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VI. EXPERIMENTS
A. LAB 1. SAMPLE COLLECTION AND FIELD MEASUREMENTS
1. OBJECTIVES:
1. Collect water samples to be used for all subsequent labs; 2. Preserve samples such that they are not compromised for future analyses; 3. Through measuring pH of samples, learn the meaning of pH and the theory and practical aspects of its measurement; 4. Through measurement of conductivity, obtain an assessment of the ionic strength of the samples; 5. Measure the suspended solids concentration and turbidity to assess the prevalence of particles; 6. Measure the dissolved oxygen content as an assay of water quality and for later assessment of air‐water exchange equilibrium. 7. Learn preliminary aspects of quality control. 2. BACKGROUND a. Sample collection: One of the objectives of this course is to help you to understand the world around you as well as to understand some of the specific practices in which you may be involved in your careers. To this end we will examine the environmental chemistry of surface and ground waters in the local area. The samples collected now will be used for all subsequent labs in this course; by the end of the course you will have acquired information concerning the composition of major and trace constituents of the waters. In the last two weeks of the course, your group will summarize one aspect of the chemical characterization of all of the samples. Most water samples will be collected during week 2. During sampling, we will measure and record water temperature, pH, dissolved oxygen, conductivity, and turbidity. Upon return to the lab, you will filter, and prepare sub‐samples for proper storage for subsequent analyses. The plastic bottles used to transport the bulk water samples back to the lab must be washed prior to use. The bottles should be rinsed three times with an aliquot of sample before being filled. All potentially important characteristics of the sampling site should be recorded, and efforts should be made to avoid contamination and to obtain a representative sample. b. Container preparation Quality control refers to the steps that are taken to assure that analytical results are accurate (i.e., give the correct value) and as precise (i.e., reproducible) as possible. One important aspect is sample contamination; it is of little value to have a sensitive, reproducible, and accurate analytical technique if samples are contaminated. Another problem is alteration of the sample during storage. Some components are likely to be consumed or excreted by microorganisms, other substances may adsorb to container walls, and others may volatilize into the air. Hence selection of the appropriate container type, proper pretreatment of the container, and appropriate storage conditions are important to maintain the integrity of any sample. The samples collected in this lab will be analyzed for a variety of substances over the 1
course of the term, and hence a number of different container types, pretreatments, blanks, and storage conditions are required. A basic pretreatment of all sample containers is washing. Containers are washed with a phosphorus‐free detergent, rinsed several times with tap water, rinsed several times more with deionized water, soaked for some period in deionized water, and finally rinsed several times with the highest purity water available. For substances that sorb strongly to container walls (e.g., trace metals, phosphate, ammonium) the containers must be soaked in dilute acid rather than deionized water. This treatment would cause contamination for other types of analyses (e.g., anions – the conjugate bases of commonly used acids are typically major anions that are found in natural waters). It is always important to fill one (or more) bottles with “pure” water that will be treated and analyzed in the same fashion as the samples. This sample “blank” is the only means of detecting contamination resulting from sample handling. c. Sample treatment An important component of sample treatment is the removal of particles. Many analytical techniques measure only the dissolved components, not those on particles. Particles actually interfere with some analytical techniques such as spectrophotometric measurements and chromatography. Furthermore, some substances tend to adsorb to particles, and bacteria generally are concentrated on particles. Removal of the particles minimizes changes in dissolved concentrations of substances that could be consumed by bacteria or adsorb to particles. Filtration is a common means of removing particles from water. In fact, the dissolved concentration of any substance is operationally defined as the concentration in a filtered water sample. Most filters are contaminated, however, and must be cleaned prior to use with