INTRODUCTION
This exercise is designed to acquaint
you with some of the basic procedures you will need to be familiar
with throughout the rest of the experiments. Some procedures
will be review for many of you, but a grasp of them all will enable
you to complete future experiments more accurately and efficiently.
At the completion of this experiment you should have grasp of
the capabilities of gas and liquid chromatography, and an idea
of the characteristics of various detectors and the use of Turbochrome
software. You should be sufficiently familiar with injection
techniques to make accurate, reproducible injections on the chromatographs.
Read the laboratory section of the syllabus prior to the laboratory
period. You will complete the exercise in the following sections,
although not necessarily in the order given.
I. Preparation of Dilutions for Selective Detectors
You will be provided with solutions of three pesticides in hexane, at a concentration of:
1) 1.0 mg/ml (for FID)
2) 10 ng/µl (for NPD and FPD)
3) 100 pg/µl (for ECD)
The pesticides are:
Heptachlor Malathion Trifluralin
Some physical properties are:
| Heptachlor | Malathion | Trifluralin | |
| MW | 373.35 | 330.36 | 335.2 |
| MP (°C) | 231 | 2.9 | 46-47 |
| Water Sol. mg/l | 0.1 | 145 | 0.5 |
| log Kow | 3.9 | 2.8 | 3.0 |
| P, Pa @ 20°C | 0.03 | 0.001 | 0.006 |
II. GC Injections
You will make one 1 µl injection
of the appropriate dilution of your solutions on instruments equipped
with the following detectors: FID, NPD, ECD and FPD in sulfur
mode. These instruments all have columns approximately matched
in stationary phase, length and flow rate. Compare the chromatograms
you obtain with the sample chromatograms by each GC. Your retention
times and peak areas should be close (± 10%) to those of
the samples. If time allows, after you have made one injection
on each detector, make a standard curve on one instrument by making
1, 2 and 3 µl injections. Determine if the detector is responding
linearly.
III. HPLC Injections
You will make one 10 µl injection
of the standard provided (0.25 mg/ml of the same 3 pesticides
in acetonitrile) on the HPLC. This will give you the experience
of using a liquid chromatograph's injector. Compare your results
(area and retention time) with the sample chromatograms.
IV. Visual Separation
The purpose of this exercise is
to provide a visual demonstration of a simple chromatographic
separation. The color additives (Fig. 1) in commercial Kool-Aid
will be separated using solid phase extraction cartridges. You
will learn more about the specifics of the separation later.
MATERIALS NEEDED:
- Grape Kool-Aid (unsweetened)
C-18 Solid Phase cartridges (3 cc)
Vacuum Manifold
Deionized water
Methanol
90:10 Methanol:Water)
Collection Vials
PROCEDURE
1. Dissolve half of the Kool-Aid® packet in approximately 30 mL deionized water.
2. Rinse the C-18 column with methanol, followed with deionized water.
3. Add approximately 1/2 mL of aqueous Kool-Aid to the column.
4. Elute the Kool-Aid until the reservoir is almost emptied.
5. Wash once with deionized water.
6. Place a collection vial in the vacuum manifold.
7. Add 1 column volume of 10:90 methanol:water to elute the color dye. Don't let the column dry, but continue adding to completely elute the dye.
8. Observe the column and collect the dye.
9. When collection is complete, remove the first vial and place a new collection vial in the vacuum manifold.
9. Add 1 column volume of 100% methanol and continue adding 100% methanol until all remaining dye is removed from column.
10. Observe and collect the second
dye.
QUESTIONS
1. What is/are the mobile phase(s) in this system?
2. What is/are the stationary phase(s)?
3. Which dye elutes first? The column consists of tiny beads coated with an octadecysilyl compound (C-18). This C-18 coating is very hydrophobic (water hating). Can you hypothesize why the dyes elute in the order they do based on the structure of the dyes and the hydrophobic character of the stationary phases? Why do the dyes not elute with pure water (step 5)?
4. How well resolved are the two dyes? (Are the two dyes completely separated?)
5. If you could monitor this separation
on a chart recorder, can you predict what the elution profiles
would look like?
V. GC AND HPLC COMPONENTS
There will be a bench set up with
many GC and HPLC components. During the times when you may be
waiting for access to an instrument, become familiar with these
elements. During discussions of theory and operation, it sometimes
helps if you know what an item looks like, and may make its function
more easily understood.
