Lab 4: Lake and River Water Chemistry
Introduction
Traditionally, chemical analyses of lakes and rivers have focused on those
elements that are necessary for the growth of algae and aquatic plants, especially the
elements phosphorus, nitrogen, silica, and carbon. In addition, dissolved oxygen is
measured as a means of determining habitat suitability for animal life and determining
metabolic rates of photosynthesis, respiration, and decomposition. Determining the
concentrations of P, and N in water samples requires several analyses that measure these
elements in all of their forms, each form having a different meaning for the ecology of
the system. For example, ammonium (NH4) is more readily used as a nitrogen source by
algae and plants than nitrate (NO3). A typical suite of chemical analyses of fresh water
would include: nitrate (NO3), nitrite (NO2), ammonium (NH4), Total Kjeldahl nitrogen
(organic nitrogen), silica (SiO2), alkalinity (for inorganic carbon), Total phosphorus (TP),
and soluble reactive phosphorus (SRP).
Concentrations of these compounds are determined mainly by two types of
analysis: spectrophotometry and titration. In this lab we will perform two analyses as
examples – Total Phosphorus by spectrophotometry, and alkalinity by titration. For
extensive discussion of chemical analyses see Limnological Analyses (Wetzel and Likens
2000)
Total Phosphorus
The chemical speciation of phosphorus in fresh waters is very complex. In general, it
is classified into analytically defined groups that may or may not have simple
interpretations. These categories include soluble reactive phosphorus-phosphate (SRP),
total dissolved phosphorus (TDP), and total phosphorus (TP). Strickland and Parsons
(1972) contains a detailed discussion of these groupings and the analysis necessary for
each.
In this procedure, we will determine Total Phosphorus (TP). The first step in the
analysis is to “digest” the sample – to use a strong oxidant to convert all of the
phosphorus in the sample (including particulate P and organic P) to SRP. Total
phosphorus can then be determined using the relatively simple procedure for SRP
Overview
In this procedure the phosphate in a filtered water sample reacts with a mixed reagent
of molybdate, ascorbic acid and trivalent antimony, forming a yellow phospho-molybdate
complex. This is then converted by reducing agents to a blue complex. The
concentration of P in the sample will be proportional to the absorbance (intensity) of the
blue color.
Reagents (These will be prepared for you ahead of class)
A. Ammonium molybdate solution: 15 g up to 500 ml DI water
B. Sulfuric acid: 140 ml added to 900 ml DI water
C. Ascorbic acid : 27 g in 500 ml DI water
(stock frozen and thawed immediately prior to analysis)
D. Potassium antimonyl-tartrate solution: 0.34 g in 250 ml DI water
Stocks that are frozen or refrigerated need to be warmed to room temperature before
using. Stock = 0.2197g KH2PO4 oven-dried (105o C, 24h) dissolved in 1L DI water,
then diluted by half.
Procedures
Digestion of Samples
1. Pour 100 ml of well-mixed samples or distilled water blank into a dry and acidwashed erhlenmeyer flask.
2. Add 2.0 ml of strong acid solution and 1.0 gram of potassium persulfate (safety
glasses/gloves; no mouth pipetting).
3. Record weight of flask and contents
4. Boil gently on a pre-heated hot plate for 30 or 40 minutes (in a fume hood), or until
an approximate volume of 10 ml is reached.
5. After cooling, add 2 drops of phenolphthalein indicator. Neutralize to a faint pink
color with drops of 1N NaOH. Add drops of dilute acid solution to just discharge
the color.
6. Add DI water to flask to return to weight recorded in #3.
Analysis (Step 1 will be done for you ahead of class)
1. Make a MIXED REAGENT solution by adding, in the alphabetical sequence only
(adding reagents out of order will ruin the test), the reagents listed above in the following
proportions. Mix thoroughly after adding each reagent. You will need 5ml of mixed
reagent per reading. Count your samples, standards, and blanks (x2 for duplicates) and
make only as much as you need.
# of readings
2
20
30
A
2 ml
20
30
B
5 ml
50
75
C
2 ml
20
30
D
1 ml
10
15
Total Volume
10 ml
100 ml
150 ml
Keep mixed reagent out of direct sunlight and let stand for 10 minutes before analyses.
The mixed reagent is stable for about 6 hours
2. STANDARDS
a. Prepare duplicate standard solutions at 5 concentrations (0, 25, 50, 100, 200 g/L)
by diluting the stock standard solution as follows:
Note the sizes of volumetric flasks that are available (250, 500, and 1000ml) and
calculate the amount of stock and DI needed to make each solution. Be
economical, you will only need 50ml of each duplicate. Fill in the volumes on the
chart below ahead of class.
Volumetric pipettes and flasks (not graduated cylinders or auto-pipettes)
should always be used when making up standards. If you think you may be off by
even a fraction of a ml in any measurement, start over!
[Stock Standard Solution: 1 ml contains 25 g of P, we don’t count the other
molecules in the KH2PO4 ]
0
25
50
100
200
g/L Blank
g/L = 1
g/L =
g/L =
g/L =
ml stock in 1000 ml DI water
ml stock in 500 ml DI water
ml stock in 250 ml DI water
ml stock in 250 ml DI water
Prepare blanks and standards. All spectrophotometer readings should be taken 10
minutes after adding the mixed reagent.
b. Prepare a reagent blank by pouring 50ml of DI (use the digested, cooled DI
prepared earlier) into an Erlenmeyer flask and adding 5ml of mixed reagent.
