Kinetics of Reactive Dye Hydrolysis
My partners and I worked the graphs, would you make sure that I have labeled
everything correctly and have answered the protocol (Bolded or highlighted in
yellow)
Did I include the right raw data or should some of that be in results/discussions
Definitely need help with the questions
Objectives:
The objectives of this experiment are:
1. To measure changes in the concentration of components of commercial textile
dyes as a function of time using liquid chromatography.
2. To determine the order and rate of dye hydrolysis from the concentration data
acquired as a function of time.
3. To compare the rate of hydrolysis to the rate of color fast dyeing using methanol
as a simulant for the hydroxyl groups on cellulosic fibers.
4. To ascertain if the hydrolysis reactions changes the spectral characteristics of the
reactive dye using UV-visible absorption spectroscopy.
Text Reference:
Skoog, Holler, and Nieman Principles of Instrumental Analysis, 6th edition,
Saunders College Publishing, Fort Worth, TX 1998.
 An Introduction to Chromatographic Separations
 Liquid Chromatography
Handbook of Instrumental Techniques for Analytical Chemistry, Frank Settle,
editor. Prentice Hall, Upper Saddle River, NJ 1997, Ch 9 & 50.
Rubinson and Rubinson, Contemporary Instrumental Analysis, Prentice Hall,
Upper Saddle River, NJ 2000, Ch 14 & 18.
Other References:
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1. O’Neill, C., F. R. Hawkes, D. L. Hawkes, N. D. Lourenco, H. M. Pinheiro,
and W. Delee. “Colour in textile effluents – Sources, measurement, discharge
consents and simulation: A review.” J. Chem. Technol. Biotechnol. 74:10091018 (1999).
2. Phillips, D. “Environmentally friendly, productive and reliable: priorities for
cotton dyes and dyeing processes.” J. Soc. Dyers Colour. 112:183-186 (1996).
3. Hao, O. J., K. Hyunook, and P. C. Chiang. “Decolorization of wastewater.”
Crit. Rev. Environ. Sci. Technol. 30:449-505 (2000).
4. U.S. Environmental Protection Agency. Notebook Project: Profile of the
Textile Industry, EPA/310-R-97-009, Office of Compliance, Washington,
D.C. (1997).
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Introduction
Reactive dyes are now the dyes of choice for cotton and cotton blend fabrics
because of their superior colorfastness, ease of application, brilliant colors and wide
variety of color shades. The growing use of cotton world-wide has resulted in a steady
increase of reactive dye usage.1 The worldwide annual use of reactive dyes is projected to
exceed 180,000 tons by the year 2004.2
Reactive dyes are intensely colored compounds that form covalent bonds with
cellulose. Reactive dyes are substances comprised of two components: a chromophore
and a reactive moiety. Commonly used chromophores are azo, anthraquinone, and
phthalocyanine compounds with sulfonic acid groups to confer sufficient solubility for
the dyeing process (Figure 1). Chlorotriazinyl, sulfatoethylsulfone and vinylsulfone
moieties are commonly used reactive groups that form covalent attachments with
hydroxyl groups on the fiber (Figure 2).
Figure 1. The azo dye reactive yellow 3 showing an azo-type chromophore
(black), a triazine reactive moiety (blue) and two solubilizing sulfonate functional groups
(red).
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Figure 2. Four commonly used reactive moieties for reactive dyes.
Typically, dyeing of cotton is performed at elevated temperature (30 to 85oC)
under high salt (25 to 100 g/L NaCl) and high caustic (pH > 11) conditions. High salt
concentration promotes the solubility of the dye and its adsorption into the fabric; high
caustic concentration promotes deprotonation of the hydroxyl groups on the fiber thereby
increasing the rate of covalent bond formation. Under these conditions, however, the
reactive group of the dye also undergoes hydrolysis. The hydrolysis product is highly
colored, has no affinity for the fabric, and accumulates in the dye bath. It is common
practice to discharge the dye bath as textile plant wastewater once the concentration of
hydrolyzed dye exceeds a critical concentration threshold. These components are nondegradable under the typical aerobic conditions found in conventional, biological water
treatment systems. Reactive dyes released into the environment cause rivers to become
colored, interfere with the river's ecosystem, and, in some instances, are toxic.3
According to the U.S. Environmental Protection Agency, an average of 1 to 2 million
gallons/day of wastewater is generated by a typical dyeing facility, most of which is
associated with dyeing and finishing processes.4
Note: This experiment involves transfer of hot caustic solutions via syringe
from one container to another. To prevent injury, goggles and gloves are to be worn at
all times. If liquids come in contact with your skin, flush the area immediately with
water and notify the TA for further instructions.