VI. VOLUMETRIC TRANSFERS AND WEIGHING
You will use one 1.00 ml and 10.00 ml pippette, one automatic pipettor, two 25 ml and one 125 ml Erlenmeyer flasks, and a suction bulb. You will be shown appropriate weighing and pipetting techniques before you begin measurements. There will be two types of balances used. The top loading balance is less accurate for small weights and will be used for the 10 ml measurements. The analytical balance will be used for the 1 ml and automatic pipette weighing.
- A. Zero the balance with the flask
on it.
B. Without removing the flask from the balance, carefully pipet 10.0 ml distilled water into the 125 ml flask and record the weight.
C. Zero again, add an additional 10.0 ml and again, record the weight. Repeat for a toaltotal of three 10 ml additions.
D. Repeat steps 3 to 5 for the 1.0 ml and automatic pipettes, dispensing them into the 25 ml flasks, using the analytical balance. Be very careful with the analytical balances.
E. For each pipette calculate the
mean, standard deviation, and the relative standard deviation
of the delivery weights.
VII. ENVIRONMENTAL SAMPLING
At some stage in Lab 0 please go
to the station marked "Environmental Sampling" and
take the directed number and size of samples and record your
"results" on the attendant sheet. The entire data set
produced by the class will be discussed in class.
To be turned in at the beginning of your next lab period.
Show all calculations!
1. You are given a solution labeled as follows:
1.0 mg/ml parathion in ethyl acetate
You use a volumetric pipette to
remove 1.0 ml of this solution and dilute this aliquot to a final
volume of 25 ml in a volumetric flask.
What is the concentration (in µg/µl)
of the resulting solution?
Would you use this solution on a
NPD? Why or why not?
2.
Answer the question in Lab 0 under Visual Separation.
3.
In Lab 0 you were given a 1.0 mg/ml, 10 ng/µl and 100 pg/µl
solutions of heptachlor, malathion and trifluralin in hexane
for injection onto the appropriate GC/specific detector. If
I gave you the 1.0 mg/ml solution show how you would prepare
the other two; you are limited to using volumetric flask not
greater than 25 mls, syringes not smaller than 100 microliters
and the lowest mass you can accurately weigh is 10 mg.
4. You are involved in the analysis of Metam-sodium from the upper Sacramento River and have obtained the following data for your standard curve. Your stock solution of Metam-sodium is 15 ng/µl.
Injection volume Area Response
5 µl 106,530
10 µl 196,442
20 µl 423,407
30 µl 637,908
What is the equation for the line
drawn through these points? How good is the linear fit?
If you injected 10 µl of a
sample of river water and obtained an area response of 327,304
for a peak that co-elutes with your Metam-sodium standard, what
would the concentration be of Metam-sodium in the river water
sample?
5.
Please look up, or calculate, the molecular weight of chlorpyrifos
and then show how you would prepare 10 mls of a 75 pg/µl
stock solution. How many ng/ml would this be? This concentration
level is appropriate for use on the ECD detector.
If instead we only had an NPD detector
would chlorpyrifos give a response? What concentration would
you prepare to inject onto an NPD detector.
6.
You are analyzing for compound Nuke' Em in water. You are given
1.5 liter of water which you extract and concentrate in extract
to 5.0 ml. You inject 2 µl of this solution on a GC and
get a response of 26060 area counts. Before injecting this sample,
you put on the following standards and got the corresponding
response.
Data Table
Standard Volume Response
(ng/ml) ml (area count)
5 1 7865
2 14923
25 1 41018
2 78251
How much (in µg) of compound
Nuke'Em was in the original liter of water? How much would this
be expressed in ppb?
7.
In Lab Zero you injected a mixture of trifluralin, heptachlor,
and malathion onto a variety of GC detectors and onto an HPLC
with UV detection.
a). Please turn in a copy of the
output from the GC with FID detection with all the peaks labelled
as to the identity. To do this you must observe the signals
received from the other detectors. Please explain, in detail,
your reasoning behind your assignments. Treat your chromatogram
like you would put it in your lab notebook with all conditions
indicated. Do the same for your HPLC output.
b). Why with the UV detector at
254 nm did you only observe one peak? What pesticide do you
think this was and why?
8. You are interested in measuring Atrazine in Lake Ontario water and learn from the literature that you would expect very low ppt levels (~20 ng/liter). If you were to do a simple liquid/liquid extraction of the lake water with dichloromethane and analyze it on a GC/NPD making 1 microliter injections please answer the following questions: 1) how much water would you need to take?; 2) after concentrating the dichloromethane what would your final optimal extract volume be?; 3) what concentration (in pg/microliter) would your 4 standards optimally be?; 4) how would you make these standards? [same limitations as in question 3 above].