samples. To clean a filter, purified water and (when appropriate) dilute acid are run through the filter prior to filtration of the sample. Again, it is important to filter the sample blank to determine if contamination occurs during filtration. The two changes that are most likely to occur during sample storage are sorption to sample walls and consumption or excretion by microorganisms. Several steps may be taken to inhibit these processes. Samples may be frozen to minimize (although not eliminate) bacterial activity. Preservatives (azide, formalin, chloroform) may be added that kill microorganisms. Acid or an excess of an appropriate ion may be added to prevent sorption to container walls. Samples may be stored in amber or foil‐covered containers to prevent photo‐induced reactions. d. Measurement of pH The measurement of pH is deceptively simple. All you do is stick the electrode in the solution and read the meter. Right? But what is pH? What is the meter? What is the electrode? How do you know whether the value you read is correct? By definition, pH is the negative logarithm of hydrogen ion activity. Hence, pH is inversely proportional to concentration, but it is not a direct measure of concentration. The relationship between activity (A) and concentration (C) is well known; A = γ • C where γ is the activity coefficient. Activity coefficients are a function of the ionic strength (I or μ) of the solution, and may be calculated according to a number of approximations such as the Debye‐
Hückel Equation (log(γ) = ‐Cz2•√I where z is the ion charge and C is a constant). These 2
approximations are most accurate in dilute solutions (I < 0.01 M). In highly concentrated solutions (I > 0.5 M) the approximations are quite imprecise. How then does one measure hydrogen ion activity? In theory it is easy. Chemical reactions respond to activities, not concentrations. Hence one could measure an equilibrium situation for a reaction involving hydrogen ions. The option usually selected is to measure the equilibrium potential between two electrodes. Electrodes allow us to measure the potential energy existing to drive a reaction by converting the chemical energy to electrical energy or an electromotive force (EMF). To measure pH, one needs to establish a chemical reaction in which the activities of all reactants except that of H+ are held constant. Most commonly this is done with a glass electrode. The glass membrane acts via a cation exchange mechanism to selectively pass H+ ions. The solution on the inside of the membrane is buffered at a constant pH. The potential across this membrane is dependent only on the activity of H+ ions in the solution. By connecting this electrode in a circuit with a reference electrode of known (constant) potential, one can measure the potential difference between these two cells which is a function only of pH. The relationship between the EMF and pH is quantified with the Nernst equation: RT
RT
E = Eo −
ln(Q) = E o − 2.303⋅
log(Q) nF
nF
in which E is the voltage drop between the two electrodes, Eo is the voltage drop under standard conditions, R is the gas constant, T is absolute temperature, n is the number of electrons involved in the electrochemical reaction at the electrode, F is the Faraday constant, and Q is the reaction quotient for the electrochemical reaction. We mentioned above that all parameters in Q are kept constant except the concentration of H+ ion. Thus the difference between voltages measured at two different pH values is: +
⎛ {H +}⎞
RT ⎛ {H1 }⎞
RT
1 ⎟ ⎜
⎜
⎟
E1 − E2 = −
ln
log
= −2.303
+
nF ⎝ {H2 }⎠
nf
⎝ {H2+}⎠
The numerical value of 2.313RT/nF is 59.16 mV; hence for a change in pH of one unit (i.e., a 10‐
fold change in {H+}), the theoretical difference in voltage readings is 59.16 mV. We refer to this value as the Nernst slope. In practice several problems arise. The reference electrode does not necessarily have a constant potential that is independent of solution conditions. The reference electrode must be in electrical contact with the glass electrode through the water (sample) solution. This connection is accomplished via a “salt bridge” or junction. The potential across this junction does not remain constant with time and is not completely independent of solution conditions. Factors such as stirring rate, sample viscosity, and measurement duration can affect the loss of salt (K+) ions across the junction and hence change the reference potential. Hence these conditions must be held constant by using calibration buffers of a composition similar to that of the samples to be measured. The most common problem is fouling (blocking) of the junction; this reduces the rate of ion movement and causes slow equilibration and drift of the electrode. All of these problems result in values of the slope that are slightly different than the theoretical Nernst value. Note that the electrode used in your laboratory sections has the reference and glass electrode combined in one unit. 3
