LABORATORY PREPARATION
Laboratory experiments are designed with the intention of being
completed during the allotted classroom time, however, some may
require more or less time depending on the pace at which you work.
In order to expedite things it is mandatory that you be prepared
prior to coming to the lab. This includes reading the lab procedure
handout thoroughly and understanding what the experiment entails.
In addition, any applicable calculations (such as dilution factors)
should be made in advance so that work may commence upon arriving
in the lab. To work most efficiently, it may be necessary at
times to be performing several steps of the experiment simultaneously
(for example: making injections on the GC for your standard curve
while preparing unknown samples). A flowchart is helpful in determining
the time steps will take and the order in which things must be
done. In addition, it is important that you refamiliarize yourself
with lab techniques learned in your chemistry classes. Be prepared!
Some experiments by their nature take longer than others, as
well as variability of instrument performance being a factor,
but the biggest determinant will be your preparation.
LABORATORY NOTEBOOK
You are required to maintain a lab notebook. This must be a
bound notebook with numbered pages, like the type used in other
chemistry labs. It is very important to get in the habit of writing
in a lab notebook. Every good lab researcher can tell you stories
about being "saved" by having kept a good notebook.
It is suggested that you write an outline of each lab in your
notebook before you start, that way you have a good overall picture
of the experiment before starting. At the end of the term
your notebook will be graded!
LABORATORY GROUPS
On the first day of lab, depending on class size, you may be
assigned to a lab group. These groups will work together the
entire semester. It is suggested that you work with your group
as much as possible in terms of understanding the labs. You may
work cooperatively on the labs to the extent of sharing information
on the different sections that were performed by different people,
but you must do your own write-up-no group lab reports.
LABORATORY SAFETY
Please remain aware that you are working with "toxicants"
in all your experiments! If handled correctly, these compounds
pose little or no danger in a laboratory setting. However, carelessness
could lead to serious problems for yourself or someone else.
With this in mind, absolutely no food or beverages will be permitted
in the lab. EYE PROTECTION MUST BE WORN BY ALL PERSONS AT
ALL TIMES IN THE LAB!!! Long pants and shoes (no sandals)
are required laboratory attire. A lab coat is highly recommended.
In addition, disposable gloves are provided for your safety during
procedures when exposure might happen. Clean up any spills immediately
and thoroughly; if you are not sure how, ASK SOMEONE! Be considerate
of the person using the scale, bench, or instrument after you.
Keep the lab benches clear of non-essential items. Backpacks,
etc., should be kept somewhere out of the way. Do not leave them
on the floor or in the aisles.
Before you leave the lab the first day, be sure you know where
the following safety devices are: 1) Eyewash station, 2) Emergency
shower, 3) Nearest telephone and number to call (8-2222), 4) Fire
Extinguisher, 5) Fire Alarm pull station. If you do have to call
for help, be sure to send someone outside the building to direct
the emergency crews in. Also, in the event of a power outage,
STOP whatever you are doing, cover all open containers and LEAVE
THE BUILDING. Power outages stop our fume hoods and thus the
labs are not vented.
WASTE HANDLING
Chemical and solvent wastes are divided into several categories:
you will be directed by the TAs where to put your waste.
Make sure you dispose of all your solutions and samples at the
end of your experiments. Also, thoroughly clean up your bench
area at the completion of your work.
INTRODUCTORY MATERIAL FOR LABS
Since much of what we will do in this lab involves some sort
of separation, it is important for you to have some basic understanding
and terminology even before we cover it in lecture. The following
is a basic introduction to chromatography (a fancy word that really
just means separation) and to the instruments we will use in lab.
You should read this before the first lab and then again after
seeing the actual equipment.
CHROMATOGRAPHY IN A NUTSHELL
At its simplest, gas and liquid chromatography are methods to
separate sample components from each other and from matrix components
and the solvent. This is accomplished by partitioning the compounds
between a stationary phase (the column packing or coating) and
a moving phase (a gas in GC and a liquid in HPLC). Compounds
with little partitioning into the stationary phase pass through
the column more swiftly and elute first. Those that spend a lot
of time in the stationary phase are retarded and elute later.
In gas chromatography, boiling point or vapor pressure is the
major factor in elution order, although polarity has a small but
important effect. In HPLC, polarity is the primary factor in
elution order.
Since elution is based on physical properties, retention time
or elution pattern is an attribute of a molecule and can be used
to determine identity. A particular compound will have a characteristic
retention time (time from injection to detection) when run on
similar columns under similar conditions. Two different compounds
may have very close or identical retention times on one column
type, but when analyzed on a column of differing polarity usually
have different retention times. This is the basis for identification
based on chromatographic retention time.
GC and HPLC are also used in quantitation. The signal from the
detector is in the shape of a peak, and the height or area of
the peak is proportional to the amount injected. Quantitation
utilizes standard curves, where known amounts from standards of
known concentration are injected into the instruments, and the
response compared to a sample of unknown concentration.
Detectors
The only type of HPLC detector we will be using is UV. It will
detect chemicals that absorb light between 210 and 700 nm. Most
often this implies that the chemical has an aromatic ring or extended
conjugation.
GC detectors will be of several types.
FID: Flame Ionization Detector. Detects oxidizable carbons.
Sensitivity range from 50 ng/ml
to 1 mg/ml
(mg/ml) solutions.
NPD: Nitrogen Phosphorus Detector. Detects compounds
containing phosphorus or nitrogen. Sensitivity range from 50
pg/ml to 1 mg/ml
solutions.