Liquid Chromatography
An HPLC system (Figure 3) will usually consist of the following six components:
a. A high-pressure pump (usually with an integrated gradient controller)
b. A valve for the injection of sample (figure 4)
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c. A column to separate the analytes, often with a smaller guard column to
protect the main column.
d. A detector to identify and quantify the separated sample components.
e. A device (normally a computer or strip chart recorder) to record and
analyze the output from the detector.
f. A waste receptacle
Figure 3. A schematic showing the major components of an HPLC system.
Figure 3. A two position, six port injector in the load (left) and inject (right) positions.
Images from VICI/Valco. Technical note 203.
HPLC systems can be run as “normal phase” (i.e. the stationary phase is more
polar than the mobile phase) or “reverse phase.” (i.e. the mobile phase is more polar than
the stationary phase). The terms “normal” and “reverse” are of historical origin, as
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“normal” phase liquid chromatography was developed first.
applications utilize reverse phase chromatography.
Currently most HPLC
In this experiment, a reverse phase system will be employed, using a C18
stationary phase. C18 columns have octadecyl functional groups chemically attached the
silica support, giving the column a hydrophobic character. The polar mobile phase will
consist of two components. The more polar component will be a 20 mM sodium
phosphate buffer (pH 6.0). The less polar component will be acetonitrile containing 8.0
g/L of tetraethylammonium bromide as an ion pairing agent.
Ion pairing agents contain two different chemical moieties. The first is a
hydrophobic portion that interacts with the stationary phase. The second is a charged
group that is complementary to the analyte of interest. The hydrophobic portion of ion
paring reagent is retained by the stationary phase, while the ionic portion interacts with
the analytes of interest. Through the interaction between the ion pairs of the ion pair
reagent and the analyte, partitioning is achieved between the stationary and mobile phase.
Ion pairing reagents are often used in the analysis or reactive dyes. This is
because the solubilizing sulfonic acid groups (pKa < 2.1) present in the dyes are ionized
at all pH values accessible to most reverse phase columns, since acid hydrolysis of the
silyl ether bond occurs at pH values below ~2.5. The use of ion-pair chromatography
increases the partitioning of the dyes between the mobile and stationary phases,
improving the chromatographic separation.
In the first of the experiment, you will determine the optimal composition of your
mobile phase. Your mobile phase consists of two solutions. The first is a 20 mM sodium
phosphate buffer (pH 6.0). The second solution is acetonitrile containing 8.0 g/L
tetrabutylammonium bromide as an ion pairing reagent. Choosing the correct mobile
phase composition provides multiple benefits:
1. Improved chromatographic separation
2. Improved analysis time
3. Improved peak shape. (this is particularly important for later-eluting
components. As the retention time increases, peaks will tend to broaden)
The benefits outlined above are not necessarily mutually supportive. For example
decreasing the analysis time can have an adverse effect to the chromatographic
separation. It will be your job, as the analyst, to choose the appropriate balance between
the benefits outlined above.
In this experiment your buffer is solution A and the acetonitrile is solution B.
This is by convention. In liquid chromatography, the aqueous buffer is almost
universally referred to as A. You will analyze four different mobile phase compositions:
1. 40% A:60% B
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2. 45% A:55% B
3. 50% A:50% B
4. 55% A:45% B
After you have determined the optimum buffer concentration, you will determine
the rate of methanolysis and hydrolysis of your dyes.
Experimental
Part I. Optimization of the mobile phase.
1. Acquire from your TA a sample that has undergone methanolysis for 30 min.
You will use this sample to optimize your mobile phase. This vial should contain
the dye, the hydrolysis product and the methanolysis product.
2. Using a blunt 50 uL syringe, inject 30-50 µL of sample into the injection port of
the LC. The amount of sample injected onto the column will be less than this.
The actual injection volume is determined by the size of the sample loop attached
to the injection port. Do not use a syringe with a pointed tip, as this will damage
the injector seal.
3. Acquire chromatographs for each of the prescribed mobile phase compositions.
After you change the mobile phase composition, you will need to wait 10 minutes
for the column to re-equilibrate.
4. Based on the data that you acquired, choose the best mobile phase composition
for your hydrolysis and methanolysis experiments.
Part II. Analysis of dye composition.
1. The following stock solutions will be provided (for each dye):
a. dye solution: ~5 mg/mL dye
b. hydrolysis solution: 2.5 mL of 4 mM NaOH in a 5.0 mL serum bottle
c. methanolysis solution: 2.5 mL of 4 mM NaOH and 60 uL/mL methanol
in a 5.0 mL serum bottle.
d. neutralizing solution: 3.0 mL of 20 mM sodium phosphate buffer in a 5.0
mL serum bottle
e. Record your unknown dye sample numbers and the concentration of each
dye.
