Gas spec Flashcards
Define gas chromatography
Seperation technique utulised for small, volatile, non-polar, thermally stable compounds.
Derivitisation required (TMS/silylation)
- Can be used for qualitative or quantitative analysis
What is seperation in gas chromatography based on?
Seperation based on boiling points and affinity/adsoprtion to stationary phase
What does dervitisation in gas chromatography do?
Converts less volatile and thermally labile substances into compounds that can be analyzed in the gaseous state
Applications of GC-MS
Toxicology:
- Drug and steroid analysis
Pharmaceuticals
- Impurities & residual solvent analysis
Food
- Analysis of pesticides, addatives and contaminates
Environmental testing
- Analysis of volatile environmental pollutants
Can you describe the compontents of GC?
- Mobile phase: carrier gas (inert gas)
- Flow regulator, contains gas filters
- Autosampler (holds sample at a high temp to allow vaporisation)
- Column oven - contains column and can be thermically programmed
What samples are apporpriate for GC?
- Mixtures of several components
- Sample is introduced as a liquid
- The analyte is often at a low conc
- Sample dissolved in volatile solvent
Describe the seperation process of GC
Sample injected (1uL) and moved by the carrier gas onto the column.
- The column is coated with wax type material with varying affinity (dipole dipole, van der Waals) for the analyte
- Seperation based on affinity
- Individual analytes are detected as they emerge from the end of the column through the detector
How does boiling point affect GC seperation?
The lower the boiling point the happier the analyte is being in the gaseous phase. Meaning it will move faster through the column.
What makes a carrier gas?
- Inert
- He (common but expensive), H2 (good seperation but not completely inert) or N2 (seperation not the best)
- Choice dictated by detector, cost and availabilit
- High purity gases required to remove any interferences
Where does vapourisation occur?
In the injector port
Purpose of injection of sample
- Deposit sample into the column in the narrowest band possible (small V done rapidly)
- Produces tall narrow peaks
- Give max resolution and sensitivity
What are the two types of injection methods?
Split and splitless
Also direct injection
What is meant by split injection?
A portion of injected solution is discarded
- Only a small portion of sample goes through the column
Used for concentrated samples
What benefits might a split injection have?
Avoids overloading of column and unresolvable peaks
What is a major disadvantage of split injection?
Contamination of the septa or septa go unnoticed, due to small sample size
What is splitless injection?
Most of the sample goes through the column. The injection is slow compared to split.
What samples are used for splitless or split injections?
Split - Concentrated samples
Splitless - Dilute samples
What is an advantage of splitless injection?
Allows for accurate determination
What is direct injection?
Sample is transferred straight onto the column. Needle is either directly into column or directly above
For what analytes are direct injections used? and what analysis?
For thermally labile compounds, e.g. pesticides, drugs
- Wide boiling poin range
- High molecular weight
What are columns made of?
Can be metal, glass, fused silica, aluminium clad, inert metal
- Now fused silica is mostly used in capillary columns
What are some column types?
Packed (preparative)
- Glass or stainless steel
- Internal diameter 5um
- Larger capacity, low resolution
Capillary (analytical)
- Thin fused silica
- 250um ID
- Smaller capacity, high resolution
- High seperation efficiency
- Easily overloaded
What forces act on the stationary phase for retention?
Van der Waals, dipole-dipole and H-bonding
How is polarity utilised in stationary phases, with regard to analytes polarity?
Polar phases for polar components
Non-polar phases for non-polar components
What is meant by film in capillary columns and what does it affect?
Amount of stationary phase coating
Affects retention and capacity
What are some consequences of thicker films?
Increases retention and capacity, but increase runtimes, higher bleeds and lower temperature limits
What types of analytes are used with thicker films?
Low boilers, gases, solvents and volatiles
When would a thin film be appropriate and how does a thin film affect the GC?
Useful for high boilers, have high efficiency, lower bleed, faster run times and higher temperature limits, however they have limited retention.
Standard capillary film thickness is?
A. 600nm
B. 0.35um
C. 15nm
D. 0.25um
D
What is meant by column capacity?
Max amount that can be injected without significant peak distortion
What affects column capacity?
