Surfactants Analysis by High Pressure Liquid Chromatography: Reversed Phase Column and
Conductivity/UV/Evaporative Light Scattering Detector
1.0 Scope and Application
The following is the analytical method to be used for aqueous surfactant solution before and after Treatability Study in Hill AFB.
2.0 Summary of Method
If samples cannot be analyzed immediately, the vials are stored at 40C before injection. Before the HPLC analysis. samples require removal of any excess phase which may clog the HPLC column. A 20 mL aliquot of samples will be injected automatically into the HPLC connected to a conductivity detector (for ionic surfactants), a UV detector (for surfactants with phenyl ring) or an Evaporative Light Scattering detector (for nonionic surfactants). The result will be calculated relative to the external standard.
3.0 Interferences
Raw HPLC data from all blanks, samples, and spikes must be evaluated for interferences. If possible these interferences must be eliminated. Common interferences for this method are listed below.
1) Contamination by carryover can occur. In order to minimize carryover, the pipets or syringes used for transferring or injecting the solution will be rinsed with nonpolar (acetone) and polar (ultrapure water) solutions before and after each transferring. For different surfactant analyses, the pipets and syringes will be soaked in the Norchromax solution (one package of Norchromax powder dissolved in 2.5 liter 98 % sulfuric acids) for 30 mm and rinsed with excess water before switching.
2) Other interferences could occur due to the high background of coelute of salt from soil or the extracted organics from soil. Backgrounds from these compounds should be carefully evaluated and subtracted. Significant interferences may require modification of conditions (i.e., change UV wavelength or mobile phase, etc.).
3) Interferences due to the impurity in commercial surfactants include either the overlapping or multiple peaks on the chromatograms. If separation of the impurity is difficult, the interpretation of the raw chromatogram should be carefully noted and recorded. 4) If any excess phase exists (e.g., NAPL, soil), clogging of the HPLC column will likely occur. A 0.2 mm Acrodisc filter (Gelman cat # LCl3 PVDF) will be used for removal of the excess phases. Any abnormal increase of pressure or dramatic decrease in surfactant retention time will be closely evaluated and inspected.
4.0 Apparatus and Materials
HPLC system- Shimadzu Low Pressure Mixing Pumping Systems (LC-10AD Solvent
Delivery Pump, FCV-10AL Valve, Mixer SUS, HDU-lA Helium
Degasser) with an SIL-10A Autoinjector.
Detector
UV- Beckman 166 Programmable Detector Module or Waters 486 Tunable
Absorbance Detector
Conductivity- Alltech 320 Conductivity Detector
Light Scattering- Alltech VAREX MKIII Evaporative Light Scattering Detector
Column- Alltech Reversed-Phase Nucleosil C18, 5 um, 150mm x 4.6mm
Guard Column- Alltech All-Guard Cartridges, Reversed-Phase, 7.5mm x 4.6mm
Integrator- Hewlett Packard 3394 integrator
5.0 Reagents
Water- SYBRON-Barnstaed double deionized water
Surfactants- The highest purity obtained from the manufacturers
Methanol- HPLC grade
6.0 Procedure
6.1 Sample preparation
Surfactant standard solutions (100, 200, 500, 1000, 2000 mg/L) will be prepared by dissolving surfactant in water and adjusting to the final concentrations with dilution. In solubilization and mobilization studies and sorption studies, the resulting solution will be centrifuged first and the supernatant will be separated and kept in 40 ml vials for analysis. If any excess phase exists (e.g., NAPL, soil), the sample will be filtered with a 0.2 mm Acrodisc filter (Gelman cat # LC13 PVDF) before analysis. Methanol and water mixtures (mobile phase) will be prepared by dissolving water into methanol in an one liter graduated cylinder.