2. Two sets of three serum bottles containing dye, the hydrolysis and methanolysis
solutions will be placed in the wells of the pre-heated heating block. A syringe
needle will be placed through the septum of each bottle to allow the hot solutions
to vent. Make sure that each bottle is surrounded with sand. This will promote
rapid heat transfer from the block to the contents of the bottle.
3. Obtain the absorbance spectrum over the range 350-750 nm for the stock dye
solutions. Note: the stock dye solution should be diluted by putting a few drops
of the dye in the bottom of the cuvette and diluting with nanopure water.
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Determine the wavelength of the absorbance maxima of each dye and use the
value in setting the wavelength of the UV-Vis detector on the liquid
chromatography system.
4. Place eight microcentrifuge tubes in the rack provided and label them 0, 10, 20,
30, 40, 50, 60, 90 and 120. Pipet 100 microliters of the neutralizing solution into
each tube. Close the lid after adding the neutralizing solution.
5. When the block has attained the designated temperature (~90ºC) peel back the
aluminum cover on the hydrolysis serum bottle and carefully insert a syringe
needle through the septum.
6. Zero the timer and withdraw exactly 0.5 mL from the dye solution with a 1.0 mL
syringe. Empty the contents of the syringe into the serum bottle containing the
hydrolysis solution and start the timer. As quickly as possible, withdraw a 100
microliter aliquot from the reaction bottle with the 100 L syringe and empty the
contents of the syringe into the tube labeled “0”. To facilitate timely withdrawal
and analysis of aliquots, one student should acquire and neutralize the aliquots;
the other student should prepare the LC, inject the neutralized aliquot, and label
the resultant chromatogram.
7. When the reaction has proceeded for 10 min, withdraw a second 100 microliter
aliquot from the reaction via syringe and empty the contents of the syringe into
the tube labeled “10”. The reaction is quenched upon addition of the dye sample
into the buffer, so you do not need to inject the dye sample into the HPLC
immediately.
8. After about 10 min you should start the methanolysis reaction in the same
manner as the hydrolysis reaction. Start the methanolysis reaction at an
appropriate time so that you will be withdrawing either methanolysis or
hydrolysis aliquots every five minutes or so.
9. Repeat step 6 until the elapsed time of reaction is 120 min. Leave the reaction
bottle in the heat block while proceeding with the second reaction. Note:
Microsoft Excel is available on each computer for data analysis. Why not enter
the data from the previous separation into the spreadsheet while waiting for the
chromatogram to be completed?
10. Label, compile, and copy the chromatograms obtained. Tabulate the percent peak
areas as a function of time. Be sure that your data are properly integrated. If you
need assistance with integrations, ask you instructor or TA for assistance.
11. Before disposing of your samples, obtain the absorbance spectrum over the range
350-750 nm for the following solutions: hydrolysis reaction product mixture and
methanolysis reaction product mixture.
Report
The report should include plots of the percent composition of the mixture as
a function of time for both hydrolysis and methanolysis. Tabulate the apparent rate
constant for the hydrolysis reaction and compare it with literature values. Include
zero, first and second order plots of your data. You should also include a table
comparing λmax for the starting material, the hydrolysis product and the
methanolysis product. Did I label the red font correctly in my graphs
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In the Discussion section, be sure to identify the rate order of the hydrolysis
reaction. Justify your assignment with sound chemical reasoning. Propose additional
experiments that should be performed to confirm your conclusion.
Questions for Discussion
1. How did the solvent polarity affect the retention times of your dye components?
How does changing the solvent polarity affect the partitioning of the dye between
the mobile and stationary phase? What was the polarity index of the solvent
mixture that you used in part II?
2. What is the structure of your reactive dye used in this experiment? Identify the
chromophore and the reactive moiety. What are the structures of the hydrolysis
product(s)?
3. How does your observed rate constant for hydrolysis of the reactive dyes compare
with literature value(s)? Account for any discrepancies. Did the presence of
methanol in solution modify the rate of hydrolysis? Justify your answer.
4. In a typical textile dyeing process, the initial concentration of reactive dye is
usually 10 mg/mL. If a dye bath containing 1000 gal of dye solution is
maintained at the temperature used in your experiment, how long will it take for
hydrolysis to reduce the concentration of active dye to 1 mg/mL?
5. Compare and contrast the spectral properties of the dyes with those of the
products of the hydrolysis and methanolysis reaction. What assumptions did you
make about the spectral properties of you hydrolysis and methanolysis products?
6. Did you find evidence for the presence of intermediates in the hydrolysis
reaction? (Hint: examine the decrease in area of the dye with the increase in
area(s) of the products.) Similarly, did you find evidence of the intermediates in
the methanolysis reaction?
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