Film thickness, temp, internal diameter, stationary phase selectivity
What are some consequences of exceeding column capacity?
Peak broadening, asymmetry, tailing
When is a shorter column more appropriate than longer columns?
Shorter is more appropriate for screening few analytes, whilst longer column are appropriate for complex mixtures (with closely eluting peaks)
What are some consequences of a short column length (<15m)?
Faster runtimes, higher efficiency seperations, good for high Mw compounds
How is resolution affected by length of column?
Resolution decreases as length decreases.
Think back to theoretical plates
What are some consequences of a longer column length (>50m)?
Higher resolution, suitable for low boilers, less active samples or complex temperature ramps
- BUT with longer run times
What is the typical standard length?
A. 45m
B. 60m
C. 30m
D. 20m
C
What is the best combination of column features for a low boiler?
Long column length with a thicker film
- volatility and activity of the sample needs to be considered for specifics
Compare the difference between using a smaller to a larger interal diameter?
Compared to larger internal diameter - A smaller internal gives better resolution for early eluting compounds.
- The smaller ID means the sample capacity is lower meaning that its easily overloaded.
Larger ID has a greater dynamic range and has sufficient resolution for complex mictures
How is flow rate affected by internal diameter?
Decreases with smaller ID
How is seperation efficiency measured in GC?
Measured using theoretical plates (N)
- Large N = more efficient
- Think back to HETP
What is significant about the column oven?
It is programmable: meaning start at low themp and gradually ramp to high temp
What are the benefirs of temp programming in column oven?
- Produces more constant peak width
- Better sensitivity for componetns retained
- Improved chromatographic resolution
- Peak refocusing at head of column
What are some common detectors in GC?
- Thermal conductivity
- Flame ionisation
- Electron capture
- Thermionic
- Mass spec
Match the detector with application
A. Thermal conductivity B. Flame ionisation C. Electron capture D. Thermionic E. Mass spec
- Drugs, steroid hormones, vitamins, metabolic products
- Universal, carbon monoxide
- Hydrocarbons, ethanol, methanol, acetone, isopropanol
- Nitrogen- and phosphorus-containing compounds
- Chlorine containing compounds
A2 B3 C5 D4 E1
Describe the sample journey in GC-MS
- Sample injected into column to seperate molecules
- Effluent from GC passes through transfer line into the ion trap/ion source
- Molecules undergo electron/chemical isolation ration
- Ions accelerated and detected according to their m/z ratio
- Electron multiplier produces a signal proportional to ions detected
What ionisation techniques are appropriate for non-volatile and volatile analytes?
Volatiles
- electron impact (EI): hard ionisation
- Chemical ionisation (CI) - soft ionisation
Non-volatiles
- Direct infusion/electrospray (ESI)
- Matrix assisted laser adsorption (MALDI)
Describe electron impact.
EI (direct/hard ionisation)
- Gas molecules enter source through heated probe or GC column
- Beam of 70 eV electrons from heated filament bombard molecules forming Molecular+ ions that fragment in unique reproducible way to form a collection of fragment ions
- EI spectra can be matched to library standards (ONLINE collections)
How can you enhance the ionisation efficiency in EI?
Using magnets to increase effiency of collision
What is chemical ionisation (soft)?
- Detect molecular ions (because less fragmentation)
- Positive and negative modes
An indirect ionisation technique, because bombarding electrons collide with reagent gases first to produce reactant ions.
- Reactant ions are formed and these collide with sample molecules to form ions
- Reagent gases - methane, ammonia
What is an electron multiplier?
This is a detector made up of dynodes.
- Discrete dynode or continous dynode EM
Results in digitised and can be further processed and displayed on the PC
Advantages of GC-MS
- Good for small, volatile, non-polar, thermally stable analytes
- Screening application in unknowns
- Sensitivity and resolution reproducible
- Easy to operate
- High speed analysis
- Low cost due to low maintenance
- Highly robust reproducible mass spectra (EI)
Limitations of GC-MS
- Analyte must be volatile or made volatile by chemical derivitisation
- Analyte or its derivative should be thermally stable
- Sample prep generally takes longer than for other methods
- Large sample volume usually
- Lack of direct sample analysis
- Molecular ion is often lost when using EI
What are some common issues and what do they signify?
