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To teach the techniques of standardization and improve your volumetric and gravimetric lab

A solution which is to be used as a titrant in titrimetric analysis must, in most cases, be one
whose concentration is known to four significant figures. Such a solution is referred to as a
standard solution. When the solution is measured in a buret to four significant figures and the
sample weighing is also made to four figures, the analyst is justified in carrying four figures in
their calculated results, as you can demonstrate yourself by calculating the standard deviation for
standardization of the NaOH in this experiment.

Standard solutions may be prepared by either the direct method or by the indirect method. In the
direct method, an exact weight of a primary standard is taken, dissolved and diluted. In the
indirect method, the solution is prepared to an approximate concentration and then standardized
by titration against a primary standard or against another solution which has itself been
standardized against a primary standard. We will use the direct method to determine the
concentration of a prepared (but not standardized) NaOH solution in this experiment.

Solid sodium hydroxide is not available in high purity because of its strong reactivity toward
CO2 of the atmosphere. The pellets of solid NaOH in the reagent bottle can be contaminated
with Na2CO3 because of the reaction

2NaOH + CO2 Na2CO3 + H2O

Sodium carbonate is fairly insoluble in a saturated solution of sodium hydroxide and any sodium
carbonate present will settle to the bottom of the container. Therefore this solution assures a CO2
free source of NaOH. At room temperature a saturated NaOH solution is about 50% NaOH and
is 18-20 M. Carbon dioxide free water should be used for dilution in preparing the 0.1 N solution
of NaOH to avoid the occurrence of the above heterogeneous reaction in solution. This water can
be obtained by boiling distilled water for a few minutes to drive out the dissolved CO2.

We will use potassium acid phtalate (KHP, MW = 204.2 g/mole) for standardization. The
structure of this compound is given below.


O- K+

When standardizing HCl and NaOH solutions, the NaOH solution is used as the titrant (the
solution kept in the buret) in order to minimize absorption of CO2 from air by the NaOH solution
and to retain the more readily perceived indicator color change from colorless to pink (rather
than pink to colorless). The concentration of the NaOH should be calculated to the 4th decimal
point since the volumes measured in the titrations are all made to four significant figures. Both of
these solutions will be used in succeeding experiments in this course so they should be kept
tightly stoppered in your desk.

Experimental Procedure for NaOH Standardization

WEAR YOUR GOGGLES at all times in this lab. Base solutions are extremely caustic to your
eyes, and will take only a few seconds to blind you. The people around you are probably more
dangerous to you than you are, so don't take them off while you are in the lab even if you aren't
doing anything yourself.

A. Preparation of approximately 0.1 NaOH (This part will be done for you)

1. Half fill the rubber stoppered liter tincture bottle in your desk with boiled distilled water and
add about 6 ml of saturated NaOH solution. This is best added by using a syringe-transfer pipet
so as to avoid the use of a graduated cylinder. Be sure to keep from disturbing the insoluble
Na2CO3 which forms a layer of sediment on top of the excess solid NaOH on the bottom of the
container. Do not allow the saturated NaOH to run down the walls of the bottle since this
solution has a great affinity for glass surfaces and is difficult to rinse away from the bottle walls.

(Your instructor may choose to dispense the saturated NaOH for you for safety reasons and in
order to speed up the process. If so, be sure to bring up your liter bottle half-filled with boiled
water, and WEAR YOUR GOGGLES!!)

2. Shake the bottle vigorously for 5 minutes to mix the NaOH thoroughly and finally dilute to
near the neck (leaving some space so you can get good mixing) with boiled distilled water.
Shake vigorously for another 5 minutes in order to mix in this water. Label the bottle
approximately 0.1 NaOH.

B. Standardization of 0.1 N NaOH.

1. Weigh out approximately 0.5 g of KHP, but record the final weight to 4 significant figures.

2. Dissolve the acid in approximately 25 mL of distilled water in a 125 or 250 mL Erlenmeyer


3. Add 2 - 3 drops of phenolphthalein indicator solution to the flask, and titrate each Erlenmeyer
flask with your prepared 0.1 N NaOH to the first pink color that persists for at least 30 seconds
(why might the pink color fade in time?). Be sure to read the initial and final volumes of NaOH
to the nearest 0.01 mL for each titration.

4. Repeat this procedure for another two samples. Note that you can wash a fraction of a drop
off the tip of your buret as well as the walls of the flask with your squirt bottle when you are near
the end point. The extra water won't hurt, and it can help the overall precision.

5. As a quick check on your precision, calculate the ratio to weight of KHP divided by volume
of NaOH for each sample titrated and determine the percent difference between the high and low
values. As you increase your skills, you should be able to produce three successive titration
results that agree to within less than 0.5% of one another (the equipment is capable of giving a
precision of 0.2%). If agreement is not better than 0.5 - 1%, you will need to improve the
agreement with more trials. In calculating the concentration you should reject only those results
which fail to meet the Q test or which come from titrations which you know you did incorrectly.
(If you know you made a mistake, you usually repeat the titration and throw out that analysis
before you do the statistics.)

