EXPERIMENT #1

Pipette Calibration, pH Measurement and Statistics

OVERVIEW

The purpose of this experiment is to become familiar with the lab by making some simple measurements with some of the tools we will use for other labs. The statistical concepts we have discussed in lecture (mean value, standard deviation, confidence intervals, etc.) will be applied to the calibration of micropipettes and to the pH measurement of several samples.

Equipment Needed

Lab Notebook, Safety Glasses, Lab Coat

Balance

Weighing boats

Teflon spatula

Deionized water from water purification system

Thermometer

pH Probe and VWR Symphony pH Meter

Wash bottle with deionized water

100 ml volumetric flask

Glass Funnel

Micropipettes and pipette tips

Test tubes and rack for holding samples

Beaker of salt (provided by teaching assistant)

Dilute sulfuric acid sample (provided by teaching assistant)

Cloud/fog samples (provided by teaching assistant)

Table of density of water (provided by teaching assistant)

Procedure

Calibration of micropipettes

The Finnpipette micropipettes used in this lab are a convenient way to measure and dispense small volumes of liquids. Disposable polyethylene tips are used with these pipettes. The teaching assistant will demonstrate how to attach the tips, how to pick up liquids and dispense them, and how to discard the used tips. In later lab exercises it will be important to be able to use these pipettes correctly in order to properly measure solutions and prevent contamination of samples.

Check that the analytical balance you will use is level. Set the 40 - 200 µl pipette to 200 µl and use it to pick up a clean tip. Place a clean weighing boat on the balance and tare the reading to zero (depending on the balance you are using, you may have to open the glass doors in order to place things on the balance; it's a good idea to close the doors when taking readings to prevent interference from air currents). Add 200 µl of deionized water (DI water) and record the reading. Repeat this for a total of five measurements (either remove the weighing boat, empty it, dry it, and re-tare it, or continue adding water to the weighing boat and re-tare it in between measurements -- which method do you think is more accurate?). Set the 200 - 1000 µl pipette to 1000 µl and record 5 measurements. Record the temperature of the DI water. The teaching assistant will have a chart of the density of water as a function of temperature. Use this chart and record the density of the water you have used for these measurements.

Calibrate pH probe

The teaching assistant will demonstrate how to connect the pH probes to the portable Orion pH meters. In order to calibrate the pH probe you will use buffer solutions. We will use pH 4.01 and pH 7 buffers. Transfer approximately 1 ml of the pH 4.01 buffer into a test tube and place it in the test tube rack. Do the same for a second tube with pH 7 buffer.

First rinse the tip of the probe with DI water using your wash bottle. Gently touch the end of the probe against the plastic beaker to remove any water adhering to the tip. Rinse the probe with ~500 µl of the pH 7 buffer. Place the probe into the test tube with the 1 ml of pH 7 buffer solution.

In order to start the calibration, first set the temperature. Turn on the pH meter. Press the setup key then press the DOWN ARROW 4 times to reach the temperature setting. Press the RIGHT ARROW key (has three boxes with arrows) twice to access the temperature setting. Use the DIGIT CHANGE KEY (left of the arrows) to select each digit and the UP/DOWN ARROWS to adjust the temperature digits as needed to match the temperature of the water you measured above. Press the RIGHT ARROW then the DOWN ARROW to exit the temperature set mode, then press the MEASURE key to exit the setup. The temperature you set will be displayed in the upper left corner of the display.

Place the pH electrode into the pH 7 buffer, then press the CALIBRATE button. The meter should recognize the buffer and set the display to the appropriate value. Press the CALIBRATE key to accept the autobuffer recognition as the first calibration point and start the second calibration point (4.01) when prompted for the second buffer. After the second buffer is autorecognized, press the MEASURE key to exit calibration mode and return to measurement mode. Before returning to measurement mode, the electrode calibration slope will be displayed. Record this in your lab book. You should find a slope close to 100%. The slope should be at least > 90%. If not, calibration should be repeated until a high enough slope is obtained. If this takes more than two tries, consult the TA.

If something went wrong with the calibration, an error code may appear and you will have to perform the calibration again. If the problem persists, consult the TA.

Checking the calibration periodically is a good idea. If one of the buffer solutions does not read within a couple one-hundredths of the calibration value, you should recalibrate. In between measurements (or when the pH portion of this experiment is completed), leave the probe soaking in the pH 4.01 buffer. Do not leave the probe for an extended period of time in DI water.

pH Measurement of the Diluted Acid Solution

Now that the pH probe has been calibrated, you can measure the pH of the diluted acid solution the teaching assistant has provided. Rinse the pH probe with 0.5 ml of the acid solution and then place it in a test tube with approximately 1 ml of the acid solution. Take three measurements of the pH of this solution. Use the same tube with a fresh portion of acid each time, rinsing the probe with the dilute acid solution between each measurement.

You now want to make three measurements of a 1/10 dilution of this acid solution. Prepare enough diluted acid (mix well!) to make three pH measurements, remembering you need to rinse the electrode with solution prior to each measurement. Make the pH measurements and record the results in your lab notebook (you should be recording all procedures and results there).

pH of Deionized Water

Measure the pH of the DI water (three times) and record the result in your lab notebook.

The teaching assistant has provided a bottle of an inorganic salt. The molecular weight of the salt is marked on the bottle. Each group should make a 0.1 M solution of this salt. When you're ready to make the solution, use one of the spatulas to transfer the salt from the bottle. Remember not to put any chemical back into the original salt container, and clean up anything you may spill on the balance. The teaching assistant can show you a way to steady your hand as you transfer chemicals from the spatula into the weighing boat.

Recheck your calibration using fresh pH 7 and pH 4.01 buffers. Measure the pH of the solution you've made and record the results in your lab notebook.

pH of Cloud/fog Samples

The teaching assistant will provide you with actual cloud/fog samples collected from two different locations. Measure the pH of each sample three times, using the same procedures you've used above with 0.5 ml for rinses and a fresh 1ml for the measurements.

Data Exchange

Before you leave the laboratory, be sure to review the list of topics below to discuss in your lab write-up. Exchange data with other groups as needed.

Discussion/Lab Write-Up

Begin this, and each lab writeup, with a brief summary of the experiment in your own words. State the purpose of the experiment. Answer each of the questions below and address the discussion points. Consider these questions/discussion items as a starting point. Expand your analysis/discussion to include other relevant or interesting points as you see fit.

Statistical Evaluation

Using the statistical methods we've discussed in class and the material in Chapters 2-4 of the Fundamentals of Analytical Chemistry text by Skoog, West and Holler, answer the following questions:

1. What are the means and the 95% confidence limits for the pipette measurements?

2. What is the 95% confidence limit of your pH measurements for cloud/fog samples with pH < 4? Use pooled statistics for data from your group and other AT 560 groups to obtain a pooled standard deviation.

3. Are the pH measurements of the two cloud/fog samples you measured different at the 95% confidence level? Be sure to perform a hypothesis test; don’t just check whether the confidence intervals overlap.

4. Compare your measurements of the cloudwater sample pH values with those obtained by other lab groups. Are your results statistically different?

Discussion

o Comment on your experience with any difficulties measuring the pH of individual samples.

o What effect did the salt addition have on your ability to measure the deionized water pH value?

o Compare the pH measurements of the original and diluted acid solutions. Is the pH difference what you expected?