Gas Chromatography

  • Created by: LBCW0502
  • Created on: 10-11-18 10:46
What is GC used to separate?
Volatile organic compounds - a technique for the separation of mixtures of thermally stable, volatile/semi-volatile solutes in the gas phase. Consists of flowing mobile phase (inert gas) and injection port (rubber septum at temp > b.p.)
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Separation is due to what kind of differences?
Differences in partitioning behaviour between mobile gas phase and stationary phase - selective retardation.
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In other types of chromatography, what does not interact with molecules of analyte?
Mobile phase - only has the function of transporting the analyte through the column. Separation column contains stationary phase with detector
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Describe features of the stationary phase
Solid/liquid coated on solid support. Two types of GC - gas-solid, gas-liquid. Stationary phase packed in column (column chromatography) or coated onto inner walls of very narrow capillary (open tubular/capillary chromatography)
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Describe features of the mobile phase
Constantly moves over stationary phase and both are in equilibrium. Carrier/eluent gas
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What is elution?
The process of passing liquid or gas through the column to remove bound components
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Outline the history of GC
1941 by Martin and Synge (also discovered LC theory). 1955 - GC apparatus on market. Today - 200,000 GC used in the world. Cost £1500-£40,000 with GC-MS +£80,000. Latest improvement: computer-automatic control, OT columns, separate analytes fast
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Outline the start of the GC process
Sample vaporised and injected onto head of chromatography column. Sample transported through column by flow of inert gaseous mobile phase. Column contains liquid stationary phase which is adsorbed onto surface of inert solid
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Compare GC to LC
GC has flowing mobile phase, injection port, separation column and detector. LC has a much slower diffusion (liquid vs gas) and theory for HPLC derived from theory for GC
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Describe the processes which take place within the GC capillary column
Voltatile samples introduced via injection import. Continuous flow of gas elutes compounds to increase distribution ratio where they pass through detector connected to recording system
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What does the GC capillary column comprise of?
Column comprises of polyimide coating (physical stability), fused silica (solid support), stationary phase (liquid/solid). 30 metres long
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What happens to the distribution ratio if analyte B moves faster than analyte A
Db << Da. A stays longer in the stationary phase compared to B. B moves through the column fast and is detected first (shown on a chromatogram by the peaks). A has a higher affinity for the stationary phase compared to B
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Why does separation depend on polarity of the stationary phase?
Higher D for stationary phase leads to increase in retention time (takes longer to reach detector). Large differences in D for solute mix leads to increase in separation (better separation of analytes)
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Why does separation depend on temperature?
Increased temperature leads to increased proportion of soluble in gas phase (decreases retention time/reaches detector quickly). Large differences in bp for soluble mix leads to increase in separation (better separation of analytes)
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Why does separation depend on the flow rate of carrier gas?
Increased flow leads to decreased retention time, decreased efficiency (N) and decreased separation power
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Why does separation depend on the amount of sample injected?
Overloading of optimum sample: SP ratio leads to decreased efficiency (N) and decreased separation power
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Why does separation depend on the the length of the column?
Longer columns give better separations but can lead to longer runtimes (GC columns can be very long)
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State features of a typical gas chromatogram
Sharp peaks for analytes detected are various times (minutes). Heigh of peak indicates signal strength. Able to identify analytes based on b.p. (increasing temperature at specific times)
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What is GC used for in pharmaceutical science?
QC, quantification of bulk drug in pharmaceutical preparations, manufacturing residues, stability studies, PK studies, drug application studies, flavouring compounds, pharmacognosy, check suitability of packaging materials, detect counterfeit drugs
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What are the classical GC components?
Mobile phase (carrier gas) reservoir, sample introduction device, GC separation column, detector, data logger/computer
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State features of a schematic of a GC instrument
Carrier gas supply, gas pressure regulator. Flow controller, flow splitter. Sample syringe, injector. Column in oven. Detector. Flow meter, data acquisition. Chromatogram. (Know how to draw schematic of GC instrument)
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Describe features of carrier gases in GC (1)
Stored in compressed cylinder, supplied 10-40 psi, slow at 2-120 mL/min. Fine control by needle valve/mass flow controller. Types of gases: N, He, H. Gas not in distribution process (analyte has no affinity for gas, just for gas phase based on bp)
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Describe features of carrier gases in GC (2)
Gas temperature used to control rate of movement along column. Gas viscosity/density can contribute. High density gas - good efficiency - faster analysis times
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Why is air/oxygen not used as a carrier gas?