6. Calculate the exact concentration of your NaOH solution expressing it to 4 figures and label
the bottle with this information.

7. Use this standardized solution for the potentiometric and colorimetric titrations of a sports

Lab Report Information

Weight KHP:

Three trials: ___________g ______________g ______________g

Volume NaOH:

Three trials: ___________mL ______________mL ______________mL

Concentration of NaOH:

Three trials: ___________M ______________M ______________M

% RSD: ___________%

Concentration of NaOH: ________________ ________________ M


To use potentiometric and/or colorimetric titrations to analyze the total acidity of a sports drink
sample. The total acidity may be due to any or all of the following species: H3Cit, H2PO4- and

Introduction to Titration Curves:

During the course of an acid base titration the pH of the solution being titrated continuously
changes. For example, in the titration of an acid with a base, the acid solution will have a pH
less than 7 at the beginning and will show a gradual increase in pH as the base titrant is added.

As the equivalence point is approached, the pH of the solution will increase more rapidly with
the addition of a given amount of titrant. When the equivalence point is reached, the pH will
increase very rapidly. After the equivalence point the pH will continue to rise as titrant is added,
but the increase will be at a slower rate. The way in which the pH changes with addition of titrant
may be readily seen by plotting pH versus volume of titrant. Usually pH is plotted on the
ordinate and mL of titrant plotted on the abscissa. Such a graph is known as a pH titration curve.
A typical titration curve for a weak acid with a strong base is illustrated in Figure 1.

Fig. 1 Titration of a Weak Acid with NaOH


8.0 mid-point



0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0

volume NaOH

When a weak acid (e.g., acetic acid) is titrated with a strong base (e.g., NaOH) the pH is higher
than for the strong acid + strong base titration at every point before the equivalence point. This is
because the un-reacted acid, being weak, does not give as much H3O+ in solution as the same
amount of strong acid would give. Figure 1 represents a typical weak acid-strong base titration
curve. At the equivalence point, the titration reaction (Equation 1) has gone to completion and
the resultant solution contains the salt ions (Na+ CH3COO-) The conjugate acid (Na+) is too weak
to react with water but the conjugate base (CH3COO-) is a weak base that reacts slightly with
water to produce OH- according to Equation 2. Therefore, the solution pH at the equivalence
point will be somewhat above 7.

CH3COOH + OH- ---> CH3COO- + H2O (1)

(titree, weak acid) titrant conj base

CH3COO- + H2O ---> CH3COOH + OH- (2)

conj. base conj. acid

In the titration of a mixture of one or more polyprotic acids, it is possible that two or more
inflection points (equivalence points) will be obtained on the titration curve. Each equivalence
point corresponds to one hydrogen atom being removed or replaced. However, when the Ka1 and
Ka2 values for a polyprotic acid are too close together, the titration will yield only one inflection

In this experiment the unknown beverage solution will first be titrated using a pH meter and the
standardized base. The titration curve will be plotted on a spreadsheet and from it an appropriate
indicator can be chosen for a triplicate colorimetric titration for total acid in the sports drink. We
will also design a more sophisticated model using spreadsheets to determine how much of each
acid is present in the sample.

If the titration curve for your beverage were to give only one inflection point (which it wont),
then the moles of the acid would be calculated as follows. Assume that the following data were
obtained for this sample:

1) Sample weight = 0.2005 g

2) Volume of 0.1000 M NaOH needed to reach the equivalence point = 19.88 mL

moles total acidic protons = MNaOH .V NaOH

Since you have primarily citric acid in your mixture along with some H2PO4- (and possibly
H2Cit-), you will determine how much total acid is present from the observed equivalence point.
The total acid is the sum of all protons that are titrated to the endpoint observed. Based on your

knowledge of acid-base chemistry, you can determine how many protons were titrated. A
spreadsheet titration model may help in your analysis.

Procedure for Potentiometric Titration:

1) pH meters, along with a magnetic stirrer will be placed in the laboratory in appropriate
locations. Standardize the meter according to the directions at the end of the lab.

2) Fill your burette with your standard 0.1000 M NaOH solution. Be sure to rinse out the burette
with one or two small portions of standard NaOH before filling. You may carefully adjust the
initial meniscus level to zero or subtract the (non-zero) initial volume from all measurements on
your spreadsheet titration graph.

3) Place a magnetic stirring bar in the beaker containing about 40.0000 g (weighed to an
accuracy of 0.0001 g) of your unknown beverage, and set it on the stirrer beneath your burette.
Turn the stirrer to a speed that gives vigorous stirring without danger of splashing the solution
out of the beaker.

