TECHNIQUE: TITANIUM (IV) COLORIMETRIC ASSAY. Presented By: Patricia González Pagán Laboratory Meeting September 27, 2018

Transcription

1 TECHNIQUE: TITANIUM (IV) COLORIMETRIC ASSAY Presented By: Patricia González Pagán Laboratory Meeting September 27, 2018

2 Introduction Titanium (IV) is one of the important constituents of alloys and it s most used form is, titanium dioxide, TiO 2. 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 2

3 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 3

4 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. 4

5 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. 5

6 Instrumentation and Materials Micropipettes 2-20 µl µl 1.5 ml Eppendorf tubes NanoDrop 2000 Spectrophotometer KimWipes Delicate Task Wipes Isotemp Fischer Scientific Vortex Mixer Computer with Microsoft Excel Program Fig. 4 Isotemp Fischer Scientific at FB-143 Fig. 5. KimWipes Delicate Task Wipes 6

7 Methodology 7

8 Methodology 25 mm Heat and vortex to increase solubility. 1.8 M sodium acetate buffer equilibrated to ph mg ligand in 1mL Buffer Sample 125 μm If the sample has a protein 1:1 Boil and sonicate for 5 min. Centrifuge Supernatant is now the sample. 30% trichloroacetic acid (TCA). Sample : TCA Sit and equilibrate for one hour 8

9 Methodology Prepare the samples and standard series using the following. Ti (um) Ligand Ti Stock (um) Ti Stock Sample Sample Buffer 30% TCA Scan the solutions at 370 nm using the NanoDrop 2000 Specrophotometer. 1.8 M Acetate Final Volume Blank Sample

10 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

11 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. 11

12 Data Analysis 12

13 Data Analysis Example 13

14 Alternate Methodology 14

15 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. 25 mm 1.8 M sodium acetate buffer equilibrated to ph mg ligand in 1mL Buffer Heat and vortex to increase solubility. Sample 125 μm 15

16 Alternate Methodology To make the standard series incorporated into the sample, follow the table below. Ti (um) Ligand Ti Stock (um) Ti Stock Sample Sample Buffer 30% TCA 1.8 M Acetate Final Volume Blank Scan the solutions at 370 nm using the NanoDrop 2000 Specrophotometer. 16

17 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. 17

18 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. 18

19 Summary Ti (IV) Assay Characteristics ƛmax= 370 nm ph = 5.2 Method limit = > 100nM (Tecan) Method limit = > 1µM (NanoDrop 2000) 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 Use an Excel worksheet to plot the calibration curve and find the Ti concentration of your samples. 19

20 Other Methods 20

21 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. Refluxing equimolar solutions of acetoacetic acid hydrazide and salicylaldehyde solutions prepared in aqueous methanol for two hours. Fig. 9 Proposed formation of the complex. 21

22 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%. 22

23 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. 23

24 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, 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, Singh, R.; Dhadke, P. Extraction and separation of titanium (IV) with D 2 EHPA and PC-88A from aqueous perchloric acid solutions. J. Serb. Chem. Soc., 2002, 67,

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