TECHNIQUE: TITANIUM (IV) COLORIMETRIC

ASSAYPresented By: Patricia González Pagán

Laboratory Meeting

September 27, 2018

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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