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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.

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