4) Calibrate the pH meter (see directions below). Carefully lower the pH electrode into the
beaker to a depth of approximately one half to three-quarter inches. Make sure that the stirring
bar cannot touch the electrode as it rotates. Also be sure the cap is off of the electrode tip, and
replace it when you are done with your titration (after rinsing tip). Measure and record the initial
pH! Take burette readings approximately every 0.2 pH units. Record the data in a column in
your lab book.

5) Continue adding increments until the 0.2 unit rise in pH requires significantly less titrant. This
will then suggest that you are approaching an equivalence point. Cut down the amount added,
seeking to maintain increments in the pH of approximately 0.2 pH units. Titrate beyond the last
equivalence point several milliliters to a pH of 10. It is often useful to run your burette out to
nearly 50 mL and to take a final pH reading after the electrode has had a chance to equilibrate for
a half a minute.

6) Plot the pH data for your beverage in Excel. Set up the first column (A) with the pH data and
the next column (B) with the corresponding titration volume data. Label cell A1 with pH and
cell B1 with vol. titrant. You may compute the derivative of the titration quite simply by setting
up a new column (C). Label C1 as deriv. In cell C2 write the formula =B3-B2. Pull down the
formula to all the corresponding cells in column C. Include this graph in your lab report.

7) On your final graph, draw the extrapolation lines (see Figure 1) which will allow you to
determine the equivalence point for your titration to 3 significant figures. Overlay the derivative
graph and blow up the region near the endpoint so that you can get a more precise value for the
equivalence point. Mark the equivalence point on your graph.

Colorimetric Determination of Total Acid (Optional)
Using the pH of your calculated equivalence point, chose an appropriate colorimetric indicator
and perform triplicate analyses (using an Erlenmeyer flask instead of a beaker) for your sample
with that indicator. Record the end point volume for each determination. Different students
might use different indicators and compare results.

Calibration of a pH meter

The pH meter is an instrument that depends on an electrode that selectively responds to the
activity of H+ ions, creating a potential (voltage) for electrons to move that would normally
neutralize the H+ if they could get to it. As the resistance to the flow of the electrons is very
large through the very thin (and fragile!) glass balloon used to sense the H+ ions, we are
measuring the potential for such movement, rather than actually having very many electrons
moving. Still, enough move to create a signal that is amplified millions of times to give the
meter reading that you record. However, this means the pH meter is a very sensitive instrument
that can also respond to charges on your fingers and static charges on your shoes, sweaters etc.
Thus, the best readings are obtained when the meter is given time to reach the correct reading,
and without too much fast stirring that can create a static charge.

To calibrate the meter, at least two solutions of known pH are needed, one at the center point (pH
= 7.00) and another one that is either acidic (typically pH = 4.00) for measuring acid solutions,
or that is basic (typically pH = 10.00) for measuring basic solutions. The calibration solutions
are "buffered", meaning that they contain enough of an acid and its conjugate base that they
maintain a constant pH even when diluted a bit. However, care should be taken to pat off
(carefully!) water droplets on the electrode after rinsing it before using the buffers so that they
maintain their accuracy. Here's the procedure.

1) Be sure the electrode contains the appropriate filling solution (liquid should be visible inside
of the electrode when you tip it). This will usually be taken care of by the stockroom. Carefully
remove the protective rubber cap that covers the glass balloon, and mount the electrode so the
glass is covered in a beaker of water. It may need to soak awhile to hydrate the glass if it hasn't
been used for some weeks, or if someone forgot to recap the electrode when they were done.

2) Place the electrode in the pH = 7.00 buffer. Adjust the calibrate knob to give pH = 7.00 with
the temperature knob set to the lab temperature (usually 22C).

3) Take out the electrode, rinse it with deionized water from your squirt bottle over a waste
beaker, pat it dry (don't rub!) with a Kimwipe, and place it in the pH = 4.00 buffer (usually we
are more interested in the acid range). Adjust the slope knob, if there is one, to give a pH = 4.00.
Most modern, hand-held pH meters have combined the temperature and slope knobs so that you
will probably vary the temperature knob instead to give the desired pH. Don't worry that the
temperature setting is no longer correct.

4) Take out the electrode, rinse, pat dry, and place it in your unknown solution. The meter
should remain calibrated for the rest of the measurements that you will make (usually its okay

for an hour or two), but if you get suspicious readings at any time, you might wish to check its
calibration. Note that the electrode does not respond well to pH below 2 or above 10, and that
high sodium (Na+) concentrations may also be interpreted as containing H+, as the electrode
response to sodium ion becomes significant at higher concentrations.

5) When you are done reading your unknowns (with rinses and dryings between each one), again
rinse off the electrode but leave it wet before covering it with the rubber cap. If you are not the
last one to use it for the day, it is better just to place the electrode back in a beaker of water
(provided by the stockroom) to keep it wet, and this reduces the chance of busting the glass
balloon, an expensive mistake.


