the distance the solute must travel (with N2 being the biggest)
Why are carrier gases, gas purifiers needed?
To help purify gases to keep them inert
What can be used as gas purifiers?
- Mositure traps
- oxygen traps
- Hydrocarbon traps
How do moisture traps work?
o Water in gas –> hydrolysis –> noisy baseline and peak tailing (due to water dipole being present).
o Moisture trap contains molecular sieves and moisture indicating absorbents
o Water can cause hydrolysis and problems with injection so isn’t wanted.
o Peak tailing occurs when more than one retention mechanism
How do oxygen traps work?
o Oxygen in carrier gas –> oxidation of liquid phase resulting in bleeding, changes in retention, loss of resolution and peak tailing
o Also, can cause oxidation of ECD
o Remove by use of oxygen trap filters. top of trap is filled with Cu compounds and the bottom with an oxygen indicating solid
How do hydrocarbon traps work?
o Organics in gas –> noisy baseline and host peaks. Trap is filled with high-capacity activated charcoal
o High SA= more mass transfer
o Activated charcoal made from burning of fossil fuels
Tell me the purpose and typical features of injector systems for GC
Injection systems for GC
* In chromatography, sample should be introduced as a narrow band (if not then split or deformed peaks are seen)
* Liquids are injected into injector port via a self-sealing rubber septum
* Gases require a large volume of gas-tight syringe or gas sampling valve
How does injection effect banding in GC?
- If process is quick, then band spreads during injection process and gets to column as already broad band. If quick, then band is sharp
- Low BP reach column with minimal broadening
- Injector is a discriminator of solutes
The injector type and sample volume can affect the peak afficiency
What is the criteria for an ideal sample introduction system in GC?
- No discrimination of solutes in terms of boiling point, polarity, molecular weight etc
* Thermal degradation, absorption, and other solute reactions should be negligible at all sample sizes
- No loss in efficiency- little dead volume- especially for cap-GC
- Changes in column operating conditions should not influence injection process
* Reproducibility should be high- peak areas/heights and retention times
- The longer the band in the injector, the more the band spreads
- E.g., If column colder than injector then should not see cold temp and should see high reproducibility
What are the types of injection systems for packed columns?
- Flash Vaporised injector
- On-column injector
What is the main principle of the flash vaporised injector and some disadvantages?
**Flash Vaporised injector **
* Principle: large volume liquid samples (0.1-10 ml) injected via septum onto glass or metal block which is constantly swept with gas. Sample vaporises and is transported in the gas stream
* Disadvantages:
* Non-volatiles accumulate on block
* union between block and column can absorb compounds
* labile compounds can decompose
* introduces dead volume into system
Tell me about the on-column injector
Samples are placed directly on top of the column bed –> more precise method of sample injection. not generally heated
Draw a split/ splitless injector and label it
Draw and label a sandwich injection and why the compoenents are loaded the way that they are
- Approach used to inject sample and clean syringe
- Washed gas tight syringe takes in air
- Washing solvent (orange) taken up- clean and wet walls of syringe to improve volumetric accuracy and remove residual interference
- Air between wash and sample is used to avoid interference or lead to loss insensitively to broadening
- Final air is to avoid ambient contamination
- Another section of air then sample plug then another section of air
- Wash solvent may or may not be added
Sandwich injection is an injection technique in which two or three aliquots are drawn into the autosampler syringe from multiple vials, and injected into the GC inlet. The result is that the aliquots are simultaneously vaporized, mixed in the liner, and transferred as a single sample onto the GC column
Explain what is shown in this graph and how the different situations can arise
How can sample overloading be overcome?
Tell me about autosamplers
- Run sample in particular sequence
- Calibration
- Tall box is the injector mechanism
- Carousel can rotate or metal arm used to deposit samples
- Vials are generally glass and sealed with injection lined cap
- Connected to MSD
- This format is often used for online sample preparation methods like solid phase microextraction (SPME)
What type of columns are column ovens used for?
What is their temperature range and how this is achieved?