Oxygen will react with the analyte and when the temperature is increased, an oxidation reaction will occur
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Describe features of injection systems for GC
Sample should be introduced as a narrow bond. Liquids injected via self-sealing rubber septum, Gases require large volume gas-tight syringe or gas sampling valve
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What is the effect of sample volume on the chromatogram?
Large volume leads to broadened peak (Gaussian/non-Gaussian). Small sample volume leads to sharp peak. Injector type/sample volume can affect peak efficiency. Broad peaks can mask two analytes present in sample
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What is a split injection? (1)
A means of reducing sample volume for open tubular columns. Only 0.1-10% of 0.1-0.2 mL injected sample reaches column (remainder discarded). Sample injected through septum into sample vaporisation zone
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What is a split injection? (2)
At split point, some of sample enters column (remainder goes through split needle valve to a waste vent). Split ratios 20:1 - 500:1. E.g. 1 mL/min to column, 99 mL/min discarded (split ratio of 100:1)
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What is a autosampler?
GC and MSD. Carousel containing sample vials (~100) and robotic arm) with needle housing
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Describe features of column ovens
Should be easily accessible. Use with both packed and capillary columns, fast heat up and cool down. Temperature range sub-ambient (-50-450 degrees Celsius). Design - vertical vs horizontal mounting
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Describe features of temperature programming
Temperature controls proportion of solutes in gas phase (controls RT). Isothermal separations > b.p. for 2-3 mins separations
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What are the observations in temperature programming?
Shorter run time, sharper peaks, increase signal: noise ratio, optimised resolution and k, useful for mixtures with broad b.p. range, slope temperature ramp effects separation
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What are the two types of gas chromatographic formats?
Gas-solid chromatography - stationary phase is solid with chemically bonded interaction phase. Gas-liquid chromatography - stationary phase is a liquid or semi-solid coated onto solid support
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What are the two types of column format? (1)
Packed column (spherical particles) 2-4 mm diameter, 1-6 m length, make of Cu/steel, poor efficiency/cheaper. Open tubular - mostly a coated inner wall of a tube/capillary, 200-300 micrometres diameter, 10-100 m length
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What are the two types of column format? (2)
OT - made of fused silica (high quality quartz) coated with polyimide or Al. Capillary columns are less robust than packed columns but working like can be extended by bonding stationary phase onto the inner walls of the column
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Describe different features of capillary columns
WCOT (wall, capillary, OT, liquid SP coated on silica wall). SCOT (support capillary OT, solid support coated with liquid SP). PLOT (porous, layer OT capillaries, solid support is the SP and has no pores)
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Describe the types of solid support in GLC (1)
Silica based - diatomaceous Earth (silica skeletons of algae), deactivation required to remove trace metals and SiOH (silanised), packed columns - silianised glass beads, OT columns - glass capillary wall
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Describe the types of solid support in GLC (2)
Polymeric sorbents - porous polystyrene, PTF - for very polar compounds
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Describe features of a liquid stationary phase
Different types of stationary phase are available. Most commonly used is dimethylpolysiloxane (DMS- non-polar). Dimethyl groups can be replaced by more polar groups e.g. phenyl groups, cyano/amine groups. Can have polyglycol phases (very polar)
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What are the different GC stationary phases of increasing polarity?
DMS - DMS/cyano, DMS-phenyl, DMS/phenyl/cyano
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What molecular interactions would you expect with DMS stationary phase?
On non-polar columns - van der Waals predominate, no H bonding or ionic interactions, substances separate in order of increasing b.p. and more volatile substances elute from the column first
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What molecular interactions would you expect with DMA and phenyl/cyano phases? (1)
On more polar columns - van der Waals interactions. Depending on degree of cross-functionalisation: emerging interactions influence separation - dipole-dipole, dipole-ID, pi-pi interactions
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What molecular interactions would you expect with DMA and phenyl/cyano phases? (2)
Substances separate in order to increasing b.p. but some can elute later than would be predicted from volatility considerations alone
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How does increasing phenyl content of SP affect elution of analytes?
Non-polar/non-aromatic analytes elute faster (lower affinity for polar SP) - shorter retention times. Analytes with aromatic groups elute longer (pi-pi interactions with SP, very weak H bonding).
