In order to determine contaminant levels in the blubber samples under examination, we used Gas Chromatagraphy--an analytical technique which separates the compounds within a sample, over time. In GC, different chemicals and pollutants are separated-out or eluted at different, known times. This allows the identification and quantification of various contaminants in our samples.
Before we could test our blubber samples by gas chromatagraphy, however, we had to go through several purification stages.
Below is the general methodology we used.
The various stages in our high-resolution gas chromatographic analysis, included:
In order to remove the contaminants from the blubber samples, we used an extraction protocol which put them, and the lipid material, into solution. First we weighed each sample for later reference, and cut the blubber samples into small pieces. These pieces were then homogenized with a mortar and pestle in sodium sulphate, to remove any water.
The homogenate was transferred to a cellulose extraction thimble, and covered with glass wool. We ran these samples in a Soxhlet extractor for 4 hours; reflux events occurred every 7-10 minutes.
A soxhlet extractor.
What is a soxhlet extractor? The sample, which has been homogenized in sodium sulphate, is covered with glass wool and contained in a porous cellulose thimble. The thimble is placed in the extraction tube, which itself sits on a flask containing an organic solvent (like hexane).
Diagrammatic representation of a soxhlet extractor.
The solvent is boiled, and its vapor travels upward through the extraction tube into the condenser tube. The cool water flowing around the outside of the condenser tube condenses the vapor, which then drips into the thimble, containing the sample.
Because the contaminants and lipid are soluble in organic solvents, they move into the condensed solvent as it accumulated in the thimble. The solution, now containing the contaminants and dissolved lipid, build up in the thimble. Once the liquid reaches the level of the bypass arm, it is siphoned back into the flask. This continuous condensation, buildup, and siphoning is known as the reflux event.
The advantage of the soxhlet is that once the contaminants and lipid material are brought into solution, and siphoned back into the flask, they stay in the flask--so that the sample in the extraction thimble is continuously re-exposed to fresh, heated solvent--thus greatly increasing the extraction rate.
After extraction, we reduced the volume of solvent in our samples by rapid rotary evaporation down to about 2mL, using a Rotovap.
Next we separated the contaminants from the lipid material, by fractioning them using GPC (gel-permeation chromatography). GPC is a size-exclusion technique, as it works by slowing the flow of smaller molecules by trapping them in pores which are too small for large molecules--thus the larger compounds are excluded (see below).
The GPC apparatus.
Separation into two fractions was acheived in our study--the first used for lipid weight determination, and the second for PCB and OC pesticide determination and quantification. Upon obtaining the first fraction (containing the much larger lipid molecules), we reduced the solvent volume to approximately 2mL, and allowed it to completely evaporate over a few days in pre-weighed vessels--leaving a lipid-only residue which could be weighed.This is important later, because as mentioned previously, contaminants such as PCBs and OCs are associated with lipid; because, however, different organisms have different proportions of lipid in their tissues (including blubber), we divide the concentrations by the amount of lipid to get a standardized measure -- allowing comparison with other data..
What is GPC? Gel permeation chromatography (GPC) is a technique used to separate molecules based on size differences. It is also referred to as gel filtration chromatography, or size exclusion chromatography.
Molecular separation occurs in the GPC column. Inside the column there are two phases:
a) The stationary phase consists of an inert gel of porous beads-- so-called because it does not move in the column
b) A mobile phase, which is the eluent or liquid which is run through the column.
Diagrammatic representation of Gel Permeation Chromatography.
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The sample is loaded onto the column with the eluent, which run through the column together. Relatively smaller molecules like PCBs and OCs are able to pass through the pores in the beads, however, relatively larger molecules like lipids cannot. Larger molecules are said to be excluded (from passing through the pores in the beads) and as a result they move more quickly through the column (and leave the column sooner) than do smaller molecules.
By collecting the liquid that leaves the column - the eluant - the researcher achieves a successful fractionation of the sample--separating in our study, lipids and contaminants.
The final stage of sample clean-up in our procedure, was sub-fractionation using silica-gel columns. As discussed above, the first fraction from our GPC separation was used for determining lipid weight. The second fraction, however, is used in this stage -- sub-fractionation -- for contaminant analysis.
Here, the contaminant fraction from GPC separation was rotovapped to ~1mL and run on a silica column.As the name suggests, the stationary phase is silica - a finely divided white solid; the mobile phase, like in GPC, is a liquid that runs through the column.
Diagrammatic representation of a Silica column.