6.2 Operating Conditions:
Shimadzu LC-1OAD Solvent Delivery Pump/SIL-bA Autoinjector
Pumping Rate: 1 ml/min
Mobil Phase: 70-80% (vol.) Methanol/Water Mixtures
Stabilize Time: 15 minutes mobile phase injection
Delivery Mode: Isocratic
Injection Mode: Automatic; Standard Injection Mode
Sample Injection
Loop: 20 ml
Autosampler Vial: 1.5 ml
UV Detector (Beckman 166 or Waters 486)-
Wavelength: 254 nm
Range, AUFS: 2.0
Alltech 320 Conductivity Detector-
Range,us: 1.0
Polarity: minus
Time Constant: fast
Ailtech MMII Evaporative Light Scattering Detector-
Drift Tube Temp: 84-92 0C
Gas Flow: 2.15-2.45 Standard Liter Per Minute (SLPM)
Hewlett Packard 3394 Integrator-
Chart Speed: 1.0 in/min
Attenuation: 4 - 10
Peak Width: 0.04-0.16
Thresh: 2 - 3
Area Reject: 1000
Run Time: 10 - 15 minutes
7.0 Calibration:
A calibration curve (five points minimum) must be developed to verify proper operation of the HPLC system. After fitting the standards, all analytes must meet acceptance criteria. These criteria include:
1) In order to determine if the HPLC is exhibiting any deteriorated response, the percent relative standard deviation should be less than 10 percent for most surfactants.
An example calibration curve and a chromatogram for DOWFAX 8390 are provided in attachments 1 and 2, respectively.
As standards are prepared by using the actual analytes, verification of operation and calibration can be easily determined. Since the sample injection is automatic, errors due to manual operation are minimum. After initial verification, at least three standards will be analyzed after 10 samples. The standards should include one at or near the quantitation limit, a mid range, and a standard at or near the highest concentration of the calibration curve. These analyses are performed to ensure that the calibration does not shift as a function of time. If the calibration checks differ by less than or equal to 10 percent, the initial calibration is assumed to be valid. If this criterion is not met for any one of the calibration checks, corrective actions must be taken.
Since a series of surfactants with different structure will be used in this study, it is very difficult to select the appropriate surrogate spikes. Instead, the matrix spike will be used to determine the percent recovery or accuracy. The matrix spike is performed by adding as following, after the separation the supernatant, the sample aliquot will be split and a 500 mg/L of the surfactant will be added into spiking and/or spiking duplicate samples for the HPLC analysis. Triplicates will be measured.
7.1 Quantitation Limits
Compound Quantitation Limit (mg/L)
Dowfax 8390 100
CA 620 100
Steol 330 50
Tween 80 200
Aerosol OT 50
SMDNS 100
8.0 Data Interpretation
8.1 Qualitative Analyses
Qualitative identification of compounds can be achieved based on retention times. The sample chromatograms are compared with the chromatogram of standard. The relative retention time of surfactant sample should not exceed +/- 0.1 retention units.
8.2 Quantitative Analyses
Quantifying surfactant concentrations will be based on the integrated area of the specific peak(s) for the surfactants.
The external calibration curve is expressed by the following equations:
Peak Area = Slope*Concentration (mg/L) + Constant
Concentration (mg/L) = (Peak Area - Constant)/Slope
In order to determine the concentration of the sample. the resulting peak area is compared with the external calibration curve.
9.0 Quality Control
The QC requirements for this project are:
1) Analytical equipment will have regularly scheduled maintenance and daily
calibration (more frequent when appropriate). In the event that injection of replicate standards results in variations exceeding the criteria levels (as described below), the source of the variation will be identified and appropriate corrective action taken.
2) Before processing any samples, the analyst should demonstrate, through the analysis of a method blank, that interferences from the analytical system, glassware, and reagents are under control.
3) A quality control reference sample containing the target surfactant is required. This reference sample will be prepared from raw surfactant materials. This reference sample is then injected into solution and recovery determined. This should be done at three concentrations; one near the quantitation limit, one mid range, and one at or near the highest concentration used for calibration curve. For each analyte, the standard deviation of the recovery (mg/L) and average recovery (mg/L) will be compared with the corresponding acceptance criteria for precision and accuracy.
Compound Average recovery Std. Dev. (%) (n=3)
DOWFAX 8390 102 10.2
CA620 101 4.5
Steol 330 98 8.0
Tween 80 101 2.4
Aerosol OT 99 2.5
SMDNS 102 6.0