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Describe the separation on very polar SPs (e.g. polyglycol phases)
H bonding can occur. Polar substances of similar b.p. to non-polar substances have longer retention times (strong H bonding with SP) and elute longer than other non-polar substances. Van der Waals between non-polar regions of SP/non-polar analytes
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What is PEG also known as
Wax phase e.g. carbowax. Structure - HO[-CH2-CH2-O]nH
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How does increasing the polarity of SP affect analytes e.g. phenol and decane?
Decane has a lower affinity for SP due to being non-polar and elutes faster. Phenol has higher affinity for SP and elutes for longer
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What needs to be considered when choosing a SP? (1)
If unsure able sample content, begin method development phase with DMS phase. Low bleed columns are more temperature stable. Use least polar phase which results in adequate separation and time (non-polar phase - longer life)
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What needs to be considered when choosing a SP? (2)
If targeted analysis, choose phase with similar polarity. Guide for GC/literature to compare results with column and conditions
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Describe features of polarity of solutes
Non-polar/medium polarity compounds are suitable for GC. Those with high polarity which are thermally labile can be derivatised to enhance volatility/stability. Increasing volatility decreases retention time (decreases b.p.)
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Give examples of functional groups with weak-intermediate polarity
Ethers, ketones, aldehydes, esters, tertiary amines, nitro compounds, nitriles
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Give examples of functional groups with strong-intermediate polarity
Alcohols, carboxylic acids, phenols, primary/secondary amines, oximes, nitro compounds, nitriles
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What are the common derivatisation reactions?
Alkylation, arylation, silylation, acylation and esterification
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Describe features of detectors used in GC
FID/TCD. Sensitive to wide range of components/conditions. FIDs more sensitive to HCs, more sensitive than TCD. Detectors robust. TCD non-destructive/operated in series before FID (destructive). Different detectors/selectivity
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What is a non-selective detector?
Responds to all compounds except the carrier gas
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What is a specific detector?
Specific detector responds to a single chemical compound
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What can detectors be grouped into?
Concentration dependent detectors and mass flow dependent detectors
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What does a concentration dependent detector do?
Signal from a concentration dependent detector is related to concentration of solute in detector and does not usually destroy the sample
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What does a mass flow dependent detector do?
Usually destroys sample and the signal is related to the rate at which a solute molecules enter the detector. The response of a mass flow dependent detector is unaffected by make-up gas
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What are the three types of detector? (1)
Flame ionisation detector (mass flow, H/air, organic compounds, 100 pg, 10^7), thermal conductivity detector (conc, reference, universal, 1 ng, 10^7)
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What are the three types of detector? (2)
Electron capture detector (conc, make-up, halides/nitrates/nitriles/peroxides/anhydrides/organometallics, 50 fg, 10^5)
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What are the ideal detector characteristics? (1)
Adequate sensitivity for application. Good stability/reproducibility. Linear response. Operable temperature range for GC applications (20-400 degrees Celsius). Short response time not limited by flow rate (peak definition/sample frequency)
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What are the ideal detector characteristics? (2)
High reliability. Similarity in response for solutes. Non-destructive of sample
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Describe how the FID works
CH reacts with O to form cation and electrons. Insensitive to non-combustable gases (oxidised organics/NO, H2S, CO/CO2). Pyrolysed in H flame to produce electrons (jet). Potential difference of 400 V put across flame and resulting current measured
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Describe how the TCD works (aka Katharometer) (1)
Electrically heated element whose resistance at constant electrical power depends on TC of surrounding gas. TC of He or N2 gases >> most organic compounds (6 fold). Detection based on decreased TC by eluting solute molecules
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Describe how the TCD works (aka Katharometer) (2)
Reference cell and measurement cell format based on Wheatstone bridge circuitry e.g. channel 1 - carrier gas only, channel 2 - carrier gas and analytes
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Describe features of Mass Spectrometric Detector
MSD detects analytes eluting from column based on mass-to-charge ratio (m/z). Measurement required analytes to be ionised and in gas phase (GC application, now LC-MS available). Common GC ionisation sources: electron impact/chemical ionisation
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State features of the schematic of a GC with a MSD
GC - [ionisation source - mass analyser - detector] - recorder ([under vacuum])
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