Maissey Flashcards
Aims of Toxicity ID Evaluation
improve control of chronic as well as acute testing
properly document effluent toxicity
ID the causes of toxicity
provide info to enable reduction or elimination of effluent toxicity
gives fantastic data for ecological relevnce
considers bioavailability
LIMITED in ability to ID toxicant
In vivo testing
Uses whole living organisms to assess toxicological effect
EDA
uses biological effects tp narrow down potential toxicants
• EDA extracts right from the outset (organic solvent extract, solid
phases extraction, etc.) with a focus on organic compounds
• This is to the detriment of potential non organic toxicants.
• By utilising pre-concentrated and purified samples, the whole
sophisticated array of instrumentation available to analytical
chemistry can be utilised.
• This Neglects bioavailability, data of minor ecological relevance.
•In vitro testing used to narrow the range of possible compounds
(quicker and cheaper testing).
In vitro testng
Outside of the living organism Looks at various known biological outcomes of toxic materials e.g. • Endocrine disruption • Estrogenic activity • Androgenic activity • Dioxin and dioxin-like compound activity • Cytochrome P-450 activity • Mutagenicity • Bioluminescence • Growth factors
TIE:
What chemicals are causing the observed toxicity?: maintain sample integrity by using
three phases: characterisation, identification and confirmation.
EDA :
What biological effects can we observe to narrow down the potential list of toxicants:
Extract using organic solvents to isolate the likely toxicants and then performs biotesting.
Bioavailability
what is it and what is it dependent on
What fraction of the toxicant exists in a form that is able to be
absorbed, metabolised or otherwise integrated into an organism
Bioavailability is significantly dependant on:
• Polarity
• Speciation
• pH
• Ionic strength
• Sample matrix (what other chemical species exist in the sample)
• Temperature
• Chelation
Phase 1 testing - Typical Fractionation steps
• Filtration (removes particulates > 0.45 mm)
• Treatment of filtrate with C18 or XAD resin (removes non-polar organics)
• Organic-free fraction subdivided and treated with some of the following treatments…
• EDTA
• cation-exchange resin
• anion exchange resin
• zeolite column
• aeration
• pH adjustment
• treatment with thiosulfate (reacts with halogens, bleaches, etc.)
Remove non-polar organics (resin or chromatography column)
• Remove metals (add EDTA)
•Exchange cations or anions (swap metal cations plus NH4
+
for
something like sodium)
•Remove volatiles (e.g. hydrocarbons, ammonia, by bubbling air
through)
•Remove acidic or basic species (e.g. change pH)
•Remove small cations, ammonia, some reactive organics (zeolite
column)
• Remove oxidising materials like halogens and bleaches , etc.
(add thiosulfate )
Phase 2 testing - examples of follow-up from phase 1
If organics are implicated
• e.g. toxicity reduced after chromatography / inorganics not implicated
• Phase II : the stuff from the chromatography column can be eluted and
a reconstituted organics solution made up & tested separately
If zeolite, aeration and pH 2 treatment all reduce toxicity
• implies a volatile, basic species is the cause
• Phase II: test specifically for NH3 or for organic amines if no ammonia
found
Purpose of chemical
analysis in toxicology
Separate complex mixtures
• Identify individual chemical constituents
• Identify secondary pollutants
• Secondary pollutants are derivatives of
primary pollutants by either
decomposition or chemical reaction.
• Quantify individual and groups of chemical
constituents.
Fit for
Purpose?
• What will the chemical information be used for? This question is crucial in
determining which chemical tests could and can be applied.
• Timing considerations? Is a result required immediately, in a few hours or
days?
• What degree of confidence in the result is required? Different contexts
require varying degrees of quality assurance (QA) and quality control (QC) .
• What is the nature of the sample available?
• Is the sample divisible?
• Is the sample homogenous making a sub-sample representative or do multiple tests
need to be performed?
• Is there sufficient sample for multiple tests to performed?
• Can some or all of the sample be destroyed?
Overview of Chromatographic Techniques
• The mobile phase (which can be liquid or gas) carries the
sample across or through the stationary phase (solid or
viscous liquid).
• The attraction of each component of the mixture to either
the mobile or stationary phase will dictate how long it
takes to pass through the system, thus separating the
components of a mixture.
Solid phase extraction
Solid phase extraction is based on the need to partition a
liquid sample into two categories.
• One which contains a target group or individual
analyte – this remains on the stationary phase.
One that does not contain the target group/individual
analyte – this portion remains in the mobile phase.
• The species on the stationary phase can then be
extracted and analysed or used in toxicity tests
(usually EDA analysis)
• The mobile phase can then be used in toxicity tests
(usually TIE analysis)
Solid phase extraction (SPE) capsules
SPE columns are used for rapid solid phase
extraction.
Some example categories extracted with SPE:
• non-polar extractions (Lichrolut® RP18 and RP18e)
• polar extractions (Lichrolut® Si and Lichrolut® CN)
• Cation Exchange extraction (Lichrolut® SCX)
• mixed mode extraction (Lichrolut® TSC)
• non-polar extractions on a polymer phase
(Lichrolut® EN) which is especially well suited for
the extraction of pesticides and phenols in water
or drugs in body fluids.”