Molecular separation in silica gel chromatography is based upon the polarity of a particular molecule. Polar molecules, like amino acids, will adsorb or stick to the silica and these are left behind in the column as the non-polar molecules are collected in the eluant. By changing the polarity of the mobile phase a researcher can remove molecules adsorbed to the silica.
We recovered two fractions for analysis. The first (A) fraction contained:
PCBs, aldrin, HCB, heptachlor, mirex, and most of the p,p' DDE (ie. primarily PCBs)
while the second (B) fraction contained:
heptachlor epoxide, HCHs, chlordanes, dieldrin, p,p' DDT, p,p' DDD, methoxychlor, and a small proportion of p,p' DDE (ie. primarily OC pesticides).
Each of these two fractions were later analysed by high resolution gas chromatography on a Varian model 3500 HRGC with splitless injection, using electron capture detection (ECD). The results of which are explained elsewhere.
Gas chromatography is one of the most widely used methods to determine the chemical make-up of a complex, volatile mixture. In order to perform this technique a researcher uses a gas chromatograph or GC.
Diagrammatic representation of a generalized gas chromatograph.
Before gas chromatography can be performed, the sample must be disolved in a volatile solvent like iso-octane. Gas chromatography begins by injecting a very small amount of the sample (e.g. 1 uL) into the GC. This small amount of sample is then volatilized by the heat of the oven and carried into the column by an inert gas like helium (He).
It is inside the column where the separation of the individual chemical components takes place. There are two "phases" inside the column which control actual separation: the mobile phase and the stationary phase.
The mobile phase is simply the carrier gas, helium, and is so-called because it moves through the column.
The stationary phase is some non-volatile liquid like silicone rubber, wax, or oil, and is so-called because it does not move through the column.
Some chemicals have a relatively high affinity for the mobile phase, and some have a relatively high affinity for the stationary phase, but each chemical is unique.
These affinities are based on the molecular weight of the chemical and its polarity. If a chemical has a high affinity for the mobile phase, it will tend to move quickly through the column. If a chemical has a high affinity for the stationary phase it will tend to move slowly through the column. Since each chemical has unique degrees of affinity, each will eventually leave the column at a different time.
Inside the GC
The column itself is a long thin tube which is coiled so that it may be contained easily within the oven. In many of the pioneering studies which used gas chromatography, columns were 2 to 3 metres long, with a diameter of 2 to 4 mm, and made of glass or metal. These columns were packed, that is they contained many small spherical particles known generally as "solid support." The solid support was coated with a liquid stationary phase and it provided a great deal of surface area for exposure to the mobile phase.
More modern studies have turned away from this method and use the higher-resolution capillary column. Capillary columns are 10 to 60 metres long, have diameters ranging from 100 to 320 um, and are made of fused silica. Capillary columns have the stationary phase coated on the inside of the tube.
In order to determine what and when chemicals leave the column, (and therefore how much of each chemical is present), the GC is equipped with a detector. Many types of detectors exist, but for our purposes an Electron-Capture Detector (ECD) was used. This detector was chosen because it is especially useful for detecting halogenated compounds like PCBs and pesticides.
The ECD works by passing the chemicals and a different gas (e.g. nitrogen) over a radioactive source as the chemicals leave the column. The radiation produces a series of events at the sub-atomic level which result in a change in electrical potential between two electrodes when chemicals of interest pass between them.
The detector reports to a recorder, and a "gas chromatogram" is produced for the researcher to inspect.
A representation of a chromatogram.
The chromatogram shown above is representative of the PCB fraction from sample #2539. The location of the peaks along the x-axis of the graph correspond to the time at which certain chemicals left the column and were detected by the ECD. These elution times are considered constant for specific chemicals and contaminants. This constancy facilitates the individual identification of specific contaminants
For example PCB congener 138 left the column 73.05 minutes after the method began; congener 194 left later, at 88.24 minutes.
While the height of a particular peak directly corresponds to the electrical charge induced by a chemical at the electrodes of the ECD, the area under the peak (that is its size) can be used to calculate how much of a particular chemical there was in the sample: the larger the peak, the more of the chemical was present.
The actual quantity of each chemical is determined with the help of computer software. Essentially, the computer takes the information given on the chromatogram and compares it to values for known concentrations--thus calibrating concentration information against standard reference data. Ultimately such software provides the concentration of each chemical in the sample.
Click here to see the results of our study.
Click here to learn about different contaminants.
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