hyphenated techniques Flashcards
what are they
sample introduction: GC, HPLC and CE
interface: EI, CI, ESI, APCI
analyser: quadrupole, triple quad, ion trap, FTICR, TOF
(Straight from CE to FTIR, PDA and NMR)
what is hyphenated techniques
qualitative and quantitative analysis of large mixtures of compounds
target compound analysis and extremely low concentration (> 1 fg, 10E-15 g)
rapid and comprehensive structural elucidation of known compounds
pros of hyphenated techniques
shorter analysis time
higher degree of automation
higher sample throughput
better reproducibility
reduction of contamination - closed system
enhanced combined selectivity and therefore higher degree of information
huge range of applications
Gas chromatography mass spectrometry (GC/MS)
most mature - 20 years old
relatively cheap bench top systems (£30-40k)
large no. configurations
best option for mixtures of volatile and semi-volatile compounds
disadvantages: need for compound volatility and thermal stability
Liquid chromatography mass spectrometry (LC/MS)
most versatile
relatively cheap (£70-150k)
if it can be dissolved it can be analysed - choices of interfaces
disadvantages: more problematic then GC/MS but not overly so
GC x GC/TOF MS
extremely powerful technique for separating massively complex mixtures of volatile and semi-volatile compounds
compounds separated on long column (1st phase) and then pulsed onto the second short column (2nd different stationary phase)
requires an extremely fast detector and high end data system
4D data set very complicate - chemometric approach useful
LC/MS SPE/NMR
- 1D NMR, 5-10 micro g per compound, problems with solvent gradients
- 1D & 2D NMR, 5-10 micro grams per compound, problems with solvent gradients
- 1D & 2D NMR, about 1000 ng per compound, no solvent issues
Practical considerations: flow rates
GC and HPLC need to deliver flow at a suitable rate for the MS without compromising the vacuum
Practical considerations: blockages and coldspots
T-pieces and complicated interfaces are prone to blockages. systems utilising a GC are prone to coldspots
Practical considerations: maintaining a closed system
need to eliminate leaks that result in loss of performance and sample. systems utilising a GC need a gas leak detector
Practical considerations: multiple detector systems
the more complicated the hyphenated technique, the more likely it is to go wrong. the weaknesses of a technique are the sum of all of the component parts
Practical considerations: running costs
more complicated techniques command larger operating costs both in terms of consumables, spare parts and manpower for operation and maintenance
Practical considerations: data analysis and storage
some techniques produce extremely large and complicated data sets. suitable IT for manipulation and secure storage of the generated data are essential
data acquisition
- data sampled at time intervals and the sum (total absorption, total ion count) plotted against time
- series of pointed converted into a single trace giving chromatographic data, may be smoothed
- spectral data for every point used to construct the chromatographic data plane is retained and may be viewed at any point and manipulated whenever required. this is a 3D data block
- rate of collection is critical - too slow = data lost, too fast = too much data
- acquisition rate NEEDED determined by the sample and method of separation, rate POSSIBLE determined by analyser
- GC ( peak widths of 0.1-5 s) vs HPLC (10-60 s)
- TOF-MS (200 mass spectra s-1) vs FT-ICR_MS (1 mass spectra s-1)
Full-scan acquisition
- scan between two extreme values of m/z
- if width of chromatographic peak at 1/2h is 10s then scan time must be < 5s
- ensure at least 1 scan lies fully within a peak
- if scanning m/z 5-550 (range of m/z 501) this gives a dwell time of 0.01 s per mass (i.e. 5/501)
- least sensitive and least selective scanning mode
- within full scan mode sensitivity can be increased by scanning a smaller mass range (loss of data) or increasing scan time (lower resolution trace)
- uses : unknown samples, initial setup conditions, less complex mixtures
dwell time
time that a particular ion is being transmitted to the detector
selected ion monitoring (SIM)
- increase sensitivity and selectivity for known compounds - target compound analysis
- dwell time for three selected masses = 5/3
- so sensitivity increase of x166 (1.66/0.01), higher in practice
- signal:noise ratio is increased since noise is time averaged
- ions are less intense in other compounds therefore selectivity is increased
- loss of qualitative information - only 3 ions
- sensitivity increased by performing SIM at high resolution
- uses: environmental monitoring, drug detection, contaminants in food
sensitivity
the instrument response for an analyte over that of the background signal i.e. signal to noise ratio
selectivity
the extent to which a method can determine a particular analyte in mixtures without interferences from other components
Full scan acquisition vs SIM
full scan = see everything
SIM = only scans key ions of target compound
selected reaction monitoring (SRM)
- more sensitive and selective than SIM
- tandem mass spectrometer (triple quadrupole or quadrupole ion trap)
- select a specific precursor ion in MS 1
- allow ion to fragment by collision induced dissociation (CID)
- use MS 3 to scan for a prominent product ion
- multiple precursor and/or product ions may be selected but the name of the technique remains SRM
- the final signal obtained is dependent on a specific precursor and product ion (s) generated by fragmentation of a molecule
- increased signal:noise ratio under MS/MS conditions
- at expense of qualitative data
- uses: highly complex samples where the target compound co-elutes with many other compounds
data analysis
- generate large amounts of data (Gb)
- work required is largely determined by the goals of the analysis
i.e. complex mixture analysis: easier, preparative work and data analysis very time consuming vs
target compound quantification: more difficult to set up and perform but data analysis relatively easy and can even be automated
generating mass spectra
- electing a point on the TIC
- better to select a range
interfering background data
- sample a part of the TIC where there is no peak
- common for GC/MS -siloxanes constitute a permanent background
- other techniques also have their own backgrounds
- removal: subtracting a range of background from the range of internet we generate clean spectrum of the compound
quantification of target compounds
- use of internal standard introduced as early as possible, should be chemically similar to target compounds
- enables pre-analysis losses to be ignored
- similar ionisation characteristics
- isotopic analogue of the target compounds is the best possible internal standard
- conduct analyses with standards and vary. the amount of target compound
- construct calibration graph, best precision achieved with a straight line and a slope equal to unity
