+1 862 207 3288 Introduction:
4-nitrotoluene (4-NT) and 4-nitroaniline (4-NA) are two of the more important breakdown products of nitrobenzene explosives, such as trinitrotoluene (TNT).The relative and absolute abundance of these substances in soils or waters contaminated with explosives residues (e.g. at abandoned army facilities) gives important information about the rates and mechanisms of explosive degradation reactions, so it is essential that there are accurate analytical methods for their determination. Due to the fact that there are many different breakdown products for explosives, gas chromatography with mass spectrometric detection (GC-MS) is a very popular method for their determination. In order to quantify each compound accurately, it is important to achieve good chromatographic separation between the individual compounds. In this lab, you will characterize the separation of 4-NT and 4-NA by GC-MS, and study parameters that influence this separation.
Prelab:
1) Describe (briefly!; target: one paragraph) how the chromatographic separation approach (ignore the detection) youíre using in this experiment works (in principle). Include the general nature of stationary and mobile phase, the general properties of the analytes, as well an explanation of how and why the analytes are separated. Then, name the (one) most important experimental parameter that influences the separation, and indicate qualitatively what effects a variation of this parameter has on the separation. Since this material has not been covered in the lectures yet, consult the corresponding chapters in the textbook, or the overview provided in the seminar.
2) Predict the elution order of your two analytes in this separation, and explain, based on the properties/structure of the analytes, and the underlying principle of the separation, how you determined the predicted elution order. Information relevant to answering this question can also be found in the same sources mentioned under 1).
3) Read the instructions for the samples that you need to prepare in week 1 of this experiment. Calculate the amounts of the neat chemicals required to make the first two stock solutions. For the subsequent dilution, assume for your calculations that you have prepared stock solutions containing exactly the target concentration. You will then modify these calculations in the lab during week 1, once you know the actual concentrations of your stock solutions. Write out step-by-step recipes for the preparation of all solutions. Your TA will check your calculations, and correct them with you, if necessary.
For tutorials on making standard solution, refer back to lab A. For instructions on using analytical balances, refer to the lab manual, the textbook, or the following video:
4) Read the instructions for the measurements you will conduct in week 2, as well as the tasks you are asked to complete for the lab report. Make a detailed list of all data you need to take home at the end of week 2 of this experiment. Your TA will check this list for completeness, and discuss with you, if necessary.
Week 1:
You will be given the two substances to be separated, 4-NT and 4-NA, as pure compounds. These substances are solids under standard conditions. From these neat substances, make twoseparate10 mL stock solutions in the appropriate solvent (which will be given to you), each containing a target concentration of 1,000 mg/L of the respective substance. For this purpose, you will be using an analytical balance, 10 mL volumetric flasks, and spatulas. It is OK to prepare solutions that are not exactly 1,000 mg/L (nominal concentration), but close to it (actual concentration). Once you have prepared these solutions, calculate their actual concentrations, and use those in the next step.
From these two stock solutions, prepare one mixed standard solution containing exactly 20 mg/L of each analyte, using 10 mL volumetric flasks and a variable micropipetter with an appropriate working range (either 20 ñ 200 or 100 ñ 1,000 µL) for this dilution.
You will also be given an unknown sample, containing both 4-NT and 4-NA in concentrations around 20 mg/L, as well as other chemicals. This will be your ìsampleî for testing the effect of the sample ìmatrixî on the separation.
Label all of your samples properly (name, date, content of container), and store them in the refrigerator until the day of analysis.
Week 2:
Analyze your samples by GC-MSunder two different chromatographic conditions (ìnormalî and ìalteredî) in the following order:
standard under normal conditions, replicate 1
standard under normal conditions, replicate 2
standard under normal conditions, replicate 3
unknown sample under normal conditions
standard under altered conditions, replicate 1
standard under altered conditions, replicate 2
standard under altered conditions, replicate 3
unknown sample under altered conditions
Make sure to bring a memory stick, and obtain electronic copies of all relevant chromatograms, including the data you will need to complete the tasks listed in the following for the lab report. Also, take note of the exact separation conditions used for the ìnormalî and the ìalteredî separation, as you will need these to complete the lab report.
Report tasks
1) determine elution time windows for each analyte
Determine the elution times for each analyte under ìnormalî conditions for each of the three replicate analyses. Then, determine the average elution time and its variability (expressed as a standard deviation) for each analyte.
2) characterize peak shapes
Using the definition presented in the lectures, calculate the peak asymmetry factor for both peaks in each of the three replicate chromatograms of the standard solution under ìnormalî conditions. In order to complete prelab question 4), you may need to look up the definition of peak asymmetry in the textbook in advance. Additionally, calculate the average asymmetry factor and its standard deviation from the three replicate analyses. Interpret all(8) determined asymmetry factors using ìtailing/frontingî terminology.
3) quantify separation
Using the definition presented in the lectures, calculate the separation between the two analytes in each of the three replicate samples. From these three results, calculate the average separation and its standard deviation under ìnormalî conditions.
4) describe influence of matrix on separation
Describe any differences you can visually detect in the chromatogram of the unknown sample vs. those of the standard. From the analysis of the unknown sample under ìnormalî conditions, determine the elution time for the two analytes, the asymmetry factors for their peaks, and the separation between the two peaks, in analogy to steps 1) ñ 3) above. Is either of these parameters different in the unknown sample than in the standards? If so, describe the effect of the matrix on the separation.
5) characterize separation under ìalteredî conditions
In analogy to steps 1) ñ 3) above, determine elution windows, characterize peak shapes and quantify separation of the two analytes under the ìalteredî chromatographic conditions. Are there any substantial differences between the two sets of separation conditions (ìnormalî vs. ìalteredî) with respect to either of these parameters? If so, explain why these changes are observed, on the basis of the fundamental mechanism of the used separation methodology, and/or the general aspects of chromatographic theory explained in the lectures.
6) determine effect of ìalteredî separation conditions on matrix tolerance
Compare the chromatogram for the unknown sample obtained under the ìalteredî separation conditions to a) the chromatogram obtained for the unknown sample under ìnormalî conditions, and b) the chromatograms obtained for the replicate analyses of the standard solution under ìalteredî conditions (in analogy to task 4) above). Describe (incl. quantitative considerations) the effects of the sample ìmatrixî on the separation under ìalteredî conditions, and compare them to such effects observed under ìnormalî conditions. How have the ìalteredî chromatographic conditions changed the analysis of the two compounds of concern, and how can you explain this based on the differences between the ìnormalî and ìalteredî separation conditions?
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