technique: titanium (iv) colorimetric assay...sodium 1, 2‐dihydroxybenzene‐3, 5‐disulfonic...
TRANSCRIPT
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TECHNIQUE: TITANIUM (IV) COLORIMETRIC
ASSAYPresented By: Patricia González Pagán
Laboratory Meeting
September 27, 2018
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Introduction
• Titanium (IV) is one of the important constituents
of alloys and it’s most used form is, titanium
dioxide, TiO2.
• The combination of good strength and high
strength-to-weight ratio makes titanium suitable
for many critical applications such as civilian and
military air- frame parts, nuclear power plants, food
processing plants, oil refinery heat exchangers,
marine components, pacemaker castings and
medical prostheses.Fig. 1 3D-printed titanium sternum and rib cage
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Introduction
• Titanium (IV) assay is a spectrophotometric
quantitative analysis of the percentage of
titanium (IV) in a sample. It can be useful to
characterize products and determine the
presence of significant amount of the metal.
• It can be done for samples that have proteins.
For this, the protein has to be digested using
trichloroacetic acid (TCA).
Fig. 2 Structure trichloroacetic acid
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Introduction
• The general procedure for the Ti Assay includes the formation of a Ti(IV) trisligand or
bisligand complex with a high-affinity ligand and a spectrophotometric analysis.
• The absorbance is taken at a wavelength where the complex has the maximum
absorbance.
• A calibration curve is plotted using a standard series. By interpolation or extrapolation,
the Ti concentration of the sample can be determined and further analysis yield the %Ti
of the original sample.
• This procedure can be used for other metals like Fe(III) by finding a high-affinity ligand
for the metal you are evaluating and setting the pH to ensure the ligand binds to the
metal.
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Introduction• This assay involves the formation of a Ti(IV) trisligand complex in order to quantify the
concentration of Ti(IV). The ligand is sodium 6,7-dihydroxynaphthalene-2-sulfonate and it serves
as a bidentate ligand with very high affinity for Ti(IV).
• The assay is performed at pH 5.2 to ensure that it is selective for Ti(IV) versus similar metal ions
such as Fe(III).
Fig. 3 The complex between Ti(IV) and the sodium 6,7-dihydroxynaphthalene-2-sulfonate.
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Instrumentation and Materials
• Micropipettes
– 2-20 µL
– 20-200 µL
• 1.5 mL Eppendorf tubes
• NanoDrop 2000 Spectrophotometer
• KimWipes Delicate Task Wipes
• Isotemp ® Fischer Scientific
• Vortex Mixer
• Computer with Microsoft Excel Program
Fig. 5. KimWipes Delicate Task
Wipes
Fig. 4 Isotemp ® Fischer
Scientific at FB-143
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Methodology
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Methodology
Sample 125 μM
1.8 M sodium acetate buffer equilibrated to pH 5.2 6.5 mg ligand in
1mL Buffer
25 mM
Boil and sonicatefor 5 min
30% trichloroacetic acid (TCA).
1:1
Sample : TCA
Heat and vortex to increase solubility.
If the sample has a protein
Sit and equilibrate for one hour
. Centrifuge
Supernatant is now the sample.
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MethodologyPrepare the samples and standard series using the following.
Scan the solutions at 370 nm using the NanoDrop 2000 Specrophotometer.
Ti (uM) Ligand
(uL)
Ti Stock
(uM)
Ti Stock
(uL)
Sample
(uL)
Sample
“Buffer”
(uL)
30%
TCA
(uL)
1.8 M
Acetate
(uL)
Final
Volume
(uL)
Blank 0 0 5 0 10 10 25 50
0 25 0 5 0 10 10 0 50
5 25 50 5 0 10 10 0 50
10 25 100 5 0 10 10 0 50
15 25 150 5 0 10 10 0 50
20 25 200 5 0 10 10 0 50
30 25 300 5 0 10 10 0 50
40 25 400 5 0 10 10 0 50
50 25 500 5 0 10 10 0 50
Sample 25 0 5 20 0 0 50
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Using the NanoDrop 2000 Spectrophotometer
• Open the NanoDrop 2000 icon on the desktop and select UV-Vis.
• Make sure to remove the Kim Wipe from the pedestal.
• Set the 370nm wavelength and run your blank.
• Add 2 µL of the solution and make sure that there are no bubbles when you add your sample. This is why it is good to run the standard series twice.
• Wipe using a Kim Wipe to clean every time you change solution.
• When you’re finished make sure you clean the area and leave a Kim Wipe in the pedestal.
• Save your work! Fig. 6 NanoDrop 2000
Spectrophotometer at FB-143.