Name: _____________________

1) Attach a plot of the titration and derivative data from Excel.

2) Based on the initial pH, draw the structure of the predominant species of citrate and phosphate
in solution at the start of the titration? Explain briefly.

3) How many moles of acidic protons were titrated? What is the confidence interval for this

PART 3: Ion Chromatographic Analysis of Chloride and Total Citrate and Phosphate

Ion chromatography utilizes ion exchange to accomplish separation of analyte ions, followed by
chemical suppression of eluent conductivity and conductivity detection. A liquid sample is introduced at
the top of the separator column. The ionic eluent pumped through the column causes differential
migration of the analyte ions down the column as a result of thermodynamic factors (e.g., size and
affinity of the ions to the active sites of the column). The analyte ions are separated into discrete bands
that are detected, most often with a conductivity detector. The chromatograph output is a plot of
conductivity versus time (a chromatogram). Each ion appears as a peak in the chromatogram.
Identification and quantification is performed by comparing the retention times (time from injection to
peak maximum) and peak heights to the standards of the sample.1

Ion-Exchange Resins
The column packings for ion chromatography consist of ion-exchange resins bonded to inert polymeric
particles. For cation separation the cation-exchange resin is usually a sulfonic or carboxylic acid, and for
anion separation the anion-exchange resin is usually a quaternary ammonium group. For cation-
exchange with a sulfonic acid group the reaction is:

-SO3- H+(s) + Mx+(aq) <===> -SO3- Mx+(s) + H+(aq)

where Mx+ is a cation of charge x, (s) indicates the solid or stationary phase, and (aq) indicates the
aqueous or mobile phase. The equilibrium constant for this reaction is:

[-SO3- Mx+]s [H+]aq

Keq = --------------------
[-SO3- H+]s [Mx+]aq

Different cations have different values of Keq and are therefore retained on the column for different
lengths of time. The time at which a given cation elutes from the column can be controlled by adjusting
the pH. Most ion-chromatography instruments use two or more mobile phase reservoirs containing
buffers of different pH, and a programmable pump that can change the pH of the mobile phase during
the separation.

Ion Detection
Ions in solution can be detected by measuring the conductivity of the solution. In ion chromatography,
the mobile phase contains ions that create background conductivity, making it difficult to measure the
conductivity due only to the analyte ions as they exit the column. This problem can be greatly reduced
by selectively removing the mobile phase ions after the analytical column and before the detector. This
is done by converting the mobile phase ions to a neutral form or removing them with an eluent
suppresser, which consists of an ion-exchange column or membrane. For cation analysis, the mobile
phase is often HCl or HNO3, which can be neutralized by an eluent suppresser that supplies OH-. The Cl-

or NO3- is either retained or removed by the suppresser column or membrane. The same principle holds
for anion analysis. The mobile phase is often NaOH or NaHCO3, and the eluent suppresser supplies H+
to neutralize the anion and retain or remove the Na+. 2 In this experiment, we will use an anion
exchange column with a NaOH eluent to measure the concentration of citrate, phosphate and chloride.

Prepare your sample by making a 0.100% w/v solution (dry sample) or 5% v/v (liquid sample) in Milli-
Q water. Weigh or pipet the sample and transfer it quantitatively to an appropriate sized volumetric
flask. A set of standards will be prepared by each group of three students. Each student group will
make stock standards of 1000 ppm phosphate (from KH2PO4), citrate (from Na3Cit2H20) and chloride
(from NaCl). Note for calculation that the concentration (in ppm) is of the anion, but you will use a salt.
Make sure you compensate for this in your calculations! Choose appropriate standard concentrations by
diluting aliquots of the stock standard. A range of mixed standards in the 1 -100 ppm (and up to ~150
ppm for citrate) give a good response by suppressed conductivity detection. For example, one mixed
standard might contain 10 ppm each of phosphate, citrate and chloride. Before you make any dilutions,
confer with your professor about your plan of attack. In addition, each group should prepare and run
one blank sample.

Each sample and standard should be added to the 5-mL autosampler containers provided for the
instrument. The professor will demonstrate the loading technique. The samples and standards will be
logged onto the instrument schedule and the analysis will then be set up to run with an autosampler.

Data Collection:
Each group member should use Excel to perform a standard linear regression analysis for one of the
three analytes (Cl-, PO43- or citrate) with appropriate error bars and error analysis. Include error bars for
the replicate calibration and unknown points and propagate your error using standard linear regression

Each group member will prepare a calibration graph of concentration vs. peak area for one of the ions.
If the calibration is linear for the standards, you may use the linear regression data for calculating the
amount of each ion in your solution. Each student in the group may do the calculations for all samples
of that ion, but the final result will be the responsibility of the students whose sample is reported (i.e.
check your partners work!). You should also calculate the limit of detection for each ion.