Column ovens
* Should be easily accessible; use with both packed and capillary columns, fast heat-up and cool down; temperature range sub ambient **(-50˚c to 450˚c) **
- Design: vertical vs. horizontal mounting
*** Use fans to add or remove heat **
How can temperature programming be used?
What are the different types?
Why may it be used?
- Temperature controls the proportion of solutes in the gas phase –> **controls retention time **
- Longer on stationary phase the longer the band has to spread
- Isothermal separations ≥ TBP for 2-30 min separations
- Ramp type gradient can be linear or curved and to add samples at set times.
- Gradient used to shorten run time, as seen in observations
What are the two types of column format?
o Packed column- spherical particles
o Open tubular (OT)- mostly a coated inner wall of a tube/ capillary
What are the different types of Open-tubular capillary GC columns?
- WCOT (Wall coated open tubular)- frequently used
- SCOT (support coated open tubular)
- PLOT (porous layer open tubular)
Representative properties of different columns used in GC
For the liquid stationary phase there are different types which can be used but what is the most used?
- Most used is dimethylpolysiloxane (DMS) (non-polar stationary phase)
- Dimethyl groups can be replaced by more polar groups e.g., phenyl groups, cyano groups, amine groups
- Can have polyglycol phases (very polar)
Draw the different variations of the DMS liquid stationary phase.
Why do we need both groups?
o Cyanol contain permanent dipole and phenyl do not
o Phenyl have delocalised electrons above and below benzene ring which can be pushed or pulled within close proximity of analyte with **permanent dipole- induced polarity. **
Advantage=when nonpolar analytes is present, don’t repel phenyl and still react via non-polar mechanisms
Aromatic compounds retain on phase very well
Polarity can be induced on phenol ring by perenmant dipole like ketone for better retention
“like retains like” in GC
Can use both phenyl and cyano if needs to be
What molecular interactions would you expect with DMS and phenyl and/or cyano phases?
**Semi-polar separation **
* On more polar columns (DMS/phenyl; DMS/cyano; DMS/ phenyl/cyano)
* Van der waals interactions will still occur **
**
* But depending on the degree of cross-functionalism…
o Emerging interactions occur which will influence separation: dipole-dipole; dipole-induced dipole will be more common; pi-pi interactions
o Substances still tend to separate in order of increasing BP but some substances will elute somewhat later than would be predicted from volatility considerations alone
What interactions are present in polar separation and how may this affect BP and retention times?
Polar separation
* On very polar Stationary Phases (SP) (polyglycol phases)
* Hydrogen bonding can occur
* Polar substances of similar BP to non-polar substances will have longer retention times (they will interact more strongly with the SP through H-bonding and hence will spend longer time associated with the SP and elute later than non-polar substances
* There will still be van der waals interactions between non-polar regions of SP and non-polar analytes/ non-polar regions of molecules
* **Van der waals are less than those in the dipole-dipole interactions **
Has the retention time of the following changed and why?
Look into…
Polarity of solutes/ analyte classes
** Generally non-polar/medium polarity compounds suitable for GC**
o Those with high polarity/ which are thermally liable can be derivatised to enhance volatility/ stability
o Increasing volatility decreases retention time (i.e., decreases BP)
o Alcohols and polyols can be considered polar
o Ketone is less polar than aldehyde
o Ether is non-ionisable
o Longer hydrocarbon chain the less polar it is
o Alkene has inducible polarty due to Pi-pi interactions, however less than benzene
o Polarity generally is logP (higher logP means the less polar it is)
What is a retention indices and the equation to calculate it?
- Retention indices relates the relative tr of a solute to a homologous series of alkanes- a means of comparing column performances for a target analyte (see Harris pg 535-537)
Kovats examples
Look up more and practice
What are the ideal detector characteristics for a GC?
