Introduction to Gas Chromatography Mass Spectrometry Flashcards
What are Gas Chromatography and Mass Spectrometer?
Gas Chromatograpy
- Separation technique for volatile organic compounds
- Can be used as a qualitative or quantitative tool
- Pharmaceuticals, environmental pollutants, drugs, metabolic compounds
Mass Spectrometer
- Used as the detector
How is the sample used for Gas Chromatography Mass Spectrometer?
- Usually a mixture of several components
- Sample usually introduced as a liquid
- Components of interest (analytes) usually in low concentrations (<1% to ppb levels)
- Samples dissolved in volatile solvent
What are key components of GC-MS?
Hardware to introduce the sample
- Injector
Technique to separate the sample into components
- Column
- Oven
- Carrier Gas: Nitrogen or Helium (1-2 mL/min)
Hardware to detect the individual components
- Detector
Data processing
What is the seperation process?
- Sample is introduced into system via hot, vaporising injector. Typically 1uL injected
- Flow of “Carrier Gas” moves vaporised sample (i.e. gas) onto column
- Column is coated with wax type material with varying affinity for components of interest
- Components are separated in the column based on this affinity.
- Individual analytes are detected as they emerge from the end of the column through the detector
How does the Chromatogram appear?
- Different analytes have different affinity for the column and so elute at different times – rt
- Response relative to amounts – qual and quant dep on application
- Detectors number of detectors – but for this MS, also FID (alcohols), TCD (CO
What is a GC step by step?
- Carrier Gas
- Injector
- Column (Capillary, Stationary Phase)
- Oven
- Detectors - Mass Spectrometer
What is the carrier gas?
Inert gases such as Helium or nitrogen
- Choice dictated by detector, cost, availability
- Pressure regulated for constant inlet pressure
- Flow controlled for constant flow rate
- Chromatographic grade gases (high purity)
What is the process of Injection of the sample?
- A GC syringe penetrates a septum to inject sample into the vaporization chamber
- Instant vaporization of the sample, 280 °C
- Carrier gas transports the sample into the head of the column
- Purge valve controls the fraction of sample that enters the column
What is the purpose of Injection?
- Deposit the sample into the column in the narrowest band possible
- The shorter the band at the beginning of the chromatographic process (tall narrow peaks) gives maximum resolution and sensitivity
- Therefore type of injection method and operating conditions is critical in obtaining precise and accurate results
What is a split injection?
Mechanism by which a portion of the injected solution is discarded. Only a small portion (1/1000 - 1/20) of sample goes through the column
- Used for concentrated samples (>0.1%)
- Can be performed isothermally
- Fast injection speed
- Injector and septa contamination not usually noticed
What is a splitless injection?
Most of the sample goes through to the column (85-100%)
Used for dilute samples (<0.1%)
- Injection speed slow
- Should not be performed isothermally
- Solvent focusing is important
- Controlled by solenoid valve. Requires careful optimisation
What is an on column injection?
All of the sample is transferred to the column. Needle is inserted directly into column or into insert directly above column
- Trace analysis
- Thermally labile compounds e.g Pesticides, Drugs
- Wide boiling point range
- High molecular weight
What is a Large Volume Injection nd the steps involved?
To enhance sensitivity in Environmental applications.
- Uses 100µL syringe: Inject up to 70 µL
- Very slow injection with injector temperature a few degrees below solvent boiling point, split open, flow at about 150 mL/ min
- Solvent vents out of split vent, thus concentrating the analytes
- Close split
- Fast temperature ramp to top column temperature +20°C
- Column programming as per sample requirements
What are types of Columns?
Packed (Preparative)
- Glass or stainless steel
- 1-5m length and 5µm ID
- Larger capacity, low resolution
Capillary (analytical)
- Thin-fused silica
- 1—100m length, 250µm ID
- Smaller capacity, high resolution
- Higher separation efficiency
- Easily overloaded by sample
What are characteristics of a capillary column to consider?
- Length (10m - 100m)
- Internal Diameter (0.1mm - 0.53mm)
- Liquid Stationary Phase
- Film Thickness (0.1um - 5um)
- Polarity (non-polar - polar)
What are advatages of shorter and longer column lengths?
Shorter (<15m)
- Screening samples with few analytes
- Faster runtimes, higher efficiency separations, good for high MW compounds
- Resolution decreases as length decreases
Longer (>50m)
- Separation of complex mixtures with closely eluting peaks
- Higher resolution as length increases, suitable for low boilers, less active samples or complex temperature ramps
- Slow run times
What are advatages of shorter and longer internal diameter?
Smaller ID
- Good resolution of early eluting compounds
- Lower sample capacity
- Limited dynamic range
- Easily overloaded
Larger ID
- Have less resolution of early eluting compounds
- Increased sample capacity
- Sufficient resolution for complex mixtures
- Greater dynamic range
What are characterisitics of Stationary Phases?