Meter
A o
g
Junction
Ag+, K+, ClReference electrode
Cl-
Buffer
H+
Glass electrode
e. Measurement of conductivity Conductivity is a measure of the ability of a solution to carry an electric current. This ability depends on the presence of ions, the atomic‐scale particles that ferry charges through solution. Each type of ion (e.g., H+, Ca2+, etc.) has a unique ability to carry charge; on the atomic scale this ability is determined by the valence (charge) and mobility of the ion. For a solution, the total conductivity is the sum of the conductances of all of the individual ions. Hence, the conductivity depends on the concentrations and types of ions present. Conversely, measurement of conductivity gives an indication of the total concentration of ions present in a solution. We will use conductivity to estimate the total ion concentration in solution in terms of the "ionic strength". Ionic strength (“I” or “µ”) is defined as: 1
2
I = ⋅ ∑ cizi i
2
where ci is the concentration (moles/L) of species i, and zi is the charge of species i. The units of ionic strength are moles/L. A correlation is generally observed between ionic strength and conductivity; one such correlation (Snoeyink and Jenkins 1980) is: I = 1.6x10‐5•Conductivity where conductivity (also known as specific conductance) is given in µmho/cm. (Note that the SI unit for conductance is the siemens; one siemens (S) equals 1 ohm‐1 or 1 mho). You may have noticed that we used two different words above, conductance and conductivity. Conductance is defined as the inverse of resistance and has units of inverse ohms or mho. The resistance of any resistor depends on its size (length and area); to normalize for size, we often speak of the specific resistance. Similarly, we normalize conductance and speak of the conductivity or specific conductance. Conductivity, κ, is defined as: l
κ = L⋅ = L ⋅ k A
4
Voltage source
R4
R3
Pt electrode
R1
Sample
where L is the conductance (μmho), and k (cm‐1) is the cell constant whose value depends on the dimensions (length, l, and area, A) of the measuring cell. The units for conductivity are, therefore, μmho/cm or μSiemens/cm. Conductivity is measured analogously to an unknown resistance. A current is passed via two electrodes of known dimensions through a controlled volume of solution; the conductivity meter measures the resistance in the solution by balancing the solution resistance against internal resistors until no further current flows. At that point, the ratio of Rsolution/R1 is identical to R3/R4; since R1 is known, the solution resistance may be calculated. This measuring device, known as a Wheatstone bridge, enables very precise measurements of resistance. 3. PROCEDURES:
a. Container preparation (done the week before field sampling) Each group will prepare the 5 container types in the table below for three samples (i.e., 15 containers per group, 3 of each type). Specific methods for washing are given after the table. After sampling, each group will filter three samples, and then pour aliquots into the bottles indicated in the table. _________________________________________________________________________ Lab Vol. Bottles# wash filter* storage __________________________________________________________________________ Sample 1‐L plastic water GF/F anions 30‐mL plastic water GF/F freeze cations 30‐mL plastic acid** GF/F refrigerate alkalinity 250‐mL plastic water GF/F refrigerate DIC/DOC 40‐mL glass water GF/F refrigerate _____________________________________________________________________ #
minimum volume 5
* Rinse the filter with water (or acid solution for samples whose bottles were acid‐washed) and sample before filtering the whole of the sample. ** The GTA will acidify samples to a pH of approximately 3.0 with 3 drops of conc. HCl. Water wash: a) wash bottles in detergent and water; b) rinse three times with tap water; c) rinse three times with deionized water; d) fill bottles with deionized water and allow to soak for at least 1 hour; e) rinse bottles with Milli‐Q water. Acid wash: Steps a‐c as above. d) Fill bottles with 1% HCl and allow to soak for at least one hour. e) Rinse three times with Milli‐Q water. b. Sample Collection Two 1‐L plastic bottles will be filled at each sampling location. Rinse the sample bottle with the sample water before filling the bottle for transport to the lab. Use a bucket to collect a water sample, and then pour the water into the 1‐L plastic bottle. Alternatively, in flowing water, you can hold the bottle in the water facing upstream and collect directly into the bottle. Collect a “representative” sample by