FPD: Flame Photometric Detector. Detects phosphorus
(in the phosphorus mode) or sulfur (in sulfur mode). Sensitivity
range from 50 pg/ml to 1 mg/ml
solutions.
ECD: Electron Capture Detector. Detects groups that
can hold an extra electron, such as aromatic rings and conjugated
systems, and particularly halogens. Sensitivity range from
10 pg/ml to 100 ng/ml
solutions.
Sensitivity ranges are for comparison only.
NOTES:
1. When using a highly sensitive detector such as ECD, NPD or
FPD, it is imperative that the syringe, glassware and solvents
be as clean as humanly possible. Use only resi--grade or redistilled
solvents, use clean or solvent-rinsed glassware, and never use
the same syringe to handle both concentrated and dilute solutions.
Also, never use chlorinated solvents or water for injection.
Clean the injecting syringe by drawing clean solvent into it
and the injecting it into a waste container several times, then
chromatograph only solvent to verify that the syringe is clean.
2. The reliability of the ECD results rests largely on three
factors: (a) cleanliness, (b) being able to inject reproducibly
on the GC (same operator makes all injections in a series) and
(c) ensuring that the detector is responding linearly. Check
the latter by injecting amounts of a standard solution in the
region of interest. Assuming your injection technique is good
the data when plotted mass vs response should provide a linear
plot. For ECD detectors, where this problem is most frequently
encountered, the linear range can vary from day to day. The newer
detectors have a much broader linear range than their predecessors,
but it is still important to check for linearity when doing any
quantitative analysis.
3. The FPD in sulfur mode is not a linear detector. Standard
curves on this detector are exponential. This will be explained
lecture. In the phosphorus mode, it is linear.
TO INJECT
HPLC Rheodyne injector: This is a fixed loop type-the
loop size is on a tag hanging from the loop. Make sure that the
needle is clean by rinsing it with solvent several times. Put
the needle into your sample solution and draw it into the syringe
and expel it again once or twice. Fill the syringe with at least
5 ml more than your desired injection
volume. Get ride of bubbles by tapping the side of the barrel
and then expel them by pushing the plunger a few ul until the
bubbles have exited the syringe. Rapidly turn the injector to
the load position, insert the needle and with a consistent and
fluid manner depress the plunger. To "inject" your
sample onto the analytical column rapidly turn the injector to
the inject position while the syringe remains in the injector.
Simultaneously, with your left hand, press START on the integrator.
Remove your syringe and rinse several times with solvent.
GC injection: If you wash a GC syringe in solvent, depress
the plunger (so the syringe is nominally empty) and then pull
the plunger back and look at the barrel, you will notice just
under 1 ul of solvent remains. This will be vaporized when the
needle gets hot upon injection, and a variable amount enters the
GC. To eliminate the variability that would be caused if this
were sample, a special technique is used to measure sample for
GC injection.
First, clean the syringe by rinsing it in solvent. Then with
the needle out of solution draw the plunger back so the end of
the plunger is at the 1 ul line. Place the needle in your sample
and draw the plunger up exactly to the amount you want to inject
(if you want to inject 2 ml, the end
of the plunger barrel is pulled up to 3). Remove the needle from
the sample, and pull the plunger back into the barrel and look
at the results. You will see the end of the plunger, a clear
area (solvent) just under 1 ul long, a shiny 1 ml
space (air), another clear area (sample) that should exactly be
the number of ml you wanted to measure,
followed by another shiny area (air). You will be delivering
exactly the amount of sample you intend, and the variable amount
of solvent that also gets injected will not change the sample
amount. After injecting, rinse the syringe with solvent many
times.
LAB REPORTS: Laboratory reports will be graded by the
following criteria.
Long reports are 100 points each: Overall Length Limited
to 5 pages or less (minus graphs, tables, and figures
etc.).
Abstract (10) (2) State objectives & scope of study
(2) Methodology described
(2) Summarize results
(2) States principle conclusion
(2) Overall clarity
Introduction (15) (5) Background information on the methods
(3) Rationale for present study (purpose)
(5) Background information on the analyte
(2) Overall clarity
Materials & Methods (20) (5) Instrumentation & conditions
(13) Experimental procedures & materials
(2) Overall clarity
Results & Discussion (40) (3) Overall description of experiment
(15) Presentation of data, tables, figures etc.
(15) Discussion of results; what do they mean?
(5) Questions from lab handout
(2) Overall clarity
Conclusions (5) (5) Overall summary
References (5) (5) Proper and thorough
Length limit (5) (5) Proper length (5 if 5 pages; 0 if greater)
Short reports are 70 points each: Overall length limited
to 4 pages or less (minus graphs, tables, and figures
etc.)
Abstract (5) (1) State objectives & scope of study
(1) Methodology described
(1) Summarize results
(1) States principle conclusion
(1) Overall clarity
Introduction (8) (3) Background information on the methods
(2) Rationale for present study (purpose)
(1) Background information on the analyte
(2) Overall clarity
Materials & Methods (10) (2) Instrumentation & conditions
(7) Experimental procedures & materials
(1) Overall clarity
Results & Discussion (35) (3) Overall description of experiment
(10) Presentation of data, tables, figures etc.
(15) Discussion of results; what do they mean?
(5) Questions from lab handout
(2) Overall clarity
Conclusions (4) (4) Overall summary
References (3) (3) Proper and thorough
Length limit (5) (5) Proper length (5 if 4 pages; 0 if greater)
NOTE: Please strive to draw connections throughout your
report between the experiment you have completed and the larger
principles/concepts in environmental chemistry.