To determine the concentration of a specific polyaromatic hydrocarbon (PAH) in a mixture. Unknown samples will contain several different PAH's. You must determine the retention time and calibration factor for your assigned "target" compound (analyte), using a standard addition technique. You will be working in groups of four, where each member has a different target analyte, so that information regarding the elution sequence for each of the components in sample mixtures can be shared.
[Instrumental procedures are specific for the particular instrument being used. Refer to Appendix 10 or 11 for the Spectraphysics 8700 or Waters Millenium systems, respectively.]
A. Equipment And Chemicals Provided
1. ternary pump and control unit
2. sample syringe (100mL)
3. priming syringe (5 mL)
4. column
5. detector
6. printer
7. injector valve
8. mobile phase bottles
9. sonicator
10. acetonitrile (filtered through .45mm filter membrane)
11. HPLC water (pre-filtered through .45mm filter membrane)
12. 70:30 acetonitrile/water (filtered through .45mm filter membrane)
13. sample mixture of naphthalene, phenanthrene, pyrene and
fluorene
14. standard naphthalene, phenanthrene, pyrene and fluorene
solutions
15. screw-capped sample vials (5 w/ septum caps; 4 w/ solid cap)
(per group of four students)
16. micropipet and disposable pipet tips
17. screw driver (only needed if group does calibration, C.3)
18. disposable plastic gloves (small and large sizes)
B. Check Out Items (One per group of four students)
1. one 1.0 ml pipet
2. one 50 ml beaker
3. one 10 ml volumetric flask with glass stopper
4. one small pipet bulb
5. one 5 mL volumetric flask (optional, for calibration procedure)
C. Sample Preparation
CAUTION: Wear the provided protective gloves before handling samples. Conduct sample and solvent transfers in a hood.
C.1. Triple rinse a 10 mL volumetric flask with 70:30 ACN/H2O. (Drain but don't dry this flask.) Also triple rinse nine sample vials (four with solid cap, five with septum caps), with pure acetonitrile (squeeze bottle). Drain and place vials and caps upside down to dry on clean lint-free tissue (Kimwipe) in the hood. Discard rinse solvent in the designated waste bottle. Retain the rinse beaker for C.3 (place upside down on a clean Kimwipe in the hood).
C.2. Bring a 10 mL volumetric flask to the lab instructor for the group's unknown sample mixture. Also bring four clean, dry sample vials (with solid cap) to your instructor for standard solutions for each of four unknowns for the group. Each student selects one of the standards and subsequently determines the concentration of this substance in the group unknown. Your instructor will tell you the concentration of each standard. Cap and mark the "standard" vial (vial A).
C.3. Bring the volume of your sample in the 10 ml volumetric flask to the mark with 70:30 ACN/H2O solvent using the beaker from C.1. Mix thoroughly.
C.4. Conduct the following procedure as quickly as possible to avoid significant volatilization of the sample solution. Withdrawing a small amount (~0.2 mL) of sample solution each time from the 10 mL volumetric, rinse the pipet three times with the sample solution. Then, pipet 1.0 mL of the sample solution to each of four septum-capped sample vials prepared in C.1, and mark each as vials C, one for each student in the group. Identify vials separately for each student. Pipet approximately 1 mL into one other septum-capped vial, and mark this as vial B, for the group. Cap all vials tightly.
C.5. With the Eppendorf micropipet adjusted to 10ml, attach a disposable pipet-tip. Wet the pipet-tip by immersing 2-3 mm into the standard solution (vial A); depress the control button to the first stop (dispense solution) and then slowly release the button (draw solution in), repeating three times; depress the button to the second stop (completely empty the tip) after the third wetting cycle. While the control button is at the second stop, remove the pipet-tip from solution; then release button completely. Depress button to the first stop; re-immerse tip (2-3 mm) in the standard solution (vial A); allow button to glide back slowly. Slide tip out along the inside of the vial. Wipe off any external droplets with lint-free tissue (Kimwipe). (Do not wipe close to the pipet-tip opening to avoid absorbing out any of the sample contents.) To dispense the 10 mL standard solution into vial C, place the pipet-tip against the dry inside surface of the vial; press the control button slowly down to the first stop and wait 1-3 sec; then continue to press the button down to the second stop; this will deliver all remaining liquid in the pipette tip. While continuing to hold the control button down, slide the tip out along the inside of the vessel. When completely removed eject the pipet-tip by depressing the control button to the final stop. Tightly screw the septum cap of vial C; shake and mix the contents. (This is the standard addition sample.)