avoiding obvious inhomogeneities such as sediments and surface scum. Label the sample bottle so that it can be clearly identified. Record in your notebook a description of the sampling site and the label used on the bottle. c. Field Measurements Measurements of temperature, pH, conductivity, dissolved oxygen and turbidity will be made in the field. An individual group will be responsible for measuring the same parameter at all 3 sites that are visited. Procedures for the field measurements are given below. Similarly, back in the lab, the same groups will measure their group’s parameters in the water samples collected previously by the TA. Groups 1 & 6 will measure pH, Groups 2 and 7 will measure conductivity and temperature, Groups 3 and 8 will measure dissolved oxygen, Groups 4 and 9 will measure turbidity, and groups 5 and 10 will actually collect the water samples. At all of the field sites, make enough measurements of each parameter so that you know the variability of that parameter in that location; this will require between 3 and 10 measurements. Record all measurements in your notebooks. Instructions for use of the pH Probe I. Calibration: 1. Plug the pH electrode into the top of the HQ40d HACH meter. 2. Turn on the power. 3. Use the up and down arrows in the center of the meter to select pH mode. 4. Press the blue button to start calibrating the pH probe. 5. The meter will prompt you to insert the probe into a calibration solution. We will be using 3 different solutions to calibrate the probe. ( pH’s of 4, 7, and 10) Remove the 6
protective vinyl sleeve from the pH electrode and place it into the pH 4 buffer solution. Press the green read button to start reading the pH of the calibration solution. 6. The pH meter will automatically recognize the pH of the solution you are measuring. Once it automatically stabilizes and selects a pH value, it will prompt you to insert the probe into another solution. The pH 4 option will now be blacked out in the display window. Rinse off the probe and insert it into the pH 7 solution. Once again, press the green button to read a value for this pH solution. 7. Once the probe has calibrated for a pH of 7, rinse off the probe and repeat the procedure for the pH 10 solution. 8. Now that all of the calibration solutions have been measured, press the Up arrow on the key pad to select done. Accept the calibration and now the probe is ready to take measurements. Sample pH measurement: 1. Set the probe to pH mode by using the up and down arrows on the key pad. 2. Once you are ready, rinse off the probe and insert it into the solution that is to be measured. 3. Press the green read button and wait for the probe to stabilize. 4. When the lock symbol appears in the upper left hand corner of the display, a stable pH for the measured solution will be displayed. Record this value as the pH of the sample. 5. Make sure that you rinse the probe with water before you take your next measurement. Instructions for use of Conductivity Probe: 1. The conductivity probe is already calibrated and is ready to use. 2. To start taking measurements, plug the Conductivity probe into the top of the HQ40d HACH meter. 3. Turn on the meter. 4. Use the up and down arrows to select conductivity mode for the meter. 5. Now you should be ready to take measurements. Insert the probe into the solution that is going to be measured. Then press the green read button to take a measurement. 6. The reading will be stable when the lock symbol appears in the top left hand corner of the display. Once this happens, record the conductivity reading that is displayed on the screen. This is the conductivity of your measured solution. 7. Be sure to rinse the probe with distilled water before you take your next measurement. 7
Instructions for Use of Dissolved O2 Probe: 1. The dissolved O2 probe that we are using is very precise and only needs to be calibrated once every year, so you will not have to do this calibration. 2. Make sure that the LDO O2 probe is plugged into the HQ20 HACH meter. 3. Once this is done, press the power button on the meter to turn the device on. 4. Remove the plastic cover from the tip of the O2 Probe. Be careful not to damage the tip of the probe. 5. Rinse the probe off with distilled water and then insert the tip of the probe into the water sample that is to be measured. 6. Press the blue check mark button to start taking a reading. Once the meter displays a stable value, record the number down. This is the concentration of dissolved O2 in the water. 7. Make sure that you rinse off the probe with distilled water before taking another measurement. Instructions for use of Turbidimeter Calibration Instructions: 1. To calibrate the turbidimeter, first press the power button on the device. 