Ion Chromatographic Analysis of Sports Beverages


Honor Code:

For each group, hand in a copy of your spreadsheet with calibration data for standards and analysis of
samples clearly labeled.

Data (fill in actual concentrations of your standards and observed areas):

Standard Calibration Data

Ion Std 1 Std 2 Std 3 Std 4 Std 5
___ ppm ___ ppm ___ ppm ___ ppm ___ ppm

Calculated Sample Concentrations

Analyte Conc.



If any ion is below the limit of detection, please note and then leave that section blank.


____________________ ___________________ ppm in diluted sample solution

sample dilution: ______________ mg/L or ml/L (circle one)

____________________ ___________________ g (by weight or volume) in beverage

Limit of detection: _____________________ ppm


____________________ ___________________ ppm in diluted sample solution

sample dilution: ______________ mg/L or ml/L (circle one)

____________________ ___________________ g (by weight or volume) in beverage

Limit of detection: _____________________ ppm


____________________ ___________________ ppm in diluted sample solution

sample dilution: ______________ mg/L or ml/L (circle one)

____________________ ___________________ g (by weight or volume) in beverage

Limit of detection: _____________________ ppm


Name: _____________________

1) Using data from Part 2 and Part 3 of the experiment and the manufacturers nutritional
information determine how much H3Cit, KH2PO4, NaCl, and either Na3Cit or KH2Cit was
required by the manufacturer to formulate your sports drink. A spreadsheet approach may work
best. Please hand in any spreadsheet calculations with your report.

2) Using Excel, prepare a distribution diagram that includes the following information as a
function of pH:

a) equilibrium concentrations: [H3Cit], [H2Cit-], [HCit2-], [Cit3-], [H3PO4]. [H2PO4-],

[HPO42-], [PO43-], [H+] and [OH-]

b) analytical concentrations: CNa, CCl, CPO4, CK, and Ccit

Use this information to develop a charge balance equation and then write a pointer function.
Directions for preparing a spreadsheet are provided. Based on the pointer function, estimate how
well your analytical calculations compare with the initial measured pH of your sports drink.

3) Compare the formulation of your beverage with others in the class and hypothesize why there
are similarities or differences.

Spreadsheet Calculations for Chemical Speciation of Sports Drinks

In this activity, you will be modeling the acid/base chemistry of polyprotic acids in a sports
drink with alpha fractions using an Excel spreadsheet. In boxes A1-A11, write the labels
Kac1, Kac2, Kac3, Kap1, Kap2, Kap3,CCit. Cp, CNa, CK and CCl. In cells C1-C6 write the labels pKac1,
pKac2, pKac3, pKap1, pKap2, and pKap3. Add the pKa values that you are assigned in cells
D1..D6. In cells B1-B6, place the formulas to calculate Ka from pKa. Highlight box B1, go to
the Insert window and highlight Name and then Define. Excel assigns the name Kac1
automatically, so press OK. Do the same for the rest of the constants. This operation defines
these values as constants rather than variables (you can also use the label of $A$1 in your
formula to show that the value in box A1 is a constant rather than a variable, but as you will
see, the define name command is easier here).

Across the 13th row, label the columns as pH, [H+], [OH-], denomC, [H3Cit], [H2Cit], [HCit],
[Cit], denom P, [H3PO4], [H2PO4], [HPO4], [PO4], pH, log [H+], log [OH-], log[H3Cit],
log[H2Cit], log[HCit], log[Cit], log[H3PO4], log[H2PO4], log[HPO4], log[PO4] and Pointer
(pH is done twice for ease of graphing). Under the pH column add numbers from 0 to 14 in
increments of 0.25. You can do this easily by typing in the values 0 and 0.25 to cells D14
and D15. Highlight these two cells and drag to box D70. The numbers will all fill in

In the appropriate (B14) cell, write a formula to determine the concentration of the H+ based
on the pH. After writing the formula, drag it down to fill in the entire column. Next do the
same for OH- and fill in the C column cells. In the appropriate box, write a formula that
represents the denominators for the alpha fraction of citrate and then one for phosphate. Fill
in the appropriate boxes with the formula. Now write formulae for the concentration based
on the alpha values of H3Cit, H2Cit, HCit, Cit, H3PO4, H2PO4, HPO4 and PO4, based on the
[H+], the appropriate equilibrium constants (that you defined earlier) and the denominator
terms that you calculated. Next, write formulas in the appropriate rows for the log[H3Cit],
log[H2Cit], log[HCit], log[Cit], log[H3PO4], log[H2PO4], log[HPO4], and log[PO4] (use the

concentrations already calculated). When you are finished drag down to all the appropriate

Pointer Function: Set up the charge balance equation below using the terms in the


Take the log(abs) of the above function to obtain the pointer function. You may wish to
scale the pointer by multiplying the log(abs) term by a constant. Note that for Cl-, Na+ and
K+ the analytical concentrations will equal the equilibrium concentrations at all pH values.