- Impurities contaminate gas line and break them down - best to use a gas filter
- Degradation of injection port septum due to high temp can sometimes be seen and may impair separation/detection
- Column bleed can occur
Match the following mass analysers to resolutions?
A. Ultra high resolution
B. High resolution
C. Low resolution
- Time of flight (TOF)
- Quadrupole, ion trap
- Ion cyclotron resonance (ICR)
A3
B1
C2
Describe the GC-MS ion trap.
Ionise analytes within the ion trap using energetic electrons
- Store ions and continue to analyse until the optimum trap capacity is reached
Increased voltage on the ring electrode of the ion trap to scan ions out of order from low to high mass - called the EI/MS Scan function
Store the mass- intensity information as a mass spectrum
In maximum capacity, how is the increasing of voltage on the ring electrode on the ion trap?
Scans ions out in order from low to high mass
GC-MS-TOF is applicable in what part of clinical biochemistry
Drug scanning
How is m/z measured using TOF?
Utilises the time taken by ions to pass along an evacuated tube as a means of measurering m/z
- Seperation is like a sprint race
What s the order of m/z arrive at the detector?
Smallest m/z arrive with increasingly m/z following
How is mass measured in poor resolutions?
We get broader peaks so we use an average mass.
What is meant by full width at half maximum (FWHM)? and how does it relate to resolution?
This is a measure of the width of a peak used as a representative of resolution.
Resolution can be calculated using = maximum resolution/change in maximum width of the peak
How does resolution impact mass accuracy?
As resolution increase the parts per million error decreases. Meaning mass accuracy increases with resolution
What are some advantages to using TOF for GC-MS?
Shorter time frame - several thousand spectra in one second.
- Leads to excellent reproducibility and better signal to noise characteristics
Continous acquisition of spectra
- Increased selectivity of correct mass
- No ion loss
- Whole mass range analysed at high resolution + sensitivity
How are isotopes determined using mass spec?
Isotopes can be distinguished since ions are seperated based on single atomic mass unit
Match the following
A. 79Br:81Br
B. 35Cl: 37Cl
- 3:1
- 1:1
A2
B1
- Intesity ratios determine the natural abundance.
M+1 peaks are often seen in the presence of what…
13C in the sample
How is drug of abuse screening done?
It uses both immunoassay and GC-MS
- The immunoassay act as the first screening method due to the rapid analysis time, but confirmation using the GC-MS and commercial libraries of drugs and metabolites are required
- Confirmation is due to the large false positive is due to interferances causing cross-reactivity
Why are the metabolites more useful in monitoring patients use of prescribed drugs?
Only the metabolite comes out in the urine so would show an indication if they have spiked the urine with small amounts of the drug to cover up non use
What are some markers of potential heroin use?
Absence of 6-MAM and codeine does not exlcude heroin use
6-MAM detected in urine for shorter period of time than morphine
Low conc of codeine - contaminant present in steel heroin due to absence of acetylcodeine in opium
What are organic acidurias?
Used as a diagnostic tool. Organic acidurias are characterised by the excretion of non-AA organic acids in urine due to metabolic defects relating to carbohydrate, FA and NA.
How is organic acdiurias examined using GC-MS?
Firstly the organic acids are extracted from acidified urine. Then derivitised using TMS.
- Using GC-MS analysis the total ions are used to produce a full spectra, which can by quantitate levels
What biochemical markers are used to determine if some one has medium-chain acyl-CoA dehydrogenase deficiency (MCADD)?
Symptomatic individuals: medium-chain dicarboxylic acids elevated, while ketones are low
Acute episodes:5-hydrozy hexanoic acid, hexanoyl glycine, phenylpropionyl glycine and suberyl glycine
MCADD is disorder of FA oxidation which impairs the bodys ability to break down medium-chain FA into acetyl CoA
Consequences of MCADD?
Hypoketotic hypoglycaemia
Metabolic acidosis due to production of lactic acid and glutaric acid
What is 2-hydroxyglutaric aciduria?
Mutation in functional enzyme breaking, causing D- or L-2-hydroxyglutarate to build up in cells
- Build up can lead to brain cell damage and cell death