2. Next, press the cal. Button to start the calibration. The turbidimeter will ask you to insert a specific vial that it wants to measure. 4 calibration vials will be used for this process. It will first ask for the vial labeled as <.1 ntu. 3. Take this vial out of the sleeve and invert it 3 times to make sure that the water is thoroughly mixed. 4. Next, wipe off the vial with a Kimwipe to make sure that the vial has no fingerprints on it. 5. Next, insert the vial into the hole in the turbidimeter while matching up the arrow on the jar with the arrow on the turbidimeter. Close the lid and press the read button to take your first measurement. 6. The turbidimeter will start to count down from 60. Once it has reached a stable value, it will stop. 7. Now you can take out the calibration sample. Now repeat steps 3 – 6 with the remaining calibration bottles. Once all bottles have been measured, press the cal button again to accept the calibration that you have just made. Then you are done. Note: Be very careful when inserting and removing the vials that you do not scratch them. Small scratches on the glass surface can ruin the calibration. Now the device will prompt you to insert the next calibration sample (20 NTU). Use of the Turbidimeter: 1. Once the calibration is complete, fill up a sample bottle with the sample that you want to measure. 2. Wipe off the bottle with a kimwipe to ensure that it is clean and insert it into the turbidimeter. 3. Press the read button on the turbidimeter and wait for the device to stabilize. 8
4. Once stable, the turbidimeter will display a value in NTU that will be the turbidity measurement of the liquid. Record the measurement, remove the sample and clean out the bottle for future measurements. d. Sample filtration, preservation, storage Each group will have three 1‐L samples to process according to the procedures below. For each sample, you will use two filters; one of these you will discard, and the other will be kept and dried for determination of suspended solids (TSS) concentrations. As you work your way through the procedure, remember that you should rinse every bottle with (filtered) sample before filling it with the sample. To avoid contamination, perform the filtration in the following order. 1. Label all of the sample bottles as directed by the GTA. 2. Assemble the filtration apparatus as demonstrated by the GTA using a glass fiber filter (GF/F). 3. Rinse the apparatus and filter by filtering a small volume (~100 mL) of Milli‐Q water and discarding it. 4. Next, filter a small volume of sample (≈100 mL) and discard this water. 5. Filter about 0.4 L of the sample through the membrane filter, rinse a 30‐mL and 250‐mL plastic bottle with some of this water, discard the rinse, and then fill the 30‐mL bottle (for anions) and fill the 250‐mL bottle (for alkalinity). Also rinse the 40‐mL glass vial with sample, and then fill the vial with sample. 6. Now filter a small volume (≈50 mL) of dilute acid solution (from the squirt bottles) through the filter, rinse the flask with the acid and discard this volume. 7. Again, filter a small volume of the sample (≈50 mL), rinse the flask and discard this volume. 8. Now filter about 50 mL of the raw sample and, after rinsing the last 30‐mL bottle with some of the filtered sample, pour the filtered sample into the 30‐mL bottle (for cation determinations). 9. Measure in a 1‐L graduated cylinder the remaining volume of sample and record this volume. You will filter the entire volume for TSS determination. 10. Remove and discard the filter that is on the filter apparatus. 11. Put the pre‐weighed filter from the labeled petri dish on the filter apparatus. Record the information on the petri dish in your notebook. 12. Filter all the remaining sample volume through this filter. 13. Transfer the filter back to the petri dish, and cap the petri dish. 14. Repeat the 13 steps above for the next two samples. 15. Give all sample bottles and petri dishes with filters to the TA. ================================================================ 9
4. DATA ANALYSIS FOR FOLLOWING LAB SESSION For the following week each group should do the following: 1. Make a table of all of the sampling locations for your lab session and the values for the field measurement that your group performed. Materials: Week 1 and 2 in lab: Sample bottles (~24 1-L plastic)
Storage bottles (~65 30‐mL plastic, 42 60‐mL plastic, 22 125‐mL plastic, 22 250‐mL plastic, 75 glass DIC vials) Field: Hach meter with conductivity, pH, LDO probes Turbidity meter Storage bottles (1‐L plastic), 2 for each site Sharpies, labeling tape Sampling bucket on rope Week 2 in lab: tape, markers filter apparatus (5) GF/F filters – half pre‐weighed for TSS forceps labels Squirt bottles with 1 % HCl solution Squirt bottles with Milli‐Q water 10