Biomaterials Lab 3 Manual Fall 2024 PDF

Biomaterials- ENGR-UH 4810 Fall 2024 Lab 3: Spectroscopy-based Characterization of Hydrogels Professor: Jeremy Teo Instructor: Shafiya Sabah Biomaterials- ENGR-UH 4810 Spectroscopy-based Characterization of Hyd...

Biomaterials- ENGR-UH 4810 Fall 2024 Lab 3: Spectroscopy-based Characterization of Hydrogels Professor: Jeremy Teo Instructor: Shafiya Sabah Biomaterials- ENGR-UH 4810 Spectroscopy-based Characterization of Hydrogels OBJECTIVE To perform spectroscopy-based characterization of hydrogels using Attenuated Total Reflectance Fourier Transform Infrared (ATR FTIR) Spectrometer. INTRODUCTION Infrared, or IR, spectroscopy is a chemical analysis technique that takes advantage of the interaction between infrared light and matter. The atoms in chemical compounds are constantly moving and vibrating in different ways. Each of these vibrations occurs at a different frequency that is unique to the chemical bond and compound. As these frequencies match those of the IR light, chemical compounds can absorb IR light which excites the vibrations in the molecules. If for example we shine IR light through some water, we can use a detector to determine which frequencies of light were absorbed as those frequencies will be “missing” from the original beam of IR light. After the IR light is detected, we can plot the information obtained from the detector to create the IR spectrum. The spectrum shows which frequencies of light were absorbed by the sample and therefore which vibrations were excited when the IR light passed through the sample. As each chemical species will have vibrations at different frequencies, the resulting spectrum of each compound will be unique. This means IR spectroscopy creates a “chemical fingerprint” that can be used to identify and quantify almost any chemical species. FTIR spectroscopy is a widely used spectroscopy technique for biomaterials characterization. FTIR analysis of polymeric biomaterials determines the functional groups in the system. By analysing absorbed or transmitted FTIR light, the molecular structure of the biomaterials can be predicted. The functional groups are necessary for biological response, biocompatibility, and biodegradability. Hence it is important to know the types and amount of these functional groups present in a polymeric biomaterials system. Furthermore, the drug-polymer interaction studies also can be carried out using FTIR and this interaction can be confirmed by disappearance or appearance of the FTIR peaks from drug and the polymer conjugate. 2 Biomaterials- ENGR-UH 4810 Spectroscopy-based Characterization of Hydrogels Figure 1: FTIR spectrum of collagen Figure 1 shows the FTIR spectrum of collagen. The spectrum shows typical amide bands derived from collagen. The peak at ~1600-1700 cm-1 are assigned to amide I derived from the 𝑪 = 𝑶 stretch of the collagen peptide bonds. Peaks at ~1500-1550 cm-1 are assigned to amide II and peaks at ~1200-1300 cm-1 are assigned to amide III due to 𝑵 − 𝑯 deformation. Agarose hydrogel is expected to show peaks associated with polysaccharides. In the range of ~2800-3000 cm-1 peak originates from the stretching vibrations of 𝑪 − 𝑯. Peaks in the range of ~1000-1150 cm-1 are characteristic of 𝑪 − 𝑶 − 𝑪 stretching vibrations, representing the glycosidic linkages in agarose. MATERIALS 1. Agarose hydrogel 2. Collagen hydrogel METHODS 1. Prepare the following hydrogels in 1.5 ml Eppendorf tubes: ✓ Agarose hydrogel: heat agarose powder with water. ✓ Collagen hydrogel: mix collagen gel solution with buffer solution and incubate at 37 ºC. 3 Biomaterials- ENGR-UH 4810 Spectroscopy-based Characterization of Hydrogels Figure 2: Samples for FTIR testing 2. Prepare the FTIR system for testing. Start the OPUS software on the computer next to the instrument. 3. Pipette a drop of water on the crystal. Click the “Background Single Channel” button, the instrument will start scanning the background. The progress status will be shown at the bottom of the screen. Figure 2: BRUKER FTIR system 4. For each hydrogel, carefully take out a small amount of the sample with the help of a spatula and place on the crystal. Run the sample spectrum by clicking on the “Sample Single Channel” button. The progress of the scan can be seen at the bottom of the screen. 4 Biomaterials- ENGR-UH 4810 Spectroscopy-based Characterization of Hydrogels Figure 3: Hydrogel sample on the crystal for FTIR testing RESULTS & DISCUSSION 1. Include the FTIR spectrum of agarose and collagen hydrogels. The y-axis should be labelled as ‘Absorbance’ and the x-axis should be labelled as ‘Wavenumber cm-1’. [Note: Open the. 𝑑𝑝𝑡 files with Notepad, copy paste data to excel and generate plots.] The x-axis should be from 4000 to 400, Right-click on the axis and select Format Axis, In the Format Axis pane, look for an option called Axis Options, Under Axis Options, check the box labelled Values in reverse order.] On the plots mark the characteristic agarose and collagen absorption peaks based on the information provided in the Introduction (page 3) of this manual. 2. Collagen is a protein, and agarose is a polysaccharide. How are the FTIR spectra of these two types of biopolymers expected to be different? 3. At what wavenumbers do the characteristic absorption peaks marked in question 1 occur for both collagen and agarose hydrogels? Do the values match the expected range of values provided in the Introduction (page 3) of this manual. 4. What are some sources of error that may influence the accuracy of recorded data values. 5 Biomaterials- ENGR-UH 4810 Spectroscopy-based Characterization of Hydrogels LAB REPORT Individual report: submit answers to results and discussion as a Pdf format. Cover page should include name, net ID, lab number and title. REFERENCES 1. "Guide to FT-IR Spectroscopy". Bruker. https://www.bruker.com/en/products-and- solutions/infrared-and-raman/ft-ir-routine-spectrometer/what-is-ft-ir-spectroscopy.html 2. S. Pradhan, S. Rajamani, G. Agrawal, M. Dash, S.K. Samal, "7 - NMR, FT-IR and raman characterization of biomaterials", Editor(s): Maria Cristina Tanzi, Silvia Farè, Characterization of Polymeric Biomaterials, Woodhead Publishing, 2017, Pages 147-173, ISBN 9780081007372, 3. C. Liu, Z. Han, and J. T. Czernuszka, "Gradient collagen/nanohydroxyapatite composite scaffold: Development and characterization," Acta Biomaterialia, vol. 5, no. 2, pp. 661-669, 2009. Available: https://doi.org/10.1016/j.actbio.2008.09.022. 4. Chang MC, Tanaka J. "FT-IR study for hydroxyapatite/collagen nanocomposite cross-linked by glutaraldehyde". Biomaterials. 2002 Dec;23(24):4811-8. doi: 10.1016/s0142- 9612(02)00232-6. 5. Jarosz, A.; Kapusta, O.; Gugała-Fekner, D.; Barczak, M. "Synthesis and Characterization of Agarose Hydrogels for Release of Diclofenac Sodium". Materials 2023, 16, 6042. https://doi.org/10.3390/ma16176042 6