- Adequate sensitivity for the application
o Is the application of a trave analysis nature or not? - Good stability and reproducibility
o Stability= read out constant in normal conditions. If not, then it’s a noisy detector. Chemical noise is due to chemical in mobile phase which could be impurity in carrier gas. drift is another type of noise which is increase/decrease overtime but no chromatographic conditions being altered
o Results in accurate and precise quantitation - Linear response
o Should extend over several orders of magnitude
o Sometimes this can be described by curvature (attempt should be made to find LOBF) - Operable temperature range for GC applications
o From room temperature(ambient) to 400˚c
o Need to be thermally stable
o Usually hotter than over to minimise band broadening - Short response time not limited by flow rate
o Peak definition and sampling frequency - High reliability
o Inexperienced uses, e.g., MS - Similarity in response for solutes
o May be limited in terms of selectivity
o Use of internal standards - Non-destructive of sample
Tell me the following about the flame ionisation detector (FID):
* detector used for what?
* insensitive to?
* How its used
* pros and cons
**Flame ionisation detector (FID) **
- Most widely used detector in GC and specific to most organic compounds
CH + O –> CHO+ + e- -
Insensitive to non-combustible gases, e.g.,
o Completely oxidised organics
o NOx, H2S, CO and CO2 - Pyrolyzed in hydrogen flame to produce electrons (jet)
- Potential difference of roughly 400V put across the flame and resulting current measured
- Mass sensitive detector rather than concentration sensitive measures the number of reduced carbon atoms in the flame
Pros
* High sensitivity (roughly 10-13 g/solute), large linear range (roughly 107), low noise and easy to use
Cons
* Destructive of sample
* Jet tip becomes dirty
* Air/ H2/ carrier gas purity
Tell me the following about nitrogen-phosphorous detectors (NPD):
* Whats used
* what the bead contains
* similarities/ differences to FID
Nitrogen-phosphorous detector (NPD)
- An electrically heated (600-800˚c) glass bead is placed above flame in an FID
- Bead contains caesium bromide or rubidium chloride
- In the presence of alkali metal atoms, organo-phosphorous or nitrogen-containing compounds burn to produce ionic species which are usually unstable
- Similar to FID
- In a normal FID, the ions produced by combustion of the sample decrease rapidly in concentration above the flame
- If the collector electron is moved away from the vicinity of the flame, little or no response is obtained under normal FID conditions.
- In contrast in the NPD, since the ions persist much longer, a selective response to N- or P- compounds is obtained
Tell me about the thermal conductivity detector (TCD):
* aka
* what it is and how it works
* what detection is based on
* features (pros/cons)
* not suitable for
**Thermal conductivity detector (TCD) **
- Sometimes referred to as a “katharometer”
- Electrically heated element whose resistance as a constant electrical power depends on the thermal conductivity(TC) of the surrounding gas
- Non-destructive of sample (no flame exists)
- Indirect detection mode
- TC of He or N2 gases»_space; most organic compounds (roughly 6 fold)
- Detection is based on reduction in TC by eluting solute molecules
- Reference cell and measurement cell format based on Wheatstone bridge circuitry i.e.,
o Channel 1: carrier gas only
o Channel 2:carrier gas + analytes - Has large linear range (roughly 105g/solute/mL)
- General detection mode for inorganic/organic species
- Non-destructive of sample
- Insensitive (roughly 10-8)
o Not suitable for trace analysis
o Not suitable for capillary GC
Draw the flow splitter for 2˚ TCD ref. cell
Tell me the following about the Electron capcture detector (ECD):
* What it is selective for
* What it is insensitive to
* What the gas is ionised by
* What the role of the electronegative analytes are
* type of response produced
* what type of approach it is based on
Electron capture detector (ECD)
- Selective for electronegative atoms
o Halogenative species, peroxides, quinones and nitro groups - Insensitive to amines, alcohols, and hydrocarbons
- Gas is ionised by H3 of Ni63 foil (beta emitter)
o Steady current
o Positive ions, radicals, and thermal electrons - Electronegative analytes capture electrons and reduce current
- Non-linear response: potential across detector is pulsed
- Narrow linear range of roughly 102
- Works on indirect approach
Tell me the following about flame photometric detector (FPD):
* What other detector it is similar to
* What compoundsit is similar t
* Typeof photons emitted
Flame photometric detector (FPD)
* Similar to FID
- Specific to Phosphor and Sulphur containing compounds
- Photos emitted of certain wavelengths pass through in line filter and signal amplified
Draw the schematic of a GC with an MSD
Draw a quadrupole schematic
Practical examples