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Data Analysis
• Plot the absorbance at 370 nm versus the metal
concentrations.
• Determine the trend line for the plot and record
the line of best fit.
• Use the trend line equation to determine the
concentration of your samples. Remember to
correct for the dilutions that you performed in
order to get your original concentration.
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Data Analysis
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Data Analysis Example
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AlternateMethodology
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Sample 125 μM1.8 M sodium acetate buffer
equilibrated to pH 5.26.5 mg ligand in
1mL Buffer
25 mM
Heat and vortex to increase solubility.
Alternate Methodology
This assay can be done by incorporating the sample into the standard series. It can be performed for
protein or non-protein samples. This method is suitable for non-protein samples since there is no
need for TCA.
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Ti (uM) Ligand
(uL)
Ti Stock
(uM)
Ti Stock
(uL)
Sample
(uL)
Sample
“Buffer”
(uL)
30%
TCA
(uL)
1.8 M
Acetate
(uL)
Final
Volume
(uL)
Blank 0 0 5 0 20 0 25 50
0 25 0 5 10 10 0 0 50
5 25 50 5 10 10 0 0 50
10 25 100 5 10 10 0 0 50
15 25 150 5 10 10 0 0 50
20 25 200 5 10 10 0 0 50
30 25 300 5 10 10 0 0 50
40 25 400 5 10 10 0 0 50
50 25 500 5 10 10 0 0 50
To make the standard series incorporated into the sample, follow the table below.
Scan the solutions at 370 nm using the NanoDrop 2000 Specrophotometer.
Alternate Methodology
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Data Analysis• Plot the absorbance at 370 nm versus the concentrations.
• Determine the trend line for the plot and record the line of best fit.
• Use the trend line equation to find the x intercept by setting y=0. This will give you the concentration
of Ti in your samples.
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Ti Stock Solutions
The Ti stock solutions are made using “Titanium atomic absorption standard solution” in 0.01 M HCl.
Fig. 7 Titanium atomic
absorption standard solution,
found in the flammable
substances cabinet.
Fig. 8 The Ti stock solutions at our lab.
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Summary Ti (IV) Assay
• ƛmax= 370 nm
• pH = 5.2
• Method limit = > 100nM (Tecan)
• Method limit = > 1µM (NanoDrop 2000)
Characteristics Procedure
• Prepare the ligand solution in a 5.2 pH
buffer.
• Prepare your samples to 125 µM.
• Measure the absorbance at 370 nm
using the NanoDrop 2000.
• Use an Excel worksheet to plot the
calibration curve and find the Ti
concentration of your samples.
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Other Methods
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Determining trace amounts of Ti
• A way to determine trace amounts of Ti(IV) is by using N'-(2-hydroxybenzylidene)- 3-
oxobutanehydrazide (HBOBH) as a reagent.
• The proposed titanium (IV) and HBOBH bisligand complex is the following.
Fig. 9 Proposed formation of the complex.
• Refluxing equimolar solutions of
acetoacetic acid hydrazide and
salicylaldehyde solutions prepared in
aqueous methanol for two hours .
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Determining trace amounts of Ti
Table. 1 Photometric and analytical characteristics pertaining to the proposed method
• It can be used to determine Ti (V) concentrations in synthetic mixtures and alloys.
• The reproducibility of the method was excellent and recoveries reported were ranging
from 102 to 104% .
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Determining Ti(IV) and Fe(III)
• Ion‐pair reversed phase liquid chromatography using sodium
1,2‐dihydroxybenzene‐3,5‐disulfonic acid (Tiron) as a precolumn chelating reagent.
The detection limits for titanium(IV) and iron(III) are 0.5 and 2.0 μg/L, respectively. The method has been applied to the simultaneous determination of titanium(IV) and iron(III) in river water samples and has furnished highly precise results.
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References
• Srilalitha, V.; Prasad, G.; Kumar, R.; Seshagiri, V.; Ravindranath, R. A new spectrophotometric
method for the determination of trace amounts of titanium (IV). FU Phys ChemTech, 2010, 8,
15-24.
• Tinoco Lab. Ti(IV) Colorimetric Assay.
• Nagaosa, Y.; Segawa, S. Reversed phase HPLC determination of titanium (IV) and iron (III) with
sodium 1, 2‐dihydroxybenzene‐3, 5‐disulfonic acid. J High Resolut Chromatogr, 1994, 17, 770-
772.
• Singh, R.; Dhadke, P. Extraction and separation of titanium (IV) with D2EHPA and PC-88A from
aqueous perchloric acid solutions. J. Serb. Chem. Soc., 2002, 67, 507-521.
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