What is ion exchange?
The exchange of ions of the same charge between
an insoluble solid and a solution in contact with it:
enables cation and anions to be separated and
examined.
Anion-exchange resin: mobile anions (in sample) are
retained by cations bonded to the stationary phase
and replaced with single, known anion.
Converse mode of exchange is also available (cationexchange
resin).
Ion exchange resins
• Micro beads of an organic polymer substrate
• Targeted ions are trapped in pores triggering release of replacement
ions of equivalent charge.
Ion Chromatography
Stationary phase : anion-exchange or cation-exchange resin (polymer) Mobile phase : Aqueous solution of an electrolyte : ions in the mobile phase compete with analyte ions for ion-exchange sites on the resin this enhances separation.
Mobile Phases- ion chromatography
The aim of chromatography is to separate the minor constituents of the
sample. The mobile phase can help facilitate this.
• Aqueous solution of an electrolyte
• e.g. for separation of anions, using Na2CO3
(aq) as CO3
2-
ions compete with
sample anions for sites on the anion-exchange resin
• e.g. for separation of cations, using HCl (aq) as H+
ions compete with sample
cations for sites on the cation-exchange resin
Mobile Phases- ion chromatography
Retention depends on
• The charge and size of the ions (more highly charged ions retained longer)
• The type and concentration of the competing ions in the mobile phase (can
be varied)
• For some ions, pH will be very important e.g. carbonate, phosphate,
carboxylates
Gas Chromatography - GC
• Mobile Phase is a carrier gas. • Stationary Phase is a solid or liquid (supported on a surface). • Stationary phase is located in a (usually) long and narrow column. • Sample introduced near beginning of column where it is vapourised. • Detector senses compounds eluting from the column. • GC is most important analytical method for volatile compounds.
GC Applications
- hydrocarbons
- organochlorines
- pesticides
- drugs
GC stages
Mobile phase supply, sample loading, separation in heated column, detection, data analysis
Carrier Gas
• Gas must be inert with respect to sample.
• Gas must not be retained significantly by
stationary phase.
• Typical gases are He, H2
and N2
Injection port
has complex gas flows to make
injection ‘sharp’ and reproducible.
The injection port is always in a heated zone
of the GC and it is usually kept hotter than the
GC column
Syringes
• Syringes require good operator technique to be reproducible • Autosamplers are robotic injectors - very reproducible - up to 100 samples. 100 mL gas syringe 5 mL liquid syringe 1 mL liquid syringe
Split/ splitless injector
• To avoid overloading the column a
fraction of the injected sample is vented
to waste via the ‘split outlet’.
• The ‘split ratio’ (e.g. 40:1) is controlled by
software
Columns and GC Oven
• Capillary (most common) and packed column types
• Columns kept in a heated oven - high temperature
needed to keep compounds in vapour form (40 –
250 ºC )
• Column temperature greatly affects speed of
analysis and how compounds are separated.
• Temperature is very carefully controlled
• Isothermal or variable temperature programs can be
used
Column Temperature and Program
• Higher column temperatures speed up the analysis but are detrimental to separation of the more volatile components of mixtures. • A temperature program allows the separation to be speeded up progressively allowing the most volatile components to separate at lower temperatures first.
Isothermal elution Isothermal elution
Same column
temperature for entire analysis.
Poor separation for complex
mixtures and takes time!
Temperature gradient elution:
improves resolution while also
decreasing retention time
Packed Columns for GC
- Usually made from 3-6 mm diameter tubing of about 1 to 3 m length
- Packed columns are inexpensive to make, have high capacity but relatively low resolution
- Require flow rates of 20-50 mL/min
Capillary (Open Tubular) Columns
• Long very narrow fused silica tubes typically 0.3
mm diameter and 10-50 m long
• Stationary phase coated on the inside wall of
the column in one of three ways
• Wall coated (WCOT)
• Support coated (SCOT)
• Porous layer (PLOT)
Order of elution is mainly determined by
volatility
- Least volatile = most retained
- Polar compounds (ex: alcohols) are the least volatile and will be the most retained on the
GC system
Detectors
• Detectors sense the elution of compounds (other than the carrier gas).
• They create an electrical signal that is recorded versus time to create the
chromatogram.
• Usually the area under a peak increases in proportion to the amount of a
particular substance eluting.
• Main detectors are:
• Flame Ionisation (FID)
• Thermal Conductivity (TCD)
• Electron Capture (ECD)
• Flame Photometric (FPD)
• Mass Spectrometer (MS)
Flame Ionisation Detector
• Analyte mixed with air and H2 • Mixture ignites in flame • Hydrocarbons generally have a response factor equal to the number of carbon atoms in their molecule. • Linear response to mass of carbon atoms in analyte (7-orders) • Can detect 10-13 g/s • Insensitive to inorganics • Cheap and easy to use
GC-Mass Spectrometry
• The mass spectrometer obtains a
characteristic fingerprint (a mass spectrum) of
compounds as they elute from the column.
