Procedure 1. Capillary Conditioning Each day before testing, the capillary tubing must be was pre-conditioned.
First, it is flushed for ten minutes with MilliQ water. Next, it is flushed for thirty minutes with 0.1 normal sodium hydroxide (0.1N NaOH) to remove adsorbates and refresh the capillary surface. Finally, it is flushed for thirty minutes with the electrophoretic solution. After pre-conditioning, a blank is run, with only the electrophoretic solution as a sample, to establish a reference baseline. The capillary should be also conditioned before each analysis. This conditioning included flushing the capillary for one minute with MilliQ water, for two minutes with 0.1N NaOH, and for three minutes with the electrophoretic solution. The electrophoretic solution in the inlet and outlet buffer vials should be replaced after five runs. 2. Sample Analysis Samples are injected hydraulically with a built-in, automatic sample injector. Injections lasted for three seconds at a pressure of 30 millibars. First, a sample containing only methanol is analyzed to establish its migration time.
Next, a sample containing methanol and Sudan III is analyzed to determine the migration time of the SDS micelles.Once this information is established, samples of each PAH standard are analyzed individually to determine the migration times of the compounds. A second series of tests is conducted to verify the migration times established in the first set of tests. First, a sample of the benzo(a)pyrene standard is analyzed and its migration time was established. Next, a sample containing benzo(a)pyrene and fluoranthene is analyzed to determine the migration time for fluoranthene. The experimenter continued with this procedure, adding one PAH compound at a time to the mixture for each subsequent analysis, until ultimately a mixture containing all five PAH compounds is analyzed. Apparatus Instrument: Hewlett Packard HP 3D CE system.
A fused-silica capillary tubing of inner diameter 50 mm and an effective length of 56 cm was used as a separation column. The temperature in the capillary was kept constant at 25oC by a built-in temperature control system, and a potential of 15 kV was applied across the capillary.
Ion detection was carried out with a diode array detector at a sample wavelength of 254 nm, with an 80 mm band width, and a reference wavelength of 314 nm, with an 80 mm band width. Data were recorded and analyzed by Hewlett Packard analysis software.
Reagents 1. PAH compounds: acenapthene, anthracene, benzo(a)pyrene, fluoranthene, and napthalene. 2. Dimethyformamide (DMF) 3. Methanol 4. Sodium hydroxide 5. Sudan III 1-{[4-(Phenylazo)phenyl]azo]-2-naphthalenol 6. g-cyclodextrin (g-CD) Sodium dodecyl sulphate (SDS) 7. Phosphate-borate buffer solution (pH 7.0) 8. Electrophoretic solution: The electrophoretic solution is prepared by dissolving SDS, the micellar phase, and g-CD, a modifier added to increase selectivity, in the phosphate-borate buffer solution. The SDS solutions are filtered through 0.45 mm pore size membranes prior to use. 9. PAH standards: The PAH standard solutions are prepared by first dissolving the compounds in 1 to 3 milliliters of DMF, subsequently diluting, with methanol, to a concentration of 100 parts per million (ppm), and finally adding 250 ppm of sudan III, to mark the elution of the SDS micelles. Acenapthene and anthracene standards are prepared at a concentration of 250 ppm. Capillary Electrophoresis The capillaries used in CE and related techniques areas almost invariably made from drawn fused silica externally coated with polyimide for strength and mechanical stability. They normally have no coating on their internal walls, however, certain substances can be bonded to the inner surface, either to eliminate sample to wall interactions or to modify the magnitude or direction of the electro-osmotic flow (EOF). Bare Silica Capillaries The capillary inner wall has a large number of surface silanol groups which, when in contact with an electrolyte solution, dissociate leading to a negative charge on the capillary wall. Consequently, in order to preserve overall electrical neutrality, there must be an excess of cations in the electrolyte immediately adjacent to the inner surface. When a voltage is applied, these cations migrate in the direction of the cathode and in so doing exert a force on the surrounding fluid causing it to flow in the same direction. This electrically induced flow is known as electro-osmotic flow (EOF). In bare silica capillaries, i.e. uncoated silica, the electro-osmotic flow is high when the electrolyte pH is greater than 6 and effectively drops to zero for pH values of 3 or less. For reproducible analyses it is important that the surface density of free silanol groups remains constant from one analysis to the next. For this reason, the capillary is often rinsed with sodium hydroxide solution (or alternatively a strong acid) followed by a further rinsing with the run buffer. For coated capillaries, the rinsing procedures are generally less harsh in order to avoid the possibility of stripping off of the inner coating of the capillary. In order to obtain a consistent EOF, new uncoated capillaries are flushed with 0.1M NaOH solution for 20minutes prior to their initial use. This procedure is carried out in order to reactivate the surface silanols. The Procedure must no be carried out with wall coated capillaries.
Capillary Background -- Uncoated CE capillaries are made from high quality drawn fused silica with a high degree of UV transparency. Capillaries have an outer diameter of 375µm and are available with internal diameters of 50µm and 75µm. For convenience of handling and easy installation, the majority of CE instruments use a cartridge, which houses the coiled capillary. There are a number of standard lengths for each instrument type. These standard lengths are given by the way in which the capillary is coiled, i.e., each length increment should be a multiple of the coiled circumference. Capillary lengths are typically 25 to75cm. The injection end of the capillary must be cleanly cut to give a flat surface. Poorly cut capillaries can lead to loose polyimide coating and a cracked or shattered fused silica edge which will lead to tailing peaks, sample carry over and a reduction in peak area precision. It is therefore of considerable benefit for the CE user to obtain expertly prepared capillaries. Capillaries are cut to a high degree of precision with the UV detection window accurately prepared and positioned. A small length of the polyimide coating is also removed from the injection end in order to improve sample introduction precision.