C.6. Before injecting your sample into the column, all suspended micro-particles should be broken up by ultrasonication. Otherwise, those solids may clog the column. Make sure the ultrasonic cleaner contains about one inch of deionized water above the bottom of the sample basket. Then place the vials B and C in a 50 mL beaker. Add deionized water to the beaker to one-quarter to one-half the height of the vials. Then place the beaker into the water of the ultrasonic cleaner. Turn the switch to "On" and sonicate for 5 seconds. Do not leave the cleaner on continuously for more than 5 minutes.
D. Data Analysis
D.1 Share information among your group to identify the elution order (retention times) of naphthalene, phenanthrene, pyrene and fluorene. The first peak observed at 214 nm is the dead volume marker (KNO3).
D.2 The peak area (in A.U.*min. units) of most peaks is printed in the integrator section of the printout. Record the peak area of your unknown from the Standard addition sample (vial C). Identify the corresponding peak from the chromatograms of the unknown sample (vial B). Record that peak area.
D.3 Calculate the concentration of your unknown in vial B by the standard addition method (see Section E). Calculate the number of theoretical plates (N) for your analyte from the chromatogram obtained with vial C (see Section F). Record results in your lab notebook and on the HPLC report sheet.
D.4 If your group was assigned to calibrate the system, record flow rate results, as well as the value of theoretical plates (N) obtained for each analyte done by the group, in the HPLC Instrument Calibration Notebook.
E. Standard Addition Method
For constant conditions of sample size (injection volume), flow rate, solvent composition, and column conditions, the area of the peak corresponding to the target analyte is proportional to its concentration in the injected sample mixture.
AB = kCB (1)
where AB and CB are, respectively, the analyte peak area and analyte concentration for sample solution B; and k is the proportionality constant for the specified experimental conditions. The peak area, AC, for the sample plus standard addition (solution C), can be related to the added quantity of analyte:
AC = kCC = k[CBVB + CSDV]/(VB+DV) (2)
where, CC is the concentration of sample-plus-standard; VB is the original sample volume (1.0 mL) containing the unknown analyte; CS and DV are, respectively, the concentration and volume (10mL) of added standard. Thus, by dividing Equation (2) by Equation (1), and solving for CB:
CB = (ABCSDV)/[AC(VB+DV) - ABVB] (3)
The concentration of analyte in the original sample, CB, can be computed from Equation (3), if the injection volumes are identical for the two chromatograms.
Figure 1. Standard Addition of component 2. Upper trace is for solution B; lower trace is solution C
The injection volumes may not be identical, but the following correction approach can be used because
D
V << VB in this procedure:Observe the area ratio of "marker" peaks (AM) for solutions B and C, where the "marker" peak is for a component whose concentration is unchanged (i.e., not the target analyte). Obtain a value of AC for the target analyte by multiplying the observed area of the analyte peak, AC', by the ratio of the areas of the "marker" peaks for chromatograms obtained from vials B and C:
AC = (AC')[(AM)B/(AM)C] (4)
Substitute AC from Equation [4] into Equation [3].
F. Determination of Number of Theoretical Plates
Use the chromatograms printed out under the spectrum index section. To your unknown peak, draw tangent lines at the inflection points of the peak, which are indicated by the first and the third dotted lines of each peak. Extrapolate the tangent lines to intersect the baseline. The width of the peak (W) is obtained by measuring the corresponding time difference between the first and the second tangent-baseline intersections. To determine N, use the equation below, with tR and W measured in the same units (min. or sec.)
Figure 2. Determination of No. Theoretical Plates, HPLC.
Results and calculations Table: Spectraphysics/Waters/PDA System DH 413
Results and calculations Sample Table: Spectraphysics/Waters/PDA System DH 413
HPLC Calibration Results Table: Specraphysics SP8700
HPLC Calibration Results Sample Table: Specraphysics SP8700
Results and Calculations Table. Waters model 510 Millenium System DH 413
Results and Calculations Sample Table. Waters model 510 Millenium System DH 413