• This is an extremely powerful combination
that is highly sensitive and selective and
allows for the identification of unknown
sample components.
• Area of peaks for unknowns and standards are compared in the
software and concentrations are reported.
• MS has very high sensitivity as a detector for GC
• On-line libraries are available for identification of experimental mass
spectra - can identify unknowns often with a high degree of certainty
IC Advantages
- Replaces several ‘wet chemistry’ techniques – faster and more sensitive
- Allows simultaneous analysis of many groups of target species i.e. alkali, alkaline earth and ammonium
- Relatively cheap (cheaper than HPLC) and easy to run
facts HPLC
- Replaces several ‘wet chemistry’ techniques – faster and more sensitive
- Allows simultaneous analysis of many groups of target species i.e. alkali, alkaline earth and ammonium
- Relatively cheap (cheaper than HPLC) and easy to run
Most common detectors for HPLC and IC in
environmental toxicology
• Conductivity meter: Any target analyte with an ionic charge (ions)
(this is the most popular detector for ion chromatography)
• UV-Vis spectrophotometer: If target species can absorb in UV or Vis
(many can and this is a cheap and easy method of detection)
• Mass Spectrometer: Especially suitable for large organic molecules,
especially when identification of secondary pollutants is required –
this is a more expensive and skilled method of detection
applications of GC
• Volatile and some semi-volatile organic compounds
• Hydrocarbons
• Organohalides
• Pesticides (though with caution – HPLC also popular for these)
• Inorganic volatile species can also be analyzed in GC but in aqueous environmental samples these
tend to be in ionic form for IC/HPLC is preferable (also they tend to be explosive and dangerous to
handle)
advantages of GC
Advantages
• highly sensitive and selective and allows for the identification of secondary pollutants (when
coupled with mass spectrometry)
• GC-MS considered the “gold standard” for many standard analytical methods of Environmental
VOCs
• Cheaper than HPLC
Most common detectors for GC in environmental
toxicology
There are LOTS of different detectors for GC. Theses are the two most common:
• Flame ionisation (FID) : Cheap and less skill to interpret, used in many
routine analysis methods
• Mass Spectrometer (MS): Especially suitable for large organic
molecules, especially when identification of secondary pollutants is
required – this is a more expensive and skilled method of detection
Applications of UV-Vis spectrophotometry in
environmental toxicology chemical analysis
Applications
• Used for the quantitative determination of organic and inorganic compounds in solution (both ionic
and non ionic)
• Generally need to know the identify of the target compound for effective analysis so not good for
qualitative analysis as a stand alone method- Very often used in tandem with HPLC
• Not good for primary analysis of complex mixtures – potential for absorption overlap
Advantages of UV Vis spec
- Very quick and relatively cheap instruments.
- Low level skill required to use.
- Portable instruments available (in situ testing)
Applications of ICP-MS/AES and AAS in
environmental toxicology chemical analysis
Application
• Typical elements of interest are heavy metals such as : Cr, Pb, Cd, Hg, As in envirotox
context
• Most of the elements in the periodic table can be assessed – organic molecules
can be assessed through combusted but not common in enviro-tox.
Advantages of ICP-MS
- Simultaneous analysis of all metals
- Can detect low or very low concentrations (g L-1 or below…ppb to ppt!!)
- Large linear range – can measure high and low concentrations reliably
- Possible to detect isotopic composition of elements
Important limitation of elemental analysis
Speciation
Does not give information about the ionic or molecular species present in the sample
Compare the usefulness of the information….
- xxx amount of Hg in the water
- xxx amount of the highly toxic methylHg in the water
-Six different forms of Hg were found, consisting of….
Speciation is important in toxicology, not just total concentrations!
elemental analysis can be modified to give speciation information but this is not easy (beyond scope of course).
Applications of FT-IR in environmental
toxicology chemical analysis
• Solvents absorb in the IR (c.f. water above) and solution-state spectroscopy is difficult with
environmental samples. (Not suitable for most TIE processes)
• Gas phase spectra can be collected in the lab (in gas cells) or in the field - where the atmosphere or
the gas from an emission stack is the sample (open-path monitoring - see www.opsis.se )
• IR spectroscopy is directly relevant to understanding greenhouse gases (see later section of course)
and is used as a tool in investigating gas-phase reaction mechanisms
• FT-IR is also very useful as a screening method for assessing unknown samples since it is quick and
needs little sample pre-treatment (identify types of plastics; organic vs. inorganic materials)
• Special accessories allow components in aqueous samples to be measured (though only at relatively
high concentrations, excluding most environmental analysis applications)
• Mostly qualitative analysis, quantitative analysis is more difficult (there are better methods, like
electric sensors)
Beer’s Law Calibration
spectrophotometric
techniques.
Calibration
needs a blank and a series of standards
• In any calibration, the standards should have a
composition that matches the sample, as far as
possible. This is to minimise matrix effects. This is
especially important for environmental samples
• Calibrations should bracket the sample concentrations
• Extra reagents may be needed to control pH or reduce
interference problems.