Plotting: Highlight the boxes pH, log [H+], log [OH-], log[H3Cit], log[H2Cit], log[HCit],
log[Cit], log[H3PO4], log[H2PO4], log[HPO4], log[PO4] and Pointer (including the labels).
Choose the Chart button and highlight a section just below the pKa values for your graph.
Follow the directions (X-Y Scatter, X-Y Scatter, 1, etc.). You now have a plot of log [HA]
as a function of pH. To make it look better, double click on any axis that you want to
change. A dialog box will come up. Click on Scale to change the scale and position of the
axes (make sure you undo the x-marked auto boxes). The suggested scale is log [x] from 0 to
-14 and pH from 0 to 14. Remove the gray background by clicking on the grey color and
highlighting automatic and then choosing a white color. When you are satisfied with the
graph, then you can print a copy. To print a copy, double click on the graph, choose the File
command, then Print Preview.

NOTE: To get a more accurate pH estimate, you may wish to plot the pointer over a
narrower range in pH increments of 0.01 pH units. This should be relatively easy if youve
designed your spreadsheet well.

Chemical Speciation Analysis of Sports Drinks by Acid-Base Titrimetry and
Ion Chromatography: A Challenging Beverage Formulation Project

Instructors Notes

Hazards: Standard precautions should be taken when handling acids and bases.

Materials & Methods:

PART 1: Standardization of NaOH

This is a standard lab that is found in almost all texts and lab manuals. There is an on-line
version available from Dan Harris Quantitative Chemical Analysis text (6th edition) web site (1).

Materials and Reagents:

50% NaOH solution
volumetric flask for diluting NaOH
plastic storage bottle (for NaOH)
volumetric pipets
50-ml graduated burette
150-ml Erlenmeyer flask
Phenolphthalein solution

PART 2: Potentiometric and Colorimetric Titration

This is a standard lab that is found in almost all texts and lab manuals. As above, there is also an
on-line version available from Dan Harris Quantitative Chemical Analysis text (6th edition) web
site (1). The only modification of significance is the amount of solid or liquid sports beverage
required. Typically, a 40-ml liquid sample of a sports beverage will require 15-20 ml of 0.1 M
NaOH. In our labs, we have students determine which indicator (pKa ~ 8-9) they might use based
on the potentiometric results. We obtained acceptable results with phenolphthalein and thymol
blue indicators.

Materials and Reagents:

Analytical balance or 40-ml pipette
Magnetic stirrer
150-ml beaker
50-ml graduated burette
150-ml Erlenmeyer flask
indicator solutions

PART 3: Ion Chromatographic Analysis of Citrate, Phosphate and Chloride
Though a number of ion chromatographs and columns are available that may work, listed below
is the equipment that we have used successfully. Standard concentrations in the range of 1 -100
ppm for each ion work well. The conductivity response of chloride is considerably higher than
phosphate and citrate. With a 5% dilution, extrapolation from the citrate standard will be
required which reduces the accuracy. Thus, citrate standards up to ~150 ppm may provide better
results. With the high degree of linearity for our calibration plots (r2>0.999) extrapolation was
not a problem. The amount required to prepare each of the stock solutions is provided in the
table below. For the most accurate results, the potassium phosphate, sodium citrate and sodium
chloride standards should be dried and desiccated prior to stock solution preparation. For
sodium citrate dihydrate care must be taken not to dry at too hot a temperature so that the water
of hydration is not lost. All reagents are of analysis grade and available from Fisher Chemicals.
Many other vendors are possible for these common reagents.

We also used potassium bromide as an internal standard but the results were less satisfactory
than analyzing without an internal standard. This is likely because we use an autosampler for
injection. Without an autosampler, injection of standards and samples at 25-minute intervals
could prove tedious.

There is a small amount of ascorbate in the Powerade sample that could also be analyzed
simultaneously with the IC conditions given below. We chose not to quantify the ascorbate at the
low concentrations present.

Preparation of stock standards

Reagent Standard MW mg/100 ml
Na3Cit2H2O 1000 ppm citrate 294.11 155.53
KH2PO4 1000 ppm phosphate 136.08 143.29
NaCl 1000 ppm chloride 58.4428 164.85

Sources for chemicals used

Reagent Vendor Catalog # Grade CAS #
Na3Cit2H2O Fisher Chemicals S/3320/53 analysis 6132-04-3
KH2PO4 Fisher Chemicals P/4800/50 analysis 7778-77-0
NaCl Fisher Chemicals S/3160/53 analysis 7647-14-5
KH2Cit N/A N/A N/A 866-83-1
NaOH Fisher Chemicals S/4845 analysis 1310-73-2
Phenolphthalein Fisher Chemicals P/2400/48 indicator 77-09-8