- Choice of phase determines selectivity
- Hundreds of phases available. Many phases give same separation
- Same phase may have multiple brand names. Stationary phase selection for capillary columns much simpler
- Like dissolves like: Use polar phases for polar components and Use non-polar phases for non-polar components
What is the Film thickness?
- Amount of stationary phase coating
- Affects retention and capacity
- lStandard capillary columns typically 0.25µm
What are the differences found between using thicker films and thinner films?
- Thicker films increase retention and capacity but typically have slower runtimes, higher bleed and lower temperature limits. Used for low boilers, gases, solvents and volatiles.
- Thin films are useful for high boilers, have high efficiency, lower bleed, faster run times and higher temperature limits, however they have limited retention.
What is column capacity?
The maximum amount that can be injected without significant peak distortion
How does column capacity increase?
Column capacity increases with :
- Film thickness
- Temperature
- Internal diameter
- Stationary phase selectivity
What happens is the column capacity is exceeded?
If exceeded, results in:-
- Peak broadening
- Asymmetry
- Tailing
What is a common issue is encoutered in GC-MS?
Column Bleed
- One of the most common issues encountered in GC-MS
- Stationary phase degrades over time; components pass through the column contributing to the signal, usually minimal and of no consequence
- Excessive bleed is often caused by something damaging the stationary phase of the column, oxygen from a leak or some component in the sample
- Can result in a higher baseline signal, peak tailing, poor reproducibility, presence of unusual fragments at high levels and ghost peaks
- Check for leaks, use inline oxygen filters on carrier gas, review sample preparation and loading requirements
What are advantages to temperature programming of the column oven?
Start at low temperature and gradually ramp to higher temperature
- More constant peak width
- Better sensitivity for components retained longer
- Improved chromatographic resolution
- Peak refocusing at head of column
How does high and low oven temperature result in?
- High - co-elution, poor resolution but faster separation
- Low - longer elution times with improved resolution
Compromise temperature to optimise program
What are the types of detector and their use?
- Thermal conductivity: Universal, Carbon monoxide
- Flame ionisation: Hydrocarbons, Ethanol, methanol, acetone, isopropanol
- Electron capture: Chlorine containing compounds
- Thermionic: Nitrogen- and phosphorus-containing compounds
- Mass spectrometer: Drugs, steroid hormones, vitamins, metabolic products
What is Mass Spectrometry?
- Mass spectrometry is a micro-analytical technique used to obtain information regarding structure and molecular weight of an analyte
- In all cases some form of energy is transferred to analyte to cause ionisation
- In principle each mass spectrum is unique and can be used as a “fingerprint” to characterise the sample
- Destructive method - sample consumed during analysis
What is GC-MS?
GC-MS is a hyphenated technique that combines the separation ability of the GC with the detection qualities of mass spectrometry
What is the process of GC-MS ?
- Sample injected onto column via injector
- GC then separates sample molecules
- Effluent from GC passes through transfer line into the Ion Trap/Ion source
- Molecules then undergo electron /chemical ionisation
- Ions are then analysed according to their mass to charge ratio
- Ions are detected by an electron multiplier which produces a signal proportional to ions detected
What are components of a Mass Spectrometer?
- Sample introduction
- Source (ion formation)
- Mass analyzer (ion separation) - high vacuum
- Detector (electron multiplier tube)
What are the fdifferent sample introduction/sources?
Volatiles
- Electron impact (EI) – hard ionisation
- Chemical ionization (CI) – soft ionisation
Non-volatiles
- Direct infusion/electrospray (ESI)
- Matrix Assisted Laser Adsorption (MALDI)
Elemental mass spectrometry
- Inductively coupled plasma (ICP)
- Secondary Ion Mass Spectrometry (SIMS)
How does Electron Impact take place?
- Gas-phase molecules enter source through heated probe or GC column
- 70 eV electrons bombard molecules forming M+* ions that fragment in unique reproducible way to form a collection of fragment ions
- EI spectra can be matched to library standards
How does chemical ionisation take place?
CI (soft ionization)
- Higher pressure of methane leaked into the source (mtorr)
- Reagent ions transfer proton to analyte
What does an electron multipier do?
- Passes the ion current signal to system electronics
- Signal is amplified
- Result is digitised
- Results can be further processed and displayed
What is the advanatge of GC-MS?
- Best technique for volatiles, non-polar analytes
- Highly robust reproducible mass spectra (EI) - Universal fragmentation conditions
- Libraries. Transferable among instruments (universal) and commercially available mass spectral libraries
- Automated identification by mass spectra through deconvolution
What is deconvolution?
Deconvolution is the process of computationally separating co-eluting components and creating a pure spectrum for each component.
What are limitations of GC-MS?
- Analyte must be volatile or made volatile by chemical derivatisation
- Analyte or its derivative should be thermally stable
- Sample preparation is generally longer
- Larger sample volume
- Lack of direct sample analysis
- Molecular ion is often lost when electron impact (EI) ionization is used