(Optional step for very low P samples) Prepare a 2x reagent blank by adding
twice the amount of mixed reagent to 50ml DI.
c. Prepare a color blank by adding all reagents except “D”, the potassium antimonyl
tartrate (4.5 ml total) to 50ml of the sample water.
d. Pour 50ml of each standard solution into labeled Erlenmeyer flasks.
e. Add 5 ml of mixed reagent to each labeled flask.
f. After 10 minutes reaction time read absorbance at 885 nm using a 10cm long
spectrophotometer cell. Read samples in order of increasing concentration. Rinse
the cell with a small amount of developed sample before filling the cell for
measurement.
3. UNKNOWNS
(Ottawa River 1, Ottawa River 2, Allens Lake 2m, Allens Lake 8m, Maumee Bay)
a. Add 50 ml of samples to Erlenmeyer Flasks.
b. Add 5 mls mixed reagent.
c. Measure as above.
4. Calculations - Using Excel, plot standard absorbance (x-axis) against standard
concentration (y-axis) and insert a linear best-fit line. Determine the slope of this line
(standard curve). Run a regression: Is R2 within acceptable limits? Using the slope
of the line, the concentration of the unknown samples in g/L is:
g/L = (sample abs - reagent blank abs - color blank abs) x (slope)
Save file on computer and print out your standard curve with R2, line equation, and
concentration of unknowns. Include with your report.
Gran Alkalinity Analysis
Overview
The term “Alkalinity” is synonymous with “Acid Neutralizing Capacity” and
essentially means the acid buffering capacity of the carbonate system in water. By
gradually adding strong acid to a water sample, we can determine at what point the
supply of buffering ions (carbonate and bicarbonate) in the sample is exhausted.
Alkalinity is an important feature of water bodies faced with increasing acidity due to
acid rain or runoff from industrial operations. In addition, alkalinity can be used to
estimate the concentration of dissolved inorganic carbon (DIC) available for use in
photosynthesis by algae and aquatic plants.
The most accurate method for determining alkalinity is by the Gran titration
method. We will use a strong acid to titrate well beyond the equivalence point. The
proportionality of acid added to the resulting pH is used to determine the equivalence
point with either graphical or mathematical methods (See Wetzel and Likens, pp117-121,
126-127).
Reagents
0.1 N sulfuric acid (for soft water use 0.01 N)
Equipment
pH meter
magnetic stir plate
Digital burette with 0.01 ml gradation
Procedure
(Perform alkalinity titrations on each of the samples: Ottawa 1, Ottawa 2, Allens Lake 2
m, Allens Lake 8m, Maumee Bay)
1. A pH meter is used throughout the analysis to determine pH. Standardize the pH
meter using buffers of 4.0 and 7.0.
2. Measure 100 ml of sample in a graduated cylinder and add to a beaker.
3. Insert pH probe and stir bar.
4. Determine initial pH.
5. Titrate sample to approximately pH 4.0 and record the volume of acid used. Note: pH
drops rapidly below pH 5. Continue to titrate in 0.10 ml increments or less and record
pH and the cumulative volume of acid after each addition. Stir the sample continually
and allow pH reading to stabilize before recording pH.
6. Titrate sample to a final pH of approximately 3.2. Ensure that you have several points
(8-10) between pH 4.0 and 3.2.
7. Mathematically the ml of titrant at the equivalence point may be determined from the
Gran F1 function.
F1 = (Vo + V) * 10(-pH)
where:
Vo = volume of sample
V = volume of acid
The X intercept of the best fit regression line for the last several data points (ml, F1) is
the acid volume necessary to neutralize the sample to the equivalent point. This may
be easily solved on excel. With F1 on the y-axis and ml acid on the x-axis, fit a linear
trendline through the last several data points and get the equation of the trendline. The
ml of acid at the equivalence point will be where the trendline crosses the x-axis.
9. The alkalinity is reported in milliequivalents (meq) per liter using the following
formula.
Alk = (ml of titrant to equivalence point) * (Normality of titrant)
(sample volume in liters)
Alkalinity is often expressed as mg CaCO3 L-1. To convert, multiply meq/L by 50.
Note: Normality
= Equivalents/liter
= mEquivalents/ml
10. Calculate dissolved inorganic carbon using:
DIC = (total alkalinity) x (pH factor [see table])
= mg carbon / L
Questions
Questions:
1. What would doing a 2x reagent blank tell you?
2. Compare TP between the different locations. What do you think accounts for the
differences in TP between the two streams of the Ottawa River, Allens Lake and Maumee
Bay?
3. Use the table (from Limnology, R.G. Wetzel) to characterize the samples from Allens
Lake and Maumee Bay as Oligotrophic, Mesotrophic, Eutrophic, or Hyper-eutrophic.
Where do the Ottawa tributaries fall on this scale?
4. How does TP in the epilimnion of Allens Lake compare to the hypolimnion? Give a
potential explanation for this.
5. Answer questions #2,4 above for your alkalinity results.
Lab Report
As usual, organize your report into typed sections consisting of:
Introduction: You may use readings from your text or Limnological Analyses (Wetzel
and Likens 2000) to introduce the reasons for analyzing water samples for TP and
alkalinity.
Methods and Materials: Can be cut and pasted from your lab documents or other sources.
Point out any changes made to the protocols.
Results: This section mainly consists of figures and tables that summarize your results.
Be sure to include figures of your standard curve for TP and your trendlines for
alkalinity. Place all TP and Alkalinity results of field samples into 1 table for easy
comparison. This section should briefly point out noteworthy features of the table and
figures.
Discussion: This section should answer the questions above and expand on your
interpretation of the results. If there were any problems with the standard curve or
analyses, discuss possible sources of error or contamination.
ADDITIONAL ASSIGNMENT FOR GRADUATE STUDENTS (optional for
undergrads): Read the “General Water Chemistry Protocol” and “Spectroscopy”
documents and submit answers to the questions.