Ion Chromatography Materials and Reagents:

5 mM NaOH in degassed Milli-Q water
100 mM NaOH in degassed Milli-Q water
degassed Milli-Q water

Pump and Detector:
Dionex GP40 Pump and ED40 Electrochemical Detector employing conductivity

Columns & Suppressor:

Dionex AS11 Anion exchange column
Dionex AS11 Guard Column
Dionex Anion Trap Column (for removing carbonate)-the ATC is installed between the
pump and injection port to remove carbonic acid in the eluents
ASRS UltraII suppressor-set at 100 mA

Milli-Q H2O

Analysis Conditions:
Flow rate: 1 ml/min
Gradient elution: The analysis time for each sample is 25 minutes with a 7-minute equilibration
time and an 18-minute run time according to the following schedule:

Time (min.) Milli-Q H2O 5 mM NaOH 100 mM NaOH

0 (equilibrate) 90% 10% 0%
7 (inject) 90% 10% 0%
9.5 90% 10% 0%
13.5 0% 100% 0%
25 0% 65% 35%

Under these conditions our last peak always eluted before 14 minutes after injection.

Figure 1. Top chromatogram: Anion analysis of a 3-anion standard with 37.6 ppm
chloride (~ 6 minutes), 25.2 ppm phosphate (~11.5 minutes) and 21.0 ppm citrate (~12.5
minutes). Middle chromatogram: Anion analysis of lemon-lime Powerade sample
diluted to 5%(v/v) with Milli-Q water. Bottom chromatogram: Anion analysis of
Watermelon Ice Gatorade sample diluted to 5%(v/v) with Milli-Q water. Analysis
Column: IonPac AS11A Guard Column AG11A; eluent: Milli-Q water + 5.0 mM
NaOH+ 100 mM NaOH; flow rate: 1.0 mL/min; detection suppressed conductivity.
Solute concentrations in 5%(v/v) Powerade : 17.6 ppm chloride, 0.99 ppm phosphate,
and 124 ppm citrate. Solute concentrations in 5%(v/v) Gatorade: 21.3 ppm chloride,
16.1 ppm phosphate, and 143 ppm citrate.

The most interesting but complex part of this lab for students is the calculations. Thus,
two spreadsheets are provided, one for Gatorade and one for Powerade analysis. The
spreadsheet calculations allow for the following inputs:

Part 1- Standardization data (same on both spreadsheets):

Input Data:
Equivalence volume of NaOH (ml)
weight of KHP used (g)

Output Data:
[NaOH] = weight of KHP(mg)/(equiv volume*MWKHP)
average and standard deviation: calculated from multiple samples by standard Excel
%RSD = 100*std deviation/average
confidence interval = t*std dev/sqrt (# samples)
t = tinv(0.05, #samples)

Part 2 Beverage Titration Data (Gatorade spreadsheet)

Input Data:
Volume of drink used (ml or g)
Volume required for endpoint (ml)

Output Data:
The spreadsheet calculates the mmoles of total acid, mmole of total acid/L, mmoleH3Cit
acid/L, mmole H3Cit/L and the mg H3Cit/L by the following expressions:

A) mmole total acid = MNaOH*endpoint volume

B) mmole acid/L = b*1000/volume of drink added
C) mmoleH3Cit acid/L = b 2*mmole H2PO4 (from IC results)
D) mmole H3Cit/L = c/3 (accounts for 3 equivalents of H+ per mole of H3Cit)
E) mg H3Cit/L =d*MW(citric acid)

Part 3- Ion Chromatography and Nutritional data

Input Data:
Ion chromatographic results for chloride, phosphate and citrate in ppm for undiluted
samples (all are determined with r2>0.99 by linear regression)
Included in the spreadsheet is also the nutritional data from the product labels.

Output Data (Nutritional information):
a) mg Na+/L = (mg Na/240 ml serving) *1000/240
b) mmole Na/L = a/AWNa
c) mg K+/L = (mg K+/240 ml serving) *1000/240
d) mmole K+/L = c*AWK
e) mg Cl-/L = (mg Cl/240 ml serving) *1000/240
f) mmole Cl-/L = e*AWCl
g) mmole Na3Cit = b d
h) mg Na3Cit = MWNa3Cit*g

Output data (calculations):

i) mmole PO4/L = ppm PO4(IC)/MWPO4
j) mg K+/L = i*AWK
k) mmole Cl-/L = ppm Cl (IC)/MWCl
l) mg Na+/L (from Cl-) = k*FWNaCl
m) mmole Na+/L (from Na3Cit) = b-k
n) mg Cit/L (from Na3Cit) = m*MWCit
o) mmole Na+ (from Na3Cit) = 3*m
p) total mmole Na+(calc) = o + k
q) total mg Na+ (calc) = p*FWNaCl

Recipe for Drink

r) mmole H3Cit = D
s) mg H3Cit = D * MWH3Cit
t) mmole KH2PO4 = i
u) mg KH2PO = i* MW KH2PO4
v) mmole Na3Cit =m
w) mg Na3Cit = m *MWNa3Cit
x) mmole NaCl = k
y) mg NaCl = k*FWNaCl

Part 2 Beverage Titration Data (Powerade spreadsheet)

Input Data:
Volume of drink used (ml or g)
Volume required for endpoint (ml)

Output Data:
The spreadsheet calculates the mmoles of total acid, mmole of total acid/L, mmoleH3Cit
acid/L, mmole H3Cit/L and the mg H3Cit/L by the following expressions:

A) mmole total acid = MNaOH*endpoint volume

B) mmole acid/L = b*1000/volume of drink added
C) mmoleH3Cit acid/L = b 2*mmole H2PO4 (from IC results)-2 mmole
H2Cit(from below)
D) mmole H3Cit/L = c/3 (accounts for 3 equivalents of H+ per mole of H3Cit)
E) mg H3Cit/L =d*MW(citric acid)

Part 3- Ion Chromatography and Nutritional data (Powerade)

Input Data:
Ion chromatographic results for chloride, phosphate and citrate in ppm for undiluted
samples (all are determined very well by linear regression)
Included in the spreadsheet is also the nutritional data from the product labels.

Output Data (Nutritional information):

a) mg Na+/L = (mg Na/355 ml serving) *1000/355
b) mmole Na/L = a/AWNa
c) mg K+/L = (mg K+/355 ml serving) *1000/355
d) mmole K+/L = c*AWK
e) mmole Cl-/L = b (Na+ is sole source of Cl-)
f) mg Cl-/L = f*AWCl

Output data (calculations):

g) mmole PO4/L = ppm PO4(IC)/MWPO4
h) mg K+/L (from KH2PO4) = g*AWK
i) mg K+/L (from KH2Cit) = c - h
j) mmole Tot Cit/L (from KH2Cit and H3Cit) = ppm Cit(IC)*MWCit
k) mmole K+ (from KH2Cit) = d h
l) mg KH2Cit = j*MWKH2Cit
m) mg K+/L (from KH2Cit) = k*AWK
n) mg Citrate/L (from KH2Cit) = k*MWCit
o) total mmole K+/L = m + h or i+h
p) total mg K+/L = o*AWK
q) mmole Cl-/L = ppm Cl-(IC)*AWCl

r) mg Cl-/L = q*AWCl
s) mmole Na+(calc from Cl-) = q
t) total mg Na+ = q*FWNa

Recipe for Drink

u) mmole H3Cit = D
v) mg H3Cit = D * MWH3Cit
w) mmole KH2PO4 = g
x) mg KH2PO4 = g* MW KH2PO4
y) mmole KH2Cit =k
z) mg Na3Cit = k*MWKH2Cit
a) mmole NaCl = q
b) mg NaCl = q*FWNaCl

The last two pages include the spreadsheet calculations for the laboratory that are
described above. These will be submitted in a template format for final copy.

Beverage Lab Grading Rubric
(160 pts. total)

Data (25 pts.):

NaOH standardization data (KHP weight and volume of NaOH) (3 pts.)
IC data (4 pts.)
Colorimetric data for beverage titration (weight/volume of beverage and volume
of NaOH) (3 pts.)
Potentiometric titration data as an Excel spreadsheet (5 pts.)
Weight of compounds used to make IC stock standards and dilutions. (3 pts.)
Calibration data for citrate, phosphate, chloride with x- and y-error bars. (5 pts.)
Initial pH of beverage (2 pts.)

Calculations (80 pts.):

Concentration of standardized NaOH w/ CI (5 pts.)
IC calculations for citrate, phosphate, chloride with confidence intervals (10 pts.)
Were CIs from linear regression lines propagated using Eqn. 5-14? (10 pts.)
Calculation of total citrate for colorimetric titration (5 pts.)
Recipe for drink w/ detailed calculations (30 pts.)
Case I t-test comparing either K+ with PO43- OR Na+ with Cl- (5 pts.)

Theoretical pH graph of citrate and phosphate with correct analytical
concentrations (15 pts.)

Questions (15 pts.):

Structures of citrate and phosphate as they exist in beverage (5 pts.)
What effect did phosphate have on the determination of citrate?
Is there any suspect systematic error?

Results (40 pts.):

Was standardization titration within 1% of the correct answer? (5 pts.)
Was %RSD less than 0.5% for standardization? (5 pts.)
Was precision on beverage analysis reasonable? (10 pts.)
Is the recipe accurate? (20 pts.)
http://www.scimedia.com/chem-ed/sep/lc/ion-chro.htm, updated 6/18/96